Journal of applied horticulture Vol 17 issue 2, 2015

Page 1

Journal of

ISSN 0972-1045 Vol. 17, No. 2, May-September, 2015

Applied

Appl Hort

Horticulture

Journal of THE SOCIETY FOR ADVANCEMENT OF HORTICULTURE

Journal

JOURNAL OF APPLIED HORTICULTURE Vol. 17, No. 2, May-September, 2015

Appl

CONTENTS Cloning and characterisation of APETALA3-like and PISTILLATA-like B class MADS-box genes from sweet cherry —Kenji Beppu, Hidemi Sumida and Ikuo Kataoka (Japan)

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165

Journal

Journal of Applied Horticulture, 17(2): 87-91, 2015

Appl

Cloning and characterisation of APETALA3-like and PISTILLATAlike B class MADS-box genes from sweet cherry Kenji Beppu*, Hidemi Sumida and Ikuo Kataoka Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan. *E-mail: beppuk@ag.kagawa-u.ac.jp

Abstract We isolated APETALA3 (AP3)-like and PISTILLATA (PI)-like cDNA clones called PaTM6 and PaPI from sweet cherry (Prunus avium). PaTM6 showed very high similarity to the TM6 lineage of AP3 of other Rosaceae species. PaTM6 contained three amino acid residues (F, T, M) within the MADS box and the (H/Q)YExM sequence near the K box, both of which are characteristic of the AP3 subfamily. A paleo AP3 motif was present at the C-terminal end of PaTM6. PaPI showed very high similarity to PI of other Rosaceae species. PaPI had the serine residue and the KHExL sequence within the MADS box and near the K box, respectively, both of which are characteristic of the PI subfamily. A PI motif was present at the C-terminal end of PaPI. Both PaTM6 and PaPI JHQHV ZHUH H[SUHVVHG VSHFLÂżFDOO\ LQ petals and stamens, the same expression patterns as those of class B MADS-box genes. These results indicated that PaTM6 and PaPI are homologues of AP3 and PI, respectively. Key words: AP3, class B gene, double pistils, PaPI, PaTM6, PI, Prunus avium

,QWURGXFWLRQ Recently, attempts have been made to produce sweet cherry in southwestern Japan to harvest fruits earlier than in the northern major production areas and to supply local markets. In this region, however, the occurrence of double fruits is a major problem (Beppu and Kataoka, 2011). This malformation is due to abnormal differentiation of pistil primordia in the previous growing season (Philp, 1933; Tucker, 1934). We demonstrated, under controlled conditions that the occurrence of double pistils in ‘Satohnishiki’ markedly increased when the trees were exposed to temperatures above 30 °C (Beppu and Kataoka, 1999). Furthermore, extremely high temperature induced the formation of not only of double pistils but also of pistil-like appendages that replaced anthers (Beppu and Kataoka, 1999; Ryugo, 1988). Thus, the B and C classes of homeotic genes, which control stamen and pistil formation, may be involved in these phenomena. Generally, the class B and C genes together induce the formation of stamens, and the class C gene is required for the formation of pistils (Weigel and Meyerowitz, 1994). To evaluate the transcriptional levels of class B and C MADS-box genes of sweet cherry grown at different temperatures, these JHQHV PXVW ÂżUVW EH LVRODWHG ,Q WKH 5RVDFHDH WKH IDPLO\ WR ZKLFK sweet cherry belongs, the class B and C genes of rose and apple have been cloned (Kitahara and Matsumoto, 2000; Kitahara et al., 2001, 2004; Yao et al., 2001; Linden et al., 2002). Based on their sequences, we previously isolated the AGAMOUS–like C class genes of sweet cherry (Beppu et al., 2015). In this study, we isolated the AP3-like and PI-like B class MADSbox genes of sweet cherry and evaluated their expression in each RUJDQ RI WKH Ă€RZHU

Materials and methods Isolation of class B MADS box gene: RNA isolation: Flower buds of ‘Satohnishiki’ sweet cherry were collected on 29 July,

when the buds were the most sensitive to high temperature inducing double pistil formation (Beppu et al., 2001). They were stored at -80 °C until use. Total RNA was isolated from J SODQW PDWHULDO E\ WKH PRGLÂżHG FHW\OWULPHWK\ODPPRQLXP bromide (CTAB) method, as described by Kotoda et al. (2000). 2QH PLFURJUDP RI WKH WRWDO 51$ ZDV XVHG IRU ÂżUVW VWUDQG F'1$ synthesis by SuperScript II RT (Life Technologies, MD) with an adapter-dT primer (5’CGA CGT TGT AAA ACG ACG GCC AGT TTT TTT TTT TTT TTT -3’) consisted of M13-20 sequence primer and oligo (dT)16. Cloning of homologues by PCR: We used a combination of UHYHUVH WUDQVFULSWLRQ 57 3&5 DQG UDSLG DPSOLÂżFDWLRQ RI F'1$ ends (RACE) techniques to isolate AP3 and PI homologues. First, partial sequences of AP3 and PI homologues were isolated by reverse RT-PCR with AP3-F1-ra (5’-CCA GAC SAA CAG GCA GGT GAC CTA-3’)/ AP3-R2-ra (5’-AGA TGA GTR ATG GAG GAG-3’) and PI-F1-ra (5’-CTC AAG YAA CAG GCA GGT GAC CTA-3’)/ PI-R2-ra (5’-TGG AGA TTT GGC TGA WTA GGC-3’). These primers were designed from conserved regions of AP3 and PI in Rosaceae [rose (Kitahara and Matsumoto, 2000; Kitahara et al., 2001) and apple (Yao et al., 2001; Linden et al., 2002; Kitahara et al., 2004)], respectively. PCR was performed using a programme of 30 cycles at 94 °C for 30 sec, 52 °C for 30 sec, and 72 °C for 1 min with an initial GHQDWXULQJ RI ƒ& IRU PLQ DQG D ÂżQDO H[WHQVLRQ RI ƒ& IRU 7 min. The PCR reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM each of dNTPs, 400 nM each of primers, 0.1 mg of template cDNA, and 1 unit of TaKaRa Ex Taq polymerase (Takara Shuzo Co., Shiga, Japan) in a 50 mL reaction volume. PCR products were subcloned into the TA cloning vector (pGEM-T Easy Vector System; Promega, WI, USA). Nucleotide sequences of several clones were determined with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) and the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan).

88

Cloning of sweet cherry class B MADS-box genes

3’- and 5’-RACE to obtain full length clones were performed. For 3’ RACE of AP3 and PI homologues, Pa-AP3-F5 (5’-AAG GTG AAG AAC TTG GAG GAAAGA-3’) and Pa-PI-F7 (5’-TAT GAG CTG CAC AAA CAG GAG ATG A-3’) primers were used, respectively, with M13-20 primer as the adapter primer. These primers were designed from the sequences obtained by RT-PCR described above. PCR condition for 3’ RACE and TA cloning was same as RT-PCR described above. 5’ RACE was carried out using a 5’-Full RACE Core Set (Takara Shuzo). First, the reaction mixtures were prepared according to the manufacture instructions. 7KHVH PL[WXUHV ZHUH IRU ÂżUVW VWUDQG F'1$ V\QWKHVLV E\ 57 XVLQJ the 5’-phosphorylated RT-primer Pa-AP3-P (5’-CAA CCG CAG ATT CAT-3’) and Pa-PI-P (5’-GGC AAA CGG TAT CTG-3’). The conditions of the RT reaction were 10 min at 30 °C, 60 min at 50 ƒ& PLQ DW ƒ& IROORZHG E\ FRROLQJ ƒ& 7KH F'1$ ÂżUVW VWUDQG was self-ligated by T4 RNA ligase according to the manufacture LQVWUXFWLRQV )RU WKH ÂżUVW DQG VHFRQG 3&5V RI WKH VHOI OLJDWHG F'1$ ÂżUVW VWUDQG WZR VHWV RI SULPHUV ZHUH GHVLJQHG )RU WKH ÂżUVW PCR the primers were Pa-AP3-S1 (5’-GGAAGT ACC ACG TGA TCAAA-3’) and Pa-AP3-A1 (5’-TCA TGA CCC AAC CTC TGC CT-3’), and Pa-PI-S1 (5’-ACAAGC AGT CCAAGT TCG TC-3’) and Pa-PI-A1 (5’-ATT CAA CCA TCT TTC CAG AG-3’). For the second PCR the primers were Pa-AP3-S2 (5’-GAA CTT GGA GGA AAG AAG AG-3’) and Pa-AP3-A2 (5’-GTA GGG CTA ATA TAC TCG TG-3’), and Pa-PI-S2 (5’-GAG CAT AAG CGC CTC ACT TA-3’) and Pa-PI-A2 (5’-CTT AAT GAT CCC ATT CCT CC Âś 7KH UHDFWLRQ PL[WXUHV RI WKH ÂżUVW DQG VHFRQG 3&5V were prepared according to the manufacturer’s instructions. The ÂżUVW 3&5 ZDV SHUIRUPHG XVLQJ D SURJUDPPH RI F\FOHV DW °C for 30 sec, 54 °C for 30 sec, and 72 °C for 1 min with an initial GHQDWXULQJ RI ƒ& IRU PLQ DQG D ÂżQDO H[WHQVLRQ RI ƒ& IRU min. The second PCR was performed similarly except the cycle number (30 cycles). The second PCR products were subcloned into the TA cloning vector and sequenced as described above. Comparison and phylogenetic analysis: The deduced amino acid sequences of AP3 homologue genes of apple (MdTM6, Accession number AB081093)㸪Barberton daisy (GDEF1, AJ009724)㸪birthwort (AeAP3-1, AF230697), grape (VvTM6, DQ979341; VvAP3, EF418603)㸪hydrangea (Hydrangea TM6, AF230703)㸪mandarin (CitMADS8; AB218614)㸪petunia (Petunia TM6, AF230704; pMADS1, X69946)㸪 poplar (LtAP3, AF052878), potato (STDEF, X67511)㸪 rapeseed (BAP3, AF124814)㸪 rose (MASAKO B3㸪AB055966; MASAKO euB3, AB099875)㸪 sweet cherry (PaTM6, AB763909, this study)㸪tobacco (NTDEF, X96428)㸪tomato (Lycopersicon TM6㸪X60759; LeAP3, AF052868) and wintersweet (CfAP31, AF230699) were aligned using the program Clustal W. Those of PI homologue genes of apple (MdPI, AJ291490), Barberton daisy (GGLO1, AJ009726),cucumber (CUM26, AF043255)㸪grape (VvPI, DQ988043)㸪 peach (PpMADS10, EU005663)㸪petunia (pMADS2, X69947)㸪 rose (MASAKO BP, AB038462)㸪 sweet cherry (PaPI, AB763910, this study) and thale cress (PI, D30807) were also aligned. Expression analysis of class B MADS box gene: Floral organs, sepals, petals, stamens and pistils and young leaves were collected at anthesis. They were stored at -80°C until use. Total RNA was isolated as described previously (Beppu et al DQG ÂżUVW strand cDNA synthesis was done as described above.

Based on the sequences of AP3-like and PI-like genes obtained DERYH VSHFL¿F SULPHU VHWV IRU HDFK JHQH ZHUH PDGH 57 3&5 was performed with the primers, AP3-RT-F (5’-GTA AAA TGC ACG AGT ATA TT-3’) and AP3-RT-R (5’-GTG GAT CCT CAG AGG CTA AT-3’) for AP3 like gene, PI-RT-F (5’-GAA AGA TGG TTG AAT ACT GC-3’) and PI-RT-R (5’-TCA TCT CCT GTT TGT GCA GC-3’) for PI like gene. Actin was used as the RT-PCR control (Yamane et al., 2003). PCR was performed as described above. The PCR products were run in 1% (w/v) agarose gels for 0.5 h at 100 V using the Mupid 2 electrophoresis system (Advance Co. Ltd., Tokyo, Japan).

5HVXOWV DQG GLVFXVVLRQ Isolation of class B MADS-box gene: The deduced amino acid sequences of Prunus avium TM6 (PaTM6) (DDBJ/EMBL/ GenBank Accession Nos. AB763909) are shown in Fig. 1 with those of other Rosaceae species. PaTM6 was 708 bp in length with an open reading frame corresponding to 235 amino acid residues. The deduced amino acid sequence of PaTM6 contained a MADS box and a K box. The MADS-box domain of PaTM6 spanned amino acids 1 to 60 and was a highly conserved region, whereas the K-box domain spanned amino acids 89 to 154 and was a moderately conserved domain. Three of four characteristic amino acid residues: phenylalanine, threonine and methionine, within the MADS box of the DEF (= AP3) subfamily (TheiĂ&#x;en et al., 1996) were represented by amino acid residues 29, 36 and 47. A highly conserved sequence of AP3 homologues (H/Q)YExM (Kramer et al., 1998) was present in the corresponding region with a slight divergence (amino acid residues 83–87). These results suggest that PaTM6 is a sweet cherry homologue of AP3. AP3 homologue genes in higher eudicots have been divided into two groups, the TM6 and euAP3 lineages (Kramer et al., 1998). The PaTM6 protein showed high similarity to proteins known to correspond to TM6 lineage members, in particular MdTM6 of apple (Malus domestica) (85%) and MASAKO B3 of rose (Rosa rugosa) (76%), both of which, like sweet cherry, are Rosaceae species (Fig. 2). PaTM6 had the classical TM6 motif: the paleo AP3 motif (YGxHDLRLA) (Kramer et al., 1998) in the C-terminal region (amino acid residues 227–235). These results indicated that PaTM6 belongs to the TM6 lineage.ŕ ‰ The deduced amino acid sequences of P. avium PISTILLATA (PaPI) (DDBJ/EMBL/GenBank Accession Nos. AB763910) are shown in Fig. 3 with those of other Rosaceae species. PaPI was 633 bp in length with an open reading frame corresponding to 210 amino acid residues. The deduced amino acid sequence of PaPI also contained a well-conserved MADS domain from amino acids 1–59 and a conserved K box from amino acids 89–150. A characteristic amino acid residue, serine, within the MADS box of the GLO (=PI)-subfamily (TheiĂ&#x;en et al., 1996) was LGHQWLÂżHG DPLQR DFLG UHVLGXH $ KLJKO\ FRQVHUYHG VHTXHQFH of PI homologues, KHExL (Kramer et al., 1998), was present in the corresponding region (amino acid residues 83–87). PaPI carried the PI motif (MPFxFRVQPxQPNLQE) in the C-terminal region between amino acid residues 193 and 208. This motif is present in almost all described PI type proteins (Kramer et al., 1998). PaPI protein had high similarity to proteins known to correspond to PI subfamily members (Table 1). In particular, PaPI showed the highest similarity to PpMADS10 of peach (P.

Cloning of sweet cherry class B MADS-box genes

89

Amino acids within the MADS-box characteristic of APETALA3-subfamily PaTM6 MdTM6 MdMADS13 MASAKO B3

MGRGKIEIKLIENHTNRQVTYSKRRNGIFKKAQELTVLCDARVSLIMLSNTGKMHEYISP MGRGKIEIKLIENQTNRQVTYSKRRNGIFKKAQELTVLCDAKVSLIMLSNTSKMHEYISP MGRGKIEIKLIENQTNRQVTYSKRRNGIFKKAQELTVLCDAKVSLIMLSNTNKMHEYISP MGRGKIEIKLIENQTNRQVTYSKRRNGIFKKAQELTVLCDAQVSLIMQSSTDKIHEYISP ************* *************************** ***** * * * ****** MADS-box region

60 60 60 60

PaTM6 MdTM6 MdMADS13 MASAKO B3

TTTTKRMYDDYQKTMGVDLWSSHYQAMKDTLWKLKEINNKLRREIRQRLGHDLNGLTHAQ TTTTKSMYDDYQKTMGIDLWRTHYESMKDTLWKLKEINNKLRREIRQRLGHDLNGLSYDD TTTTKSMYDDYQKTMGIDLWRTHEESMKDTLWKLKEINNKLRREIRQRLGHDLNGLSFDE TTTTKKMFDLYQKNLQIDLWSSHYEAMKENLWKLKEVNNKLRRDIRQRLGHDLNGLSYAE ***** * * *** (H/Q)YExM sequence K -box region

120 120 120 120

3D70 /56/('.0$66/(9,5(5.<+9,.747(76...9.1/((55*10/+*<̻̻̻ELASEDPQY 177 0G70 /56/('.0466/'$,5(5.<+9,.747(77...9.1/((55*10/+*<̻̻̻EAASENPQY 177 MdMADS13 LASLDDEMQSSLDAIRQRKYHVIKTQTETTKKKVKNLEQRRGNMLHGYFDQEAAGEDPQY 180 0$6$.2 % /4'/((7064694,,5'5.<+9/.74$(775..9.1/((5161/0+*<*̻̻APGNEDPQY 178 * * * * ** ***** *** ** ******* * * *** * *** 3D70 *<991(*(<(6$9$/$1*$61/)7,+/+4',5̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻'+6 0G70 &<9'1(*'<(6$/9/$1*$11/<7)4/+516'4/̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻++3 0G0$'6 *<('1(*'<(6$/$/61*$11/<7)+/+̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻̻+5 MASAKO B3 GYVDNEGDYESAVALANGASNLYFFNRVHNNHNLDHGHGGGSLVSSITHLQNPNNHGNHN 238 * *** **** * *** ** * PaTM6 NLHHHGGSSLGSSITHLHDLRLA MdTM6 NLHHHRGSSLGSSITHLHDLRLA 0G0$'6 1/++*̻*66/*66,7+/+'/5/$ MASAKO B3 LENGHGGGSLISSITHLHDLRLA * ** ************ paleo-AP3 motif

235 237 232 261

Fig. 1. Deduced amino acid sequences of AP3 homologue genes in Rosaceae species, sweet cherry [PaTM6 (this study)], apple [MdTM6 (Kitahara et al., 2004), MdMADS13 (van der Linden et al., 2002)] and rose [MASAKO B3 (Kitahara and Matsumoto, 2000)].

persica) (97%), in the same genus as sweet cherry, followed by MdPI of apple (88%). These results suggest that PaPI is a sweet cherry homologue of PI.

1994). The detection of PaTM6 and PaPI transcripts in petals and stamens supports the hypothesis that PaTM6 and PaPI are homologues of AP3 and PI, respectively.

Expression analysis of class B MADS-box gene: In Arabidopsis, expressions of AP3 and PI LQ PDWXUH ÀRZHUV DUH UHVWULFWHG WR petals and stamens (Jack et al., 1992; Goto and Meyerowitz, 1994). The same expression patterns have been reported in Rosaceae species such as rose and apple (Kitahara et al., 2001; Linden et al., 2002). In the present study, the expression patterns of PaTM6 and PaPI were characterised by RT-PCR analysis. )UDJPHQWV RI WKH H[SHFWHG VL]H ES ZHUH DPSOL¿HG E\ 57 PCR from petals and stamens with AP3-RT-F and AP3-RT-R primers (Fig. 4). RT-PCR with PI-RT-F and PI-RT-R primers also DPSOL¿HG D IUDJPHQW RI WKH H[SHFWHG VL]H ES IURP SHWDOV and stamens. No RT-PCR fragment was observed in leaves, sepals and pistils with these primer sets. These results suggested that both PaTM6 and PaPI JHQHV ZHUH H[SUHVVHG VSHFL¿FDOO\ LQ SHWDOV and stamens, thus displaying the same expression patterns as AP3 and PI, respectively (Jack et al., 1992; Goto and Meyerowitz,

Based on sequence similarity and tissue-specific expression patterns, PaTM6 and PaPI were inferred to be homologues of APETALLA3 and PISTILLATA, respectively. Next we plan to analyse transcription levels of the class B genes PaTM6 and PaPI and the class C genes PaAG and PaSHP (Beppu et al., 2015) in WKH ÀRZHU EXGV RI VZHHW FKHUU\ JURZQ DW GLIIHUHQW WHPSHUDWXUHV with the aim of clarify their involvement in pistil doubling.

Acknowledgement This work was partially supported by JSPS KAKENHI Grant Number 23580041.

References Beppu, K. and I. Kataoka, 1999. High temperature rather than drought stress is responsible for the occurrence of double pistils in `Satohnishiki’ sweet cherry. Sci. Hort., 81: 125-134.

90

Cloning of sweet cherry class B MADS-box genes

PaTM6 MdTM6 MASAKO-B3 HydrangeaTM6 TM6 lineage

VvTM6 PetuniaTM6 LycopersiconTM6 GDEF1 STDEF LeAP3 pMADS1 NTDEF CitMADS8

euAP3 lineage

VvAP3 BAP3 MASAKOeuB3 CfAP3-1 LtAP3

paleoAP3 lineage

AeAP3-1 0.1

Fig. 2. Phylogenic trees of the deduced amino acid sequences of PaTM6 (box) and other AP3 homologues (see Materials and methods).

Serine residue within the MADS-box characteristic of PISTILLATA -subfamily PaPI PpMADS10 MdPI MASAKO BP

MGRGKIEIKRIENSSNRQVAYSKRRNGIIKKAKEITVLCDAKVSLVIFASS*.09(<&6̻ MGRGKIEIKRIENSSNRQVTYSKRRNGIIKKAKEITVLCDAKVSLVIFASS*.09(<&6̻ MGRGKVEIKRIENSSNRQVTYSKRRNGIIKKAKEITVLCDAKVSLIIYSSS*.09(<&6̻ MGRGKIEIKRIENSSNRQVTYSKRKNGIIKKAKEITVLCDAKVSLIIIASSGKMVEYCSG 60 ***** ************* **** ******************** * ********** MADS-box region

PaPI PpMADS10 MdPI MASAKO BP

PSVTVTDILDKYHGQAGKKLWDAKHENLSNEVDRVKKDNDSMQVELRHLKG(',76/7+. PSVTVTDILDKYHGQAGKKLWDAKHENLSNEVDRVKKDNDSMQVELRHLKG(',76/7+. PSTTLTEILDKYHGQSGKKLWDAKHENLSNEVDRVKKDNDSMQVELRHLKG(',76/1+9 PQETRMKILDKYHSQSGKRLWDAKHENLCNEVDRVKKDNDGMQIELRHLKGEDITSLNHV 120 * * ****** * ** ********* *********** ** ************* * KHExL sequence K-box region

3D3, (/0$/(($/(1*/$615'.46.)9*0/,(1*5$/(((+.5/7<(/+.̻4(0KIEENVREL 178 3S0$'6 (/0$/(1$/(1*/$615'.46.)9'0/5(1(5$/(((+.5/7<(/+.̻4(0KIEENVREL 178 MdPI ELMALEEALENGLTSIRDKQSKFVDMMRDNGKALEDENKRLTYELQKQQEM.,.(19510 0$6$.2 %3 '/0$/(($,(1*/$6,5'506.<0'$95(115$/('(1.5/$<4/+.̻​̻00KSEENLRDM 178 ***** * **** * ** ** * *** * *** * * * ** ** * 3D3, (1*<+45/*̻​̻​̻​̻1<114,3)$)5943,431/4(50 3S0$'6 (1*<545/*̻​̻​̻​̻1<114,3)$)5943,431/4(50 MdPI ENGYHQRQLGNYNNNQQQIPFAFRVQPIQPNLQERI 0$6$.2 %3 1̻​̻​̻​̻​̻​̻​̻​̻​̻​̻​̻<11174,3)$/59431431/+'50 * ***** **** **** * PI motif

210 210 215 203

Fig. 3. Deduced amino acid sequences of PI homologue genes in Rosaceae species, sweet cherry [PaPI (this study)], peach [PpMADS10 (Zhang et al., 2008)], apple [MdPI (Yao et al., 2001)] and rose [MASAKO BP (Kitahara and Matsumoto, 2000)].

Cloning of sweet cherry class B MADS-box genes

91

Table 1. Identities of deduced amino acid sequences of PaPI and other PI homologues (see Materials and methods) (%) PpMADS10 MdPI MASAKO-BP VvPI CUM26 pMADS2 GGLO1

PI

PaPI PpMADS10 MdPI

97

88

76

76

75

70

63

59

88

77

76

76

70

65

60

78

72

74

68

67

62

66

69

66

62

54

77

70

69

59

72

72

60

71

59

MASAKO-BP VvPI CUM26 pMADS2 GGLO1

Fig. 4. RT-PCR analyses of PaTM6 and PaPI genes in different tissues. Lane 1, leaves; 2, sepals; 3, petals; 4, stamens; 5, pistils. Beppu, K. and I. Kataoka, 2011. Studies on pistil doubling and fruit set of sweet cherry in warm climate. J. Jpn. Soc. Hort. Sci., 80: 1-13. Beppu, K., T. Ikeda and I. Kataoka, 2001. Effect of high temperature H[SRVXUH WLPH GXULQJ Ă€RZHU EXG IRUPDWLRQ RQ WKH RFFXUUHQFH RI double pistils in ‘Satohnishiki’ sweet cherry. Sci. Hort., 87: 77-84. Beppu, K., H. Yamane, H. Yaegaki, M. Yamaguchi, I. Kataoka and R. Tao, 2002. Diversity of S-RNase genes and S-haplotypes in Japanese plum (Prunus salicina Lindl.). J. Hort. Sci. Biotech., 77: 658-664. Beppu, K., H. Sumida and I. Kataoka, 2015. Sweet cherry MADS-box genes ‘PaAG’ and ‘PaSHPÂś KRPRORJRXV WR FODVV & Ă€RUDO LGHQWLW\ genes. Acta Hort., (In press). Goto, K., E.M. Meyerowitz, 1994. Function and regulation of the Arabidopsis Ă€RUDO KRPHRWLF JHQH PISTILLATA. Genes Dev., 8: 1548-1560. Jack, T., L.L. Brockman and E.M. Meyerowitz, 1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell, 68: 683-697. Kitahara, K. and S. Matsumoto, 2000. Rose MADS-box genes ‘MASAKO C1 and D1Âś KRPRORJRXV WR FODVV & Ă€RUDO LGHQWLW\ JHQHV Plant Sci., 151: 121-134.

59 Kitahara, K., S. Hirai, H. Fukui and S. Matsumoto, 2001. Rose MADSbox genes ‘MASAKO BP and B3Âś KRPRORJRXV WR FODVV % Ă€RUDO identity genes. Plant Sci, 161: 549-557. Kitahara, K., T. Ohtsubo, J. Soejima and S. Matsumoto, 2004. Cloning and characterization of Apple Class B MADS-box genes including a novel AP3 homologue MdTM6. J. Jpn. Soc. Hort. Sci., 73: 208-215. Kotoda, N., M. Wada, S. Komori, S. Kidou, K. Abe, T. Masuda and J. 6RHMLPD ([SUHVVLRQ SDWWHUQ RI KRPRORJXHV RI Ă€RUDO PHULVWHP identity genes LFY and AP1 GXULQJ Ă€RZHU GHYHORSPHQW LQ DSSOH J. Amer. Hort. Sci., 125: 398-403. Kramer, E.M., R.L. Dorit and V.F. Irish, 1998. Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics, 149: 765-783. Van der Linden, C.G., B. Vosman and M.J.M. Smulders, 2002. Cloning and characterization of four apple MADS box genes isolated from vegetative tissue. J. Exp. Bot., 53: 1025-1036. Philp, G.L. 1933. Abnormality in sweet cherry blossoms and fruit. Bot. Gaz., 94: 815-820. Ryugo, K. 1988. Fruit Culture - Its Science and Art. Wiley, New York. 7KHL‰HQ * - 7 .LP DQG + 6DHGOHU &ODVVLÂżFDWLRQ DQG SK\ORJHQ\ RI WKH 0$'6 ER[ PXOWLJHQH IDPLO\ VXJJHVW GHÂżQHG UROHV RI 0$'6 box gene subfamilies in the morphological evolution of eukaryotes. J. Mol. Evol., 43: 484-516. Tucker, L.R. 1934. Notes on sweet cherry doubling. Proc. Amer. Soc. Hort. Sci., 32: 300-302. :HLJHO ' DQG ( 0 0H\HURZLW] 7KH $%&V RI Ă€RUDO KRPHRWLF genes. Cell, 78: 203-209. Yamane, H., K. Ikeda, K. Ushijima, H. Sassa and R. Tao, 2003. A pollenexpressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry, Prunus cerasus and P. avium. Plant Cell Physiol., 44: 764-769. Yao, J.L., Y.H. Dong and B.A.M. Morris, 2001. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADSbox transcription factor. Proc. Natl. Acad. Sci. USA, 98: 1306-1311. Submitted: November, 2014; Revised: February, 2015; Accepted: March, 2015

.

Journal

Journal of Applied Horticulture, 17(2): 92-95, 2015

Appl

7KUHH FULWHULD IRU FKDUDFWHUL]LQJ Ă RZHU RSHQLQJ SURĂ€OHV DQG GLVSOD\ YDOXHV LQ FXW VSUD\ W\SH FDUQDWLRQ Ă RZHUV So Sugiyama, Shigeto Morita1 and Shigeru Satoh1* Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan. Kyoto Prefectural Institute of Agricultural Biotechnology, Seika Town, Kyoto 619-0224, Japan. *E-mail: ssatoh@agr.ryukoku.ac.jp 1

Abstract 3UHYLRXVO\ ZH KDYH GHYHORSHG D PHWKRG ZKLFK XVHV WZR FULWHULD ¾WLPH WR ÀRZHU RSHQLQJœ DQG ¾YDVH OLIHœ IRU FKDUDFWHUL]LQJ ÀRZHU RSHQLQJ SUR¿OHV LQ FXW VSUD\ W\SH ÀRZHUV RI FDUQDWLRQ 7KHVH WZR FULWHULD ZHUH XVHG WR HYDOXDWH WKH DFWLYLWLHV RI ÀRZHU SUHVHUYDWLYHV ZKLFK DFFHOHUDWH ÀRZHU EXG RSHQLQJ UHVXOWLQJ LQ VKRUWHQLQJ WKH WLPH WR ÀRZHU RSHQLQJ DQG GHOD\ VHQHVFHQFH UHVXOWLQJ LQ H[WHQVLRQ RI YDVH OLIH ,Q WKH SUHVHQW VWXG\ ZH GHYHORSHG WKH WKLUG FULWHULRQ ¾JURVV ÀRZHU RSHQLQJœ ZKLFK FKDUDFWHUL]HV WKH DELOLW\ RI ÀRZHU buds to open. Using this criterion the activity of analogs of pyridinedicarboxylic acids was successfully evaluated in addition to the SUHYLRXVO\ UHSRUWHG HYDOXDWLRQ RI WKHLU DFWLYLW\ RI DFFHOHUDWLRQ RI ÀRZHU EXG RSHQLQJ DQG H[WHQVLRQ RI YDVH OLIH Key words: Flower bud opening, display value, pyridinedicarboxylic acid, senescence, spray-type carnation, vase life.

,QWURGXFWLRQ &DUQDWLRQ LV RQH RI WKH PRVW SRSXODU FXW ÀRZHUV DQG RI KLJKHVW economic importance in the floriculture industry in many FRXQWULHV &XW ÀRZHUV RI FDUQDWLRQ DUH XVHG LQ WZR IRUPV RU categories, i.e., the standard type in which carnations have one ÀRZHU RQ D VWHP DQG WKH VSUD\ W\SH LQ ZKLFK FDUQDWLRQV KDYH PXOWLSOH ÀRZHUV RQ D VWHP ,Q UHFHQW \HDUV VSUD\ W\SH FDUQDWLRQ ÀRZHUV KDYH EHFRPH SRSXODU EHFDXVH WKH\ FDQ EH JURZQ ZLWK less labor and meet modern consumer’s demand. Carnation FXOWLYDUV GLIIHU LQ WKH OHQJWK RI WKH YDVH OLIH RI FXW ÀRZHUV 1XNXL et al., 2004), which is one of the characteristics determining the FRPPHUFLDO YDOXH RI RUQDPHQWDO ÀRZHUV 8VXDOO\ WKH YDVH OLIH RI FDUQDWLRQ ÀRZHU KDV EHHQ GHWHUPLQHG E\ REVHUYLQJ VHQHVFHQFH SUR¿OHV i.e., in-rolling of petal margin and wilting of whole petals as well as ethylene production. This method has been XVHG VXFFHVVIXOO\ IRU FXW FDUQDWLRQ ÀRZHUV RI WKH VWDQGDUG W\SH +RZHYHU LQ VSUD\ W\SH FDUQDWLRQ ÀRZHUV WKH YDVH OLIH RI WKH ÀRZHUV LV GHWHUPLQHG E\ WKH VXP RI WKH ÀRZHULQJ SHULRG RI HDFK ÀRZHU ZKLFK UHTXLUHG GHYHORSPHQW RI DQRWKHU PHWKRG GLIIHUHQW IURP WKDW IRU WKH VWDQGDUG W\SH ÀRZHUV

VWDQGDUG W\SH Ă€RZHUV RI Âľ:KLWH 6LPÂś FDUQDWLRQ 6DWRK et al. FRQÂżUPHG WKDW XVLQJ WKH DERYH GHVFULEHG PHWKRG IRU GHWHUPLQLQJ WKH YDVH OLIH RI FXW VSUD\ W\SH FDUQDWLRQ Ă€RZHUV 3'&$ OHQJWKHQHG WKH YDVH OLIH RI FXW VSUD\ W\SH Ă€RZHUV RI ‘Light Pink Barbara (LPB)’ carnation, as well as it inhibited ACC oxidase action using a recombinant enzyme produced in E. coli cells from a carnation ACC oxidase gene (DcACO1). Sugiyama and Satoh (2015) revealed that PDCA analogs in addition to 3'&$ FRXOG DFFHOHUDWH Ă€RZHU RSHQLQJ RI Âľ/3%Âś FDUQDWLRQ which was demonstrated by observing the shortened time to Ă€RZHU RSHQLQJ LQ DGGLWLRQ WR WKH SUHYLRXVO\ VKRZQ H[WHQVLRQ RI YDVH OLIH )RU FKDUDFWHUL]LQJ WKH DFFHOHUDWLRQ RI Ă€RZHU RSHQLQJ E\ 3'&$V WKH\ GHYHORSHG DQRWKHU FULWHULRQ ÂľWLPH WR Ă€RZHU opening’, which was determined by the number of days from the start of the experiment until the time when the percentage of IXOO\ RSHQ DQG QRQ VHQHVFHQW Ă€RZHUV UHDFKHG 7KLV VHFRQG criterion was successfully used to describe the activity of PDCA DQDORJV WR DFFHOHUDWH Ă€RZHU EXG RSHQLQJ 6XJL\DPD DQG 6DWRK 2015). Interestingly, moreover, Sugiyama and Satoh have noticed at the same time that PDCA analogs markedly increased the gross QXPEHU RI RSHQ Ă€RZHUV XQSXEOLVKHG UHVXOWV 7KH LQFUHDVH ZDV SUREDEO\ FDXVHG E\ WKH HOHYDWLRQ RI DELOLW\ RI Ă€RZHUV EXGV WR RSHQ E\ 3'&$ DQDORJV DV ZHOO DV E\ WKH DFFHOHUDWLRQ RI Ă€RZHU (bud) opening and the delay of onset of senescence. Therefore, in the present study, we aimed to establish a method to determine WKH DELOLW\ RI Ă€RZHU EXGV WR RSHQ DQG WR HYDOXDWH 3'&$ DQDORJV LQ HOHYDWLQJ WKH DELOLW\ RI Ă€RZHU EXGV LQ FXW VSUD\ W\SH Ă€RZHUV of carnation.

Previously, Satoh et al.(2005) established a method to determine WKH YDVH OLIH RI VSUD\ W\SH FDUQDWLRQ ÀRZHUV E\ REVHUYLQJ WKH FKDQJH LQ WKH SHUFHQWDJH RI RSHQ ÀRZHUV WR WKH WRWDO QXPEHU RI LQLWLDO ÀRZHU EXGV ,Q PRUH GHWDLO WKH YDVH OLIH LQ GD\V ZDV determined by the number of days during which the percentage of IXOO\ RSHQ DQG QRQ VHQHVFHQW ÀRZHUV ZDV RU PRUH 7KH YDVH life determined by this method was successfully used to evaluate the action of preservatives, such as sucrose and 1,1-dimethyl SKHQ\OVXOIRQ\O VHPLFDUED]LGH RQ FXW VSUD\ W\SH ÀRZHUV RI carnation.

Materials and methods

Recently, Vlad et al. (2010) reported that 2,4-pyridinedicarboxylic acid (2,4-PDCA) could suppress ethylene production from FDUQDWLRQ ÀRZHUV E\ LQKLELWLQJ WKH DFWLRQ RI DPLQRF\FORSURSDQH 1-carboxylate (ACC) oxidase, and prolong the vase life of cut

Original experiments and data: Data used for analysis of gross flower opening, which was defined in the present study and described later, came from those obtained previously and shown in two separate papers, Satoh et al. (2014) and Sugiyama and

7KUHH FULWHULD IRU FKDUDFWHUL]LQJ ÀRZHU RSHQLQJ SUR¿OHV DQG GLVSOD\ YDOXHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV 6DWRK %ULHÀ\ D carnation cultivar, Dianthus caryophyllus L. ‘Light Pink Barbara (LPB)’, which blooms spray-type ÀRZHUV ZDV XVHG 7UHDWPHQW RI FDUQDWLRQ ÀRZHUV ZLWK DQDORJV of pyridinedicarboxylic acid (PDCA) was conducted as follows: WKUHH EXQFKHV RI ÀRZHU VWHPV WULPPHG WR FP ORQJ HDFK KDYLQJ ÀRZHU EXGV EXGV LQ WRWDO SHU EXQFK ZHUH SXW LQ 0.9 L glass jars with their stem end in 300 mL of test solutions RQH EXQFK SHU JODVV MDU 7KH ÀRZHUV ZHUH OHIW XQGHU FRQWLQXRXV OLJKW IURP ZKLWH ÀXRUHVFHQW ODPSV Pmol m-2 s-1 PPFD) at 23°C and 40–70% relative humidity for 24 days, and during this period the distilled water (control) was replaced every week and PDCA-containing test solutions were replenished as necessary. )XOO\ RSHQ DQG QRQ VHQHVFHQW QRW ZLOWHG DQG WXUJLG ÀRZHUV ZKLFK ZHUH UDQJLQJ IURP 2V WR 6V RI ÀRZHU RSHQLQJ VWDJHV (Harada et al., 2010; Morita et al., 2011), were counted daily DQG WKH SHUFHQWDJH RI WKHVH ÀRZHUV WR WKH WRWDO QXPEHU RI LQLWLDO ÀRZHU EXGV SHU EXQFK ZDV FDOFXODWHG 'DWD ZHUH SUHVHQWHG as changes of the percentages of fully open and non-senescent ÀRZHUV GXULQJ GD\V )ORZHU EXQFKHV KDYLQJ RU PRUH IXOO\ RSHQ DQG QRQ VHQHVFHQW ÀRZHUV ZHUH UHJDUGHG DV WKRVH having a display value. Determination of gross flower opening: In addition to the SUHYLRXVO\ GH¿QHG WZR FULWHULD IRU GHVFULELQJ ÀRZHU RSHQLQJ SUR¿OHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV YDVH OLIH 6DWRK et al., DQG WLPH WR ÀRZHU RSHQLQJ 6XJL\DPD DQG 6DWRK WKH WKLUG FULWHULRQ JURVV ÀRZHU RSHQLQJ ZDV GH¿QHG WR GHVFULEH WKH DELOLW\ RI ÀRZHU EXGV WR RSHQ 7KH JURVV ÀRZHU RSHQLQJ ZDV calculated by the sum of percentages at 40% or more of fully open DQG QRQ VHQHVFHQW ÀRZHUV GXULQJ LQFXEDWLRQ 7KH WHUP µVFRUHV¶ ZDV HPSOR\HG DV WKH XQLW IRU JURVV ÀRZHU RSHQLQJ

93

WKH QXPEHU RI RSHQ ÀRZHUV FRPSDUHG ZLWK WKDW LQ WKH FRQWURO 7KHUHIRUH ZH GH¿QHG µJURVV ÀRZHU RSHQLQJ¶ DV D PHDVXUH RI WRWDO QXPEHU RI RSHQ ÀRZHUV GXULQJ H[SHULPHQWV DQG FDOFXODWHG its values by the sum of percentages at 40% or more during experiments, as described in Materials and methods. The gross ÀRZHU RSHQLQJ WKH XQLW LV µVFRUHV¶ RI WKH ÀRZHUV WUHDWHG ZLWK 2,4-PDCA at different concentrations was 187 at 0 mM (control), 258 at 0.3 mM, 504 at 1 mM, and 604 at 2 mM (Table 1). The ODWWHU WZR ZHUH VLJQL¿FDQWO\ GLIIHUHQW IURP WKH FRQWURO 7KHVH observations suggested that 2,4-PDCA treatment elevated the DELOLW\ RI ÀRZHU EXGV WR RSHQ :H FRQVLGHUHG WKDW WKLV SURFHGXUH IRU GHWHUPLQLQJ µJURVV ÀRZHU RSHQLQJ¶ LV XVHIXO WR HYDOXDWH WKH DELOLW\ RI ÀRZHU EXGV DV DIIHFWHG HOHYDWHG E\ WUHDWPHQW ZLWK chemicals such as 2,4-PDCA. Recently, Sugiyama and Satoh (2015) showed that not only 2,4-PDCA but also other PDCA analogs, including 2,3-, 2,5-, DQG 3'&$ KDG DFWLYLWLHV WR DFFHOHUDWH ÀRZHU RSHQLQJ DV ZHOO DV WKRVH WR OHQJWKHQ WKH YDVH OLIH LQ FXW ÀRZHUV of ‘LPB’ carnation. In the present study, we determined the gross ÀRZHU RSHQLQJ ZKHQ WUHDWHG ZLWK UHVSHFWLYH FRPSRXQGV XVLQJ the original data (Fig. 4 and Table 1 in Sugiyama and Satoh, 2015). The results are shown in Table 2, with the data for time WR ÀRZHU RSHQLQJ DQG YDVH OLIH ZKLFK ZHUH DGRSWHG IURP 7DEOH 1 of Sugiyama and Satoh (2015). In the experiment with PDCA DQDORJV DW P0 WKH FRQWURO P0 ÀRZHUV KDG WKH JURVV ÀRZHU opening of 45 scores. All the PDCA analogs at 1 mM increased WKH JURVV ÀRZHU RSHQLQJ ZKLFK UDQJHG IURP WR VFRUHV depending on each PDCA analog, although there was no statistical VLJQL¿FDQFH ,Q WKH H[SHULPHQW ZLWK 3'&$ DQDORJV DW P0 WKH

Statistical analyses: Statistical analyses were carried out by Steel’s, Williams’ or Dunnett’s multiple range tests using an online statistical analysis program, MEPHAS (http://www.geninfo.osaka-u.ac.jp/testdocs/tomocom/; January 30, 2015).

5HVXOWV DQG GLVFXVVLRQ Previously, two criteria, vase life (Satoh et al., 2005) and time to ÀRZHU RSHQLQJ 6XJL\DPD DQG 6DWRK ZHUH GHYHORSHG IRU FKDUDFWHUL]LQJ ÀRZHU RSHQLQJ SUR¿OHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV DV GHVFULEHG LQ ,QWURGXFWLRQ ,Q WKH SUHVHQW VWXG\ ZH GH¿QHG WKLUG FULWHULRQ µJURVV ÀRZHU RSHQLQJ¶ IRU GHVFULELQJ DQRWKHU ÀRZHU RSHQLQJ SUR¿OH WKH DELOLW\ RI ÀRZHU EXGV WR RSHQ and tried to use it for evaluating the action of PDCA analogs in FXW VSUD\ W\SH ÀRZHUV RI µ/3%¶ FDUQDWLRQ Fig. 1 shows changes in the percentage of fully open and nonVHQHVFHQW ÀRZHUV IRU FXW µ/3%¶ ÀRZHUV WUHDWHG ZLWK 3'&$ DW GLIIHUHQW FRQFHQWUDWLRQV WKLV ¿JXUH FDPH IURP )LJ RI 6XJL\DPD and Satoh (2015) and Fig. 4 of Satoh et al. (2014). The time to ÀRZHU RSHQLQJ ZDV GD\V IRU WKH FRQWURO DQG LW ZDV VKRUWHQHG by treatment with 2,4-PDCA to 4.3 days at 0.3 mM, 3.3 days at 1 mM, and 3.8 days at 2 mM, although only the treatment with 1 P0 3'&$ ZDV VLJQL¿FDQWO\ GLIIHUHQW IURP WKH FRQWURO 7DEOH 6XJL\DPD DQG 6DWRK 7KH YDVH OLIH ZDV VLJQL¿FDQWO\ lengthened by treatment with 2,4-PDCA; from 8.3 days of the control to 12.7, 17.5 and 19.5 days at 0.3, 1.0 and 2.0 mM 2,4PDCA, in this order (Table 1; Satoh et al., 2014). Data in Fig. 1 also suggested that 2,4-PDCA treatment increased

Fig. 1. Three criteria for characterizing the flower opening profiles in cut spray-type carnation flowers. In addition to previously defined two criteria, time for flower opening (1) and vase life (2) (Sugiyama and Satoh, 2015), the third criterion, gross flower opening (3), was defined for evaluating 2,4-PDCA which improves displaying values of cut spray-type carnation flowers. The vase life of the cut flowers in days was defined as the duration when the percentage of fully open and non-wilted flowers was 40% or more. The time to flower opening was defined as the time in days from the start of the experiment until the percentage of open flowers reached 40%. Whereas, the gross flower opening (the unit is ‘scores’) was determined by the sum of percentages at 40% or more from the day when the percentage reached or surpassed 40% through the day when they declined to or fell below 40%. This figure came from the original and modified drawings that appeared as Fig. 4 in Satoh et al. (2014) and Fig. 2 in Sugiyama and Satoh (2015), respectively. Each point is the mean of 3 replicates, each with 5 flower stems with 5 flower buds IORZHU EXG IRU HDFK UHSOLFDWH Æ” &RQWURO P0 3'&$ ż P0 3'&$ Æ‘ P0 3'&$ ', 2 mM 2,4-PDCA.

94

7KUHH FULWHULD IRU FKDUDFWHUL]LQJ ÀRZHU RSHQLQJ SUR¿OHV DQG GLVSOD\ YDOXHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV

Table 1. Evaluation by three criteria of 2,4-PDCA which improves display value of cut flowers of ‘Light Pink Barbara’ carnation PDCA (mM ) 0 (control ) 0.3 1.0 2.0

7LPH WR ÀRZHU opening (days) 4.4 4.3 3.3* 3.8

Decrease (%) – 2 25 14

Vase life (days) 8.3 12.7* 17.5* 19.5*

Increase (%) – 53 111 135

*URVV ÀRZHU opening (scores) 187 258 504* 604*

Increase (%) – 38 170 223

The open-flower stage good for display was defined as the duration when 40 % or more buds are in the fully-open and non-senescent stage [Os 6–Ss 2 (Harada et al., 2010; Morita et al., 2011)]. Data for the time to flower opening came from Fig. 2 in Sugiyama and Satoh (2015), and those for the vase life from Table 1 in Satoh et al. (2014). Data are shown as the mean of triplicate bunches, each with 5 flower stems with 5 buds (25 buds in total per bunch). * shows a significant difference from the control (0 mM) by Steel’s multiple range test (MRT) (P < 0.05) for time to flower opening and by Williams’ MRT(P< 0.05) for vase life and gross flower opening. Table 2. Evaluation by three criteria of PDCA analogs which improve display value of cut flowers of ‘Light Pink Barbara’ carnation Chemicals Experiment 1 (1 mM PDCA) Experiment 2 (2 mM PDCA) 7LPH WR Ă€RZHU Vase life (days) *URVV Ă€RZHU RSHQLQJ 7LPH WR Ă€RZHU Vase life (days) *URVV Ă€RZHU RSHQLQJ opening (days) (scores) opening (days) (scores) Control 2,3-PDCA 2,4-PDCA 2,5-PDCA 2,6-PDCA 3,4-PDCA 3,5-PDCA

5.5 (100) 3.3* (60) 4.3*(78) 5.0*(91) 4.3*(78) 8.0(145) 5.3* (96)

4.0(100) 14.0*(350) 14.0*(350) 17.0*(425) 12.7*(318) 8.0*(200) 14.3*(358)

45 (100) 244 (542) 189 (189) 348 (773) 201 (447) 101 (224) 206 (458)

9.0 (100) 4.0*(44) 4.0*(44) 4.7*(52) 5.3*(59) 5.7*(63) 4.7*(52)

8.3 (100) 14.7*(177) 14.7*(177) 13.0*(157) 9.3*(112) 14.7*(177) 15.5*(187)

40 (100) 531*(1328) 423*(1058) 412*(1030) 283* (708) 441*(1103) 506*(1265)

The original data for the time to flower opening and vase life were adopted from Fig. 4 and Table 1 in Sugiyama and Satoh (2015).Data are shown as the mean of triplicate bunches, each with 5 flower stems with 5 buds (25 buds in total per bunch), but data for time to flower opening and vase life in the control for Experiment 1 are the mean of 2 replicates since the remaining third replicate did not attain 40% open flowers. Figures in the parentheses show the percentages to the control.*shows asignificant difference from the control in each column by Dunnett’s multiple range test (P< 0.05).

JURVV ÀRZHU RSHQLQJ ZDV VLJQL¿FDQWO\ LQFUHDVHG IURP WKH FRQWURO value of 40 scores to much higher values from 283 to 531 scores depending on each PDCA analog. 2,3-PDCA gave the highest HOHYDWLRQ RI JURVV ÀRZHU RSHQLQJ LQ WKLV H[SHULPHQW -XGJLQJ from the combined results at 1 mM and 2 mM PDCA analogs, we suggest that PDCA analogs had activity to increase the gross ÀRZHU RSHQLQJ UHVXOWLQJ LQ WKH HOHYDWLRQ RI WKH DELOLW\ RI ÀRZHU buds to open. The order among PDCA analogs in terms of their DFWLRQ WR HOHYDWH WKH JURVV ÀRZHU RSHQLQJ ZDV QRW FOHDU DOWKRXJK it was evident that 2,3-PDCA gave the highest elevation in both concentrations. Previously, it was suggested that 2,3-PDCA and 2,4-PDCA were most effective among PDCA analogs for the DFFHOHUDWLRQ RI ÀRZHU RSHQLQJ DQG WKH H[WHQVLRQ RI YDVH OLIH 6XJL\DPD DQG 6DWRK LQ FXW ÀRZHUV RI ¾/3%œ FDUQDWLRQ 7KH SUHVHQW UHVXOWV RQ WKH JURVV ÀRZHU RSHQLQJ SDUWO\ DJUHHG WR the previous ones. We did not study further the reason for the difference of chemical structures of PDCA analogs among three actions, i.e. DFFHOHUDWLRQ RI ÀRZHU RSHQLQJ H[WHQVLRQ RI YDVH OLIH DQG LQFUHDVH RI JURVV ÀRZHU RSHQLQJ DQG LWV HOXFLGDWLRQ UHPDLQV as a subject of future investigation. In the original data (Fig. 3 in Sugiyama and Satoh, 2015), the PD[LPXP YDOXHV RI WKH SHUFHQWDJH RI IXOO\ RSHQ ÀRZHUV RI WKH untreated control, which slightly surpassed 40%, were much ORZHU WKDQ WKRVH REWDLQHG SUHYLRXVO\ ZLWK ¾/3%œ ÀRZHUV IRU example, 66–88% (Satoh et al., 2014) and 100% (Satoh et al., 7KH ORZ PD[LPXP SHUFHQWDJH RI RSHQ ÀRZHUV PLJKW KDYH UHVXOWHG IURP GLIIHUHQFHV LQ WKH DELOLW\ WR RSHQ DPRQJ ÀRZHU samples, which were cultivated and harvested in different years and seasons. It is likely that the low ability might be caused by shortage of sugars after harvest, which resulted from their consumption for respiration and reduced supply by depressed photosynthesis under low lightening condition. In fact, it is known that sugar accumulation in petal cells reduces the petal

ZDWHU SRWHQWLDO DQG SURPRWHV ZDWHU LQÀX[ LQWR WKH SHWDO FHOOV UHVXOWLQJ LQ FHOO HQODUJHPHQW DQG FXOPLQDWLQJ LQ ÀRZHU RSHQLQJ (Evans and Reid, 1988; Ho and Nichols, 1977; Ichimura et al., 2003). Exogenously-applied sugars, such as glucose, fructose and sucrose (after being hydrolyzed by invertase forming glucose and IUXFWRVH UHVXOW LQ WKH SURPRWLRQ RI ÀRZHU RSHQLQJ 3URPRWLRQ RI ÀRZHU EXG RSHQLQJ E\ H[RJHQRXVO\ DSSOLHG VXJDUV ZDV DOVR found in our previous studies, in which 0.1 M sucrose slightly LQFUHDVHG WKH QXPEHU RI IXOO\ RSHQ DQG QRQ VHQHVFHQW ÀRZHUV LQ FXW VSUD\ W\SH ¾/3%œ ÀRZHUV 6DWRK et al., 2005) and sucrose DQG SDODWLQRVH ERWK DW DFFHOHUDWHG ÀRZHU EXG RSHQLQJ LQ ¾/3%œ ÀRZHUV 6DWRK et al., 2013). Some PDCA analogs at 2 mM DFFHOHUDWHG ÀRZHU EXG RSHQLQJ DQG LQFUHDVHG WKH JURVV ÀRZHU RSHQLQJ WKH SHUFHQWDJH RI RSHQ ÀRZHUV VXUSDVVHG UHVXOWLQJ LQ RSHQLQJ RI DOPRVW DOO ÀRZHU EXGV 6XJL\DPD DQG 6DWRK These results suggested that the low ability of buds to open in FXW VSUD\ W\SH ¾/3%œ ÀRZHUV LV QRW DOZD\V FDXVHG E\ VKRUWDJH RI VXJDUV LQ WKH ÀRZHU WLVVXHV 6XJDUV PD\ DFW DV D VLJQDO WR LQGXFH WKH RSHQLQJ RI ÀRZHU EXGV LQ DGGLWLRQ WR DFW DV DQ HQHUJ\ VRXUFH IRU ÀRZHU RSHQLQJ 7KH DFWLRQ PHFKDQLVP RI VXJDUV DV ZHOO DV PDCAs remains to be elucidated in the future. The present study established a method to determine the gross ÀRZHU RSHQLQJ ZKLFK HYDOXDWHV WKH DELOLW\ RI ÀRZHU EXGV WR RSHQ IRU FKDUDFWHUL]LQJ WKH ÀRZHU RSHQLQJ SUR¿OHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV :H WKLQN WKH WKUHH FULWHULD WKH SUHVHQW ¾JURVV ÀRZHU RSHQLQJœ DQG RWKHU WZR SUHYLRXVO\ GH¿QHG FULWHULD ¾WLPH WR ÀRZHU RSHQLQJœ DQG ¾YDVH OLIHœ ZLOO EH XVHIXO WR FKDUDFWHUL]H ÀRZHU RSHQLQJ SUR¿OHV LQ VSUD\ W\SH ÀRZHUV RI RWKHU RUQDPHQWDOV as well as carnation. On the other hand, from the point of view of ÀRZHU SUHVHUYDWLYHV WKH WKUHH FULWHULD ZRXOG EH XVHIXO WR HYDOXDWH end-points of the chemicals, i.e., to determine physiological steps RI ÀRZHU RSHQLQJ LQ ZKLFK WKH\ ZLOO DIIHFW DQG to elucidate WKH PHFKDQLVPV IRU HOHYDWLRQ RI DELOLW\ WR RSHQ RI ÀRZHU EXGV

7KUHH FULWHULD IRU FKDUDFWHUL]LQJ ÀRZHU RSHQLQJ SUR¿OHV DQG GLVSOD\ YDOXHV LQ FXW VSUD\ W\SH FDUQDWLRQ ÀRZHUV DFFHOHUDWLRQ RI ÀRZHU EXG RSHQLQJ DQG H[WHQVLRQ RI YDVH OLIH RI RSHQHG ÀRZHUV .HHSLQJ WKLV QRWLRQ LQ PLQG ZH DUH FXUUHQWO\ WHVWLQJ WKH HIIHFWV RI 3'&$ DQDORJV RQ ÀRZHU RSHQLQJ SUR¿OHV RI VRPH RUQDPHQWDOV ZLWK VSUD\ W\SH ÀRZHUV RWKHU WKDQ FDUQDWLRQ

Acknowledgements :H WKDQN ¿QDQFLDO VXSSRUW E\ D *UDQW LQ $LG WR 6 6DWRK IRU 6FLHQWL¿F 5HVHDUFK IURP WKH -DSDQ 6RFLHW\ IRU WKH Promotion of Science.

References Evans, R. Y. and M. S. Reid, 1988. Changes in carbohydrates and osmotic potential during rhythmic expansion of rose petals. J. Amer. Soc. Hort. Sci., 113: 884-888. Harada, T., Y. Torii, S. Morita, T. Masumura and S. Satoh, 2010. 'LIIHUHQWLDO H[SUHVVLRQ RI JHQHV LGHQWL¿HG E\ VXSSUHVVLRQ VXEWUDFWLYH K\EULGL]DWLRQ LQ SHWDOV RI RSHQLQJ FDUQDWLRQ ÀRZHUV J. Exp. Bot., 61: 2345-2354. Ho, L. C. and R. Nichols, 1977. Translocation of 14C-sucrose in relation to changes in carbohydrate content in rose corollas cut at different stages of development. Ann. Bot., 41: 227-242. Ichimura, K., Y. Kawabata, M. Kishimoto, R. Goto and K. Yamada, 2003. Shortage of soluble carbohydrates is largely responsible for VKRUW YDVH OLIH RI FXW µ6RQLD¶ URVH ÀRZHUV J. Japan. Soc. Hort. Sci., 72: 292-298.

95

Morita, S., Y. Torii, T. Harada, M. Kawarada, R. Onodera and S. Satoh, 2011. Cloning and characterization of a cDNA encoding sucrose V\QWKDVH DVVRFLDWHG ZLWK ÀRZHU RSHQLQJ WKURXJK HDUO\ VHQHVFHQFH in carnation (Dianthus caryophyllus L.). J. Japan. Soc. Hort. Sci., 80: 358-364. Nukui, H., S. Kudo, A. Yamashita and S. Satoh, 2004. Repressed ethylene SURGXFWLRQ LQ WKH J\QRHFLXP RI ORQJ ODVWLQJ ÀRZHUV RI WKH FDUQDWLRQ µ:KLWH &DQGOH¶ UROH RI J\QRHFLXP LQ FDUQDWLRQ ÀRZHU VHQHVFHQFH J. Exp. Bot., 55: 641-650. Satoh, S., Y. Kosugi, S. Sugiyama and I. Ohira, 2014. 2, 3\ULGLQHGLFDUER[\OLF DFLG SURORQJV WKH YDVH OLIH RI FXW ÀRZHUV RI spray carnations. J. Japan. Soc. Hort. Sci., 83: 72-80. Satoh, S., M. Miyai, S. Sugiyama and N. Toyohara, 2013. PalatinoseK\GURO\]LQJ DFWLYLW\ DQG LWV UHODWLRQ WR PRGXODWLRQ RI ÀRZHU RSHQLQJ in response to the sugar in Dianthus species. J. Japan. Soc. Hort. Sci., 82: 337-343. Satoh, S., H. Nukui and T. Inokuma, 2005. A method for determining WKH YDVH OLIH RI FXW VSUD\ FDUQDWLRQ ÀRZHUV J. Appl. Hort., 7: 8-10. Sugiyama, S. and S. Satoh, 2015. Pyridinedicarboxylic acids prolong the YDVH OLIH RI FXW ÀRZHUV RI VSUD\ W\SH µ/LJKW 3LQN %DUEDUD¶ FDUQDWLRQ E\ DFFHOHUDWLQJ ÀRZHU RSHQLQJ LQ DGGLWLRQ WR DQ DOUHDG\ NQRZQ action of retarding senescence. Hort. J., 84: 172-177. Vlad, F., P. Tiainen, C. Owen, T. Spano, F.B. Daher, F. Oualid, N.O. Senol, D. Vlad, J. Myllyharju and P. Kalaitzis, 2010. Characterization of two carnation petal prolyl 4 hydroxylases. Physiol. Plant., 140: 199-207. Received: March, 2015; Revised: April, 2015; Accepted: April, 2015

Journal

Journal of Applied Horticulture, 17(2): 96-100, 2015

Appl

(YDOXDWLRQ RI VHQVRUV IRU VHQVLQJ FKDUDFWHULVWLFV DQG ÀHOG RI view for variable rate technology in grape vineyards in North Dakota Ganesh C. Bora*, Purbasha Mistry1 and Dongqing Lin Department of Agricultural and Biosystems Engineering, North Dakota State University, Fargo, USA, 1Natural Resource Program, North Dakota State University, Fargo, USA. *E-mail: ganesh.bora@ndsu.edu

Abstract Sensors have been used to detect tree sizes for agrochemical and fertilizer applications in grape vineyards. Rugged and reliable sensors are required to measure the size and quality of tree canopy volume for variable rate fertilizer application. Real time sensing is important as size of the tree changes with time due to biological factors and management practices. This study evaluated ultrasonic VHQVRU RSWLFDO VHQVRU DQG D ODVHU VHQVRU IRU WKHLU VHQVLQJ FKDUDFWHULVWLFV DQG ÂżHOG RI YLHZ )R9 LQ D UDQJH RI FRQGLWLRQV 7KH )R9 was established by moving targets perpendicular to the centerline on both sides. The maximum sensig range of sensors varied from 6 to 8 m with ultrasonic sensor having the highest range. The beam widths for ultrasonic sensors were found to be wide (maximum 950 mm) whereas optical sensor has a narrow maximum beam width of 70 mm. The laser sensor has a sharp beam and did not work well in outdoor environment with plant materials. Statistical analysis was also done for sensors and found that P value is lower than 0.001 and R2 YDOXH FORVHU WR ZKLFK LQGLFDWHV VLJQLÂżFDQW EHWWHU UHVXOW LQ WKH YLQH\DUG IRU VHQVLQJ FKDUDFWHULVWLFV Key words: Vineyards, sensors, variable rate technology (VRT), tree-sensing

,QWURGXFWLRQ 3UHFLVLRQ IDUPLQJ DOORZV RQH WR PDQDJH FURSV LQ D VLWH VSHFLÂżF manner. Recently various aspects of precision farming are JHWWLQJ SRSXODU DPRQJ WKH IDUPHUV GXH WR LWV EHQHÂżFLDO HIIHFWV on crops. One of the precision agriculture technologies is variable rate technology (VRT) which allows differential application of fertilizer throughout the field based on the requirement. Therefore, primary goal of using VRT is to PD[LPL]H SURÂżW WR LWV IXOOHVW SRWHQWLDO FUHDWH HIÂżFLHQFLHV LQ LQSXW application, and ensure sustainability and environmental safety. There are different kinds of variable rate technologies available which can be used with or without a GPS system. The two basic technologies are either map-based or sensor-based. A sensor based VRT system mainly detects heights or determine canopy volume of trees to apply different pre- determined variable rates of fertilizer with no prior mapping or data collection involved. VRT fertilization in vineyards has potential economic and HQYLURQPHQWDO EHQHÂżW 5HDO WLPH VHQVRUV PHDVXUH WKH GHVLUHG properties like growth of the trees by measuring heights. Measurements made by such a system are then processed and used immediately to control a variable-rate applicator (VRA Tools). The disadvantage of using tree height is that there may be absence of leaves in some trees or presence of damaged leaves. Due to this steps are being taken to quantify the tree canopy volume for variable rate fertilization (Zaman et al., 2005b). It has been noted that, yield also has direct relationship with tree canopy volume (Wheaton et al., 1995 and Whitney et al., 1999). Zaman et al. (2005a) suggested incorporating temperature compensation system in the sensor while quantifying the sources of error including different environmental factors like air temperature in ultrasonic measurement.

Most of the commercially available sensors like ultrasonic, photoelectric, optical and laser sensors are basically developed for industrial purposes and indoor environment. However, recently they are being used in the agricultural sectors also because of their SRWHQWLDO IRU XVH LQ VHQVLQJ GHWHFWLQJ DQG SURÂżOLQJ WUHHV :HL DQG Salyani (2005) studied a laser scanning system to quantify foliage density of citrus trees and density. Tumbo et al. (2001) compared citrus tree canopy volume measurements by manual, laser and by ultrasonic sensors. Schumann and Zaman (2005) used ultrasonic sensor and photoelectric sensor to measure tree canopy volume and tree heights to apply pre-determined amounts of fertilizer to a single tree. Escola et al. (2011) checked the overall performance of ultrasonic sensor in apple tree canopies. Jeon et al. (2011) also tested ultrasonic sensors for accuracy in measuring distances to VLPXODWHG FDQRSLHV RI ÂżHOG FURSV 0LOOHU et al. (2003) tested a variable rate granular fertilizer spreader with both GPS-guided prescription mapping and real-time tree size measurement with photoelectric sensors in citrus groves. The main focus of this study was to evaluate and compare different sensors that are available in the market and verify whether they are suitable for sensing grapevines for use in variable rate fertilizer application.

Materials and methods Three different types of sensors (Ultrasonic, optical, laser) namely Banner Ultrasonic sensor, Banner optical sensor and Balluff laser sensor were tested for this study according to their VSHFLÂżFDWLRQV 7KH VHQVRUV ZHUH PDLQO\ VHOHFWHG WR FKHFN WKHLU SHUIRUPDQFH LQ WHUPV RI WKHLU VHQVLQJ UDQJH ÂżHOG RI YLHZ DQG WKHLU VHQVLQJ FKDUDFWHULVWLFV LQ WKH YLQH\DUGV 7KH ÂżHOG RI YLHZ or beam pattern is the angle over which objects are detected by

Evaluation of sensors for variable rate technology in grape the sensor. In some cases FoV is also presented as angle of view which describes the angular extend of area covered by the sensor. The FoV of the sensors were established by measuring the sensing width at different distances. The experiment was done in both indoor and outdoor environment to assess any difference. For the indoor experiment, the sensor was mounted on a stand and a tall 2-inch diameter white PVC pipe was moved towards the center line perpendicularly and marked on the ground when the target was detected. Similar points were established on the other side of the center line. The beams were detected both sides of the center OLQH WR GRFXPHQW WKH ÂżHOG RI YLHZ DOVR NQRZQ DV EHDP SDWWHUQ After the sensor is excited by 12V battery, signal was transmitted. A circular white PVC pipe (2 in dia) was used to detect the signal. The pipe was moved both side of the center line, till the voltage was recorded. The voltage on the center line at different distance was also recorded. The schematic of experimental set-up is shown in Fig. 1. Care was taken for engaging proper dip switches in the ultrasonic sensors and the output wirings in all the sensors. The LQGRRU H[SHULPHQW KHOSV WR YHULI\ WKH VSHFLÂżFDWLRQV RI WKH VHQVRUV by the manufacturer.

97 Target

Sensor

Centerline

Beam Edges

Fig. 1. Experimental set up

Lab test: A 2.03 m high and 1.30 m wide portable screen was used as a moving target shown in Fig. 2. When the sensor signals hit the target, the distance was measured and the corresponding output voltage was recorded. The procedure was repeated in indoor and outdoor environments. Water was sprayed at the sensor to see if moisture affected performance. Field test: The outdoor experiment was conducted in the Red trail Vineyard which is in the south central North Dakota (46o 54’ 09� N, 97o 29’ 42� W). The three sensors were placed at vineyards rows with some changes for the sensor placement. The VDPH H[SHULPHQW VHW XS ZDV HVWDEOLVKHG LQ WKH ¿HOG FRQGLWLRQV DV VKRZQ LQ )LJ 7KH ¿HOG WHVW ZDV UHSHDWHG WKUHH WLPHV WR JHW better results.

Fig. 2. Set-up for lab test

Statistical analysis was done for the output of ultrasonic sensor. 7KH FRHIÂżFLHQW RI GHWHUPLQDWLRQV 52) of the linear output along ZLWK FRHIÂżFLHQW RI YDULDWLRQ &9 DQG URRW PHDQ VTXDUH HUURU (RMSE) were observed. CV, expressed in percentage, is the statistical measure of deviation of a variable from its mean and is calculated by dividing standard deviation by the mean value. RMSE is the root mean square deviation from the average. Ultrasonic Sensor: Banner Ultrasonic Sensor (U-GAGE QT50ULBQ6), is from Banner Engineering Corp. (Minneapolis, Minn.) and is a waterproof, compact, long range, temperature compensated with operating condition range of -20 oC to 70 oC (-4 to 158 oF) and sensing range from 200 mm (8 in) to 8m (26 ft) with 1 mm resolution. The signal frequency is 75 kHz and supply

Fig. 3. Set-up for field test

Table 1. Manufacturers’ sensor specifications 6SHFL¿FDWLRQV

Photoelectric

Ultrasonic

Laser

Sensing range

6 m (20 ft)

8 m (26 ft)

6 m (20 ft)

Supply voltage

10-30 VDC

10-30 VDC

15-30 VDC

Output response time

1 ms

100 ms

5 ms

Operating frequency

75 kHz

Wave lengths

Infrared (880 nm)

Output

Discrete

Operating temperature

Red light Laser (650‌670 nm) Analog (0-10V)

o

o

-20 to 55 C (-4 to 131 F)

o

Analog (0-10V) o

-20 to 70 C (-4 to 158 F)

-10 to 55 oC (12 to 131 oF)

98

Evaluation of sensors for variable rate technology in grape

voltage is 10 to 30 VDC. It has a scalable analog output with adjustable minimum and maximum limits and output can be selected 0-10 VDC or 4-20 mA via DIP switches. For this test, the output was set for 0-10 VDC. The sensor has 9 m (30 ft) long color coded 5-wire cable with shield. Optical Sensors: 7KH GLIIXVH UHÀHFWDQFH SKRWRHOHFWULF optical sensors are from Banner Engineering Corp. (Minneapolis, Minn.) and are commonly known as Banner eyes. These are compact, self contained sensors in metal die cast housing and have an infrared sensing beam (880 nm) with supply voltage of 10-30 VDC at less than 40 mA. The sensor has an output response time of 1 ms (on & off) and works at a temperature range of -20 oC to 55 oC (-4 to 131 o F). Its output type is discrete (NPN) and has a sensing range of 6m (20 ft). The sensor has two status LEDs; yellow for signal and green for power. This is currently used in VRT spreaders available in Florida to detect heights of citrus trees. Laser Sensor: The BOD 63M is a photoelectric sensor for distance measurement with laser as emitter light source and is marketed by Balluff Inc., Florence, KY. The sensor is basically used for industrial purpose and provides output voltage that is directly proportional to distances between targets and the sensor. It is a compact rugged metal-housed sensor with a class II red light laser (650‌670 nm) sensing beam. The sensing range is 500 mm (1.6 ft) to 6 m (20 ft) and has analog and discrete output. The supply voltage is 15-30 VDC with current consumption of less than 100 mA. The light spot diameter is 5 mm at 3 m and 10 mm at 6 m sensing distance. The working temperature range is -10oC to 55 oC (14 oF to 131 oF). It has three status LEDs, green – supply voltage indicator, yellow – output indicator, and the red – stability indicator. During this test, 24 V were supplied from a battery and the analog output (voltage) was measured at different sensing distances using terminals three and ¿YH WR UHFRUG WKH RXWSXW YROWDJH IURP WKH YROWPHWHU ZKLFK ranged 0-10V.

manufacturer did it with 1 inch pipe and 500 mm plate. The experiment was done with 2 inch pipe, because larger target like tree crop is being used. The beam widths for ultrasonic sensors were found to be wide ZLWK D PD[LPXP RI PP $OWKRXJK WKH PDQXIDFWXUHU VSHFL¿HG WKH maximum width would be around 1600 mm. the actual maximum angle GHJHHV ZDV VPDOOHU WKDQ WKH VSHFL¿HG PD[LPXP EHDP DQJOH RI 45 degrees. The sensor provided better result in the vineyard (outdoor) IRU VHQVLQJ FKDUDFWHULVWLFV 7KH JUDSK LOOXVWUDWLQJ WKH ¿HOG RI YLHZ angle of view and sensing range of the sensor was found to be quite VLPLODU ZLWK WKH PDQXIDFWXUHUœV VSHFL¿FDWLRQV ZKLFK DUH VKRZQ LQ )LJ 4, 5 and 6, respectively. SAS program procglm was administered to DQDO\]H WKH RXWSXW DQG IRXQG WKDW WKHUH ZDV QR VLJQL¿FDQW GLIIHUHQFH LQ all conditions. R2 value of the linear output along with CV and RMSE in given in Table 2. The sensor signal stabilizes during the period of testing and repeatability was great. Optical Sensors: The Banner optical sensor was tested for its sensing range and FoV in different light detection conditions. The sensor has two indicators; a green LED shows power that the sensor is on and the yellow LED indicates that the sensor is detecting a target. If the yellow LQGLFDWRU ÀDVKHV WKHUH LV PDUJLQDO H[FHVV JDLQ LQ OLJKW FRQGLWLRQ 7KH )R9 LV HVWDEOLVKHG E\ ¿QGLQJ WKH EHDP ZLGWK RI WKH VHQVRU LQ ERWK conditions and is shown in Fig. 7. When light is sensed, it could detect a target till the sensing range of 4.9 m with highest beam width of 47 PP :LWK PDUJLQDO H[FHVV JDLQ \HOORZ LQGLFDWRU ÀDVKLQJ WKH UDQJH ZDV P ZKLFK ZDV VLPLODU WR WKH PDQXIDFWXUHUœV VSHFL¿FDWLRQ RI

5HVXOWV

Fig. 4. Ultrasonic Sensor. Field of View from Experiment

Ultrasonic Sensor: The data were recorded for both outdoor and indoor environment. Although according to the manufacturers it was rated that the sensor can sense up to 8 m, actually it only sensed up to 7.2 m. Hence, the maximum limit was set at that point while data were recorded in the outdoor environment. The voltage output from the sensor was similar in outdoor and indoor. But it was different, when the sensor was wet with water. Water was added to it because; the manufacturer claimed that it will work in rain. The sensor provided reliable signal in the wet condition. The experiment was conducted by 2 inch PVC pipe which was XVHG DV WKH WDUJHW LQ RUGHU WR ÂżQG RXW WKH ÂżHOG RI YLHZ 7KH

Fig. 5. Angle of view for the Banner ultrasonic sensor

Table 2. Statistical analysis of output of Banner ultrasonic sensor Environment R2 Rep to Rep CV (%)

RMSE

P value

Indoor

0.9999

1.14

0.0447

<.001

Outdoor

0.9999

0.53

0.0211

<.001

Wet

0.9998

1.95

0.0829

<.001

All together

0.9969

5.41

0.2185

<.001

Evaluation of sensors for variable rate technology in grape

99

6 m. The maximum beam width was 70 mm in comparison to the PDQXIDFWXUHU¶V VSHFL¿FDWLRQ RI PP ZLWK LV DQ HOOLSWLFDO VKDSH The beam width was narrow compared to the ultrasonic sensor.

Fig. 6. Ultrasonic Sensor characteristics-Sensing range vs. voltage output in different conditions

Laser sensor: The evaluation test was conducted in an outdoor and indoor environment. The sensing range was different for differed colored target and also for indoor and outdoor environment as shown in Table 3 and Fig. 8. The statistical analysis of the signal output is presented in Table 4. The sensing range for grape leaf as target in indoor environment is shown in Fig. 8 and in the outdoor environment is shown in Fig. 9. The laser sensor did not work well in the vineyards. Signal from laser VHQVRU ZDV HUUDWLF GXH WR LWV FRQÀLFW ZLWK VXQ VKLQH ,W GLG QRW GHWHFW DQ\ REMHFW ZLWK SODQW OHDYHV DV WKH ODVHU EHDP JHWV UHÀHFW and refract by the leaves. Table 3. Sensing ranges of Balluff Laser Sensor for different colored targets Target color

Sensing range (m) Indoor

Fig. 7. Optical Sensor. Field of View from Experiment

Outdoor

White

6.46

6.42

Black

6.29

6.20

Green

6.43

6.34

Brown

6.46

6.47

Gray

6.40

6.22

Yellow

6.45

6.51

'LVFXVVLRQ

Fig. 8. Trend of output for the Balluff laser sensor

Fig. 9. Trend of analog output of Balluff laser sensor with leaf as target in an outdoor environment

Banner Optical Sensors and ultrasonic sensor worked well in vineyards and gave reliable results. Optical sensor has narrow FoV whereas the ultrasonic has a wide FoV. The beam widths were maximum 950 mm and 70 mm for ultrasonic and optical sensor respectively. So, both of them are suitable for detecting a tree. There were problem with the laser sensor and thus a reliable output cannot be determined. Both optical and ultrasonic sensor FDQ EH XVHG WR ¿QG WKH FDQRS\ YROXPH RI WKH YLQH\DUG EDVHG on distances and eventually predetermined amount of fertilizer can be applied according to the height of each tree. Here, the ultrasonic sensor has a higher sensing range of 7.2 m (Li et al., 2002). Therefore, it can be said from this study that: (i)Ultrasonic sensor and optical sensor worked well and can be used for VRT of tree crops (vineyards); (ii) The Banner ultrasonic sensor is waterproof and provides reliable signal even in wet condition (iii) Pre-determined amount of fertilizer according to canopy volume or height for each tree (iv) FoV and sensing range should be calibrated for individual tree crop; (v) The laser sensor did not perform at outdoor environment (beam passed through leaves).

Table 4. Statistical analysis of analog output of Balluff Laser Sensor R2

CV (%)

RMSE

P-value

0.999985

0.30

0.016681

<0.001

PVC pipe (Outdoor)

0.999903

0.89

0.047697

<0.001

PVC pipe (Both Environment)

0.999810

0.96

0.053896

<0.001

Leaf (Indoor)

0.999967

0.46

0.023469

<0.001

All together

0.999646

1.30

0.066358

<0.001

Environment PVC pipe (Indoor)

100

Evaluation of sensors for variable rate technology in grape

References Banner Engineering Crop, 2005. QMT42 Long-Range Diffuse Sensors. Minneapolis, MN. Banner Engineering Crop, 2005. U-gage QT50U Series sensors with analog output. Minneapolis, MN. Balluff Inc, 2005. BOD 63M Series Sensors with analog output. Florence, KY. EscolĂ , A., S. Planas, J.R. Rosell, J. Pamar, F. Camp, F. Solanelles, F. Gracia, J. Llorens and E. Gil, 2011. Performance of an ultrasonic ranging sensor in apple tree canopies. Sensors (Basel), 11: 24592477. Jeon, H.Y., H. Zhu, R. Derksen, E. Ozkan and C. Krause, 2011. Evaluation of ultrasonic sensor for variable-rate spray applications. Computer and Electronics in Agr., 75: 213-221. Li, B., J.D. Whitney, W.M. Miller and T.A. Wheaton, 2002. Ultrasonicbased canopy volume measurements of citrus trees for precision agriculture. ASAE Paper No. 021053. St. Joseph, Mich.: ASAE. Miller, W.M., J.D. Whitney, A.W. Schumann and S. Buchanon, 2003. A test program to assess VRT granular fertilizer applications for citrus. ASAE Paper No. 031126. St. Joseph, Mich.: ASAE. Schumann, A.W. and Q.U. Zaman, 2005. Software development for real-time mapping of ultrasonic tree canopy size. Computer and Electronics in Agr., 47(1): 25-40.

Tumbo, S.D., M. Salyani, J.D. Whitney, T.A. Wheaton and W.M. Miller, 2001. Laser, ultrasonic and manual measurements of citrus tree canopy volume. ASAE paper no. 011068, St. Joseph, Mich.: ASAE. Wei, J. and M. Salyani, 2005. Development of a laser scanner for measuring tree canopy characteristics: Phase 2. Foliage density measurement. Transactions of the ASAE, 48(4): 1595-1601. Wheaton, T.A., J.D. Whitney, W.S. Castle, R. P. Muraro, H.W. Browning and D.P.H. Tucker, 1995. Citrus Scion and rootstock, topping height, and tree spacing affect tree size, yield, fruit quality, and economic return. J. Amer. Soc. Hort. Sci., 120(5): 861-870. Whitney, J.D., W.M. Miller, T.A. Wheaton, M. Salyani and J.K. Schueller, 1999. Precision farming applications in Florida citrus. Appl. Eng. Agr., 15(5): 399-403. Zaman, Q.U., A.W. Schumann and H.K. Hostler, 2005a. Quantifying sources of error in ultrasonic measurement of citrus orchards. ASAE Paper No. 051223. St. Joseph, Mich.: ASAE. Zaman, Q.U., A.W. Schumann and W.M. Miller, 2005b. Variable rate nitrogen application in Florida citrus based on ultrasonically-sensed tree size. Appl. Eng. Agr., 21(3): 331-335. Submitted: October, 2014; Revised: January, 2015; Accepted: January, 2015

Journal

Journal of Applied Horticulture, 17(2): 101-105, 2015

Appl

6KDGH HIIHFWV RQ JURZWK Ă RZHULQJ DQG IUXLW RI DSSOH S.S. Miller, C. Hott and T. Tworkoski* USDA-ARS, 2217 Wiltshire Road, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA. *E-mail: tom.tworkoski@ars.usda.gov

Abstract Light is a critical resource needed by plants for growth and reproduction. A major portion of the apple (Malus x domestica Borkh.) tree’s canopy is subjected to shade during most daylight hours each day and such shade may affect productivity. The current research GHWHUPLQHG HIIHFWV RI PRUQLQJ DIWHUQRRQ DQG DOO GD\ VKDGLQJ RQ SURFHVVHV WKDW DUH VLJQL¿FDQW WR RUFKDUG SURGXFWLYLW\ ,Q ¾*LQJHU *ROGœ 0 DSSOH WUHHV ZHUH SODQWHG LQ WKH ¿HOG QHDU .HDUQH\VYLOOH :9 DQG VKDGH WUHDWPHQWV ZHUH LPSRVHG IURP WR Trunk and branch growth were reduced consistently by morning shade (MS) compared to no shade (NS) and full shade (FS) and afternoon shade (AS) had intermediate effects. Total branch growth from 2002 to 2005 was 164, 168, 145, and 157 cm for FS, NS, MS, and AS, respectively. Although shade affected yield inconsistently from year-to-year, total yield from 2002 to 2005 was 7.8, 201.6, 72.5, and 110.6 kg/tree for FS, NS, MS, and AS, respectively. Time of shading clearly affected yield with full shade causing the greatest reduction, followed by partial shade treatments, MS and AS. Concentrations of soluble carbohydrates, particularly sorbitol, were greater in leaves of AS compared to MS. It is postulated that MS may have adversely affected photosynthesis at a time of day that was most conducive to KLJK QHW DVVLPLODWLRQ 3ODQWLQJ DQG WUDLQLQJ DSSOH WUHHV WR PLQLPL]H VKDGH HVSHFLDOO\ PRUQLQJ VKDGH PD\ EHQH¿W RUFKDUG SURGXFWLYLW\ Key words: Apple, carbohydrate, fruit quality, productivity

,QWURGXFWLRQ $SSOH JURZHUV DUH VHHNLQJ PRUH HI¿FLHQW VXVWDLQDEOH SURGXFWLRQ methods that employ reduced inputs to remain globally competitive, insure consistent annual production, and to meet the increasing demand for locally grown fresh fruits. Many IDFWRUV LQÀXHQFH FURSSLQJ DQG IUXLW TXDOLW\ SRWHQWLDO LQFOXGLQJ the environment, cultural practices, canopy growth habit, crop history, and endogenous plant hormones (Faust, 1989; Maib, 1996). A better understanding of these factors and how they LQWHUDFW LV QHFHVVDU\ IRU IXUWKHU DGYDQFHV LQ SURGXFWLRQ HI¿FLHQF\ Considering that more than 90% of plant dry weight is derived IURP SKRWRV\QWKHWLFDOO\ ¿[HG FDUERQ WKH LPSRUWDQFH RI FDUERQ assimilation and carbon partitioning in optimizing production is evident (Flore and Lakso, 1989; Forshey and Elfving, 1989). The role of light and photosynthesis in tree fruit production, particularly apple, has been well documented (Flore and Lakso, 1989; Lakso, 1994). Much of the research has focused on altering tree size, shape, and planting systems (Robinson, et al., 1991) to increase light (photosynthetic active radiation, PAR) interception and thereby maximize carbon assimilation with an ultimate goal of increased yields and enhanced fruit quality. While recognizing the importance of these methods for increased light interception, it remains that a major portion of the apple tree’s canopy is subjected to shade during most daylight hours HDFK GD\ 6KDGLQJ WR RI IXOO VXQOLJKW UHGXFHG ÀRZHU DQG IUXLW QXPEHUV WRWDO \LHOGV DQG IUXLW GU\ ZHLJKW LQ ¿YH \HDU ROG apple trees subjected to two training systems (Chen et al., 1997). Several days of continuous shade soon after bloom can reduce fruit set (Byers et al., 1990). Studies with partial shade are very limited (Moran and Rom, 1991) and little is known concerning WKH HIIHFW RI OLPLWHG VKDGH GXULQJ D VSHFL¿F GLXUQDO SHULRG RQ IUXLWLQJ DQG JURZWK 6KDGH ZKLFK UHGXFHV ÀRZHU EXG IRUPDWLRQ

DQG IUXLW VHW PD\ DOVR LQWHUDFW ZLWK FURS ORDG WR DIIHFW ÀRZHU formation (Jackson and Palmer, 1977). Results reported by Jackson and Palmer (1980) indicate that the crop size necessary WR UHGXFH ÀRZHULQJ LV OHVV XQGHU ORZ OLJKW OHYHOV 7KLV FRXOG EH important in areas like the eastern U.S. that have many partly cloudy and hazy days. When Lakso and Musselman (1976) investigated the effect of cloudiness on interior light in apple canopies, they found that diffuse light was greater under partly cloudy conditions compared to cloudless conditions resulting in increased PAR levels to the interior canopy. Whole-canopy photosynthesis measurements of apple trees have determined the general light response and demonstrated effects of cropping and heat stress (Wßnsche and Palmer, 1997; Whiting and Lang, 2001; Glenn et al., 2003). None of these studies have examined the plant response as a function of long-term seasonal whole-tree shading. There is a need to optimize carbon assimilation in orchard systems and manage carbon partitioning to improve cropping and fruit quality. The objective of the current research was to determine effects of morning, afternoon, and all-day shading on processes WKDW DUH VLJQL¿FDQW WR RUFKDUG SURGXFWLYLW\ 7KH K\SRWKHVLV ZDV WKDW VKDGH DSSOLHG DW VSHFL¿F LQWHUYDOV GXULQJ WKH GD\ DQG WKURXJKRXW WKH growing season will alter dry matter production and partitioning in apple and affect yield. The long-term goal of this and related work is to optimize light interception and carbon partitioning to fruit for consistent annual cropping and high fruit quality.

Materials and methods ‘Ginger Gold’/M.9 apple trees were planted in a solid block of three rows with 20 trees per row in 1996. Border trees, consisted of ‘Pink Lady’, ‘Gold Rush’, and ‘Liberty’, each on M.9 rootstock, that were planted as a single row on both sides of the test block

102

6KDGH HIIHFWV RQ JURZWK ÀRZHULQJ DQG IUXLW RI DSSOH

and on the end of each test row. Trees were spaced 2.4 m apart in rows spaced 4.9 m apart and were oriented in a north-south direction. Trees were headed to about 76 cm at planting and trained as a central leader with a metal pole supported by one wire at a height of 1.5 m above the ground. All trees were dormant pruned annually by the same individual to maintain uniformity to the extent possible. Once the basic central leader form was established (after the second growing season), pruning cuts were primarily (ca. 99%) thinning type cuts. Pollination was enhanced by the placement of two active bee hives within the immediate vicinity of the test block. Crop load was adjusted by hand following “June dropâ€? to space fruit approximately 15 cm apart on limbs. Trees received the local recommended cultural and pest management program throughout the study (Pfeiffer, 1998). A weed-free strip was maintained under the tree canopy on both sides of the URZ IURP WKH WUXQN WR WKH GULS OLQH ,Q WKH ÂżUVW JURZLQJ VHDVRQ D mechanical rotary hoe was used to obtain the weed-free strip and recommended herbicides were used in the following years. In 2002, trees were subjected to shade with 73% shade cloth from 0700 to 1330 HR (morning shade, MS) or 1330 to 2000 HR (afternoon shade, AS) daily from 2 weeks after full bloom (WAFB) to 10 WAFB and again 16 through 23 WAFB. A 24 hr constant 73% shade treatment (full shade, FS) and a non-shade treatment (no shade, NS) were included. In years 2003-05 95% shade cloth was used for all shade treatments. In all years, periodic shade treatments were applied daily to replicate trees using specially constructed shelters controlled by electronic time clocks (Fig. 1). The shade shelters measured 2.43 m wide by 5.67 m long and 3.23 m tall at the highest point in the center. A black polypropylene shade fabric (Hummert International, St. Louis, MO) providing 73% or 95% actual shade was used. Shade cloth was applied over the top of the shade shelters and extended down the sides to within about 45 cm of the ground. For the FS shelters all four sides were enclosed (Fig. 1A), but only the sides parallel to the row were covered in the partial shade shelters (Fig. 1 B). Metal poles were secured to each end of the shelter’s roof extending about 1.0 m down the tree row and the shade cloth was placed over these poles forming an awning (Fig. 1B). The partial shade shelters were connected to a cable and winch system operated by a time clock that was designed to pull the shelter over the test tree plots at the designated time each day. At the end of the day or at dawn the next day the partial shade shelters were manually

repositioned at the end of each row in preparation for the daily shade treatments. Treatment and sampling dates varied from year-to-year based on phenological development of the trees (Table 1). Response variables measured annually included trunk diameter (30 cm above the graft union), bloom cluster count before and after pruning, fruit count and dry weight (dw) at harvest, internal fruit quality (firmness, soluble solids, and starch index rating) at harvest, current season shoot length, and leaf and shoot carbohydrate levels (Stutte et al., 1994). Total soluble carbohydrate concentrations were calculated as the sum of glucose, fructose, sucrose, sorbitol and glucose-6-phosphate. <LHOG HIÂżFLHQF\ <( DQG EORRP HIÂżFLHQF\ ZHUH FRPSXWHG DV a function of trunk cross-sectional area (TCSA) data. Branch growth was the mean of 20 terminal current-year shoots selected at random per tree. Bloom cluster count was the total number of blossom clusters per tree taken between pink and full bloom. Fruit was harvested in August when the starch index rating reached a 3.0 level (Blanpied and Silsby, 1992). The four shade treatments were randomly assigned to 4 reps and each experimental unit was composed of 3 or 4 tree subsamples. Data was analyzed with the Proc Mixed Model of SAS (SAS Institute, Cary, NC).

5HVXOWV DQG GLVFXVVLRQ Growth: In most years, unshaded trees (NS) had the most or were among the most TCSA and branch growth (Table 2). Compared to NS, no shade treatment consistently reduced branch growth but TCSA growth was reduced by MS and FS in 2003 and 2005. TCSA did not differ between NS and AS trees but branch growth was reduced by AS from 2003 to 2005. In previous work, Miller (2001) found that 27% of full sun imposed by shading for a fourhour period each day from 3 wks after full bloom (FB) until 6 wks after harvest had minimal effects on growth and fruiting. In the current study 95% shade reduced growth but timing of shade WUHDWPHQW HDFK GD\ KDG VLJQL¿FDQW HIIHFWV 5HGXFHG VXQOLJKW in MS treatments may have reduced carbon assimilation at a time of the day when temperatures were lower and conducive to stomata opening and to photosynthesis. The AS-treated trees may have enabled photosynthesis by reducing sunlight and leaf temperatures and thus reduced stress in the afternoon. Flowering: 7KH QXPEHUV RI ÀRZHU FOXVWHUV SHU FP2 TCSA were

Fig. 1. Shade shelters used to apply full shade (FS) (A) or partial shade (MS, AS) (B) to ‘Ginger Gold’/M.9 apple trees. Structures were the same dimensions except rubber tired wheels were added to the partial shade shelters to facilitate positioning at sSHFLÂżF times each day

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Table 1. Time of bloom, imposed shade, sampling for carbohydrates, and harvest. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS). Shade cloth was 73% in 2002 and 95% in 2003-05 Year Bloom Harvest Shade treatment Sample dates FS MS AS 2002 Apr. 18 Aug. 14-15 73% May 6-Oct. 2 73% May 6-Oct. 2 73% May 6-Oct. 2 Jul. 1, Jul. 31, Aug. 27, Oct. 1 2003

Apr. 22

2004

Apr. 21

2005

Apr. 25

Aug. 21-22 95% May 13-Jul. 1 then Aug. 19-Sep. 17 Aug. 16-19 95% May 14-Jul. 7 then Sep. 1-Oct. 22 Aug. 15-17 95% May 18-Jul. 11 then Sep. 1-Oct. 19

95% May 13- Sep. 17

95% May 13- Sep. 17 Jun. 5, Jul. 1, Jul. 29

95% May 14- Oct. 22

95% May 14- Oct. 22 Jun. 9, Jul. 6, Aug. 9, Aug. 31

95% May 18- Oct. 19

95% May 18- Oct. 19 May 17, Jun. 16, Jul. 11

counted to evaluate the biological cropping potential before SUXQLQJ DQG UHGXFHG FURS ORDG WKDW PD\ UHĂ€HFW D PDQDJHG WUHH after pruning (Table 3). Number of clusters before pruning rose and fell from one year to the next, with trees receiving NS, MS, and AS treatments (Table 3). The MS and AS were in positive synchrony and varied together from year-to-year; they were in opposite flowering cycle with NS. This biennial bearing obfuscated effects of shading. For example, in 2005 one might interpret MS and AS to have decreased the number of blooms per cm2 TCSA (4.5 and 4.3, respectively) compared to NS (17.7) (Table 3). However, the reduced number of clusters may also be due to high bloom in 2004 resulting in reduced bloom in 2005. Heavy bloom years often follow light bloom years leading to alternate years of high yield (Westwood, 1978). The number of clusters before pruning appeared to be declining with time in FS trees. The number of clusters following pruning generally followed the same trends as the cluster count before pruning. Fruit 1R IUXLW GHYHORSHG XQGHU )6 $V ZLWK Ă€RZHU FOXVWHUV the density of fruit per cm2 TCSA appeared to be following a biennial pattern under NS, MS, and AS. From 2002 to 2004 the NS treatment had the largest number of fruit per tree. Fruit number and weight per tree were both reduced by and did not differ between MS and AS treatments (Table 4). In contrast, vegetative growth was reduced more by MS than AS suggesting that fruit sinks were more sensitive to shade than vegetative sinks as described by Bapete and Lakso (1998) (Tables 2 and 4). Previous Table 2. Trunk cross-sectional area (TCSA) and branch growth of ‘Ginger Gold’ apple trees grown under four shade treatments from 2002 to 2005. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS) Shade Year treatment 2002 2003 2004 2005 TCSA (cm2) FS 28.0 b* 34.4 b 37.1 b 40.4 b NS 42.1 a 52.8 a 58.1 a 73.8 a MS 30.2 b 32.7 b 38.4 b 42.0 b AS 46.2 a 50.7 a 61.2 a 65.3 a TCSA growth (cm2/yr) FS 6.4 ab 4.2 b 2.7 b 3.2 b NS 10.0 a 10.6 a 5.2 b 15.7 a MS 2.5 b 2.5 b 5.6 b 3.5 b AS 11.3 a 4.5 b 10.4 a 4.0 b Branch length (cm) FS 41.6 a 35.9 b 43.4 a 43.2 b NS 35.3 c 42.2 a 43.9 a 46.8 a MS 33.8 c 34.7 b 40.0 b 36.7 c AS 38.8 b 35.9 b 40.6 b 41.7 b * Within each variable and year, means followed by the same letter do not differ in the Proc. Mixed procedure ( P=0.05)

ZRUN GHPRQVWUDWHG WKDW YHJHWDWLYH JURZWK PD\ UHWDUG Ă€RZHU RU fruit growth due to shade. Early season shading of more than 60% full sunlight decreased partitioning of carbohydrates to fruit and vegetative stem growth was favored over fruit in ‘Empire’ trees (Bepete and Lakso, 1998). Shade reduced fruit retention and yield in both the year of shading and the following year (Jackson and Palmer, 1977). Three days of 80% shade resulted in up to 70% fruit abscission (McArtney et al., 2004). In 2003 and 2005, the largest fruit weight was associated with NS even though crop load was greater than or as great as MS and AS (Tables 3 and 4). Total dry weight of apples can be reduced by 12% and 30% with 73 % shade applied for partial and all day, respectively (Moran and Rom, 1991). 6KDGLQJ GLG QRW FRQVLVWHQWO\ DIIHFW IUXLW TXDOLW\ )UXLW ÂżUPQHVV starch and soluble sugars tended to be greatest in NS but fruit quality measures differed between MS and AS (Table 5). Fruit ÂżUPQHVV WHQGHG WR EH ORZHU DQG VWDUFK LQGH[ KLJKHU LQ 06 WKDQ AS, suggesting that fruit maturity advanced more quickly in fruit under MS than AS. Carbohydrates: Stem and leaf samples were collected at the same time each day, always just before shade was removed from MS and before shade was installed in AS. So, AS and NS were exposed equally to sun each morning when samples were collected. A second sampling note is that in May 2005 leaves and stems were sampled before shade was installed. Table 3. Bloom and fruit load of ‘Ginger Gold’ apple trees grown under four shade treatments from 2002 to 2005. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS) Shade Year treatment 2002 2003 2004 2005 Cluster (number / cm2 TCSA) before pruning FS 22.8 a* 12.5 a 8.0 bc 5.8 b NS 10.8 c 14.9 a 3.0 c 17.7 a MS 19.2 ab 1.0 b 16.6 a 4.5 b AS 14.4 bc 0.9 b 10.2 ab 4.3 b Cluster (number / cm2 TCSA) after pruning FS 13.9 a 7.6 a 4.2 b 4.2 b NS 5.5 c 8.4 a 0.7 c 9.2 a MS 10.4 ab 0.7 b 9.4 a 3.5 b AS 7.0 bc 0.2 b 4.6 bc 2.9 b Fruit (number / cm2 TCSA) FS 1.2 c 0.0 b 0.0 b 0.0 c NS 8.7 a 4.7 a 6.1 a 1.6 b MS 2.7 bc 4.9 a 0.6 b 3.2 a AS 2.9 b 4.0 a 0.6 b 3.1 a * Within each variable and year, means followed by the same letter do not differ in the Proc. Mixed procedure ( P=0.05).

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104

Table 4. Yield of ‘Ginger Gold’ apple trees grown under four shade treatments from 2002 to 2005. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS) Shade Year 2002 2003 2004 2005 treatment Fruit harvested (no. / tree) FS 30.7 c* 0.0 c 0.0 b 0.0 b NS 362.5 a 246.5 a 340.5 a 139.6 a MS 82.7 bc 176.7 b 23.7 b 133.2 a AS 132.5 b 243.5 ab 36.7 b 210.7 a Fruit harvested (kg / tree) FS 7.8 c 0.0 d 0.0 b 0.0 b NS 66.6 a 47.2 a 57.1 a 30.7 a MS 19.5 bc 24.4 c 4.9 b 23.7 a AS 32.3 b 35.8 b 6.6 b 35.9 a Fruit weight (g / fruit) FS 256.9 a 0.0 c 0.0 b 0.0 c NS 190.1 b 195.5 a 171.4 a 221.1 a MS 240.0 a 140.1 b 172.0 a 179.2 b AS 245.8 a 149.8 b 163.1 a 178.7 b * Within each variable and year, means followed by the same letter do not differ in the Proc. Mixed procedure (P=0.05).

Table 5. Fruit characteristics of ‘Ginger Gold’ apple trees grown under four shade treatments from 2002 to 2005. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS) Shade Year treatment 2002 2003 2004 2005 Fruit resistance to pressure (kg) FS 7.6 a* no fruit no fruit no fruit NS 7.3 ab 8.1 a 6.4 b 8.7 a MS 7.2 b 7.5 c 6.3 b 7.8 b AS 7.4 a 7.9 b 6.8 a 8.0 b Fruit starch index (low=9, high=1) FS 2.5 b no fruit no fruit no fruit NS 3.9 a 2.3 c 5.7 a 1.7 a MS 3.7 a 3.5 a 5.4 ab 1.6 a AS 2.3 b 2.9 b 4.9 b 1.7 a Fruit soluble sugars (%) FS 12.0 b no fruit no fruit no fruit NS 12.4 a 12.3 a 11.4 a 12.7 a MS 11.9 b 11.0 b 11.5 a 10.6 b AS 11.4 c 10.8 b 11.6 a 10.5 b * Within each variable and year, means followed by the same letter do not differ in the Proc. Mixed procedure (P=0.05).

Table 6. Soluble sugar concentration in leaf and stem of ‘Ginger Gold’ apple trees grown under four shade treatments from 2002 to 2004. Shade treatments included Full Shade (FS), No Shade (NS), Morning Shade (0730 – 1330, MS), and Afternoon Shade (1330 – 2000, AS) Soluble sugar concentration (mg / g) Sample date

Shade treatment

Leaf Sorbitol

Total

82 a 82 a 56 b 76 a

220 a 205 b 191 c 213 ab

Glucose-6phosphate 42 a 43 a 43 a 48 a

Stem Sorbitol

Total

36 a 33 a 35 a 35 a

96 ab 93 b 93 ab 99 a

July 1, 2002

FS NS MS AS

Glucose-6phosphate 109 a* 95 b 112 a 110 a

July 31, 2002

FS NS MS AS

105 a 92 b 112 a 108 a

68 b 58 b 59 b 80 a

208 b 179 c 207 b 230 a

36 ab 35 b 40 a 39 ab

33 a 29 b 35 a 35 a

82 bc 77 c 90 a 88 ab

Aug 27, 2002

FS NS MS AS

87 b 90 b 115 a 111 a

69 a 64 ab 53 b 77 a

200 b 194 b 199 b 233 a

30 b 30 b 38 a 39 a

32 a 36 a 37 a 36 a

80 b 83 ab 90 a 90 a

Aug 31, 2004

FS NS MS AS

108 bc 100 c 122 a 113 ab

71 ab 77 a 49 c 63 b

212 a 209 ab 189 c 199 bc

36 a 35 a 36 a 35 a

35 a 35 a 26 b 26 b

89 a 86 a 73 b 73 b

May 7, 2005

FS NS MS AS

116 a* 108 a 109 a 109 a

66 a 74 a 70 a 73 a

212 a 216 a 212 a 212 a

90 a 94 a 98 a 99 a

55 a 62 a 58 a 59 a

179 a 189 a 192 a 192 a

June 6, 2005

FS NS MS AS

126 a 107 b nm 116 ab

31 c 74 b nm 85 a

165 c 204 b nm 221 a

79 a 54 c 61 b 61 b

16 c 39 a 32 b 34 b

105 b 112 ab 110 ab 114 a

July 11, 2005

FS 114 a 39 b 170 b 65 a 17 b NS 105 ab 76 a 214 a 38 b 34 a MS 104 ab 44 b 174 b 41 b 30 a AS 101 b 80 a 213 a 40 b 33 a * Within each variable and year, means followed by the same letter do not differ in the Proc. Mixed procedure ( P=0.05).

92 a 86 a 86 a 88 a

6KDGH HIIHFWV RQ JURZWK ÀRZHULQJ DQG IUXLW RI DSSOH On July 1, 2002, total carbohydrate (TC) in tree leaves was greatest in FS and least in MS treatments (Table 6). By August 2002, TC was greatest in AS and was less but did not differ among the other shade treatments. Sorbitol is the main transport carbohydrate in apple and sorbitol was low in leaves of MS-treated trees during July 1 and August 27, 2002. In June and July 2005, leaf TC was VLJQL¿FDQWO\ OHVV LQ )6 DQG 06 WKDQ 16 DQG $6 WUHDWHG WUHHV As in 2002, elevated levels of TC in leaves were associated with greater levels of sorbitol during June and July 2005. During 2002 total carbohydrate (TC) in stems was similar among all shade treatments (Table 6). However, the partial shade treatments generally had the highest TC which was associated with higher glucose-6-phosphate on July 31 and August 27, 2002. *OXFRVH SKRVSKDWH LV D VLJQL¿FDQW SUHFXUVRU WR VXFURVH VRUELWRO and starch (Zhou and Cheng, 2008). Elevated concentrations of glucose-6-phosphate may indicate increased sugar transport and storage carbohydrate (starch) which may provide a necessary energy reserve for trees under shade. It is possible with a greater crop load, such as with the NS, MS, and AS treatments in June and July, 2005 (Table 4), that shading would diminish carbohydrates (Table 6). That does not appear to have occurred in this study. However, storage carbohydrates such as starch that were not measured in this study may be an important buffer. A major portion of an apple tree’s canopy is subjected to shade during most daylight hours each day and such shade may affect productivity. Shading may result from competition between trees and between growing meristems on the same tree. Canopy complexity may accentuate such intra-canopy competition. In the current experiment vegetative growth and yield were studied when shade was applied to the whole canopy. Full shade eliminated the crop and morning shade reduced growth and yield more than afternoon shade. Soluble carbohydrates in stems and leaves were inconsistent but were generally higher with no shade. Partial-day shade, notably afternoon shade, often had growth and yield that was equivalent to no shade. This suggests that morning shade may have adversely affected photosynthesis at a time of day that was most conducive to high net carbon assimilation. Training systems that reduce intra-canopy shading may help maximize yield although suppression of elevated temperatures may be necessary.

Acknowledgements We thank Lee Carper for his help on many occasions to reset the shade shelters. Disclaimer: Mention of trade names or commercial products in WKLV SXEOLFDWLRQ LV VROHO\ IRU WKH SXUSRVH RI SURYLGLQJ VSHFLÂżF information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

References Bepete, M. and A.N. Lakso, 1998. Differential effects of shade on earlyseason fruit and shoot growth rates in ‘Empire’ apple. HortScience, 33: 823-825. Byers, R.E., J.A. Barden, and D.H. Carbaugh, 1990. Thinning of spur ‘Delicious’ apples by shade, terbacil, carbaryl, and ethephon. J. Amer. Soc. Hort. Sci., 115: 9-13.

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Blanpied, G.D. and K.J. Silsby, 1992. Predicting harvest date windows for apples. Cornell Coop. Ext. Publ., Inform. Bul. 221, 12 pp. Chen, K., G.Q. Hu and F. Lenz, 1997. Training and shading effects on vegetative and reproductive growth and fruit quality of apple. Gartenbauwissenschaft, 62: 207-213. Faust, M. 1989. Physiology of Temperate Zone Fruit Trees. Wiley, New York. Flore, J.A. and A.N. Lakso, 1989. Environmental and physiological regulation of photosynthesis in fruit crops. Hort. Rev., 11: 111-157. Forshey, C.G. and D.C. Elfving, 1989. The relationship between vegetative growth and fruiting in apple trees. Hort. Rev., 11: 229-287. *OHQQ ' 0 $ (UH] * - 3XWHUND DQG 3 *XQGUXP 3DUWLFOH ÂżOPV affect carbon assimilation and yield in ‘Empire’ apples. J. Amer. Soc. Hort. Sci., 128: 356-362. Jackson, J.E. and J.W. Palmer, 1977. Effects of shade on the growth and cropping of apple trees. II. Effects on components of yield. J. Hort. Sci., 52: 253-266. Jackson, J.E. and J.W. Palmer, 1980. A computer model study of light interception by orchards in relation to mechanized harvesting and management. Scientia Hort., 13: 1-7. Lakso, A.N. and R.C. Musselman, 1976. Effects of cloudiness on interior light in apple trees. J. Amer. Soc. Hort. Sci., 101: 642-644. Lakso, A.N. 1994. Apple. In: Handbook of Environmental Physiology of Fruit Crops Vol. I Temperate crops. B. Schaffer and P.C. Andersen (eds.). CRC Press, Boca Raton, FL. p 3-42. Maib, K. (Ed.). 1996. Tree Fruit Physiology: Growth and Development. Good Fruit Grower, Yakima, Washington. McArtney S., M. White, I. Latter and J. Campbell, 2004. Individual and combined effects of shading and thinning chemicals on abscission and dry-matter accumulation of ‘Royal Gala’ apple fruit. Journal of Horticultural Science & Biotechnology, 79: 441-448. Miller, S.S. 2001. The effect of continuous and periodic whole-tree shade on the performance of ‘Ginger Gold’/M.9 apple trees. Proc. 77th Cumberland-Shenandoah Fruit Workers Conf. 77: 397-405. Moran, R.E. and C. Rom, 1991. The effects of diurnal light on photosynthesis and growth of greenhouse grown apple. HortScience, 26: 497. Abstr. Pfeiffer, D.G. 1998. Virginia-West Virginia-Maryland Commercial Tree Fruit Spray Bulletin. Virginia Cooperative Extension Publication. Blacksburg,VA, USA. 456–419. Robinson, T.L., A.N. Lakso and Z. Ren, 1991. Modifying apple tree FDQRSLHV IRU LPSURYHG SURGXFWLRQ HIÂżFLHQF\ HortScience, 26: 1005-1112. Stutte, G.W., T.A. Baugher, S.P. Walter, D.W. Leach, D.M. Glenn and T. J. Tworkoski, 1994. Rootstock and training system affect dry-matter and carbohydrate distribution in ‘Golden Delicious’ apple trees. J. Amer. Soc. Hort. Sci., 119(3): 492-497. Whiting, M.D. and G.A. Lang, 2001. Canopy architecture and cuvette flow patterns influence whole-canopy net CO2 exchange and temperature in sweet cherry. HortScience, 36: 691-698. : QVFKH - 1 DQG - : 3DOPHU 3RUWDEOH WKURXJK Ă€RZ FXYHWWH system for measuring whole-canopy gas exchange of apple trees in WKH ÂżHOG HortScience, 32: 653-658. Westwood, M.N. 1978. Temperate-Zone Pomology. W.H. Freeman and Co., San Francisco. Zhou, R. and L. Cheng, 2008. Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate. Journal of Plant Physiology, 165(9): 903-910. Submitted: January, 2015; Revised: February, 2015; Accepted: March, 2015

Journal

Journal of Applied Horticulture, 17(2): 106-108, 2015

Appl

Black rot control and bud cold hardiness of ‘Noiret’ winegrape Eric T. Stafne*, Becky Carroll1 and Damon Smith2 Coastal Research and Extension Center, Plant and Soil Sciences Department, Mississippi State University, Poplarville, MS, USA 39470, 1-601-403-8939. 1Agricultural Hall, Department of Horticulture, Oklahoma State University, Stillwater, OK, USA 74078, 1-405-744-6139. 2495 Russell Labs Building, 1630 Linden Dr., Department of Plant Pathology, University of Wisconsin, Madison, WI, USA 53706, 1-608-262-1410. *E-mail: eric.stafne@msstate.edu

Abstract Black rot, caused by Guignardia bidwellii (Ellis) Viala and Ravaz, and bud cold hardiness are both management issues in eastern U.S. viticulture. Black rot infections lead to vine stress, resulting in premature defoliation and rotten fruit, potentially compromising cold acclimation of the vine. No studies have targeted bud cold hardiness in relation to severity of prior season black rot infection. Thus, in 2011, ‘Noiret’, a hybrid winegrape, was subjected to four black rot control treatments: conventional (C), organic 1 (O1), organic 2 (O2), and no spray (N). Leaves and fruit were scored for black rot severity. The O1 and N treatments had the highest level of leaf and IUXLW GLVHDVH VHYHULW\ DQG ZHUH QRW VLJQLÂżFDQWO\ GLIIHUHQW 7KH & WUHDWPHQW KDG WKH OHDVW DPRXQW RI OHDI DQG IUXLW GLVHDVH VHYHULW\ DQG WKH 2 WUHDWPHQW ZDV LQWHUPHGLDWH DQG VLJQLÂżFDQWO\ GLIIHUHQW IURP WKH 2 1 DQG & WUHDWPHQWV %XG VDPSOHV ZHUH WDNHQ LQ -DQXDU\ February, and March 2012 and exposed to subzero temperatures (-21 °C, -23 °C, -26 °C, -29 °C) in an ethylene glycolbath to assess if prior season black rot infection impacted primary bud hardiness. In January and March nearly all buds were still alive at -21°C and ƒ& EXW ƒ& FDXVHG PRUH GDPDJH %ODFN URW FRQWURO WUHDWPHQWV ZHUH QRW D VWDWLVWLFDOO\ VLJQLÂżFDQW IDFWRU LQ WKH EXG KDUGLQHVV experiment. This could be due to black rot severity being below a critical threshold for impact or the vines had enough time to recover in late summer and fall to reach full mid-winter hardiness. Key words: Disease control, disease severity, Guignardia bidwellii LQWHUVSHFLÂżF K\EULG RUJDQLF YLQH VWUHVV Vitis spp.

,QWURGXFWLRQ Black rot, caused by Guignardia bidwellii (Ellis) Viala and Ravaz, requires constant disease management during the summer and it can lead to vine stress (Louime et al., 2010), leaf drop, and rotten fruit, potentially compromising overall vine ability to prepare for FROG WHPSHUDWXUHV Âľ1RLUHWÂś DQ LQWHUVSHFLÂżF K\EULG ZLQH JUDSH (Vitis spp. L.) cultivar released in 2006 (Reisch et al., 2006) has the potential to be important in the Oklahoma wine industry. It has been tested extensively in New York, Indiana, and other states (Reisch et al., 2006), where it was reported to be susceptible to black rot in Indiana and slightly susceptible in New York. Previous studies have not examined bud cold hardiness in relation to severity of prior season black rot infection. Monitoring bud cold hardiness in the winter is a concern for grape growers in the eastern U.S. Bud cold hardiness experiments using differential thermal analysis showed that the LTE50 mid-winter primary bud cold hardiness for ‘Noiret’ in New York was -25.7 °C (Pool et al., 1990). Mid-winter cold hardiness is stable in most locations if temperatures remain consistently low (Ferguson et al., 2011) KRZHYHU 2NODKRPD RIWHQ KDV H[WUHPH WHPSHUDWXUH Ă€XFWXDWLRQV during the vine dormancy period, thus the variation in bud cold KDUGLQHVV FRXOG FKDQJH LQ UHVSRQVH WR WKH Ă€XFWXDWLRQV LQ DPELHQW temperatures (Hubackova, 1996; Ferguson et al., 2011). *UDSH FXOWLYDUV GLIIHU LQ WKHLU UHVSRQVH WR Ă€XFWXDWLQJ PLG ZLQWHU temperatures (Mills et al., 2006; Ferguson et al., 2011). Cultivars may also respond differently when grown in different locations (Howell, 2000). A majority of the vine response is genetically controlled and can be due to the grape species involved in the cultivar’s parentage (Howell, 2000; Londo and Johnson, 2014).

Vitis species respond differently during dormancy to ambient temperatures (Jiang and Howell, 2002; Fennell, 2004; Londo and Johnson, 2014) and possibly other factors that impact cold hardiness levels. Studies to assess mid-winter primary bud hardiness have largely been conducted in more northern areas, where cold temperatures are more consistent (Pool et al., 1990; Hubackova, 1996; Jiang and Howell, 2002; Rekika et al., 2005; Ferguson et al., 2011). Poor vine management that leads to vine stress can also reduce vine cold hardiness (Howell, 2000), including injury from diseases which can interact with other vine stressors to reduce cold hardiness (Zabadal et al., 2007). This study was designed to test the efficacy of organic and conventional black rot control in relation to bud cold hardiness. The following hypotheses were tested: higher levels of black rot would result in elevated primary bud mortality due to enhanced vine stress and fungicide treatments would reduce vine stress and provide increased protection against primary bud mortality when subjected to freezing events.

Materials and methods The trial was conducted at the Cimarron Valley Research Station located in Perkins, OK, USA. The vineyard was planted in 2008 on Konawa loamy fine sand with Teller fine sandy ORDP LQWUXVLRQV Âľ1RLUHWÂś DQ LQWHUVSHFLÂżF K\EULG FXOWLYDU WKDW has some susceptibility to black rot was chosen for this study (Reisch et al., 2006). The vines were not grafted to a rootstock. The experimental design was a randomized complete block with four replicates. Single vine plots were separated by at least one non-treated vine. Plants were spaced 2.4 m apart in-row with a between-row spacing of 3.7 m. Recommended maintenance

Black rot control and bud cold hardiness of ‘Noiret’ winegrape Table 1. Black rot control treatments applied to ‘Noiret’ grape within an 8-spray program in 2011a. Spray regimen and rateb

107

Times applied

Application sequence

Conventional Mancozeb (DithaneRainshieldÂŽ 75DF 0.29 L¡ ha-1) + Quinoxyfen (QuintecÂŽ 2.08SC 0.29 L¡ ha-1) 1 1 Tebuconazole (EliteÂŽ 45 WP 0.29 L¡ ha-1) 3 2, 5, 8 7ULĂ€R[\VWURELQ )OLQWÂŽ 50 WG 0.14 L¡ ha-1) 2 3, 6 Mycobutanil (NovaÂŽ 40 WP 0.36 L¡ ha-1) 2 4, 7 Organic 1 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) 1 1 4 2, 4, 6, 8 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) + Bacillus subtilis (Serenade MaxÂŽ 14.6 WP 3.36 kg¡ ha-1) 3 3, 5, 7 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) + Bacillus pumilus (SonataÂŽ ASO 9.35 L¡ ha-1) Organic 2 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) 1 1 4 2, 4, 6, 8 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) + Bacillus subtilis (SerenadeÂŽ ASO 1.34F 14.03 L¡ ha-1) 3 3, 5, 7 %DVLF &RSSHU 6XOIDWH &XSURÂż[ 8OWUDÂŽ 40 DF 0.22 L¡ ha-1) + Bacillus pumilus (SonataÂŽ ASO 9.35 L¡ ha-1) No Spray 0 0 a Spray regimen previously described in Smith et al. (2012). bTrade names are used only for illustrative purposes and it is not implied as an endorsement of these products to the exclusion of other suitable products by Mississippi State University, Oklahoma State University, or the University of Wisconsin.

practices were followed throughout the growing season (Stafne, 2010). Four black rot control treatments (conventional (C), organic 1 (O1), organic 2 (O2), and no spray (N)) were imposed on the vines (Table 1). Fungicides were applied with a CO2-pressurized wheel barrow sprayer with a vertical boom equipped with three 7; Ă€DW IDQ QR]]OHV 7KH V\VWHP ZDV FDOLEUDWHG WR GHOLYHU L ha–1 )XQJLFLGH DSSOLFDWLRQV ZHUH ÂżUVW PDGH RQ $SU DQG continued on 14-day intervals until veraison for a total of eight sprays (Table 1). Ratings of leaf severity (average percent of leaf area with symptoms of black rot) and fruit severity (average percent of cluster area with symptoms of black rot) were taken on 22 June 2011 based on the methods described by Nutter et al. (2006). Five individual bud samples, replicated four times for each month and treatment temperature, were taken at approximately 3-week intervals beginning 19 January and ending 1 March 2012 and exposed to sub-zero temperatures (-21°C, -23 °C, -26 °C, and -29 °C) in a temperature-controlled ethylene glycol (C6H6O2) EDWK 7KHUPR )LVKHU 6FLHQWLÂżF 8/7 1HZLQJWRQ 1+ 86$ to assess if previous season black rot infection had an impact on primary bud hardiness (Rekika et al., 2005). Due to sampling error, the data for February was not included in the analysis. Buds were collected and separated by month and vine from which they were collected. A single bud from each cutting was placed in a labeled plastic bag with four bud bags per numbered vine per month. The bags were labeled 1 through 4 for each vine corresponding to the temperatures at which they were removed IURP WKH EDWK $IWHU WKH EDJV ZHUH ÂżOOHG WKH\ ZHUH VHSDUDWHG E\ the number indicated on the bag. All treatments were placed in D MDU FRYHUHG ZLWK SDUDÂżOP WR KHOS VHDO WKH OLGV $ WHPSHUDWXUH sensor (Watchdog model A110, Spectrum Technologies, Aurora, IL, USA) was double bagged and placed in a plastic container with four weights. The container was placed in the bottom center of the bath to record the bath temperature.The initial temperature was held at 1°C for 48 h, and then the temperature decreased 5 °C each hour until -21°C was reached. The temperature was then held for 24 h, after which it dropped 1°C each 30 min to the next programmed test temperature. Each time a set temperature (-21 °C, -23 °C, -26 °C, and -29 °C) and incubation period (24 h) was reached, the appropriate jar of buds was removed and replaced ZLWK DQ HPSW\ MDU FRQWDLQLQJ ÂżYH ZHLJKWV WR PDLQWDLQ Ă€XLG OHYHOV in the bath. After a jar was removed, it sat at room temperature for 3-6 h before buds were examined under a microscope.

All disease and bud data were arcsine-transformed for statistical analysis. Results presented are based on back-transformed means. Data were analyzed by analysis of variance (P<0.05) using JMP 9.0 (SAS Institute, Cary, NC, USA). Transformed treatment means were compared by Tukey’s HSD (P<0.05).

5HVXOWV DQG GLVFXVVLRQ Black rot control treatments: 6LJQLÂżFDQW GLIIHUHQFHV LQ OHDI DQG IUXLW GLVHDVH VHYHULW\ ZHUH LGHQWLÂżHG ZLWK WKH KLJKHVW OHYHO of disease recorded in the N plots (Table 2). The O1 and N treatments had the highest level of leaf and fruit lesion severity DQG ZHUH QRW VLJQLÂżFDQWO\ GLIIHUHQW 7DEOH 7KH & WUHDWPHQW had the least amount of leaf and fruit lesion severity and the O2 WUHDWPHQW ZDV LQWHUPHGLDWH DQG VLJQLÂżFDQWO\ GLIIHUHQW IURP WKH O1, N, and C treatments. Thus, the conventional spray regimen was better than the organic spray in the control of black rot. There were differences between organic spray treatments, showing that a change in product formulation (wettable powder (WP) YV DTXHRXV VROXWLRQ $6

FDQ UHGXFH VSUD\ HIÂżFDF\ 7KH 2 treatment was no different than the N treatment, suggesting that it did not provide a reduction in black rot disease severity. Table 2. Leaf and fruit severity ratings for black rot infection on ‘Noiret’ grape during the 2011 growing season in Perkins, Oklahoma Treatment Leaf severity ratinga (%) Fruit severity ratinga(%) 26.3 a Organic 1 17.5 ab No spray 16.3 a 23.8 a Organic 2 7.5 b 11.3 b Conventional 1.7 c 0.0 c a Ratings of leaf and fruit severity are the average percent of leaf area and cluster area with symptoms of black rot, respectively as described by Nutter et al. (2006). Data presented are from back-transformed means. b Means separated by the same letter within a column are not significantly different based on Tukey’s HSD (P<0.05).

Primary bud cold hardiness: Primary bud response to cold temperatures did differ among temperatures within months. At -21°C and -23°C for buds taken in January and March nearly all were still alive (Table 3). Buds exposed to colder temperatures of -26°C and -29°C had greater mortality, especially for January (Table 3). As in many eastern U.S. states, the continental climate prevalent in Oklahoma has a strong impact on vine growth and development, especially fluctuating winter and spring temperatures (Stafne, 2007). For January and March, weather was dry and unseasonably warm with an average temperature of 5°C

108

Black rot control and bud cold hardiness of ‘Noiret’ winegrape

14 days prior to sampling and no rainfall. Average temperature 14 days before the March sampling was 9 °C with only 2.8 mm RI UDLQ 7KHUH ZHUH QR VLJQLÂżFDQW LQWHUDFWLRQV RI EODFN URW FRQWURO treatment and temperature for the bud hardiness experiment. %ODFN URW FRQWURO WUHDWPHQW PDLQ HIIHFW ZDV DOVR QRW VLJQLÂżFDQW for January (P = 0.0518) and March (P = 0.2994). Temperature was found to be the dominant factor that led to bud mortality in this study. Hubackova (1996) stated that hardiness of primary buds in grapevine were dependent upon the maximum and mean temperatures to which they were exposed prior to imposed controlled freeze testing; however, even though unseasonably warm temperatures prevailed during this experiment, the primary bud hardiness of ‘Noiret’ was found to be slightly different than reported by others (Pool et al., 1990; Reisch et al., 2006). This may be attributable to the difference in methods used to determine bud hardiness or to the different environmental conditions under which the vines were grown. Drought conditions during the winter may have led to a decrease in tissue water content (Jiang and Howell, 2002) and a subsequent increase in cold hardiness, but other studies have not found consistent results in establishing a UHODWLRQVKLS EHWZHHQ ZDWHU GHÂżFLW DQG SULPDU\ EXG KDUGLQHVV LQ grapevines (Basinger and Hellman, 2006). Table 3. ‘Noiret’ grape primary bud cold hardiness at four subzero temperatures taken in January and March 2012 Treatment temperature Alive primary buds, Alive primary buds, (°C) January (%) March (%) -21 100.0 aa 100.0 a -23 100.0 a 100.0 a -26 70.7 b 85.3 a -29 61.3 b 70.7 b a Means separated by the same letter within a column are not significantly different based on Tukey’s HSD (P<0.05).

Interaction effects of black rot and temperature on cold hardiness: 7KHUH ZDV QR VWDWLVWLFDOO\ VLJQLÂżFDQW LQWHUDFWLRQ RI EODFN rot control treatment and temperature in relation to bud hardiness ( P=0.37 and P=0.98 for January and March, respectively). This could be due to black rot severity being below a critical threshold for impact or the vines had enough time to recover in late summer and fall to reach full mid-winter hardiness. This may be due to ‘Noiret’ having a moderate susceptibility to black rot (Reisch et al., 2006). In our trials in 2011, low to moderate levels of black rot leaf severity were observed (26% severity in the Nvines). Zabadal et al. (2007) stated that black rot may not be as serious a factor in potential cold hardiness reduction as late season fungi like powdery mildew (Uncinula necator (Schw.) Burr.) and downy mildew (Plasmopara viticola), so the vines may have had time WR UHFRYHU VXIÂżFLHQWO\ LQ WKLV VWXG\ WR DYRLG GHWULPHQWDO LPSDFWV Under conditions that favor greater black rot disease pressure that lead to more leaf and fruit disease severity, the results may have been different. So, even though in this study black rot had no effect on primary bud cold hardiness, further studies that incorporate disease as a factor should be conducted. Black rot control was not a statistically significant factor in the bud hardiness experiment. This could be due to the low to moderate levels of observed black rot severity being below a critical threshold for impact on ‘Noiret’ or the infections were no longer impacting vines later in the summer and they had enough time to recover to reach full mid-winter hardiness. Based on these data, primary bud mortality never reached 50% for ‘Noiret’

when subjected to subzero temperatures as low as -29 °C which ZDV VOLJKWO\ KDUGLHU WKDQ ZDV UHSRUWHG ZKHQ WKH FXOWLYDU ZDV ÂżUVW released (Reisch et al. %XGV EHJDQ WR VKRZ D VLJQLÂżFDQW loss in cold hardiness between -23 and -26 °C. More work can be done in this area to assess potential negative impacts on bud cold hardiness as it relates to disease infection in the prior season. Differences were observed between the two organic spray WUHDWPHQW VXJJHVWLQJ WKDW SURGXFW IRUPXODWLRQ FDQ LPSDFW HIÂżFDF\ We determined that low to moderate levels of black rot infection did not affect ‘Noiret’ primary bud cold hardiness in the following winter, rather temperature was the driving factor in bud mortality.

References %DVLQJHU $ 5 DQG ( : +HOOPDQ (YDOXDWLRQ RI UHJXODWHG GHÂżFLW irrigation on grape in Texas and implications for acclimation and cold hardiness. Intl. J. Fruit Sci., 6(2): 3-22. Fennell, A. 2004. Freezing tolerance and injury in grapevines. J. Crop Improv., 10: 201-235. Ferguson, J.C., J.M. Tarara, L.J. Mills, G.G. Grove and M.Keller, 2011. Dynamic thermal time model of cold hardiness for dormant grapevine buds. Ann. Bot., 107: 389-396. Howell, G.S. 2000. Grapevine cold hardiness: Mechanisms of cold acclimation, mid-winter hardiness maintenance, and spring deacclimation. Proc. ASEV 50thAnniv. Mtg., 35-48. Hubackova, M. 1996. Dependence of grapevine bud cold hardiness RQ Ă€XFWXDWLRQV LQ ZLQWHU WHPSHUDWXUHV Amer. J. Enol.Viticult., 47: 100-102. Jiang, H. and G.S. Howell, 2002. Correlation and regression analyses of cold hardiness, air temperatures, and water content of Concord grapevines. Amer. J. Enol.Viticult., 53: 227-230. Londo, J.P. and L.M. Johnson, 2014. Variation in the chilling requirement and bud burst rate of wild Vitis species. Environ. Expt. Bot., 106: 138-147. Louime, C., H.K. Vasanthaiah, S.M. Basha and J. Lu, 2010. Perspective of biotic and abiotic stress research in grapevines (Vitis spp.). Intl. J. Fruit Sci., 10(1): 79-86. Mills, L.J., J.C. Ferguson and M. Keller, 2006. Cold-hardiness evaluation of grapevine buds and cane tissues. Amer. J. Enol.Viticult., 57: 194-200. Nutter, F.W., P.D. Esker and R.A.Coelho Netto, 2006. Disease assessment concepts and the advances made in improving the accuracy and precision of plant disease data. Euro. J. Plant Pathol., 115: 95-103. Pool, R.M., B.I. Reisch and M.J. Welser, 1990. Use of differential thermal analysis to quantify bud cold hardiness of grape selections and clones. Vitis(special issue) Proc. 5th Intl.Symp. Grape Breeding, 318-329. Reisch, B.I., R.S. Luce, B. Bordelon and T. Henick-Kling, 2006. ‘Noiret’ grape. NY State Agr. Expt. Sta. Bul., 160. Rekika, D., J. Cousineau, A. Levasseur, C. Richer, H. Fisher and S. Khanizadeh, 2005. The use of a bud freezing technique to determine the hardiness of 20 grape genotypes. Small Fruits Rev., 4(1): 3-9. Smith, D.L., J.L. Lyles and A.F. Payne, 2012. Evaluation of reduced-risk and organic fungicide programs for control of black rot of grape in Oklahoma, 2011. Plant Dis. Mgt. Rep., 6: SMF012 doi: 10.1094/ PDMR06. Stafne, E.T. 2007. Indices for assessing site and winegrape cultivar risk for spring frost. Intl. J. Fruit Sci., 7(4): 121-132. Stafne, E.T. (Ed.), 2010. Handbook of Oklahoma Vineyard Establishment and Management. Okla. Coop. Ext. Serv., E-1015. Wolf, T.K. and M.K. Cook, 1992. Seasonal deacclimation patterns of three grape cultivars at constant, warm temperature. Amer. J. Enol. Viticult., 43: 171-179. =DEDGDO 7 - , ( 'DPL 0 & *RIÂżQHW 7 ( 0DUWLQVRQ DQG 0 / &KLHQ 2007. Winter injury to grapevines and methods of protection. Michigan State Univ. Ext. Bul., E2930. Submitted: May, 2015; Revised: May, 2015; Accepted: May, 2015

Journal

Journal of Applied Horticulture, 17(2): 109-114, 2015

Appl

+LJK HIĂ€FLHQF\ Agrobacterium-mediated transformation of sour orange (Citrus aurantium L.) using gene encoding Citrus Tristeza Virus coat protein Mohammad Mehdi Sohani1*, Mohammad Hosein Rezadoost1, Amir Hosein Zamani1, Mohammad Reza Mirzaei1 and Alireza Afsharifar2 1

Biotechnology Department, College of Agricultural Sciences, University of Guilan, Rasht, Iran. 2Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran. *E-mail: msohani@guilan.ac.ir

Abstract &LWUXV WUHHV DUH ZLGHO\ JURZQ LQ WURSLFDO DQG VXEWURSLFDO FOLPDWHV GXH WR WKHLU OXVFLRXV WDVWH QXWULWLRQDO DQG PHGLFDO EHQHÂżWV &LWUXV fruits are native to southeastern Asia and are among the oldest fruit crops domesticated by humans. Breeding programs including the incorporation of genetic resistance to insect pests and diseases are necessary in this crop. Citrus tristeza virus (CTV) is of particular importance due to its rapid epidemic resulting in severe plant damage. The present research aimed to transform Citrus aurantinum with a gene encoding coat protein of CTV through Agrobacterium-mediated transformation. The p25 coat protein gene was isolated from two QDWLYH &79 LVRODWHV 7ZR FRQVHUYHG UHJLRQV IURP WKH WZR LVRODWHV ZHUH LGHQWLÂżHG DQG VXEFORQHG DV D VLQJOH FKLPHU LQWR D S)*& silencing vector. Epicotyls-originated explants of C. aurantium were transformed by EHA105 strain of Agrobacterium tumefaciens. Some of the effective factors in gene transformation were examined by inoculation methods with Agrobacterium such as Acetosyringon effect (0, 50, and 100 ÎźM), inoculation time (5, 10, 15, 20, and 25 min), and co-cultivation period (1, 2, 3 and 4 days). Based on our results, maximum number of transformed plants (13.7%) were obtained under combined treatment of 50 ÎźM acetosyringone after 15 min inoculation time and 2 days of co-cultivation with Agrobacterium. One of the advantages of the current protocol is regeneration of explants through direct organogenesis which avoid callus phase and consequently somaclonal variation. Key words: Acetosyringon, vir gene induction, virus-induced gene silencing, Citrus tristeza virus

,QWURGXFWLRQ Citrus, belonging to the family Rutaceae, is one of the world’s most important fruit crops. Regarding the fact that world-wide production of citrus fruits is about 115 million tons (FAO, 2007) breeding programs are important for developing superior varieties for increasing the production and quality of the fruit. However, conventional breeding in Citrus species faces several limitations such as high heterozygosity, apomixis, self-incompatibility, and long juvenile period (Grosser and Gmitter, 2005). As a result, Agrobacterium-mediated transformation has been considered by various researchers. Hidaka et al ZHUH WKH ¿UVW WR produce Washington and Trovita transformed orange using A. tumifaciens. This method was then used for transformation of Carrizo citrange (Moore et al., 1992; Kaneyoshi et al., 1994). This approach was later improved using changes in some factors such as inducing longitudinal incision (Yu et al., 2003), using GFP marker (Ghorbel et al., 1999) and Acetosyringone (AS; Gelvin, <HW WKH HI¿FLHQF\ RI FLWUXV WUDQVIRUPDWLRQ KDV DOZD\V been reported low. Some of the underlying reasons reported are: low rate of transformed plants rooting, adverse effects following transformation (Gutierrez et al., 1997), and gene escape. Among citrus, Citrus aurantium is taken to be one of the species, resistant against genetic transformation and manipulation. Citrus tristeza virus (CTV) is the main viral diseases of citrus. This disease, so far, has ruined millions of citrus trees (Whiteside et al., 1988). C. aurantium stalk and the presence of Aphis gossypii – CTV vector – are the potential factors of outbreak of this disease

in citrus areas of Iran (Barzegar et al., 2006). Due to its potential growth in saline and limy soils and to create desirable taste and ÀDYRU LQ IUXLWV C. aurantium is among suitable rootstocks for citrus cultivation. Nevertheless, this rootstock is especially susceptible to Citrus tristeza virus. It has been shown that substituting other rootstocks for C. aurantium results in the reduction of fruit yield and quality (Ghorbel et al., 2000). Accordingly, C. aurantium genetic transformation for further resistance against CTV becomes considerably important in breeding programs. Some citrus genotypes are successfully transformed to induce resistance against CTV including, C. aurantium (Gutierrez et al., 1997; Ghorbel et al., 2000), C. aurrantifolia (Dominguez et al., 2000), C. paradisi (Febres et al., 2003), C. sinensis (L.) (Muniz et al., 2012), and poncirus (Zou et al., 2008). In present study, C. aurantium was transformed by a silencing vector, encoding part of 2 CTV-P25-CP genes to generate transgenic sour orange with induced constitutive resistance against CTV isolates.

Materials and methods CTV isolates and bioinformatics analysis: The nucleotide sequences of CTV-CP-p25 gene from two native isolate (provided by Plant Virology Research Center; Shiraz, Iran) were used in this study. For this purpose, 10 CTV-CP-p25 sequences, which were obtained from different native isolates in this center were aligned by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and further analysed by clustalW2 phylogenic program (http://

Agrobacterium-mediated transformation using gene encoding citrus tristeza virus coat protein

110

opposite insertion into the silencing vector. The inverted repeat is assembled directly in a binary vector by a 2-step cloning SURFHVV XVLQJ WKH LQWURGXFHG UHVWULFWLRQ HQ]\PH VLWHV ,Q WKH ÂżUVW cloning step, the PCR product is cleaved at the inner restriction sites, AscI and SwaI, and ligated to cleaved AscI and SwaI sites in pFGC5941. The cloned fragment was sequenced using gene VSHFLÂżF SULPHUV WR FRQÂżUP WKHLU LGHQWLW\ ,Q WKH VHFRQG VWHS the resulting PCR product is cleaved with BamHI and XbaI and inserted into the BamHI and XbaI-cleaved template plasmid based RQ PDQXIDFWXUHU PDQXDO 7KHUPR 6FLHQWLÂżF DQG QDPHG DV 3 and P2, respectively. This yields an inverted repeat separated by the ChsA intron.

www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/). This ZDV DLPHG WR ÂżQG VHTXHQFHV ZLWK PD[LPXP GLIIHUHQFH LQ this set. At the same time, Blast search (http://blast.ncbi.nlm. nih.gov/Blast.cgi) was applied to search for CTV sequences in database similar to Shiraz sequences. It was intended to determine sequences from Shiraz set with maximum homology with CTV LGHQWLÂżHG VHTXHQFHV LQ GDWDEDVH $V D UHVXOW DPRQJ CTV isolates collected from southern Iran, two sequences (CTV1 and CTV2) were selected. These 2 sequences, cloned in PTZ57R/T plasmid, had minimum homology with each other and maximum homology with the existing data in gene bank. CTV-1 and CTV-2 sequences were amplified using specific forward and reverse primers (Table 1). Forward CTV1 and reverse CTV2 primers carried XhoI restriction site. For CTV-CP-P25 DPSOLÂżFDWLRQ UHDFWLRQV ZHUH FDUULHG RXW DW ƒ& IRU V ƒ& IRU V ƒ& IRU V IRU F\FOHV SOXV D ÂżQDO H[WHQVLRQ DW ž& IRU PLQ $PSOLÂżHG '1$ ZDV GHWHFWHG E\ XOWUDYLROHW illumination after electrophoresis on 2% (w/v) agarose–ethidium bromide gels. PCR fragments were digested by Xhol enzyme DFFRUGLQJ WR PDQXIDFWXUHUÂśV PDQXDO 7KHUPR 6FLHQWLÂżF 7ZR SLHFHV ZHUH OLJDWHG XVLQJ 7 HQ]\PH 7KHUPR 6FLHQWLÂżF EDVHG on Sambrook and Russell (2001) instruction and and a DNA fragment of about 400 bp was resulted. After purifying the respective (CTV1 and CTV2 CP genes) mosaic fragment, it was used as template in further PCR reactions (Zamani, 2009).

Silencing vector characteristics: pFGC5941 vector (GenBank: AY310901.1) was a gift from Arabidopsis Biological Research center (ABRC; CD3-447). It is a binary vector specific to dicotyledonous plants, and gene transfer is based on ligase and restriction enzymes. Selection markers for bacteria as well as plant are kanamycin and basta (glufosinate-ammonium), respectively. Its dsRNA promoter is CaMV35S and its spacer is ChsA intron with repetitive reverse sequence (Fig. 1). &RQÂżUPDWLRQ RI &79 PRVDLF LQVHUWLRQ LQ VLOHQFLQJ YHFWRU First, the enzyme double digestion of pFGC5941 silencing vector (ABRC) was carried out using BamHI and XbaI restriction enzymes EDVHG RQ PDQXIDFWXUHU PDQXDO 7KHUPR 6FLHQWLÂżF WR OLQN 3 gene. In the next stage, silencing vector containing P2 fragment underwent enzymatic digestion by AscI and XbaI and P1 fragment ligated as described for P2 fragment based on Sambrook & Russell (2001). Finally, the construct was transformed into E. coli cells using electroporation method (Bio-Rad). Evaluating and examining colonies and transformed bacteria cultures were conducted by the aid of PCR. Plasmid was minipreped from positive colonies

Synthesis of a mosaic inserts (P1 and P2): The resulting mosaic fragment was used as a DNA template for producing 2 inserts and their cloning into the silencing vector. In this section, a 340 ES IUDJPHQW RI '1$ WHPSODWH ZDV DPSOLÂżHG XVLQJ ) PRVDLF and R-mosaic primers (Table 1). Each primer contained 2 different restriction sites, which allowed directional cloning yet

Table 1. Characteristics and sequence of the primers used in cloning and PCR analysis of the transformants Primers F-CTV1

Sequences 5’- CGCCTCGAGGCTAAACGATACAACATCATAGC- 3’

Restriction sites XhoI

R-CTV1

5’- GCTAAACGATACAACATCATAGC -3’

-

F-CTV2

5’- CCACTTCAATACCCTCCCG -3’

XhoI

R-CTV2

5’- CGCCTCGAGCGACTCTGATAGCGATGAACG- 3’

-

F-mosaic

5’- GCTCTAGAGGCGCGCCTGCTGCTGAGTCTTCTTTCG -3’

XbaI, AscI

R-mosaic

5’- GCGGATCCATTTAAATCCGTGGTGTCATCATCACTT -3’

SwaI, BamHI

BAR-F

5’-GAAGTCCAGCTGCCAGAAAC-3’

-

BAR-R

5’-AGTCGACCGTGTACGTCTCC-3’

-

CTV-F

5’-CGCCATGGACGAAACAAAG-3’

-

CTV-R

5’-CCACTTCAATACCCTCCCG-3’

-

KAN-F

5’-ATGTTGCTGTCTCCCAGGTC-3’

-

KAN-R

5’-GAAAGCTGCCTGTTCCAAAG-’

-

2500 LB T-DNA BIpR MAS promoter

5000 | chsA instron |

| CTV CaMV 35S

CTV

75000

10,000 ori

| RB T-DNA Ml 3 fwd

pV31 StaA

KanR

pV31 RepA bom

Fig. 1. Schematic representation of the T-DNA from the binary Plasmid pFGC5941, engineered to carry double copies of the gene encoding coat protein from CTV: LB: left border repeat from nopaline C58 T-DNA; a kanamycin resistance (kanR) gene for bacterial selection, a basta resistance (BAR) gene for plant selection, a CaMV 35S promoter to drive the expression of the inverted repeat target sequence, and a 1,352 bp ChsA intron (from the petunia Chalcone Synthase A gene) to stabilize the inverted repeat of the target gene fragment. RB: right border repeat from nopaline C58 T-DNA (the map was created by SnapGene).

Agrobacterium-mediated transformation using gene encoding citrus tristeza virus coat protein (Bioneer). Accordingly, 2 viral DNA mosaic fragment with 340 bp length and similar sequences were integrated in 2 different parts of silencing vector and in 2 opposite directions. Chalcone synthase intron (~1400bp) is located between these 2 pieces (Fig. 1). 7R IXUWKHU FRQ¿UP WKH FORQLQJ RI 3 DQG 3 LQVHUWV GRXEOH enzymatic digestion reactions was performed. Namely, extracted silencing plasmid containing respective pieces underwent enzymatic digestion either by AscI and SwaI or BamHI and XbaI in separate reactions. In both reactions, it was expected that a piece of destination silencing vector with ~400bp length to get separated and observed on gel. 7R IXUWKHU FRQ¿UP WKH FORQLQJ PRVDLF IUDJPHQW LQ GHVWLQDWLRQ vector, the same pFGC591 silencing construct was digested by AscI and XbaI. As a result, it was expected to detect a 2200bp piece including chalcone synthase intron plus 2 ~400 bp mosaic fragment on gel. Plant material: Ripe citrus fruits were provided from Ramsar Citrus Research Center (Ramsar, Iran). Seeds were collected and disinfected using sodium hypochlorite solution (2.5%) and rinsed 3 times by sterilized distilled water. The disinfected seeds were cultured in Murashige and Skoog (MS; Duchefa) medium and were kept for 4 weeks in darkness followed by 10 days under 16 h-daylength at 28 °C (Almedia et al., 2002). To prepare explants and carry out transformation, in vitro grown epicotyls were cut in 1 cm pieces. A 1 mm longitudinal incision was made in both ends of the explants. A 1-mm longitudinal incision was made at both ends of the explants and then the explants were transferred to liquid pre-culture medium containing MS salts, B5 vitamin, 500 mg/L malt extract, 2.5 mg/L BAP, 0.05 mg/L 1-Naphthaleneacetic acid (NAA; Sigma), 4.7% w/v sucrose (Merck), and pH was adjusted to 5.8. They were slowly stirred for 2 days in darkness at 28 °C.

111

mg/L malt extract, 2.5 mg/L BAP, 0.05 mg/L NAA, 4.7% w/v sucrose, 8 g/L agar, and pH 5.8. To select transformed plant cells, 50 ΟM BASTA herbicide (Bayer Crop Science) and to remove Agrobacterium 500 mg/L cefotaxime were added to the medium. Media were placed in 16-h photoperiod for 5 weeks at 28 °C till 1 mm seedlings were observed on explants. Explants were transferred to a shoot growth medium. This medium was applied to accelerate shoots longitudinal growth. It contained salts, MS vitamin, 3% w/v sucrose, 0.5 mg/L Gibberellic acid (GA3), 8 g/L agar with pH 5.8. When shoots further grew, sampling was carried out for DNA extraction. Transgenic shoot analysis: To verify transformation and evaluate LWV HI¿FLHQF\ '1$ ZDV H[WUDFWHG IURP \RXQJ OHDYHV DQG 3&5 was performed using 3 pairs of primers. Primers were related to herbicide-resistant genes (BAR) and CTV mosaic, which are located at T-DNA region of pFGC5941 Vector (Table 1). Besides, Kanamycin resistance gene (kan), located at the outside of T-DNA of the vector, was used in PCR to examine and determine whether positive results of PCR on tested genes were originated from Agrobacterium contamination of explants and shoots or from real transformed plant genome. Some PCR products were chosen UDQGRPO\ DQG VHTXHQFHG XVLQJ JHQH VSHFL¿F SULPHUV Seedlings DNA extraction: DNA extraction was carried out based on Edward et al. (1991) protocol. First, 20 mg of upper leaves were collected and grinded in 1.5 mL tube by liquid nitrogen. Then, 1 mL Edward buffer (200 mM Tris, 250 mM NaCl, 0.5% w/v SDS, 25 mM EDTA) was added to the grinded tissue. After 5s vortex, it was placed at room temperature for 2 min centrifugation for 2 min at 13000 rpm. 500 ΟL supernatant was transferred to new 1.5 mL tube, containing 500 ΟL cold isopropanol and kept at room temperature for 2 min before tube was centrifuged at 14000 rpm for 5min. When pellets dried, 50 ΟL TE buffer (10 mM Tris, 1 mM EDTA) was added to pellets. Samples were kept in freezer at -20 °C till being used.

Agrobacterium preparation: Agrobacterium tumefaciens (EHA105 strain) was transformed by electroporation to receive recombinant plasmids. A. tumefaciens EHA 105 is a disarmed derivative of A. tumefaciens A281, which is supervirulent in citrus (Cervera et al. 1998b). To select transformed bacteria, they were cultured on solid yeast extract peptone dextrose (YEP) medium containing 100 mg/L kanamycin, 50 mg/L rifampicin, 7 g/L agar and pH 6.8 at 28 °C for 48 h. A colony was cultured in liquid YEP medium with the same concentrations of antibiotic for 16 h at 28 °C. Bacterium suspension with an OD600 = 1 was centrifuged at 6000 rpm for 5 min. Its pellet was dissolved in liquid MS medium containing 0, 50, and 100 ΟM Acetosyringone (AS; 3,5-methoxy4-hydroxyacetophenone; Sigma). The same concentration of AS were also applied in co-cultivation period.

5HVXOWV DQG GLVFXVVLRQ

Transformation, selection and regeneration: Epicotyloriginated explants were submersed in MS inoculation liquid containing Agrobacterium for 5, 10, 15, 20, and 25 min at 28 ƒ& 6XEVHTXHQWO\ WKH\ ZHUH GULHG RQ ÂżOWHU SDSHU VR WKDW H[WUD bacteria are removed from explants. After inoculation, explants were cultured in co-cultivation medium including MS salts, B5 vitamin, 500 mg/L malt extract, 2.5 mg/L BAP, 0.05 mg/L NAA, 0, 50, and 100 ÎźM AS, 4.7% w/v sucrose, 8 g/L agar, at pH 5.8. They were kept for 1, 2, 3 and 4 days in darkness at 28 °C.

&RQÂżUPLQJ GRXEOH LQWHJUDWLRQ RI &79 0RVDLF IUDJPHQW LQ pFGC5941 Vector: As a result of performing PCR reactions E\ PHDQV RI PRVDLF VSHFLÂżF SULPHUV SLHFH RI a ES IURP genes encoding CTV1 and CTV2 DPSOLÂżHG UHVSHFWLYHO\ )LJ )ROORZLQJ DPSOLÂżFDWLRQ RI WKH 3&5 SURGXFWV GRXEOH GLJHVWLRQ and ligation performed. A piece of ~400bp length chimer was obtained when the ligation product used as a DNA template in PCR reaction. It proved mosaic fragment synthesis as well as ligation of CTV-1 and CTV-2 fragments (Fig. 3).

After the end of co-cultivation period, explants were transferred to regeneration medium containing MS salts, B5 vitamin, 500

Further, recombinant silencing plasmid extracted from E. coli was double digested by AscI and SwaI for P1 isolation as well

Statistical Analysis: 2QO\ VKRRWV SRVLWLYHO\ LGHQWL¿HG E\ WZR PCR reactions to contain CTV mosaic and BAR genes along with negatively to kan gene were considered as transformed plant. Statistical analysis was carried out in complete randomised block design in 3 replications (each in Petri dish with 6-8 explants). The experiment was carried out in an independent and stepwise approach. In each test, only 1 factor was examined independently. Actually, the best treatment was applied to next experiment. Data was analyzed using SAS 9.2 software. Means were compared by Duncan’s Multiple Range Test.

Agrobacterium-mediated transformation using gene encoding citrus tristeza virus coat protein

112

as XbaI and BamHI for P2 isolation in separate reactions. After electrophoresis of enzymatic digestions products, 400bp fragments (P1 and P2) observed from each reaction (Fig. 4). In addition, enzymatic double digestion of recombinant silencing vector was performed by AscI and XbaI. As a result, ~2200bp fragment was observed in the electrophoresis. This fragment included sum of 2 400bp mosaic from CTV and 1400bp pieces from chalcone synthase intron (data not shown). The effect of acetosyringone concentration on transformation HI¿FLHQF\ In this test, applying 50 ΟM AS in inoculation and cocultivation media resulted in 28 % shoot regeneration (14 out of 50 explants) from which, 9.3 % of them proved to be transformed. Applying 100 ΟM AS led to the bacterium overgrowth and consequently explants death. However, at this AS concentration, 1 out of 22 regenerated seedlings found to be transformed. There ZDV QR VLJQL¿FDQW GLIIHUHQFHV EHWZHHQ —0 DQG —0 $6 treatment regarding transformed shoots (Table 2). Seedlings considered as transformed if only the PCR results of CTV and BAR genes were positive (Fig. 5A and 5B). A PCR reaction from each gene were chosen randomly and sequenced in order to prove the genuinity of PCR products. 7KH HIIHFW RI LQRFXODWLRQ WLPH RQ WUDQVIRUPDWLRQ HI¿FLHQF\ In present study, 2-day co-cultivation led to the best results M

1

2

3

4

in producing transformed seedlings (13.7 %). In 3-day cocultivation, number of transformed shoots versus total shoots resulted in the slightly lower figure compare to 2 days coFXOWLYDWLRQ ZKLFK KDG D VLJQL¿FDQW GLIIHUHQFH ZLWK DQG GD\ periods (Table 4). In the end, transgenic shoots (Fig. 5C and Fig. 5D) were propagated by bud grafting onto sour orange seedlings as rootstocks since sour orange is vigorous and thus, ensures fast growth of the transgenic scion (Fig. 5E). Transplanted explants were kept in liquid MS medium at 28 °C for 16 h photoperiod. To adapt with environmental condition, they were transformed to pots containing 1:1:1 ratio of vermicompost, peat moss, and vermiculite. Then, they were kept under 28 °C for 16 h photoperiod. The effect of co-cultivation period on transformation efficiency: In present study, 2-day co-cultivation led to the best results in producing transformed seedlings (13.7%). In 3-day co-cultivation, number of transformed shoots versus total VKRRWV UHVXOWHG LQ WKH VOLJKWO\ ORZHU ¿JXUH FRPSDUH WR GD\V FR FXOWLYDWLRQ ZKLFK KDG D VLJQL¿FDQW GLIIHUHQFH ZLWK DQG 4-day periods (Table 4). Table 2. Effects of acetosyringon concentration on transformation efficiency of epicotyl-originated explants Acetosyringone Responsive explants/ PCR+ shoots/total shoots conc. (ΟM) total explants WUDQVIRUPDWLRQ HI¿FLHQF\

0 8/50 (b) 1/18 (5.55%) 50

~200 bp

~400 bp

Fig. 2. Amplification of genes encoding CTV1 and CTV2. M = ladder 100 bp.; Lane 1 and 2 = CTV1; Lane 3 and 4 = CTV2.

Fig. 3. Confirmation of mosaic fragment synthesis using R- and F-mosaic primers. PCR products indicates ligation of 2 CTV1 & CTV2 fragments (lane 1-4), molecular weight marker (M).

Fig. 4. Confirmation regarding insertion of 2 copies of CTV fragments in pFGC5941 destination vector. 2 types of products in lane 1 and 2 are the result of double digestions of silencing vector using SwaI - AscI and XbaI - BamHI, respectively. Lane 3: undigested plasmid along with M: marker.

14/50 (a)

3.32 (9.37%)

100 10/50 (b) 1.22 (4.54%) /RZHUFDVH OHWWHUV LQGLFDWHV VLJQLILFDQW GLIIHUHQFHV 3” LQ response to each treatment Table 3. Effects of inoculation time on transformation efficiency of epicotyl-originated explants. Positive PCR shoots regarding CTV and BAR genes plus negative PCR results relating to kan considered as transformed Inoculation period (min)

PCR+ shoots/ Total shoots (transformation HIÂżFLHQF\

5

0/7 (0.0%)

5/50 (d)

10

3/32 (9.37%)

14/50 (b)

15

4/45 (11.11%)

19/50 (a)

20

1/22 (4.5%)

10/50 (c)

25

1/16 (6.25%)

8/50 (c)

Responsive explants/ Total explants

/RZHUFDVH OHWWHUV LQGLFDWHV VLJQLILFDQW GLIIHUHQFHV 3” LQ response to each treatment Table 4. Effects of co-cultivation period on transformation efficiency of epicotyl-originated explants. Highest transformation efficiency was obtained from 2 days co-cultivation. Shoots with positive PCR results regarding CTV and BAR genes and negative PCR results concerning kan- were considered as transformed Days Responsive explants/ PCR+ shoots/total shoots Total explants 7UDQVIRUPDWLRQ HI¿FLHQF\

1 6/50 (c) 0/6 (0.0%) 2

26/50 (a)

10/73 (13.7%)

3

19/50 (b)

5/45 (11.11%)

4

7/50 (c)

0/12 (0.0%)

/RZHUFDVH OHWWHUV LQGLFDWHV VLJQLÂżFDQW GLIIHUHQFHV LQ UHVSRQVH WR HDFK treatment

Agrobacterium-mediated transformation using gene encoding citrus tristeza virus coat protein In the end, transgenic shoots (Fig. 5C and Fig. 5D) were propagated by bud grafting onto sour orange seedlings as rootstocks since sour orange is vigorous and thus, ensures fast growth of the transgenic scion (Fig. 5E). Transplanted explants were kept in liquid MS medium at 28 °C for 16 h

A

B

C

113

photoperiod. To adapt with environmental condition, they were transformed to pots containing 1:1:1 ratio of vermicompost, peat moss, and vermiculite. Then, they were kept under 28 °C for 16 h photoperiod. After 4 months growing in the growth chamber, all transgenic plants showed a normal phenotype, identical to that of control non-transgenic sour orange plants.

'LVFXVVLRQ The explants were culturepHd on solid regeneration medium after co-cultivation in order to induce shoot. Many aspects of cellular differentiation and organogenesis in tissue and organ cultures have been found to be controlled by an interaction between cytokinin and auxin concentrations (van Staden et al., 2008). With this regard, shoot formation could be induced predictably using relatively low levels of auxin and a high level of cytokinin in the growth medium. The main intention in this experiment was to generate a balance between 2 growth regulators to induce adventive shoots on explants without going through a callus phase. These so called “direct organogenesisâ€? helps to eliminate risk of somaclonal variation (Rezadoost et al., 2013). Almeida et al UHSRUWHG WKDW WUDQVIRUPDWLRQ HIÂżFLHQF\ GHFUHDVHG ZKHQ $6 ZDV DSSOLHG 7KLV GLIIHUHQFH EHWZHHQ RXU ÂżQGLQJ DQG $OPHLGDÂśV LV GXH WR VHFUHWLRQ RI SRO\SKHQROLF compounds from cut ends of explants to pre-culture medium. Accordingly, due to the lack of polyphenolic compounds in co-cultivation medium, application of an external phenolic compound like AS, might be the reason for further vir genes induction.

D

E

Cervera et al. (1998a) found 100 ΟM AS to be the best possible amount for Washington navel orange transformation. In the transformation of Poncirus triliantus epicotyls, Zou et al. (2008) noted that 50 ΟM AS had the best results. Luth and Moore (1999) also noted that 100 ΟM AS is appropriate for the transformation of Citrus paradisi explants. Bond and Roos (1998) also reported that 200 ΟM AS was the best in Washington navel (Citrus sinensis L. Osbeck) WUDQVIRUPDWLRQ 5HSRUWV RQ WKH XVH RI $6 FRQFHQWUDWLRQV DUH YDULHG $SSDUHQWO\ WKH ¿QGLQJ ¿JXUHV GHSHQG RQ WUDQVIRUPDWLRQ PHWKRG WR D JUHDW H[WHQW In the bacterial cell culture protocol, which was adopted from Gelvin (2006), 3 steps of culture (culture in YEP medium, AB medium, and induction medium) were used to induce vir genes of Agrobacterium. The vir region includes 6 operons, which have been designated virA, virB, virC, virD, virE, and virG. The VIR gene products act in trans to mobilize the transferred DNA (T-DNA) element from the bacterial Ti plasmid to the plant genome (Stachel et al., 1986). The expression of the virulence genes except for virA LV VSHFL¿FDOO\ LQGXFHG E\ SKHQROLF compounds, including AS, which are released from the wounded plant cells (Das et al., 1986). AS also acts as a strong inducer for vir gene expression between pH 5.0 and pH 5.5. The optimal induction of vir gene was attained at acidic level (~5.2-6.0) (Yuan et al., 2008). The temperature optimum for vir gene induction (~25 °C) is generally lower than that optimal for vegetative growth of Agrobacterium (28-30 °C; Alt-Moerbe et al., 1988). %RQG DQG 5RRVH VKRZHG WKDW WUDQVIRUPDWLRQ HI¿FLHQF\ GHFUHDVHG RYHU PLQ &HUYHUD et al. (1998) introduced 15 min as the optimum time for the inoculation of Carrizo citrange explants. Many researchers suggested 20 min as optimum inoculation time for transformation (Ghorbel et al., 2000; Pena et al., 2004; Zou et al., 2008; Silva et al., 2010). However, in this study, the number of PCR+ shoots mainly decreased after 15 min due to Agrobacterium over growth. Perez-Molphe and Alejo (1998) found maximum time for citrus explants inoculation was 45 min. in Citrus aurantifolia internodes transformation via co-cultivation by Agrobacterium rhizogenes.

Fig. 5. Development and analysis of putative transformed seedlings. Confirmation of transgenic plants by PCR using genes located within T-DNA region including CTV (A) and BAR (B). N: non-transformed wild type plant; P: destination vector with insert, 1-3 putative transformed seedlings. Adventitious bud formation (C) and direct shoots organogenesis (D) from epicotyloriginated explants on selective medium in C. aurantium, Putative transgenic seedling grafted in vitro (E).

Production of shoots in selection medium does not necessarily mean that they are transformed. In our experiments many of the shoots that regenerated on Basta selective medium were “escapes�, i.e., were not CTV+. The insensitivity to selective agent may be due to protection of nontransformed cells from the selective agent by the surrounding transformed cells (Gutierrez et al., 1997). In previous reports, escape is also introduced as the most critical challenge in citrus transformation (Moore et al., 1992; Pena et al., 1995). However, attempt was made here to minimize the use of these materials to control the level of error. One of the advantages of the current protocol is regeneration of explants through direct organogenesis which avoid callus phase and consequently somaclonal variation and reduces

114

Agrobacterium-mediated transformation using gene encoding citrus tristeza virus coat protein

the risk of genetic deterioration of genetic stocks. Agrobacteriummediated transformation of mature sour orange embryos is a promising approach to make a shortcut to generate transgenic citrus in future experiments.

Acknowledgment This research was supported by a grant from Biotechnology board of The University of Guilan to M.M. Sohani.

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Gutierrez, E.M.A., D. Luth and G.A. Moore, 1997. Factors affecting Agrobacterium-mediated transformation in citrus and production of Sour orange (Citrus aurantium) plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep., 16: 745-753. Hidaka, T., M. Omura, M. Ugaki, M. Tomiyama, A. Kato, M. Ohshima and F. Motoyoshi, 1990. Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells. Jpn. J. Breed., 40: 199-207. Kaneyoshi, J., S. Kobayashi, Y. Nakamura, N. Shigemoto and Y. Doi, $ VLPSOH DQG HIÂżFLHQW JHQH WUDQVIHU V\VWHP RI WULIROLDWH RUDQJH Plant Cell Rep., 13: 541-545. Luth, D. and G. Moore, 1999. Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tiss. Org., 57: 219-222. Moore, G.A., C.C. Jacono, J.L. Neidigh, S.D. Lawrence and K. Cline, 1992. Agrobacterium mediated transformation of citrus stem segments and regeneration of transgenic plants. Plant Cell Rep., 11: 238-242. Muniz, F.R., A.J. De Souza and L.C.L. Stipp, 2012. Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection. Biol. Plantarum, 56: 162-166. Pena, L., M. Cervera, J. Juarez, A. Navarro, J.A. Pina, N. Duranvila and L. Navarro, 1995. Agrobacterium-mediated transformation of Sweet orange and regeneration of transgenic plants. Plant Cell Rep., 14: 616-619. Pena, L., R.M. Perez, M. Cervera, J.A. Juarez and L. Navarro, 2004. Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann. Bot. (Lond), 94: 67-74. Perez-Molphe, E. and N. Ochoa-Alejo, 1998. Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues. Plant Cell Rep., 17: 591-596. Rezadoost, M.H., M.M. Sohani, A. Hatamzadeh and M.R. Mirzaii, 2013. In vitro regeneration of sour orange (Citrus aurantium L.) via direct organogenesis. Plant Knowledge J., 2: 150-156. Sambrook, J. and D.W. Russell, 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, New York, USA: CSHL Press. Silva, R.P., A.J. Souza, B.M.J. Mendes and F.A.A. Mourao Filho, 2010. Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type. Rev. Bras. Frutic., 32: 1-8. Stachel, S.E. and P. Zambryski, 1986. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell, 47: 155-157. van Staden, J., E. Zazimalova and E.F. George, 2008. Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists. In: Plant Propagation by Tissue Culture, The Background. M.A. George, M.A. Hall, G.J. De Klerk (eds). 3rd ed. Dordrecht, The Netherlands: Springer, pp. 205-226. Whiteside, J.O. 1988. Compendium of Citrus Diseases, APS Press. Yu D., B. Fan, S.A. MacFarlane and Z. Chen, 2003. Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Mol. Plant Microbe, 16: 206-216. Yuan, Z.C., P. Liu, P. Saenkham, K. Kerr and E.W. Nester, 2008. 7UDQVFULSWRPH SURÂżOLQJ DQG IXQFWLRQDO DQDO\VLV RI Agrobacterium tumefaciens reveals a general conserved response to acidic conditions (pH 5.5) and a complex acid-mediated signaling involved in Agrobacterium-plant interactions. J. Bacteriol., 190: 494-507. Zamani, A.H. 2009. Cloning of gene encoding coat protein of citrus tristeza virus (CTV). MSc, University of Guilan, Rasht, Iran (dissertation in Farsi with an abstract in English). Zou, X., D. Li, X. Lou, K. Luo and Y. Pei, 2008. An improved procedure for Agrobacterium mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf) via indirect organogenesis. In vitro Cell Dev. B-Pl, 44: 169-177. Submitted: December, 2014; Revised: January, 2015; Accepted: January, 2015

Journal

Journal of Applied Horticulture, 17(2): 115-120, 2015

Appl

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.) Fenghua Wang*, Guangyuan Li, Shuangchen Chen, Yan Jiang and Shaoxian Wang Forestry College, Henan University of Science and Technology, Luoyang 471003, China. *E-mail: fenghua123668@126.com

Abstract A compound-leaf mutant of radish was induced by treatment of ethyl-methane sulfonate. We analyzed the photosynthetic, agronomic, microstructural, and quality traits of the mutant and compared them with those of wild-type. Net photosynthetic rate was approximately 30 % higher, and total chlorophyll content was approximately 36 % higher in mutant than in wild-type. However, the root weight of the mutant was only half of the wild-type. Compared with wild-type, the mutant showed 75 % higher vitamin C content, 39 % higher total soluble solids content, and 12 % lower soluble sugar content. The stomatal density was higher in compound leaves than in simple OHDYHV &RPSRXQG OHDYHV FRQWDLQHG VL[ FKORURSODVWV SHU JXDUG FHOO ZKLOH RQO\ ÂżYH LQ VLPSOH OHDYHV 7KH GHJUHH RI VWRPDWDO RSHQLQJ was greater in compound leaves. Compared with simple leaves, compound leaves showed thinner and looser vascular bundles and SKORHP FHOOV VPDOOHU SHWLROH GLDPHWHU DQG KLJKHU GHQVLW\ RI SDUHQFK\PD FHOOV $ VHTXHQFH UHODWHG DPSOLÂżHG SRO\PRUSKLVP DQDO\VLV showed that ethyl-methane sulfonate induced DNA mutations at several loci. Key words: Radish, mutant, compound leaf, microstructure, SRAP

,QWURGXFWLRQ Leaves of seed plants can be classified as either simple or compound, depending on their degree of complexity (Sattler DQG 5XWLVKDXVHU $OWKRXJK VLJQLÂżFDQW SURJUHVV KDV EHHQ made in understanding the mechanisms that regulate simple leaf development, those that regulate compound leaf development are poorly understood (Brand et al., 2007). Research on the molecular mechanisms of compound leaf development began in the late 1990s. Expression of KNOX in tomato, Arabidopsis, tobacco, cotton, poplar, and dandelion resulted in leaf variations, some even changed leaves from simple to compound (Barth et al., 2009). Results of Peng et al. (2011) showed that .12; SURWHLQ WRRN SDUW LQ OHDĂ€HW GHYHORSPHQW RI Medicago Truncatula. Shani proposed that the role of KNOX was to delay leaf maturity (Shani et al., 2009). ARP, another important gene responsible for development of compound leaf was expressed in compound phyllopodia, and participated in the morphogenesis of compound leaves (Kim et al., 2003). KNOX and ARP genes function antagonistically with each other in the development of simple leaf, however they co-express in compound leaves. Overlapping expression of ARP and KNOX1 in phyllopodium was an important character for formation of compound leaf (Nishii et al., 2010). Besides KNOX and ARP, NAM/CUC3 gene family was also important in development of compound leaf (Blein et al., 2008). The separation of edge of leaf blade was associated with expression of these genes. The further studies demonstrated that QXPEHU RI OHDĂ€HWV GHFUHDVHG ZKHQ WKHVH JHQHV H[SUHVVLRQV ZHUH minimized (Blein et al., 2008). Wang pointed out that CUC2-like gene took part in partition of leaf blade of Lotus japonicus, and multiple components were integrated to determine the complexity of leaf in Lotus japonicus (Wang et al., 2013). Recent research indicated that Trifoliate (Tf) gene encoded an MYB transcription factor that modulated leaf and shoot architecture in tomato (Naz et al., 2013). Brassinosteroid (BR) is one of the auxins. The

UHFHQW UHVHDUFK VKRZHG WKDW %5 LQĂ€XHQFHG WKH GHYHORSPHQW RI compound leaf. BR was important for formation of leaf blade boundary. Activation of BR signaling repressed CUP-SHAPED COTYLEDON(CUC) gene expression and caused organ fusion phenotypes (Gendron et al., 2012). CaĂąo-Delgado indicated that BR played an important role in formation of blade boundary and was down regulated by lateral organ boundaries (LOB). The activated BR could inhibit expression of genes responsible for lateral organ fusion 1 (LOF1) and CUC in phyllopodium (CaĂąoDelgado and BlĂĄzquez, 2013). Besides above genes, hormones, including cytokinins and auxins, also played important roles in development of compound leaf through regulating expressions of KNOX, STIP, and WU (Anna and Wu, 2011; Bartrina et al., 2011; Skylar and Wu, 2011). Generally, leaf of radish is characterized with either pinnate or entire leaf edge, and both types of leaves are simple (http: //www. shucaiyuan.com/Technology/43/15592.shtml). Radish mutants with compound leaves were obtained by ethyl-methane sulfonate (EMS) induction during breeding. The mutants showed compound and simple leaves on the same plant (Fig. 1). This trait is easy to identify at the seedling stage, it may be a good marker for hybrid seed production in radish. Furthermore, the presence of compound and simple leaves on the same plant makes these lines interesting materials for research on development of compound leaf. We analyzed the physiological and microstructural characteristics of one of the mutants, and examined the mutation by sequenceUHODWHG DPSOLÂżHG SRO\PRUSKLVP 65$3 PDUNHU

Materials and methods Plant materials: Mutants that produced compound leaves as well as simple leaves were obtained by treatment of EMS (Fig. 1). Seeds of wild-type (control, simple leaves) and mutants were VHOHFWHG DQG VRZQ LQ WKH ÂżHOG DW +HQDQ 8QLYHUVLW\ RI 6FLHQFH DQG Technology, China, in 2010 and 2011. Plants were irrigated every

116

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.)

2 days and fertilized (N : P2O5 : K2O = 1 : 0.6 : 0.6) at a rate of approximately 50 kg N ha-1. Determination of net Pn and Chl content: We used the mutant ZLWK WZR OHDĂ€HWV PXWDQW LQ WKLV VWXG\ 1HW 3n was determined with a LI-6400 portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). Chl content was measured by following method. Approximately 0.2 g leaf material was homogenized in 80 % acetone at 4°C. The homogenate was FHQWULIXJHG DQG Ă€XRUHVFHQFH ZDV PHDVXUHG DW DQG QP ZLWK D Ă€XRUHVFHQFH VSHFWURSKRWRPHWHU 0RGHO 6KDQJKDL 3UHFLVLRQ 6FLHQWLÂżF ,QVWUXPHQW &R /WG &KLQD Measurement of agronomic characters of the radishes: Plant height, plant width, leaf area, leaf number, root length, root diameter, and root weight were measured when plants were three months old. Measurement of quality traits of the radishes: Fresh radishes were sampled to measure quality traits. VC content was determined by 2, 6-dichloro-indophenol titration. Soluble protein content was determined spectrophotometrically using BSA as the standard. The soluble sugar content was determined by anthracenone method. All experiments were carried out according to the methods of Zhang et al. (2007). TSS was determined on juice using a hand held refractometer (ATC-1 Atago, Tokyo, Japan) with automatic temperature compensation. Observation of microstructure of the radishes: The epidermis was carefully peeled from the leaf, placed on a slide, stained with 1 % I2-KI for 20 min, and then observed under an Olympus BX51 microscope. Stomatal density was expressed as number mm-2. 6DPSOHV ZHUH Âż[HG LQ IRUPDOLQ IRU K DW ƒ& 7KHQ samples were dehydrated in 70 % ethanol for 1 h (3 times); 80 % ethanol for 1 h; 95 % ethanol for 1 h; a mixed solution (100 % ethanol : xylene = 1 : 1) for 1 h; and then in xylene for 1 h. 7KH GHK\GUDWHG VDPSOHV ZHUH HPEHGGHG LQ SDUDIÂżQ ZD[ DQG FXW into 5 Îźm sections with a microtome. The sections were placed LQ SDUDIÂżQ ULEERQ LQ D ZDWHU EDWK DW ƒ& 6HFWLRQV ZHUH mounted on slides, dried for 10 min, and then stained with 1 % safranin. The slides were covered with a cover slip, mounted with Canada gum, and dried at 40 °C. The slides were examined under an Olympus BX-51 microscope. SRAP analysis of the radishes: Total DNA was extracted as described by Wang et al. (2008). Bulked DNA pools from mutant and wild-type radish were constructed with DNA from 15 individual plants mixed in equimolar quantities. Sequences of SRAP primers were designed according to Li and Quiros (2001) and synthesized by Shanghai CASB Biotechnology Table 1. Photosynthetic characteristics of wild and mutant radish Radish Leaf 3Q Č?PRO Chla content (m-2 s-1) (mg g-1) Wild type Simple leaf 14.53Âą1.43a 1.81Âą0.12a Mutant

Compound leaf

20.67Âą1.22b

3.54Âą0.21b

Co., Ltd (Shanghai, China). The PCR protocols were adopted from Li and Quiros (2001). Amplified PCR products were separated by 3 % (w/v) agarose gel electrophoresis and visualized by EB staining. Statistical analysis: All data were statistically analyzed by oneway ANOVA, followed by Tukey’s test, using SPSS 10 statistical VRIWZDUH 'LIIHUHQFHV ZHUH FRQVLGHUHG VLJQL¿FDQW DW 3 ”0.05.

5HVXOWV Characters of mutants: Mutants with compound leaf were LQGXFHG ZLWK (06 7KH FRPSRXQG OHDI KDV WZR OHDĂ€HWV RQ RQH SHWLROH 7KH ZLOG W\SH KDG RQO\ VLPSOH OHDYHV ZLWK RQH OHDĂ€HW per petiole (Fig. 1). The mutants had only one compound leaf, the rest of the leaves on the plant was simple, like those of wild-type. In this study, we analyzed the characteristics of mutant. Photosynthetic characteristics of the mutant: Net photosynthesis of the compound leaf radish was approximately 30 % higher than that of a simple leaf radish. The total Chl content was about 36 % higher in radish with compound leaf than in with a simple OHDI 7KHUH ZHUH VLJQLÂżFDQW GLIIHUHQFHV LQ &KO a content, but no differences in Chl b content, between compound leaf mutant and wide type (Table 1). These results suggested that increase in photosynthesis in this mutant relied mainly on Chl a. Agronomic characteristics of the mutant: There were no differences in leaf number, leaf area, plant width, root length, and root diameter between the wild-type and the mutant. However, WKHUH ZDV D VLJQLÂżFDQW GLIIHUHQFH LQ IUHVK URRW ZHLJKW ZLWK WKDW of the mutant being only half that of the wild-type (Table 2). Quality characteristics of the mutant: The VC content was approximately 75 % higher in the mutant than in the wildtype, suggesting that this mutant was a suitable resource for improvement of VC content in radish. The mutant showed 39 % higher in TSS content, 12 % lower in soluble sugars content than the wild type (Table 3). Characteristics of guard cells of the mutant: There were QR VLJQLÂżFDQW GLIIHUHQFHV LQ WKH VL]H RI JXDUG FHOOV EHWZHHQ compound and simple leaves. The stomatal density of compound leaf was (512Âą15 mm-2), while it was (451Âą22 mm-2) in simple leaf (Fig. 2A, B). The former is about 13.53 % higher than the latter. The number of chloroplasts per guard cell also differed. These were six per cell on average in compound leaves while only ÂżYH SHU FHOO LQ VLPSOH OHDYHV 7KH IRUPHU LV DERXW KLJKHU WKDQ WKDW RI WKH ODWWHU 7KH VWRPDWDO DSHUWXUH ZDV VLJQLÂżFDQWO\ different between compound leaf and simple leaf (Fig. 2C, D), the former was 2.13Âą0.11 Îźm, while the latter was 1.52Âą0.13 Îźm on the average. Chlb content (mg g-1) 2.33Âą0.42a

Chla+b (mg g-1) 4.14Âą0.23a

Carotenoids (mg g-1) 0.55Âą0.01a

2.41Âą0.31a

5.95Âą0.31b

0.51Âą0.02a

Table 2. Agronomic characters of wild and mutant radish Radish Leaf number Leaf area Plant height (cm2) (cm) Wild type 8.58Âą1.01a 323.35Âą2.3 5a 14.32Âą1.21a

Plant width (cm) 21.67Âą1.35a

Root length (cm) 19.43Âą2.32a

Root diameter (cm) 6.05Âą0.29a

Root weight (g) 850.98Âą23.36b

Mutant

22.95Âą1.86a

15.64Âą2.21a

5.42Âą0.58a

426.23Âą31.68a

8.26Âą1.12a

345.52Âą3.25ab

15.19Âą1.30a

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.) Table 3. Quality characters of the radishes Radish Vitamin C content (mg g-1) Wild type 47.56±2.34a Mutant

83.23±3.87b

117

Soluble sugar content (mg g-1) 9.20±0.12b

Soluble protein content (mg g-1) 3.57±0.08a

TSS % 8.90±0.22a

8.13±0.16a

3.65±0.11a

12.36±0.35b

Fig. 1. Mutant and wild-type radish plants (after 30 days of sowing). A: Wild-type; B: Mutant

Fig. 2. Stomatal characteristics of compound and simple leaves. A: Stomatal density (compound leaf); B: Stomatal density (simple leaf); C: Stomatal aperture (compound leaf); D: Stomatal aperture (simple leaf).

118

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.)

Fig. 3. Microstructural characteristics of simple and compound leaf. TS or VS of leaf and petiole. A: Vascular bundle in compound leaf; B: Phloem in simple leaf; C: Phloem in compound leaf; D: Vascular bundle in simple leaf; E: Petiole in compound leaf (cross section); F: Petiole in simple leaf (cross section)

Fig 4. SRAP analysis of mutant and wild-type radish. 1.3.5.7.9.11.13.17.19.21.23: Mutant; 2.4.6.8.10.12.14.16.18.20.22.24: Wild-type; M: marker

Microstructural characteristics of the mutant: Vascular bundles and phloem cells were thinner, looser, and more regular in the simple leaf than those in compound leaf (Fig. 3A-D). The diameter of the petiole in compound leaves was about 2.23Âą0.15 mm, which was smaller than that in simple leaves, about 3.16Âą0.23 mm (Fig. 3E, F). These results suggested that

compound leaves may have a lower ability to transport water, mineral nutrients, and photosynthates compared with that of simple leaves. SRAP analysis of the mutant: Twelve pairs of SRAP primers were used to analyze polymorphisms between the mutant and

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.) wild-type. Eight pairs of primers showed polymorphisms (Fig. 4). In total, 105 bands were produced, 34 of which were polymorphic. This suggested that there were mutations at several loci. In other words, the compound leaf character must be controlled by more than one gene. The next step is to identify and clone these genes to clarify the mechanisms of compound leaf development.

'LVFXVVLRQ Leaves can be simple, compound, or one of numerous intermediate forms.The development of plant leaf can be roughly divided into three continuous phases: leaf initiation, organogenesis, and histogenesis (Holtan and Hake, 2003). The special attributes that characterize monocotyledonous leaves have led to morphological interpretations like the phyllode theory, leaf base theory, and the unifacial concept. All of them aimed to interpret monocotyledonous leaves in terms of dicotyledonous leaves and to establish morphological differences between them. The mutant characterized in the present study showed simple and compound leaves existed on the same plant, which may suggest that formation of simple and compound leaves occured via essentially the same developmental process, as suggested by Bharathan et al. (2002). Besides morphology, many researches focused on molecular mechanisms of development of compound leaf. Up to now, several key genes and genetic regions involved LQ WKH FRQWURO RI OHDI VKDSH VL]H KDYH EHHQ LGHQWLÂżHG LQFOXGLQJ microRNA-regulated genes (Usami et al., 2009), ribosomerelated genes (Fujikura and Horiguchi, 2009), and a chromosomal segment (Horiguchi et al., 2009). In recent decades, studies on development of compound leaf have focused on two model plants, tomato and pea. There were two key gene families involved in the development of compound leaf, the KNOX and ARP gene families (Nishii et al., 2010). Many studies have sought to determine the roles and regulation of these two interesting gene families (Barth et al., 2009; Shani et al., 2009; Nishii et al., 2010; Peng et al., 2011). However, many important points are still to be addressed. There may be other gene families involved in regulating development of compound leaf. The SRAP analysis in this study indicated that mutations at several genetic loci contributed to the leaf phenotype. Further research is required to determine whether these genetic loci contained KNOX family genes, ARP family genes, or both, or novel genes. All types of Chl molecules function as light-harvesting pigments. Cells with higher Chl contents could collect and transfer more light energy (Wang et al., 2008). Consequently, they showed higher photosynthetic efficiency and often, higher yield as well. However, we observed that the mutant had much higher Chl content, especially Chl a content. With higher Chl content, photosynthates will be higher, thus the weight will be higher, too. However the results showed that root weight of the mutant was only half of the wild-type. Why does this happen? What was the fate of the photosynthates? Further research is required to answer this question. Nevertheless, the results showed that there was not a simple, positive relationship among Chl content, photosynthesis, and economic yield. The complexity of this relationship has become a problem for plant breeding programs. Therefore, in addition to being an interesting research material for study on compound leaf development, this mutant will also be useful to study the relationships among Chl content, photosynthesis, and yield.

119

Acknowledgements This work was supported by the National Key Technology R&D Program of China (2011BAD12B03) and the National Natural Science Foundation of China (30700002, 31101536).

References Anna, S. and X.L Wu, 2011. Regulation of meristem size by cytokinin signaling. J. Integr Plant Biol., 56: 446-454. Barth, S., T. Geier, K. Eimert, B. Watillon, R.S. Sangwan and S. Gleissberg, 2009. KNOX overexpression in transgenic Kohleria (Gesneriaceae) prolongs the activity of proximal leaf blastozones and drastically alters segment fate. Planta, 230: 1081-1091. Bartrina, I., E. Otto, M. Strnad, T. Werner and T. Schmulling, 2011. &\WRNLQLQ UHJXODWHV WKH DFWLYLW\ RI UHSURGXFWLYH PHULVWHPV ÀRZHU organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. Plant Cell, 23: 69-80. Bharathan, G., T.E. Goliber, C. Moore, S. Kessler, T. Pham and N.R. Sinha, 2002. Homologies in leaf form inferred from KNOXI gene expression during development. Science, 296: 1858-1860. Blein, T., A. Pulido, A. Vialette-Guiraud, K. Nikovics, H. Morin, A. Hay, I.E. Johansen, M. Tsiantis and P. Laufs, 2008. A conserved molecular framework for compound leaf development. Science, 322(5909): 1835-1839. Brand, A., N. Shirding, S. Shleizer and N. Ori, 2007. Meristem maintenance and compound-leaf patterning utilize common genetic mechanisms in tomato. Planta, 26: 941-951. Caùo-Delgado, A.I. and M.A. Blåzquez, 2013. Spatial control of plant steroid signaling. Trends Plant Sci., 18(5): 235-236. Fujikura, U.G. and M.R. Horiguchi, 2009. Coordination of cell proliferation and cell expansion mediated by ribosome-related processes in the leaves of Arabidopsis thaliana. Plant J., 59: 499-508. Gendron, J.M., J.S. Liu, M. Fan, M.Y. Bai, S. Wenkel, P.S. Springer, M.K. Barton and Z.Y. Wang, 2012. Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis. Proc. Natl. Acad. Sci. USA, 51: 21152-2157. Holtan, H.E. and S. Hake, 2003. Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines. Genetics, 165: 1541-1550. Horiguchi, G., N. Gonzalez, G.T.S. Beemster, D. Inze and H. Tsukaya, 2009. Impact of segmental chromosomal duplications on leaf size in the grandifolia-D mutants of Arabidopsis thaliana. Plant J., 60: 122-133. Kim, M., S. McCormick, M. Timmermans and N. Sinha, 2003. The expression domain of PHANTASTICA GHWHUPLQHV OHDÀHW SODFHPHQW in compound leaves. Nature, 424: 438-443. /L * DQG & ) 4XLURV 6HTXHQFH UHODWHG DPSOL¿HG SRO\PRUSKLVP (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor. Appl. Genet., 103: 455-461. Naz, A.A., S. Raman, C.C. Martinez, N.R. Sinha, G. Schmitz and K. Theres, 2013. Trifoliate encodes an MYB transcription factor that modulates leaf and shoot architecture in tomato. Proc. Natl. Acad. Sci. USA, 6: 2401-2406. Nishii, K., M. MÜller, C. Kidner, A. Spada, R. Mantegazza, C.N. Wang and T. Nagata, 2010. A complex case of simple leaves: indeterminate leaves co-express ARP and KNOX1 genes. Dev. Genes. Evol., 220: 25-40. Peng, J.L., J.B. Yu, H.L. Wang, Y.Q. Guo, G.M. Li, G.H. Bai and R.J. Chen, 2011. Regulation of compound leaf development in Medicago Truncatula by fused compound leaf1, a class M KNOX gene. Plant Cell, 11: 3929-3943. Sattler, R. and R. Rutishauser, 1992. Partial homology of pinnate leaves DQG VKRRWV RULHQWDWLRQ RI OHDÀHW LQFHSWLRQ Bot. Jahrb. Syst., 114: 61-79.

120

Characterization of a new leaf-compound radish mutant (Raphanus sativus L.)

Shani, E., Y. Burko, L. Ben-Yaakov, Y. Berger, Z. Amsellem, A. *ROGVKPLGW ( 6KDURQ DQG 1 2UL 6WDJH VSHFLÂżF UHJXODWLRQ RI Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins. Plant Cell, 21: 3078-3092. Skylar, A. and X.L. Wu, 2011. Regulation of meristem size by cytokinin signaling. J. Integr. Plant Biol., 6: 446-454. Usami, T., G. Horiguchi, S. Yano and H. Tsukaya, 2009. The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty. Development, 136: 955-964.

Wang, F.H., G.X. Wang, X.Y. Li, J.L. Huang and J.K. Zheng, 2008. Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.). J Plant Physio., 165: 324-330. Wang, Z., J. Chen, L. Weng, X. Li, X. Cao, X. Hu, D. Luo and J. Yang, 2013. Multiple components are integrated to determine leaf complexity in Lotus Japonicus. J. Integr. Plant Biol., 55(5): 419-433. Zhang, L.J. and J.J. Fan, 2007. Experiments for Plant Physiology. Beijing: China Agricultural University Press (in Chinese) Submitted: December, 2014; Revised: February, 2015; Accepted: March, 2015

Journal

Journal of Applied Horticulture, 17(2): 121-128, 2015

Appl

Effect of various factors on shoot regeneration from citrus epicotyl explants Randall P. Niedz*, Joseph P. Albano and Mizuri Marutani-Hert Agricultural Research Service, U.S. Horticultural Research Laboratory, 2001 South Rock Road Ft. Pierce, FL34945-3030, USA. *E-mail: randall.niedz@ars.usda.gov

Abstract The effect of various treatments on shoot organogenesis from seedling epicotyl explants from various scion and rootstock polyembryonic citrus types was determined. Treatments included water source, gelling agent, explant insertion, seed size, light intensity, malachite green, nonionic surfactants, and sodium sulphate. Tap water, with the highest levels of SO42-, Ca2+, K+, Mg2+, and Na+, resulted in the most shoots compared to the other 5 sources, suggesting a mineral nutrient effect. Carrageenan produced fewer shoots than agar and gellan gum. Explants inserted into the medium produced more shoots than those cultured on the surface, presumably because of better exposure to water and nutrients. Seed size, light intensity, malachite green, and sodium sulphate had no effect on the number of shoots UHJHQHUDWHG 7ULWRQ ; DW UHVXOWHG LQ VLJQLÂżFDQWO\ IHZHU VKRRWV RWKHUZLVH QRQLRQLF VXUIDFWDQWV KDG QR HIIHFW Key words: Water, nonionic surfactants, gelling agents, malachite green, sodium sulphate, Citrus sinensis L. Osbeck. x Poncirus trifoliata L. Raf., C. sunki Hort. ex Tanaka. x Poncirus trifoliata L. Raf., C. paradisi Macf., C. sinensis L. Osbeck

,QWURGXFWLRQ

Materials and methods

Citrus species are a major fruit crop worldwide and are consumed fresh as fruits, and processed, generally as juice. A citrus tree is typically grown on a rootstock. Some advantages of using a rootstock include tolerance to local biotic and abiotic conditions, FRQWURO RI WUHH VL]H HDUOLHU ÀRZHULQJ DQG IUXLWLQJ DQG HQKDQFHG fruit quality. One of the earliest references to the use of rootstocks in citrus is the use of lemon as a rootstock to grow citrons in the Palestine area (Mudge et al. %HFDXVH RI WKH VLJQL¿FDQW interaction between the scion and the rootstock, modern plant breeding programs include the development of both scion and rootstock types. Thus, plant breeding methods must be useful for both citrus scion and rootstock variety development.

Plant material, explant source, and culture conditions: Seed of Carrizo citrange (Citrus sinensis ‘Washington’ L. Osbeck. x Poncirus trifoliata L. Raf.), Duncan grapefruit (C. paradise Macf.), Hamlin sweet orange (C. sinensis L. Osbeck) grapefruit (C. paradise Macf.), and US-812 (Citrus sunki Hort. ex Tanaka. x P. trifoliata L. Raf.) (Bowman and Rouse, 2006) were surface disinfested as follows: after removal of the seed coat, the seeds were soaked for 30 min in 50 mL of a 30 % bleach (5.25 % w/v sodium hypochlorite) solution with 3 drops of Tween 20. Seeds were then rinsed 3 times with sterile water, allowed to soak for 18 h in water, placed on the surface of MT basal medium (Murashige DQG 7XFNHU VROLGLÂżHG ZLWK Z Y 8OWUDSXUH 7\SH $ bacteriological agar (USB Corporation, Cleveland, OH, USA) in Magenta GA-7-3 vessels (Magenta Corporation, Chicago, IL, USA), and then incubated in the dark at 27 oC for 3-4 week. Onecm-long explants were excised from the epicotyl of the etiolated seedlings. Shoot regeneration experiments were conducted in growth cabinets at 27 °C over 6-2 week in the dark followed by ZN LQ WKH OLJKW /LJKW ZDV SURYLGHG E\ FRRO ZKLWH Ă€XRUHVFHQW ODPSV Č?PRO P-2 s-1) with a 16-h photoperiod.

In vitro methods are used in plant breeding to achieve objectives that are impossible or difficult to achieve via conventional methods. For example, adventitious shoot formation or the de novo formation of meristems is an in vitro method often used in citrus breeding to produce transgenic plants (Moore et al., 1992; Pena et al., 1995; GutiĂŠrrez-E et al., 1997; PeĂąa et al., 1997; Cervera et al., 1998; Cervera et al., 1998; Luth and Moore, 1999; Costa et al., 2002; Yu et al., 2002; Almeida et al., 2003; Almeida et al., 2003; Li et al., 2003; Khawale et al., 2006; Cervera et al., 2008; Dutt and Grosser, 2009; He et al., 2011; Marutani-Hert et al. 2UERYLĂź et al. 7KH HIÂżFLHQF\ RI VKRRW IRUPDWLRQ YDULHV ZLGHO\ DFURVV citrus species and types, and is an important factor that affects the UHFRYHU\ HIÂżFLHQF\ RI SURGXFLQJ WUDQVJHQLF SODQWV 8QGHUVWDQGLQJ the conditions that affect adventitious shoot formation is required WR LQFUHDVH VKRRW UHJHQHUDWLRQ HIÂżFLHQF\ DFURVV D EURDG UDQJH RI genotypes and, consequently reduce the cost and broaden the applications that utilize adventitious shoot formation. We report the results from a series of experiments that tested various treatments reported in the plant tissue culture literature to improve adventitious shoot formation.

Responses measured and replication used: Shoot regeneration was measured by counting the number of shoots > 2 mm, the minimum size for micrografting, on each epicotyl explant. For all experiments each treatment was measured from four 100 x PP FXOWXUH GLVKHV ZLWK HDFK GLVK FRQWDLQLQJ ÂżYH HSLFRW\O explants derived from a single seedling (1 seedling per dish). A UHSOLFDWH ZDV D VHFRQG VHW RI IRXU GLVKHV ZLWK ÂżYH H[SODQWV SHU dish. Effect of water source: Six water sources were tested using Carrizo. The experiment was as a single-factor, water source, design with six levels that included 1) tap from the lab, 2) drinking (Walmart, Bentonville, AR, USA), 3) distilled (Walmart,

122

Effects of various factors on shoot regeneration from citrus epicotyl explants

Bentonville, AR, USA), 4) laboratory glass distilled, 5) Milli Q, and 6) reverse osmosis. The data was analyzed by one-way ANOVA followed by the Tukey’s multiple comparison test. The PLQHUDO QXWULHQW SURÂżOH RI HDFK ZDWHU VRXUFH ZDV GHWHUPLQHG E\ ion chromatography and included anions (B(OH)4-, Cl-, F-, NO2-, NO3-, PO43-, SO42-) and cations (Li+, Na+, NH4+, K+, Mg2+, and Ca2+) as described (U.S. Environmental Protection Agency, 1997). Effect of gelling agent (agar, carrageenan, gellan gum): Three gelling agents were tested using Hamlin and US-812. The experiment was a single-factor design with gelling agent set at three levels that included 1) Ultrapure, Type A, bacteriological agar (Affymetrix, Santa Clara, CA, USA), 2) Gelcarin GP 812 carrageenan (PhytoTechnology Laboratories, Shawnee Mission, KS, USA), and 3) Culturegel™ Type I gellan gum (PhytoTechnology Laboratories, Shawnee Mission, KS, USA). The data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Effect of explant insertion: Explant insertion into the culture medium was tested. Explants were positioned horizontally “inâ€? or “onâ€? the medium. An explant “inâ€? the medium was pushed into the medium so that the top of the explant was even with the surface of the medium. An explant “onâ€? the medium was laid on the surface of the medium without insertion. The experiment was a 2-factor design of insertion (in, on) and cultivar (Hamlin, Carrizo). The data was analyzed by 2-way ANOVA. Effect of seed size: Seed size was tested using Carrizo. First, a frequency distribution histogram was generated from the ZHLJKWV RI ÂżYH KXQGUHG &DUUL]R VHHG 6HFRQG WKH H[SHULPHQW was a single-factor design with bin set at four levels that included 0-0.1, 0.1-0.2, 0.2-0.3, and 0.3-0.4 g. The data was analyzed by one-way ANOVA. Effect of light intensity: Following the 2-week dark incubation SHULRG OLJKW ZDV WHVWHG DW WKUHH OHYHOV DQG Č?( PĂ­ sĂ­ ) using Hamlin. The data was analyzed by one-way ANOVA. Effect of malachite green: The aniline dye malachite green (Sigma-Aldrich, St. Louis, MO, USA) was tested at 4 concentrations (0, 0.001, 0.01 and 0.1 mM) using Hamlin. The data was analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test that compared each non-zero concentration to 0 mM. Effect of nonionic surfactants: Three nonionic surfactants were tested using Hamlin. The experiment was a single-factor that included a 0 % surfactant control and eleven surfactant-

concentration combinations as follows: Control (0 % surfactant), Pluronic F-68 (0.01, 0.1, and 1 %), Triton X-100 (0.0001, 0.001, 0.01, and 0.1 %), and Tween 20 (0.0001, 0.001, 0.01, and 0.1 %). The data was analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test that compared each of the eleven surfactant-concentration treatments to the 0 % surfactant control. Effect of sodium sulphate: Sodium sulphate, Na2SO4, was tested at 4 concentrations (0, 0.1, 1, and 2 mM) using Hamlin. The data was analyzed by one-way ANOVA.

5HVXOWV DQG GLVFXVVLRQ Effect of water source: The source of water used to make plant tissue culture medium is probably the most basic component of any medium. Thus, determining the effects of various available water sources is an important initial quality control step. Six water sources were used and their ionic composition determined (Table 1). Sources that had levels of ions >= 1 mM included tap water (SO42-, Ca2+, K+, Mg2+, and Na+) and Walmart drinking water (Na+ and Cl-). The remaining four sources had levels that were either “not detectableâ€? or < 1 mM. A one-way ANOVA was conducted to compare the effect of the water samples on the number of shoots/explant of a size suitable for convenient shoot tip grafting (Table 2). The effect of water VRXUFH RQ VKRRWV SURGXFHG ZDV VLJQLÂżFDQW P<0.0001). The mean separation analysis by Tukey’s test revealed that tap water SURGXFHG VLJQLÂżFDQWO\ PRUH VKRRWV FRPSDUHG WR HDFK RI WKH RWKHU ÂżYH VRXUFHV ZKLFK GLG QRW VLJQLÂżFDQWO\ GLIIHU IURP HDFK other (Fig. 1). Tap water produced 40 % more shoots/explant than the average of the other water sources. This result suggested that mineral QXWULWLRQ VSHFLÂżFDOO\ KLJKHU OHYHOV RI 6242-, Ca2+, K+, Mg2+, and/ or Na+ were responsible for the greater number of shoots produced in media made with tap water. Because a complete analysis of the water for all ions and compounds was not done, an experiment WKDW YDULHG DPRXQW RI WKH LRQV PXVW EH FRQGXFWHG WR FRQÂżUP their effect. Though tap water was the “bestâ€? source, its use is problematic due to repeatability issues. If tap water is used, an ionic composition analysis should be provided. Reconstructing tap water would require the use of software such as ARS-Media (http: //www.ars.usda.gov/services/software/download.htm?sof twareid=148#downloadForm) that uses the linear programming algorithm previously reported (Niedz and Evens, 2006).

Table 1. The mineral nutrient profile of 6 water sources as determined by ion chromatography Anions (mg L-1)

Water Sources

Cations (mg L-1)

B(OH)4-

Cl-

F-

NO2-

NO3-

PO43-

SO42-

Ca2+

K+

L+

Mg2+

Na+

NH4+

Tap

n.d.

n.d.

<1

<1

<1

<1

48

23

3

<1

6

50

1

Distilled (Walmart)

n.d.

<1

n.d.

n.d.

n.d.

n.d.

n.d.

<1

<1

n.d.

<1

<1

<1

Distilled (lab glass)

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<1

<1

<1

<1

<1

<1

Drinking (Walmart)

n.d.

8

n.d.

n.d.

<1

n.d.

n.d.

<1

<1

n.d.

<1

9

n.d.

Milli-Q

n.d.

<1

n.d.

n.d.

n.d.

n.d.

n.d.

<1

<1

n.d.

<1

<1

<1

Reverse Osmosis

n.d.

<1

n.d.

n.d.

n.d.

n.d.

n.d.

<1

<1

n.d.

<1

<1

<1

Effects of various factors on shoot regeneration from citrus epicotyl explants

123

control step. Three biopolymers, two algal and one bacterial, were selected and included agar, a “linear polysaccharide made XS RI DOWHUQDWLQJ Č• DQG ÄŽ OLQNHG JDODFWRVH UHVLGXHV´ carrageenan, a “linear, sulphated polysaccharide based on a UHSHDWLQJ GLVDFFKDULGH VHTXHQFH RI Č• ' JDODFWRS\UDQRVH UHVLGXHV linked glycosidically through various positionsâ€? and, gellan gum, a “linear, anionic heteropolysaccharide based on a tetrasaccharide UHSHDW XQLW Č• ' JOXFRVH Č• ' JOXFRXURQLF DFLG Č• ' JOXFRVH DQG ÄŽ / UKDPQRVH 6WHSKHQ et al., 2006).

Fig. 1. The effect of water source on the number of shoots produced per explant from Carrizo explants that were >= 2 mm. Mean separation by Tukey’s multiple comparison test where bars with different letters were significantly different. Bars expressed as mean + standard deviation.

Fig. 2. The effect of 3 gelling agents and 2 citrus types on the number of shoots produced per explant that were >= 2 mm. Bars expressed as mean + standard deviation.

Fig. 3. The effect of explant insertion and variety on the number of shoots per explant >= 2 mm that were regenerated from Hamlin and Carrizo epicotyl explants. Bars expressed as mean + standard deviation.

Effect of gelling agent (agar, carrageenan, gellan gum): The substrate used to support in vitro explants is another basic component of a plant tissue culture system. Determining the effects of various substrates is another important initial quality-

A two-way ANOVA was conducted to determine the effects of 3 gelling agents and 2 citrus types on the number of shoots/explant 7DEOH 7KH HIIHFWV RI JHOOLQJ DJHQW DQG YDULHW\ ZHUH VLJQL¿FDQW (P=0.0153 and P=0.0111, respectively), but the effect of the LQWHUDFWLRQ EHWZHHQ JHOOLQJ DJHQW DQG YDULHW\ ZDV QRW VLJQL¿FDQW (P=0.7025). The mean separation analysis by Tukey’s multiple comparison test on each set of gelling agents within each variety revealed only the carrageenan vs. gellan gum contrast of Hamlin VZHHW RUDQJH ZDV VLJQL¿FDQW DW DOSKD 7DEOH +DPOLQ VKRRW UHJHQHUDWLRQ ZDV UHGXFHG RQ FDUUDJHHQDQ )LJ 7KH VLJQL¿FDQW variety effect on shoot regeneration was due to a greater number of shoots/explant produced by US-812 (Fig. 2). The high shoot organogenic capacity of US-812 relative to Hamlin may be because it is a P. trifoliate hybrid. Though we are not aware of studies that directly compare the shoot organogenic capacity of P. trifoliata to other citrus types, P. trifoliata hybrids typically have high shoot organogenic capacity when compared across studies (Burger and Hackett, 1986; Duran-Vila et al., 1989; Sim et al., 1989; Maggon and Deo Singh, 1995; PÊrez-Molphe-Balch and Ochoa-Alejo, 1997; García-Luis et al., 1999; Van Le et al., 1999; Bordón et al., 2000; Moreira-Dias et al., 2000; Moreira-Dias et al., 2001; Almeida et al., 2002; Costa et al., 2004; Da Silva et al., 2005; Ali and Mirza, 2006; García-Luis et al., 2006; Molina et al., 2007; da Silva et al., 2010; Marques et al., 2011; Niedz and Evens, 2011). Because there are a large number of available gelling agents, each with complex colloidal effects, predicting WKHLU HIIHFWV LV GLI¿FXOW (PSLULFDO WHVWLQJ PD\ EH WKH FXUUHQW best method to determine which ones work well for a particular species and application. Effect of explant insertion: A two-way ANOVA was conducted to determine the effects of explant insertion, explants positioned on or in the medium, and citrus type on the number of shoots/ explant that were produced (Table 5). The effects of explant LQVHUWLRQ DQG YDULHW\ ZHUH VLJQL¿FDQW P=0.0026 and P<0.0001, respectively), but the effect of the interaction of explant insertion [ YDULHW\ ZDV QRW VLJQL¿FDQW P=0.4178). Explants inserted into the medium produced more shoots, and Carrizo produced more shoots/explant than Hamlin (Fig. 3). Insertion of explants into the medium resulted in the complete exposure of the cut ends to the culture medium and, consequently, better exposure of the cells to water, nutrients, and growth factors. The high shoot organogenic capacity of Carrizo may be due to it being a P. trifoliata hybrid like US-812. Effect of seed size: Five hundred Carrizo seed were individually weighed. Seed weight ranged from 8.2 mg to 601 mg. A frequency distribution was generated using 4 bins that were each 100 mg wide (Fig. 4). Eleven seed exceeded the maximum size of the 300400 mg bin and were not used; the single largest seed was 601 mg.

124

Effects of various factors on shoot regeneration from citrus epicotyl explants

A one-way ANOVA was conducted to compare the effect of seed size bins on the number of shoots/explant that were produced (Fig. 5). The effect of seed size bins on shoots produced was not VLJQL¿FDQW P=0.6116); thus, sorting seeds by size is not required to improve shoot regeneration. Seed size can affect germination and growth (Keddy and Constabel, 1986; Zammit and Zedler, 1990; Bretagnolle et al., 1995; Eriksson, 1999; Soltani et al., 2002; Khurana and Singh, 2004), but there are few studies on the effect of seed size on in vitro responses. A study of the effect of Table 2. ANOVA of the effect of six sources of water on the numbers of shoots >= 2 mm that were regenerated from Carrizo epicotyl explants Factor SS df MS F P-value Water sourcea 120 5 24 9 (5, 66) < 0.0001 Error 177 66 2.7 Total 297 71 a – Six sources that included 1) tapwater, 2) drinking (Walmart), 3) distilled (Walmart), 4) distilled (laboratory glass), 5) Milli Q, and 6) reverse osmosis. Table 3. ANOVA of the effect of gelling agent on the number of shoots per explant >= 2 mm that were regenerated from Hamlin and US-812 epicotyl explants Factor Gelling agenta b

Variety Gelling agent x Variety Error Total

P-value

SS

df

MS

F

8.78

2

4.39 5.40(2, 17) 0.0153

6.60 0.59 13.83 29.80

1 2 17 23

6.60 8.12 (1, 17) 0.0111 0.29 0.36 (2, 17) 0.7025 0.81

large vs small seeds in barley on embryogenic callus induction and subsequent shoot regeneration in barley, observed that large VHHGV SURGXFHG VLJQL¿FDQWO\ PRUH FDOOXV DQG VKRRWV WKDQ VPDOO seed (Özgen et al., 2007). Effect of light intensity: Light intensity can sometimes affect shoot regeneration as reported for a diverse array of plant species such as evergreen azalea (Hsia and Korban, 1998), muskmelon (Niedz et al., 1989), sugarcane (Sengar et al., 2011), apple (Magyar-Tábori et al., 2010), and cotton (Gupta et al., 2000). To the best of our knowledge, the effect of light intensity on shoot regeneration from citrus tissue explants has not been reported. However, a dark incubation period prior to incubation in the light was required for shoot regeneration from adult internode explants of sweet orange, grapefruit, and a citrange (Marutani-Hert et al., 2012). Following the dark incubation period, light was varied from 20 to 89 μE. A one-way ANOVA was conducted to compare the effect of light intensity on the number of shoots/explant that were produced from Hamlin epicotyl explants. The effect of light LQWHQVLW\ ZDV QRQ VLJQL¿FDQW P=0.1831) (Fig. 6); thus, the range of 20 to 89 μE produced equivalent numbers of shoots/explant. The effect of malachite green: The aniline dye malachite green was reported to promote shoot regeneration in raspberry leaf explants when used at less than 20 mg L-1(0.0548 mM) and

a – Three gelling agents included 1) agar, 2) carrageenan, and 3) gellan gum. b – Two citrus varieties included 1) Hamlin sweet orange, and 2) US812 citrange.

Table 4. Tukey’s multiple comparison test of gelling agents within Hamlin sweet orange and US-812 citrange Mean difference

6LJQL¿FDQFH

agar vs. carrageenan

1.05

ns

agar vs. gellan gum

-0.75

ns

carrageenan vs. gellan gum

-1.80

*

agar vs. carrageenan

0.300

ns

agar vs. gellan gum

-0.98

ns

carrageenan vs. gellan gum

-1.28

ns

Comparisons Hamlin

US-812

Fig. 4. Frequency distribution of five hundred Carrizo seed weights using 4 bins that were each 100 mg wide. Eleven seed exceeded the maximum size of the 300-400 mg bin and were not included in the experiment.

Table 5. ANOVA of the effect of explant insertion and variety on the number of shoots per explant>= 2 mm that were regenerated from Hamlin and Carrizo epicotyl explants Factor

df

MS

F

P-value

Explant insertiona

1

33.84

11.82(1, 20)

0.0026

Varietyb

1

67.20

23.49(1, 20)

<0.0001

Explant insertion x Variety

1

1.96

0.68(1, 20)

0.4178

Error

20

2.86

Total

23

a – Two horizontal insertion depths included 1) no insertion where the explant was laid on the surface of the medium, and 2) completely inserted where the top of the explant was even with the surface of the medium. b – Two citrus varieties included 1) Hamlin sweet orange, and 2) Carrizo citrange.

Fig. 5. The effect of seed size bins on the number of shoots/explant from Carrizo epicotyl explants that were produced. Bars expressed as mean + standard deviation.

Effects of various factors on shoot regeneration from citrus epicotyl explants

Fig. 6. The effect of light intensity on the number of shoots/explant from Hamlin epicotyl explants that were produced. Bars expressed as mean + standard deviation.

125

for the propagation of blackberries (Herman, 1995); this is the RQO\ UHSRUW ZH FRXOG ¿QG RQ WKH XVH RI PDODFKLWH JUHHQ LQ SODQW tissue culture. Malachite green is used extensively as a biocide in aquaculture to control protozoan and fungal infections (Srivastava et al., 2004). To examine its effects in citrus a one-way ANOVA was conducted to compare the effect of malachite green at four concentrations (0, 0.001, 0.01, and 0.1 mM) using Hamlin epicotyl explants (Table 6). The effect of malachite green was VLJQL¿FDQW P=0.0018). A mean separation analysis by Dunnett’s multiple comparisons test compared each concentration to the P0 OHYHO DQG UHYHDOHG WKDW VLJQL¿FDQWO\ IHZHU VKRRWV ZHUH produced from the 0.1 mM treatment compared to 0 mM (Fig. 7KHVH UHVXOWV DUH SDUWLDOO\ FRQVLVWHQW ZLWK WKH ¿QGLQJV IRU raspberry. No enhancement of shoot regeneration was observed, but the reduction in shoot regeneration occurred at 0.1 mM or 36.4 mg L-1, greater than the 20 mg L-1threshold reported for raspberry. Effect of nonionic surfactants: Nonionic surfactants can enhance shoot regeneration (Khatun et al., 1993; Khatun et al., Table 6. ANOVA of the effect of Malachite Green on the numbers of shoots/explant >= 2 mm that were regenerated from Hamlin epicotyl explants SS

Df

MS

F

P-value

27.7

3

9.3

9.4 (3, 12)

0.0018

Error

11.8

12

0.98

Total

39.5

71

Factor a

Malachite Green

a

– Malachite Green tested at 4 levels – 0, 0.001, 0.01, and 0.1 mM.

Table 7. ANOVA of the effect of eleven non-ionic surfactantconcentration combinations on the numbers of shoots >= 2 mm that were regenerated from Carrizo epicotyl explants Factor

SS a

Fig. 7. The effect of the aniline dye malachite green on the number of shoots/explant from Hamlin epicotyl explants that were produced. Bars expressed as mean + standard deviation.

df

MS

Nonionicsurfactants

154

11

14

Error

84

35

2.4

Total

238

46

F

P-value

5.85(11, 35) < 0.0001

a

– Three non-ionic surfactants and a control that included 1) Pluronic F-68 (0.01, 0.1, 1%), 2) Triton X-100 (0.0001, 0.001, 0.01, 0.1%), 3) Tween 20 (0.0001, 0.001, 0.01, 0.1), and 4) control (no non-ionic surfactant).

Fig. 8. The effect of 11 nonionic surfactants/concentration combinations on the number of shoots/explant from Hamlin epicotyl explants that were produced. Bars expressed as mean + standard deviation.

Fig. 9. The effect of sodium sulfate (Na2SO4) on the number of shoots/ explant from Hamlin epicotyl explants that were produced. Bars expressed as mean + standard deviation.

126

Effects of various factors on shoot regeneration from citrus epicotyl explants

1993; Davey et al., 2003), including citrus (Cancino et al., 2001; Curtis and Mirkov, 2011). Though the mechanism is unknown, but may relate to the hydrophilic-hydrophobic balance (HLB) value that determines how easily the detergent can interact with the membrane’s lipid component (Helenius and Simons, 1975; Curtis and Mirkov, 2011). The HLB numbers ranged from a high of 29 Pluronic F-68 to a low of 13.5 for Triton X-100; the HLB number of Tween 20 is 16.7. A one-way ANOVA was conducted to compare the effect of the nonionic surfactants/ concentration combinations on the number of shoots/explant that were produced from Hamlin epicotyl explants (Table 7). The HIIHFW RI QRQLRQLF VXUIDFWDQW RQ VKRRWV SURGXFHG ZDV VLJQL¿FDQW (P<0.0001). The mean separation analysis by Dunnett’s multiple comparisons test compared each surfactant/concentration to the zero surfactant control. The analysis revealed that Triton X-100 WUHDWPHQW SURGXFHG VLJQL¿FDQWO\ IHZHU VKRRWV )LJ The variance between our results and those previously reported IRU FLWUXV VXJJHVWV WKH H[LVWHQFH RI V\VWHP VSHFL¿F HIIHFWV 7KXV preliminary empirical testing is recommended. The effect of sodium sulphate (Na2SO4): Sodium sulphate increased the number of shoots/nodal explant in the Indian medicinal plant Vitex negundo (Chandramu, 2003). A one-way ANOVA was conducted to compare the effect of Na2SO4 at four concentrations (0, 0.1, 1, and 2 mM) on the number of shoots/ explant that were produced from Hamlin epicotyl explants (Table 8). The effect of Na2SO4 ZDV QRW VLJQL¿FDQW P=0.84) and revealed that supplementing MS medium with Na2SO4 up to 2 mM did not affect the number of shoots produced (Fig. 9). However, the effect on Vitex negundo may have been due to an interaction with sucrose. Though the authors did not analyze their data for interaction effects, they mentioned the effect and the data they presented in Fig. 2 does suggest a strong interaction between Na2SO4 and sucrose, where the increase in shoot number was only observed in the region of 0.29 mM Na2SO4 and 5-6 % sucrose. Because single factor experiments cannot detect interactions, our experiment would have not detected a Na2SO4 x sucrose interaction effect. Table 8. ANOVA of the effect of sodium sulfate (Na2SO4) on the numbers of shoots/explant >= 2 mm that were regenerated from Hamlin epicotyl explants Factor

SS

Df

MS

F

P-value

Na2SO4a

2.5

3

0.8

0.29 (3, 12)

0.84

Error

34.6

12

2.9

Total

37.1

15

a

– Na2SO4 tested at 4 levels – 0, 0.1, 1, and 2 mM.

The study indicate that water source, gelling agent, explant LQVHUWLRQ LQĂ€XHQFH VKRRW RUJDQRJHQHVLV IURP VHHGOLQJ HSLFRW\O explants. Variable results under different factors suggest need for optimization of media and culture conditions and the results can be used for conducting further studies.

Acknowledgements We thank Mr. Eldridge Wynn for his careful preparation of the media formulations, growth of the plant cultures, and setup and collection of the data for this study.

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Journal

Journal of Applied Horticulture, 17(2): 129-139, 2015

Appl

Suitable and available land for cashew (Anacardium occidentale L.) in the island of Lombok, Indonesia Widiatmaka1*, Wiwin Ambarwulan2, Atang Sutandi1, Kukuh Murtilaksono1, Khursatul Munibah1 and Usman Daras3 Department of Soil Soil Science and Land Resources, Bogor Agricultural University, Indonesia. 2Geospatial Information Agency, Indonesia. 3Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture, Indonesia. *E-mail: widi.widiatmaka@yahoo.com 1

Abstract Cashews have a potential economic value for local people, and as a conservation plant that is appropriate for small islands, which usually have limited resource capacities. The research for this paper was conducted on Lombok Island, Indonesia with the objective to delineate the potential areas for cashew, based on land availability and land suitability. Land availability was analyzed by taking into DFFRXQW WKH ODQG XVH DQG ODQG FRYHU PDSV LQWHUSUHWHG IURP 6327 LPDJHU\ D )RUHVW $UHDV 6WDWXV PDS DQG D PDS IURP WKH 2I¿FLDO Spatial Land Use Plan. The evaluation of the land’s suitability for cashews was conducted at a land mapping unit resulting from a soil survey, carried out at a scale of 1:25,000. The suitability analysis was done using a maximum limitation method, where the suitability OHYHO ZDV GH¿QHG E\ WKH ORZHVW VRLO FKDUDFWHULVWLFV ZKLFK GHWHUPLQHG WKH SODQWœV UHTXLUHPHQWV 7KH ODQG HYDOXDWLRQ FULWHULD ZHUH established in previous research, which included this island as an area of criteria establishment. The research results show that the land on this island has suitability status for cashews ranging from S2 (moderately suitable) to N (not suitable). The limiting factors include water availability, nutrient retention, available nutrients and rooting media, some of which can be improved. According to the DYDLODEOH DQG VXLWDEOH ODQG DQ DUHD RI KD FDQ EH DVVLJQHG DV ¿UVW SULRULW\ KD DV VHFRQG SULRULW\ DQG KD DV third priority for cashew expansion areas. Key words: Geographic information system, horticultural crop, land evaluation, land use planning

,QWURGXFWLRQ Cashew (Anacardium occidentale L.) is an important exportoriented horticultural crop (Rejani and Yadukumar, 2010; Rupa et al., 2013) and its cultivation has increased in Indonesia during recent years. Previously in Indonesia, this plant was used as a greening plant; however, as its economic potential has increased, the plant has been cultivated for commercial purposes. In Indonesia, small land holders conduct 95% of its cultivation, while the state or private estates operate only on 5% (Statistics Indonesia, 2013a). The data of the Indonesian Directorate General of Plantation (2014) show that the cashew area in Indonesia in 2008 was 573,721 ha and that it increased to 598,503 ha in 2013. The data of Statistics Indonesia (2013b) indicate that Indonesian cashew production increased from 84,200 tons in 2000 to 117,400 tons in 2013. This commodity has apparently attracting farmers’ attention because of its high economic value, and as a perennial crop, it does not require as intensive maintenance as many other food crops. One of the problems agriculture faces all over the world is land availability. The extensive and rapid conversion of productive lands around the world in response to multiple demands for land raises the concern that globally, we risk running out of productive land (Lambin, 2012). In the world, land is limited, whereas the number of people who need the land continues to grow. From 2010 to 2014, the average population growth from 212 countries was at the speed of 1.325% per year (The World Bank, 2014); and even in developing countries, its speed reached more than 3%

per year (Soubbotina, 2004; The World Bank, 2014). Increased development and population pressure in future will impact the ODQG FRPSHWLWLYHQHVV FDXVLQJ ODQG XWLOL]DWLRQ FRQÀLFWV /DQG allocation for various purposes thus needs to be regulated. This land allocation setting is also part of sustainable land resources utilization (Ma et al., 2011; Feizizadeh and Blaschke, 2012; Akinci et al., 2013). In this setting, the selection of available land ZLWK KLJK VXLWDELOLW\ IRU D VSHFL¿F XVH LV LPSRUWDQW 7KLV LV YDOLG for cashew because of its long-lived perennial nature, planting in an unsuitable land will lead to lower production, which means an XQRSWLPDO LQYHVWPHQW IRU D ORQJ WLPH LQHI¿FLHQW XVH RI FDSLWDO LQYHVWPHQW DV ZHOO DV LQHI¿FLHQW ODQG XWLOL]DWLRQ +DOODP et al., 2001; Bell, 2013). Another problem Indonesia faces in cashew cultivation is low productivity. Currently, the average productivity of cashews in Indonesia is less than 500 kg ha-1 (Statistics Indonesia, 2013b). This productivity is much lower than the productivity of cashews in other countries (Widiatmaka et al., 2014a). In India, cashew productivity has reached 1,180 kg ha-1 (Rao, 2013), and even in Nigeria the cashew productivity has reached 1,970 kg ha-1 (FAO, 2011). Indonesia’s low productivity can indeed be caused by many factors such as seeds, fertilizer, plant maintenance, eradication of pests and diseases (Nair, 2010), and other factors, but also land VXLWDELOLW\ 7KH VXLWDEOH ODQG IRU D FRPPRGLW\ VKRXOG EH UHÀHFWHG in high productivity (FAO, 1976; Nair et al., 2010). Therefore, when agricultarist expand area under a crop, there is a need to consider land suitability as an important factor in site selection for high productivity.

130

Land suitability for cashew plants in the island of Lombok (Indonesia)

Indonesia has many cashew production regions, among which is West Nusa Tenggara Province. In this province, Lombok Island, which had an actual total area of cashews in 2012 of 21,834.8 ha (Statistics of West Nusa Tenggara Province, 2013), has the potential to expand the planting of cashews, especially on land which is available for use. In this region, the cashew plant has a high economic contribution for local people, so its expansion areas is of great interest to the local people. Natural resource use on small islands needs to be managed on a sustainable basis (WCED, 1987; Lallianthanga and Sailo, 2013) because small islands have limited resource capacities (Cushnahan, 2001; Reenberg et al., 2008). Small islands must also contend with ongoing developmental pressures in addition to growing pressures from risks associated with global environmental change and economic liberalization that threaten their physical and economic security (Pelling and Uito, 2001). The sustainability of resources on small islands is very dependent on the asset management of their resources, which are generally under pressure (UNGA, 1994). Therefore, efforts should be made to avoid usage that exceeds their natural carrying capacities. When agriculturalists want to plan cultivation, they should choose a commodity that provides not only economic gain, but also ecological protection to the island. Cashew plants, in addition to SURYLGLQJ HFRQRPLF EHQH¿WV DUH DOVR FRQVHUYDWLRQ SODQWV ZLGHO\ used in Indonesia for greening and afforestation (Daras, 2007; Hadad, 2008) because they can adapt to various agroclimatic conditions; therefore, the ecological aspect of sustainability is reached. Thus, the development of cashews on a small island, in DGGLWLRQ WR SURYLGLQJ HFRQRPLF EHQH¿WV ZLOO DOVR HQKDQFH WKH social aspect, as well as the island’s ecological protection, in line with the concept of sustainable agriculture (Salazar-Ordonez et al., 2013; Fauzi and Oxtavianus, 2014). The objectives of this study were to analyze the potential expansion areas for cashew plants on Lombok Island based on the following: (i) land availability, (ii) land suitability for cashew plants, and (iii) spatial delineation and prioritization of potential land for cashew expansion areas, based on land availability and land suitability.

Fig. 1. The research areas of Lombok Island, West Nusa Tenggara Province and the soil sampling point, including soil fertility and soil profile sampling (Widiatmaka et al., 2013, 2014b)

Materials and methods The study was carried out on Lombok Island, West Nusa Tenggara Province, Indonesia (Fig. 1). Geographically, the island is located in 115.46°-116.20°E, and 8.25°-8.55°S. This island covers an area of 5,435 km². Lombok Island has an average rainfall of 1,586 mm year-1, with fairly diverse regions. The highest annual rainfall of 2,855 mm year-1 is reached in Sembalun District in East Lombok, while the lowest annual rainfall of 281 mm year-1 is recorded in Keruak District in East Lombok (Statistics of East Lombok Regency, 2012). The topography of the island is dominated by Mount Rinjani in the northern part, whose height reaches 3,726 meters above sea level, making it the third highest mountain in Indonesia. 7KH VRXWKHUQ SDUW RI WKH LVODQG FRQVLVWV PRVWO\ RI Ă€DW ODQG WKDW can be used for agriculture. Data: Land use and land cover were delineated using SPOT-6 imagery from 2013, with an accuracy of 2.5 m (Widiatmaka et al., 2013; 2014b). Image interpretation and classification was performed by supervised classification using ERDAS ,PDJLQH VRIWZDUH IROORZHG E\ ÂżHOG FKHFNLQJ )LHOG FKHFNLQJ was performed for each type of land use and land cover. The WHFKQLTXH RI ÂżHOG FKHFNLQJ LQYROYHV WKH REVHUYDWLRQ RI ODQG XVH DQG ODQG FRYHU FKDUDFWHULVWLFV LQ WKH ÂżHOG ZKLFK ZHUH WKHQ matched with their appearance in the image. Interviews with the community were also conducted to gather information that could QRW EH LGHQWLÂżHG IURP WKH LPDJH VXFK DV WKH W\SH RI SODQWV SODQW FDQRS\ VWUDWD DQG KLVWRULFDO ODQG XVH FKDQJHV 7KH UHVXOWV RI ÂżHOG checks were used as a basis for reinterpretation in order to obtain WKH ÂżQDO ODQG XVH DQG ODQG FRYHU PDS ,PDJHU\ LQWHUSUHWDWLRQ of SPOT-6 produced 28 kinds of land use and land cover based on standard national of imagery interpretation (SNI, 2010), but IRU WKH SXUSRVHV RI VLPSOLÂżFDWLRQ LQ WKLV DUWLFOH ODQG XVH DQG land cover are grouped into 14 kinds of land use and land cover. (VSHFLDOO\ IRU SDGG\ ÂżHOGV YHULÂżFDWLRQ ZDV GRQH XVLQJ ,.2126 imagery from 2012, as interpreted by the Indonesian Ministry of Agriculture (Widiatmaka et al., 2014b). Land Mapping Units (LMUs) were obtained from a soil survey and land evaluation project conducted in 2013 on the initiative of the Geospatial Information Agency, Indonesia. The soil survey was performed in an area of 289,049 ha outside the forest area and settlement area. Morphological observation of the soil was conducted by auger observation following the soil survey method of Soil Survey Division Staff (1993). In total, there were 842 morphological observations of the soil, which were input to delineate LMUs’. The 103 soil samples representing LMUs were then taken for laboratory analysis (Fig. 1.). Physical and chemical soil properties were analyzed in the laboratory of the Department of Soil Science, Bogor Agricultural University using standard laboratory methods (Tan, 2009). The survey results were outlined in a soil map at a scale of 1:25,000, which is currently available in the Geospatial Information Agency, Indonesia (Widiatmaka et al., 2013, 2014b). Spatial data of the areas with different forest status was obtained from a map of Forest Area Status (FAS) at a scale of 1:250,000 and was provided by the Ministry of Forestry (Forestry Planning Agency, 2002). In this map, various states of the forest areas are presented, including information about the different areas’

Land suitability for cashew plants in the island of Lombok (Indonesia)

131

The land suitability analysis for cashews was conducted using Automated Land Evaluation System (ALES) (Rossiter, 2001). The maximum limitation method (FAO, 1976; De la Rosa and van Diepen, 2002) was used for land evaluation. In this method, the degree of limitations of land use is imposed by land characteristics on the basis of permanent properties, a criterion is so needed (De la Rosa and van Diepen, 2002).

Fig. 2. Research steps followed to define suitable and available land for cashew plants in the island of Lombok, Indonesia

DOORZDQFH IRU FXOWLYDWLRQ 7KH RIÂżFLDO ODQG XVH SODQ ZDV REWDLQHG IURP VSDWLDO GDWD IURP WKH GRFXPHQW RI 2IÂżFLDO 6SDWLDO /DQG 8VH Plan (OSLUP) of West Nusa Tenggara Province, available at a scale of 1:100,000 (Regional Goverment of West Nusa Tenggara 3URYLQFH ,Q WKH 26/83 ODQG KDV EHHQ RIÂżFLDOO\ DOORFDWHG for different uses. Analysis: The analysis procedure used in this research is described in Fig. 2. Land availability analysis was done by overlaying land use and land cover maps resulting from the SPOT6 interpretation, a map of FAS and a map of OSLUP. The land suitability analysis for cashews was conducted using soil survey data. Both were then spatially overlaid to obtain spatial data for the suitability of the available land.

Theoretically, there are two approaches in land evaluation, i.e. direct land evaluation and indirect land evaluation (FAO, 1976). In direct land evaluation, the assessment is done directly to the growth or production of plants: lands in which a plant produces a high yield are described as suitable for such a plant, while lands in which plants produce lower yields are described as less VXLWDEOH 7KHVH FODVVLÂżFDWLRQV FDQ IXUWKHU EH GHOLQHDWHG DV YHU\ suitable, suitable, marginally suitable and not suitable. In indirect ODQG HYDOXDWLRQ WKH VXLWDELOLW\ RI VSHFLÂżF ODQGV IRU VSHFLÂżF SODQWV is described according to land quality, based on experience and previously known empirical data. In this case, land suitability DVVLJQPHQWV QHHGHG D FULWHULD RI D ODQGÂśV UHTXLUHPHQW IRU D VSHFLÂżF plant, which criteria was established based on experience in various other places. Assessment was then carried out, using the maximum limitation method, by matching the criteria of land characteristics of the area being assessed, compared to the land requirements of the plant (De la Rosa and van Diepen, 2002). Such a method is the most widely practiced in Indonesia because it can be done quickly (Ritung et al., 2007). Nevertheless, one of the weaknesses in land evaluation in Indonesia is the lack of criteria. Several criteria are available; however, they have not been

Table 1. Land suitability criteria for cashew Land Quality/ Land Characteristics Temperature -Elevation (m asl) Water availability - Rainfall (mm)

Symbol

Land suitability class Very Suitable (S1)

Moderately Suitable (S2)

Marginally Suitable (S3)

Not Suitable (N)

<195.6

195.6-324.4

324.4-456.2

>456.2

987-2.247

827-987 2,247-3,197 3.9-5.1 9.8-10.5 <0.5 3.3-4.5

601-827 3,197-4,926 <3.9 10.5-11.4

<601 >4,926 >11.4

4.5-8.4

>8.4

clay loam, sandy clay loam, loam >39.7

sandy clay, clay loam, sandy loam 21.1-39.7

clay, silty clay, silty clay loam 6.6-21.1

heavy clay, silt, loamy sand, sand <6.6

>12.40 5.4-6.4 >0.78 >65.7

8.54-12.40 5.1-5.4 6.4-6.9 0.49-0.78 <65.7

2.56-8.54 4.6-5.1 6.9-7.7 0.11-0.49

<2.56 <4.6 >7.7 <0.11

>0.072 >39.69 >0.37

0.052-0.072 10.84-39.69 0.27-0.37

0.029-0.052 1.02-10.84 0.10-0.27

<0.029 <1.02 <0.10

<11.9 <14.5

11.9-23.1 14.5-28.8

23.1-77.4 28.8-75.5

>77.4 >75.5

t w

- Dry month (number)

5.1-9.8

- Wet month (number)

0.5-3.3

Rooting media - Texture - Effective depth (cm) Nutrient retention - CEC (cmol(+) kg-1 - Water pH (1:5)

r

f

- Organic-C (%) - Base Saturation (%) n Available nutrient - Total-N (%) - Avail.-P (ppm) - Exch.-K (cmol (+).kg-1) p Terrain condition - Slope (%) - Surface rock (%) Source: Widiatmaka et al. (2014a)

132

Land suitability for cashew plants in the island of Lombok (Indonesia)

Surveyed region Fig. 3. Survey region for establishing the land suitability criteria for cashews (Widiatmaka et al., 2014a). Lombok Island includes sampling locations, resulting in criteria that were then used to evaluate land suitability for this paper.

built based on empirical knowledge of production as required by the FAO (1976). As a result, often the use of inaccurate criteria causes diagnostic errors in land evaluation. Frequently, it has been found that commodities grow and produce well in a region, but the evaluation using improper criteria produce low suitability classes, and vice versa. Such phenomena are often found in land evaluation (Sutandi and Barus, 2009; Widiatmaka et al., 2014a). To avoid such an error in land evaluation, various studies have been done to develop criteria that are relevant to crop production (Ritung et al., 2007; Sutandi and Barus, 2007). In the case of the cashew plant, a criterion that relates to land characteristics and production has been built and recently published (Widiatmaka et al., 2014a). In that research, the criterion was built with a soil survey, confronted with cashew production. Cashew plantations LQ ÂżYH SURYLQFHV DQG UHJHQFLHV ZHUH VDPSOHG /RPERN ,VODQG is one of the areas that was used for establishing the criteria (Fig. 3). The data for production per tree per year were obtained from farmers, while the soil was sampled and analysed in the laboratory. Age-adjusted cashew production was used as the yield response and plotted against land characteristics. The criteria were then established using a projection of the intersection between the boundary line and yield interval. The resulting criterion (Table 1) is considered appropriate to be used in this study because it was constructed in the area that includes Lombok Island as a location of data retrieval for confrontation between land characteristics and production. Using such criteria, we used the land evaluation concept of the )$2 IRU WKLV UHVHDUFK ZKHUH ODQG ZDV FODVVLÂżHG DV HLWKHU S1 (very suitable), S2 (moderately suitable), S3 (marginally suitable), or N (not suitable). ALES ver. 4.65e, ArcGIS 10.2 DQG 0LFURVRIW 2IÂżFH ZHUH XVHG DV VRIWZDUH WRROV $QDO\VLV ZDV performed by integrating Arc-GIS and ALES. The land evaluation model using ALES consists of several steps

(Rossiter, 2001). The Land Use Type (LUT), which in this case is WKH FDVKHZ ZDV HVWDEOLVKHG ÂżUVW /DQG 8VH 5HTXLUHPHQW /85 for this LUT was then established. In the next step, choice and establishment of Land Characteristics (LCs) for each LUR and LUT were done. Finally, a Decision Tree (DT) was made according to the criteria. The land characteristics used for the land evaluation were stored in the ALES database. The land suitability evaluation was done for each LMU. The results of ALES analysis were then transferred to the ArcGIS 10.2 for geographical reference and are described in the form of maps and tables.

5HVXOWV DQG GLVFXVVLRQ Land use and land cover: The results of the land use and land cover analysis are presented in Fig. 4a and Table 2A. The main land use and land cover in Lombok Island are the following: IRUHVW SDGG\ ÂżHOG VKUXE GU\ ODQG DJULFXOWXUH DQG SODQWDWLRQ Forest cover is the widest, located in the northern part of the island from the hilly part of the region, climbing to the direction of Mount Rinjani. The south part of the region is dominated by farms and plantations. Settlements are concentrated in the capital, Mataram, and the surrounding region. Soil and land mapping unit: The overall area of Lombok Island is divided into 52 LMUs. The components of LMU used in this VWXG\ LQFOXGH WKH VRLO FODVVLÂżFDWLRQ LQ WKH VXE JURXS FDWHJRULHV parent material, slope, and physiography (USDA, 2010). A map of Lombok Island based only on soil sub-group is presented in Fig. 4b. A summary of the soil distribution in the study area is presented in Table 2B. The soil in Lombok Island comprises ÂżYH VRLO RUGHUV 86'$ LQFOXGLQJ $OÂżVROV ,QFHSWLVROV Entisols, Andisols and Aridisols, and there are 13 soil subgroups. Inceptisols occupy the largest area, covering 268,226.2 ha, or 58.7% of the area. Another soil order that is also quite ZLGHO\ VSUHDG LV $OÂżVROV ZKLFK FRYHUV DQ DUHD RI KD

Land suitability for cashew plants in the island of Lombok (Indonesia)

a

133

b

Fig. 4. Map of (a) the land use and land cover, interpreted from SPOT-6 imagery, and of (b) the soil class up to sub-group categories in the areas outside of Forest Area Status (Widiatmaka et al., 2013, 2014b)

RU 7KH GRPLQDQF\ RI ,QFHSWLVROV DQG $OÂżVROV LV UHODWHG WR VRLO GHYHORSPHQW ZKLFK KDV EHHQ LQĂ€XHQFHG E\ WKH ORFDO climate (Widiatmaka et al E $OÂżVROV LV FKDUDFWHUL]HG E\ an accumulation of clay in a sub-soil horizon, namely the argillic horizon, and it has a high base saturation (>35%) (USDA, 2010). Inceptisols is a soil that is relatively young; the soil development is not very advanced. The development of Inceptisols and $OÂżVROV LV UHODWHG WR WKH UHODWLYHO\ GU\ FOLPDWH DUHDV 7DQ Widiatmaka et al., 2014b).

and hence their protection is important, not only for the country but also for the earth’s sustainability. This law, accompanied by the hard efforts of the Ministry of Forestry in suppressing the forest degradation, has resulted in diminishing the rate of forest degradation to about 1.08 million ha for the last three years from its level of about 2.8 million ha in the 1990s (Kusmana, 2011). The planning in this research is intended to be sustainable, and thus the regulatory status of these forests must be considered. For this reason, the areas that can be used for farming, including for cashew plantations, only include those of the ‘Area for Other Uses’ status in FAS. In a more detailed description, ‘Area for 2WKHU 8VHVÂś PHDQV DQ DUHD ZKLFK LV QRW RIÂżFLDOO\ LQFOXGHG LQ RWKHU types of FAS such as protected forest, research forest, national park as well as production forest area. In the case of Lombok Island, this ‘Area for Other Uses’ includes 295,140.9 ha.

Forest area status: The FAS map is presented in Fig. 5a, while the area of each forest’s status within the map is presented in Table 3A. Land utilization for non-forest purposes in Indonesia is regulated by the Forestry Law No. 41/1999. The cultural activity (settlement, agriculture, industry etc.) is prohibited in forest areas. This regulation is intended to preserve the forests in Indonesia. These forests have a mega biodiversity status in the tropical zone

It should be noted, however, that actually, maps of FAS are

Table 2. Area distribution of Lombok Island according to (A) land use and land cover, interpreted from SPOT-6 imagery, and (B) soil class up to sub-group category in the area outside of Forest Area Status (Widiatmaka et al., 2013, 2014b) A No

Land Use/Land Cover

B Area

No

Soil Sub-Group

Area

ha 123,687.8

% 27.1

ha 651.7

1

Lithic Udivitrands

% 0.6

755.4

0.2

2

Typic Durustepts

110

0

3

Typic Endoaquents

10,242.2

9.9

1,615.4

1.6

38.6

0

4

Typic Endoaquepts

9,639.7

2.1

5

Typic Eutrudepts

785.8

0.2

6

Typic Fluvaquents

106.5

0.1

51,738.5

11.3

7

Typic Fragiudepts

12,005.4

11.6

1

Forest

2

Mangrove forest

3

Built area

4

Bare land

5

Grass land

6

Sand

7

Plantation

8

Settlement

20,855.6

4.6

8

Typic Haplocalcids

4,108.0

4.0

9

,UULJDWHG SDGG\ ÂżHOG

106,248.0

23.3

9

Typic Hapludalfs

26,674.0

25.8

10

5DLQIHG SDGG\ ÂżHOG

21,524.4

4.7

10

Typic Haplustalfs

4,094.9

4.0

11

Shrub

58,335.7

12.8

11

Typic Hidraquents

12

Pond

36.9

0

12

Typic Udorthents

13

Dry land agriculture

57,891.6

12.7

13

Typic Ustipsamments

14

Water body

5,226.5

1.1

456,874.6

100

Total

Total

312.6

0.3

25,915.7

25.0

979.9

0.9

16,277.5

15.7

538.6 103,522.4

0.5 100

134

Land suitability for cashew plants in the island of Lombok (Indonesia)

a

b

Fig. 5. Maps of (a) forest areas, according to the map of Forest Area Status (Forestry Planology Agency, 2002), and of (b) Official Spatial Land Use Plan (Government of West Nusa Tenggara Province, 2010)

available throughout Indonesia only at a scale of 1:250,000. Therefore, using such a small-scale map in this study, delineation had to be carefully viewed and only at the planning level. 2SHUDWLRQDO XVH LQ WKH ¿HOG FRQFHUQLQJ IRUHVW ERXQGDULHV QHHGV to be detailed in coordination with the Ministry of Forestry.

The results of the land suitability analysis show that the land suitability classes for cashews range from S2 (moderately suitable) to N (not suitable). In terms of actual land suitability, the total amount of suitable land for cashews (class S2 and S3) is 61,214.2 ha, consisting of 22,242.8 ha of S2 class and 38,971.4 ha of S3 class.

$OORFDWLRQ LQ RI¿FLDO VSDWLDO ODQG XVH SODQ: Planners should refer to the OSLUP, according to Law No. 26/2007. The map of OSLUP is presented in Fig. 5b and Table 3B. According to OSLUP, the areas that are allocated as cultural areas include 60.8% of the island area, and consist of plantation, agricultural and settlement areas. The rest, 39.2% of the area, is allocated as non-cultural areas, which include protected areas, natural resources-preserved areas, production forest areas and water bodies. The areas that can still be used for cashew expansion are the plantation areas and the agricultural areas. On Lombok Island, the total area of these two areas is 273,537.1 ha.

The land qualities that cause land to be as S2 class are those of water availability (w), nutrient retention (f), and available nutrients (n). For the land quality of water availability, the land characteristics that become limiting factors are rainfall and number of dry months. Lombok Island has a very wide range of climates, the rainfall ranges from very low to very high. Research indicates that climate factors relate to moisture availability variation in soil (Tolla, 2004). Management and soil moisture regimes are factors that determine the cashew plant’s yield variability (Rejani and Yadukumar, 2010), the temporal variation of available soil moisture explained the yield variability of the cashew nuts. Gopakumar et al. (2005) also indicated that there was a decline in cashew productivity due to the warmest and drought conditions as a result of a decline in rainfall and an increase in temperature. 7KH GU\ VHDVRQ LV JHQHUDOO\ FRLQFLGHV ZLWK FDVKHZ ÀRZHULQJ DQG nut development, when crop water requirements reach maximum values, and so, low nut yields are commonly associated with years of low rainfall (Oliveira et al., 2006).

Land Suitability: The results of the soil analysis, used as land qualities and land characteristics in land evaluation are presented in Table 4, with only the analysis summary presented. The actual and potential land suitability maps were presented only in the areas that are allowed to be used for agricultural purposes according to FAS and OSLUP.

Table 3. Distribution of (A) forest areas, according to the map of Forest Area Status (Forestry Planology Agency, 2002), and of (B) Official Spatial Land Use Plan (Government of West Nusa Tenggara Province, 2010) A B No Forest Area Status Area No 2I¿FLDO 6SDWLDO /DQG 8VH 3ODQ Area ha % ha % 1 Protected forest 70,798.2 15.5 1 Protected area 103,814.5 22.7 2

Research forest

3

National park

4

Natural tourism park

5

Community plant forest

6

Production forest

7

Limited production forest

8

Area for other uses

9

Water Total

364.8

0.1

2

Natural resources preserved area

40,421.4

8.8

34,057.5

7.5

3

Production forest area

32,611.3

7.1

6,804.5

1.5

4

Plantation area

139,673.8

30.6

133,863.3

29.3

3,925.5

0.9

209.2

0

5

Agricultural area

30,096.1

6.6

6

Settlement

17,547.3

3.8

7

295,140.9

64.6

1,856.2

0.4

456,874.6

100

Water/water park Total

2,564.9

0.6

456,874.6

100

Land suitability for cashew plants in the island of Lombok (Indonesia)

135

Table 4. Results of the soil analysis used as land characteristics for land evaluation for cashew plants Soil Order

n

$O¿VRO

8

Andisol

2

Aridisol

2

Entisol

11

Inceptisol

29

Min Ave Max Min Ave Max Min Ave Max Min Ave Max Min Ave Max

CEC cmol Base satu(+).kg-1 ration (%) 7.6 92.9 22.6 99.1 47.5 100.0 16.8 99.2 17.8 99.6 18.8 100.0 31.3 100.0 34.7 100.0 38.0 100.0 2.6 52.7 18.1 93.3 43.5 100.0 2.2 89.4 11.7 99.1 31.5 100.0

pH 6.0 6.7 7.9 5.9 5.9 6.0 6.7 7.2 7.6 4.7 6.8 8.2 5.4 6.2 8.5

Organic-C (%) 0.6 0.9 1.4 0.9 0.9 1.0 1.1 1.3 1.6 0.2 0.8 1.3 0.4 1.4 4.6

For land quality of nutrient retention (f), the determining land characteristics can be the soil’s Cation Exchange Capacity (CEC), its base saturation, its C-organic content and its pH (Table 1). The analysis results indicate that the dominant limiting factor for cashew growth on Lombok Island is a low soil CEC. There was only one sampling point that had too high pH, and two sampling points that had too low a quantity of C-organic content as limiting factors and putting this land into the S2 class. However, there was not a sampling point that indicated the soil’s base saturation as a limiting factor. On Lombok Island, the soil’s CEC generally is not high enough to enter into class S1 for cashews. The pH appeared to be a limiting factor for assigning the land to the classes of S3 and N for cashews. A high pH can be a serious limiting factor and even make the land not suitable (N) for cashews. However, a too low pH could be a limiting factor for cashews as well. Ngatunga et al. (2003) observed that continued use of sulphur on the Makonde plateau is likely to result in a decline of the soil pH, which affects the cashew nut production. Furthermore, Owaiye and Olunloyo (1990) reported that the best growth of cashews is obtained between the pH ranges of 4.5 and 5.0, a pH of 4.5 being optimal. The similar tendency has been found in whole Nusa Tenggara Province by Widiatmaka et al. (2014a) For the land quality of available nutrient (n), the land characteristics

N-Total (%) 0.05 0.08 0.13 0.07 0.08 0.09 0.11 0.13 0.15 0.02 0.07 0.11 0.04 0.13 0.41

P2O5 (ppm) 1.97 34.69 94.28 4.61 20.20 35.78 15.36 17.68 20.00 5.88 22.03 73.18 4.81 26.35 81.40

K 2O (ppm) 36.0 146.4 201.0 168.4 173.0 177.5 163.0 222.8 282.6 52.3 151.6 347.5 40.7 88.8 259.3

Sand 17 34 46 43 44 45 16 20 24 22 51 94 26 56 84

Texture (%) Silt 28 38 47 29 30 30 41 44 47 26 53 8 27 50

that were taken into account were nitrogen (total-N), phosphorous (available-P) and potassium (exchangeable-K). The analysis results indicated that the land characteristics that limit cashew growth from being in the S2 class on Lombok Island were generally total-N. There was only one point sample that had a low value of P2O5, making these samples S2 class, and there were no samples that showed exchangeable-K as a limiting factor. Even in classifying the land as S3 or N class, exchangeable-K remained not a limiting factor. The soil on Lombok Island appears to have VXI¿FLHQW .2O levels for the growth of cashews. For classifying S3 and N classes, the total N together with P2O5 content appear to be limiting factors. The research of O’Farrel (2010) showed that high yields of cashew nuts can be achieved from intensively managed trees. This was achieved when N was applied at the rate of 17 g N/m2. The timing of N fertilizer is critical, and should be applied during the main vegetative growth period. Nitrogen appears necessary during the vegetative phase of the cashew tree. Application of a high-nitrogen fertilizer was responsive in this vegetative phase. In cashews, phosphorus is an essential component of the genetic material of the cell nucleus. Phosphorus deficiency causes stunting, delayed maturity, and shriveled seeds (Thompson and Troeh, 1978; Aikpokpodion et al., 2009; Widiatmaka

Table 5. Land suitability for cashew plants on Lombok Island: (a) actual land suitability, and (b) potential land suitability No Actual Land Suitability Area No Potential Land Suitability ha % ha Sub-Class 1 S2-f 3,276.8 0.7 1 S1 4,075.6 2 S2-wf 2,233.5 0.5 2 S2-w 18,167.3 3 S2-wn 566.4 0.1 3 S2-f 149.9 4 S2-fn 798.7 0.2 4 S2-n 1,993.2 5 S2-wfn 15,367.4 3.4 5 S2-fn 210.9 6 S3-f 149.9 0.0 6 S3-r 36,617.4 7 S3-n 1,993.2 0.4 7 S3-f 4,611.4 8 S3-r 32,251.0 7.1 8 N 39,710.7 9 S3-rf 2,149.7 0.5 9 na 351,338.3 10 S3-rn 2,216.7 0.5 Total 456,874.6 11 S3-fn 210.9 0.0 12 N 44,322.1 9.7 13 na 351,338.3 76.9 Total 456,874.6 100.0

na: not available area

Clay 9 28 55 25 27 28 35 36 37 5 22 44 4 17 36

Area % 0.9 4.0 0.0 0.4 0.0 8.0 1.0 8.7 76.9 100.0

136

Land suitability for cashew plants in the island of Lombok (Indonesia) /DQG VXLWDELOLW\ FDQ EH GHÂżQHG DV DFWXDO ODQG VXLWDELOLW\ DQG potential land suitability (FAO, 1976). Potential land suitability is land suitability when limiting factors in actual land suitability are improved (FAO, 1976). However, several limiting factors are permanent as some soil textures cannot be improved. On Lombok Island in general, the improvement of actual land suitability to become potential land suitability increases the extent of the suitable area by only a small amount. The total actual suitable land is 61,214.2 ha (58% of land being assessed for its suitability), while the total potential suitable land is 65,825.7 (62% of land being assessed for its suitability) (Table 5 and Fig. 6). This is because the good land on Lombok Island has mostly been cultivated for planting cashews.

a

b

Fig. 6. Map of land suitability for cashew: (a) actual land suitability, and (b) potential land suitability

et al., 2014a). Ibiremo et al. (2012) reported that the rock SKRVSKDWH WUHDWPHQW VLJQL¿FDQWO\ LPSURYHG QXW VL]HV LQ ,EDGDQ soil. Additionally, Sathiyamurthi (2013) indicated the need of improving the soil’s phosphorus in cashew orchards’ soil. In the S3 class, the land qualities that become limiting factors are rooting media (r), nutrient retention (f), and available nutrients (n). Land qualities that determine an N class are rooting media (r) and nutrient retention (f). For the S3 and N classes, other than the limiting factors of pH, total-N and P2O5, the soil texture emerges as the dominant limiting factor. This result shows that cashew is not tolerant to poor soil drainage. Previous studies indicated that soil texture is critical for cashew production (Duncan, 2001). Cashew production relates to well-drained soil with a sandy ORDP WH[WXUH 'XQFDQ &DVKHZV VHHP WR ÀRXULVK LQ D free-draining and light-textured soil (Widiatmaka et al., 2014a).

Area priority for cashew expansion: Land availability for cashew expansion areas was analyzed using a matrix of land use allocation (Table 6). In this allocation matrix, according to FAS, the only available land for cashew development planning fell under Area for Other Uses forest status. Based on the OSLUP, the lands that allowed for cashew development were agricultural and plantation areas. Based on the existing land use, land that can be developed is land in which the current land use/land cover is bare land, shrub, and dryland agriculture. Forest and mangrove forests are not recommended to be used in order to protect the environment. Law No. 26/ 2007 contains a provision that in any region, at least 30% of the area should be forest area. In the case of Lombok Island, the current forest cover is 124,443 ha, or only 27.3%, although based on the FAS, the forest area is 159,878 ha or 35%. The existing forest land cover must be maintained so as not to be used for cultivation. The existing plantation is also not suggested in the calculation of the area that can be used for the expansion. Pasture is not recommended for cashew development because Lombok Island is also a breeding area in Indonesia, ZKLFK UHTXLUHV VXIÂżFLHQW VRXUFHV IRU DQLPDO IHHG 3DGG\ ÂżHOGV ERWK LUULJDWHG DQG UDLQIHG SDGG\ ÂżHOGV DUH QRW UHFRPPHQGHG for use for food security reasons. Thus, in this case of cashew expansion, there was an area of 103,522.4 ha, taking FAS, OSLUP and existing land use into account for the available land for agricultural development. Prioritizing the land utilization for cashew plantations is based on land suitability for cashews. First priority is given to land with potential land suitability for cashews S1, second priority is given to land with potential land suitability for cashews S2, and third priority is land with potential land suitability for cashews S3. Land with N potential land suitability for cashews is not suggested to be used for cashew development. With this analysis, an area of 4,075.6 ha can be assigned as priority I for cashew extension, a land area of 18,167.3 ha can be assigned as priority II, and a land

Table 6. Matrix of land use allocation for cashew expansion area Land Use/Land Cover

2IÂżFLDO 6SDWLDO /DQG 8VH 3ODQ

Forest Area Status

Bare Land, Shrub, Dryland Agriculture

Plantation Area, Agricultural Area

Area for Other Uses

Forest, Mangrove Forest, Grassland, Sand, Plantation, Settlement, Irrigation Paddy Field, Rainfed Paddy Field, Pond, Water Body

Protected Area, Natural Area, Production Forest Area, Settlement Area, Water

Protected Forest, Production Forest, Limited Production Forest, National Park, Natural Park, Community Forest Plant, Research Forest, Water

Land Suitability S1 S2 S3 N

Land Use Recommendation Priority I Priority II Priority III Not recommended

Not recommended

Land suitability for cashew plants in the island of Lombok (Indonesia) Table 7. Area priority of cashew expansion on Lombok Island No 1 2 3 4

Use direction for cashew Priority I Priority II Priority III Not Recommended Total

Area ha 4,075.6 18,167.3 43,582.8 391,049.0 456,874.6

% 0.9 4.0 9.5 85.6 100.0

area of 43,582.8 ha can be assigned as priority III. Thus, in total on Lombok Island, the extension of cashew can be developed in the total potential area of 65,825.7 ha (Table 7 and Fig. 7). Based on the concept of economic land suitability (FAO, 1985; Rossiter, 2001), land suitability class S1 has the potential to produce 80 to 100% of the maximum production; land suitability class S2 has the potential to produce 60 to 80% of the maximum production. Land suitability class S3 has the potential production of 24 to 60% of the maximum production (Widiatmaka et al., 2014a). When referring to the highest production potential in Nusa Tenggara, based on the data of Statistics Indonesia (2013b) that is 367 kg ha-1 at the maximum production rate, so if using a lower limit on the production of each class of land suitability, additional potential production will be obtained from lands of S1, S2 and S3, respectively, with the potency of 1,196 tons.year-1 on land S1, 4,000 tons year-1 on S2 land and 3,838 tons year-1 in S3 land, or a total additional production of 9,036 tons year-1. As information, the actual production of cashew in Lombok Island is 3,116 tons year-1 (Statistics of West Nusa Tenggara Province, 2013). This rough estimation can be added to the potential use of the degraded forest land, which may also be used for the plant because cashew nut is one of the greening plants (Hadad et al., 2008). However, it is necessary for other more detailed analysis. The analysis presented is the potency of Lombok Island from point of view of land suitability and land availability. Certainly for the realization, many other parameters still need to be considered, one of them being the use of land for other commodities. According to the land tenure regime in Indonesia, the land is RZQHG ZLWK OHJDO FHUWL¿FDWH E\ LQGLYLGXDOV E\ HQWLWLHV RU E\ the State. The ability to use land owned by individuals or entities would depend on the owners’ economic interest. The analysis from this point of view is not included in the scope of this article;

137

however, such consideration can be used for further land use planning and analysis. Finally, it can be highlighted that the analysis in this research, conducted on the small island of Lombok, Indonesia has been performed by taking into account the available land for agriculture LQ WHUPV RI IRUHVW DUHD VWDWXV RIÂżFLDO VSDWLDO ODQG XVH SODQQLQJ and existing land utilization. Taking into account such factors is important, considering that land is a non-renewable resource which is needed by many sectors. Our research calculates that there was 103,522.4 ha of available land. From this available land, the actual suitable land with different suitability classes for cashews could then be delineated using land suitability analysis; in this case it was 61,214.20 ha. The choice of cashew is guided by the fact that this plant meets the requirements for sustainable use in a small island in terms of ecology and economy as well as the needs of the population. From the land suitability analysis, the limiting factors for cashew development were obtained by analysing the data from soil characteristics resulting from a soil survey. These consist of the land characteristics of water availability (based on rainfall and dry months in several regions), nutrient retention (soil CEC and soil pH), nutrient availability (total N and P2O5 content) and rooting media (soil texture). Several limiting factors can be improved to increase the number of suitable land areas for cashews. The priority area for cashew expansion was then established according to its potential land suitability. In total, an area of 65,825.7 ha with different classes of land suitability for cashews can be recommended. With the addition of these cashew areas, the potential cashew production from this island can increase. The preciseness of this mapping methodology has to be also taken into consideration. This research used different maps at various precisions; the soil map and topographical map were at a scale of 1:25,000, while the land use was interpreted using SPOT-6 imagery at a precision of 2.5 m. Through this interpretation, the REWDLQHG UHVXOW FDQ EH GHÂżQHG DV RSHUDWLRQDO IRU ODQG XVH SODQQLQJ at a middle scale of 1:25,000. Since however, the OSLUP map used was scaled at 1:100,000 and the FAS map at 1:250,000, the ÂżQDO UHVXOWV UHFHLYHG VKRXOG EH YLHZHG FDXWLRXVO\ 7KH XQFHUWDLQW\ at the middle scale land use planning may be derived from these two maps. Consequently, more attention and consideration have WR EH JLYHQ WR WKH ERUGHU RI WKH IRUHVW DV ZHOO DV WKH RIÂżFLDO ODQG use plan border when the results for the middle scale land use planning are used for operational purposes. The research steps shown in this paper can act as a step-by-step guide for other areas. It is important to note the importance of selecting suitable land, and on the other hand, to take into account WKH DYDLODEOH ODQG DFFRUGLQJ WR RIÂżFLDO UHJXODWLRQ RI ODQG XVH planning.

Acknowledgement 7KH ÂżQDQFLDO VXSSRUW IRU WKLV UHVHDUFK ZDV JLYHQ E\ *HRVSDWLDO Information Agency, Indonesia, through the program of Multisector Land Suitability and Land Capability Mapping during ÂżQDQFLDO \HDU RI

References Fig. 7. Map of priority area for cashew expansion according to land suitability and land availability on Lombok Island

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Journal

Journal of Applied Horticulture, 17(2): 140-144, 2015

Appl

Effects of pre-harvested N-(2-chloro-4-pyridinyl)-N’-phenylurea &338 VSUD\LQJ RQ WKH LPSURYHPHQW RI Ă RZHU TXDOLW\ RI Dendrobium Sonia ‘Earsakul’ S. Abdullakasim*, K. Kaewsongsang, P. Anusornpornpong and P. Saradhuldhat Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen, Nakhon Pathom-73140, Thailand. *E-mail: fagrsds@ku.ac.th

Abstract ,PSURYHPHQW RI Ă€RZHU TXDOLW\ LV D PDMRU FRQFHUQ ZKLFK SOD\V D SDUW LQ WKH HQKDQFHPHQW RI WKH PDUNHWDELOLW\ RI WKH ÂľDendrobium’ cut Ă€RZHU ,Q WKLV VWXG\ ERWK V\QWKHWLF F\WRNLQLQV N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU) and N-6-benzyladenine (BA), were foliar sprayed at rates of: 1, 5 or 10 mg L-1 and 100, 200 or 400 mg L-1, respectively, on current pseudobulbs of the Dendrobium Sonia ‘Earsakul’ with 45-50 cm in length. The treatments were applied three times at fortnight intervals, prior to terminal bud initiation. The results revealed that an application of 10 mg L-1 &338 VLJQLÂżFDQWO\ LQFUHDVHG WKH QXPEHUV RI LQĂ€RUHVFHQFH SHU SVHXGREXOE IURP WR LQĂ€RUHVFHQFH DQG WKH QXPEHU RI Ă€RZHU RQ DQ LQĂ€RUHVFHQFH ZDV LQFUHDVHG IURP WR Ă€RZHUV 7KH OHQJWK DQG WKH GLDPHWHU RI LQĂ€RUHVFHQFH KDYLQJ PJ /-1 CPPU application, also increased from 49.4 cm to 55.1 cm, and 0.57 cm to 0.66 cm, UHVSHFWLYHO\ ,Q DGGLWLRQ WKH ODUJHVW Ă€RZHU ZLGWK DQG WKH KLJKHVW IUHVK LQĂ€RUHVFHQFH ZHLJKW ZHUH DOVR REWDLQHG ZLWK DSSOLFDWLRQ RI mg L-1 CPPU treatment. Despite the application of BA, at 400 mg L-1 HQKDQFLQJ WKH KLJKHVW DPRXQW RI Ă€RZHU FRXQWV RI LQĂ€RUHVFHQFH DW Ă€RZHUV RI WKRVH LQĂ€RUHVFHQFH REWDLQHG DW OHDVW RQH GHIRUPHG Ă€RZHU 2YHUDOO WKH UHVXOWV VXJJHVW WKDW &338 VSUD\ KDV D KLJKHU SRWHQWLDO WR HOHYDWH Ă€RZHUV DORQJ ZLWK WKH LQĂ€RUHVFHQFH TXDOLWLHV RI Dendrobium Sonia ‘Earsakul’. Furthermore, according WR WKLV VWXG\ &338 KDV ORZHU HIIHFWV XSRQ DEQRUPDO Ă€RZHU VKDSHV DQG WKHLU WLPHV RI KDUYHVW Key words: Dendrobium F\WRNLQLQ %$ LQĂ€RUHVFHQFH SVHXGREXOE GHIRUPHG Ă€RZHU

,QWURGXFWLRQ Dendrobium 6RQLD ¾(DUVDNXOœ LV D IDPRXV K\EULG IRU FXW ÀRZHU SURGXFWLRQ DQG FDQ SURGXFH ÀRZHUV DOO WKH \HDU URXQG \LHOGLQJ high number of inflorescence. Thailand annually exports ODUJH YROXPHV RI WKLV RUFKLG DQG TXDOLW\ RI ÀRZHU LV WKH PDLQ criteria to be considered when exporting cut orchids. Grading standards of Dendrobium orchids generally evaluate from KHDOWK\ LQÀRUHVFHQFH SHVW DQG SDWKRJHQ IUHH ZKLFK LQFOXGHV ÀRZHU VKDSH OHQJWK RI LQÀRUHVFHQFH QXPEHUV RI ÀRZHUV SHU LQÀRUHVFHQFH DQG YDVH OLIH HWF ,Q RUGHU WR SURPRWH JURZWK DQG WKH GHYHORSPHQWDO SURFHVVHV RI ÀRZHULQJ SODQWV VRPH SODQW growth regulators are usually applied. Cytokinin, a plant growth regulator, plays a role in: 1) the cell cycle process, regulating cell division (Zhang et al., 2005), 2) the function for nutritional signal transduction (Takei et al., 2001; Sakakibara, 2006), 3) the regulation of leaf senescence (Gan and Amasino, 1995; Wingler et al., 1998), 4) the involvement of vegetative- to-reproductive phase transitions (Corbesier et al., 2003), and 5) concerns pertaining to the development of reproductive organs (Bartrina et al., 2011). In Arabidopsis, increased levels of cytokinins in leaf tissues, and shoot apical meristem, have been detected GXULQJ HDUO\ SKDVH RI ÀRZHU WUDQVLWLRQ &RUEHVLHU et al., 2003). Exogenous application of cytokinins has been reported to induce ÀRZHU LQLWLDWLRQ DQG LQFUHDVH ÀRZHU QXPEHUV LQ VHYHUDO SODQW species. For example, in lychee (Litchi chinensis) dormant buds KDYH EHHQ VWLPXODWHG WR SURPRWH ÀRZHU EXG LQLWLDWLRQ DIWHU being treated with kinetin (Chen, 1991). In Protea cv. Carnival, the application of N-6-benzyladenine (BA) can also promote

RII VHDVRQ Ă€RZHULQJ +RIIPDQ et al., 2009). Furthermore, the application of BA has been found to increase a female-to-male Ă€RUDO UDWLR RI MDWURSKD UHVXOWLQJ LQ DQ LQFUHDVH LQ IUXLW DQG WRWDO seed yields (Pan and Xu, 2011). In parallel, exogenous BA application has been found to enhance Ă€RZHU EXGGLQJ LQLWLDWLRQ DQG LQFUHDVHG VSLNLQJ SHUFHQWDJHV in several orchid species, such as the genera of: Phalaenopsis, Doritaenopsis and Dendrobium (Blanchard and Runkle, 2008; Nambiar et al., 2012; Wu and Chang, 2012). Increasing the spike percentages (from 58 to 98 %) has been reported in Phalaenopsis Luchia Pink ‘244’, treated with 70 mg L-1 BA, on days 1 and 14, after the plants were incubated at low-temperatures (26/18oC). +RZHYHU VRPH GHIRUPHG LQĂ€RUHVFHQFH ZDV GHWHFWHG :X DQG Chang, 2009). In Dendrobium Angel White, the application of 200 mg L-1 %$ SURPRWHG HDUO\ Ă€RZHULQJ LQFUHDVHG VSLNLQJ SHUFHQWDJHV DQG LPSURYHG Ă€RZHU TXDOLW\ LQ H[WHQGLQJ OHQJWK RI LQĂ€RUHVFHQFH DQG WKH QXPEHU RI Ă€RZHUV SHU LQĂ€RUHVFHQFH (Nambiar et al., 2012). In addition, BA plays a role in promoting in vitro Ă€RZHULQJ RI D. Madame Thong-In, D. Chao Praya Smile and D. Sonia17 (Sim et al., 2007; Hee et al., 2007; Tee et al., 2008). 1RQ %$ F\WRNLQLQV DOVR SOD\ UROH LQ WKH UHJXODWLRQ RI Ă€RZHU development in orchids. Application of 2-iso-pentenyl adenine (2iP) (150 mg L-1) and kinetin (300 mg L-1) to whole plants, grown under low temperatures (26 oC/18 o& LQFUHDVHG Ă€RZHU GLDPHWHUV of Phalaenopsis Sogo Yudian ‘V3’. Furthermore, kinetin (200 mg L-1 DOVR LQFUHDVHG WKH QXPEHU RI LQĂ€RUHVFHQFH SHU SODQW RI WKH Phalaenopsis Sogo Yudian ‘V3’ (Wu and Chang, 2009). CPPU

Effects of pre-harvested N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU) spraying on Dendrobium (Forchlorfenuron, (N-(2-chloro-4-pyridinyl)-N’-phenylurea) is a synthetic cytokinin. Only low concentrations of exogenous CPPU applications can affect the growth of plants, such as: CPPU promoted fruit sets of Japanese persimmon, muskmelon fruit (Sugiyama and Yamaki, 1995; Hayata et al., 2000), and enlarge the sizes of fruit in kiwifruit, grapes and Japanese persimmon (Antognozzi et al., 1993; Zabadal and Bukovac, 2006; Sugiyama and Yamaki, 1995). Although CPPU is widely used in fruit tree SURGXFWLRQ LWV HIIHFWV XSRQ WKH SURPRWLRQ RI FXW Ă€RZHU TXDOLW\ KDYH QHYHU EHHQ UHSRUWHG VSHFLÂżFDOO\ IRU RUFKLGV 7KHUHIRUH WKH objective of this study was to investigate the effects of CPPU XSRQ LQĂ€RUHVFHQFH DQG Ă€RZHU GHYHORSPHQW RI FRPPHUFLDOO\ FXW orchids, namely D. Sonia ‘Earsakul’, in contrast to widely-used BA as cytokinin.

Materials and Methods Two year old plants of Dendrobium Sonia ‘Earsakul’ were used as plant materials in this study. The plants were grown in coconut husks under 70% shade, watered once a day, and fertilized once a week. This is the general practice for orchid commercial production at the ‘Jittrakarn orchids farm’ in Karnchanaburi SURYLQFH 7KDLODQG 7HUPLQDO Ă€RZHU EXGV RI D. Sonia ‘Earsakul’ are generally developed from new developing current pseudobulbs (frontal bulbs), when they grow to approximately 50-60 cm in height. In this experiment, two synthetic cytokinins: BA, at 100, 200 or 400 mg L-1, and CPPU, at 1, 5 or 10 mg L-1, were foliarly sprayed to the current pseudobulbs (45-50 cm in height), exactly EHIRUH Ă€RZHU LQLWLDWLRQ VWDJH (DFK SODQW ZDV JLYHQ DQ DSSOLFDWLRQ of 70 mL BA or CPPU liquid solutions mixed with surfactant solution. The BA or CPPU applications were reapplied fortnight intervals for three times. The experiment was randomized in ÂżIWHHQ UHSOLFDWLRQV ZLWK VHYHQ WUHDWPHQWV $ ÂľOHDVW VLJQLÂżFDQFH difference’ (LSD) test was used to compare the treatment effects. Thereafter, the BA and CPPU applied D. 6RQLD Âľ(DUVDNXOÂś Ă€RZHUV ZHUH KDUYHVWHG ZKHQ HDFK LQĂ€RUHVFHQFH KDG IRXU IXOO\ RSHQHG Ă€RZHUV 'DWD FROOHFWLRQ LQFOXGHG JURZWK DQG \LHOG SDUDPHWHUV the number of inflorescence per pseudobulb, lengths and GLDPHWHUV RI LQĂ€RUHVFHQFH QXPEHUV RI Ă€RZHUV SHU LQĂ€RUHVFHQFH DQG WKH IUHVK DQG GU\ ZHLJKWV RI FRPSOHWH LQĂ€RUHVFHQFH ,Q DGGLWLRQ WKH SHUFHQWDJHV RI LQĂ€RUHVFHQFH REWDLQHG IRU DW OHDVW RQH DEQRUPDO Ă€RZHU DQG WKH QXPEHUV RI GD\V WDNHQ DIWHU %$ or CPPU applications until harvesting, were also recorded. The experiment was carried out between January and April 2012.

141

5HVXOWV Inflorescence development of D. Sonia ‘Earsakul’: Application of 10 mg L-1 CPPU to D. Sonia ‘Earsakul’ before flower bud initiation resulted in significantly increased inflorescence numbers per pseudobulb, and the number of LQĂ€RUHVFHQFH LQFUHDVHG IURP WR LQĂ€RUHVFHQFH SHU EXOE However, BA concentration (100, 200 or 400 mg L-1) had less HIIHFW XSRQ WKH LQĂ€RUHVFHQFH QXPEHU 7DEOH ,QWHUHVWLQJO\ CPPU applications (1, 5 or 10 mg L-1) encouraged and enhanced the inflorescence length which increased from 49.4 cm to 52.6-55.1 cm, whilst the BA application, at 100, 200 or 400 mg L-1 KDG QR HIIHFW XSRQ WKH OHQJWK RI LQĂ€RUHVFHQFH 7DEOH 1). Furthermore, the 10 mg L-1 CPPU application promoted the highest inflorescence diameters, and they significantly increased from 0.57 to 0.66 cm (Table 1). All BA treatments (100, 200 or 400 mg L-1 SURGXFHG KLJK DPRXQWV RI Ă€RZHUV SHU LQĂ€RUHVFHQFH DQG WKHUH ZHUH IURP WR Ă€RZHUV SHU LQĂ€RUHVFHQFH ZKLOH WKH QRQ WUHDWHG FRQWURO SURGXFHG RQO\ Ă€RZHUV $OWKRXJK WKH %$ WUHDWPHQWV SURPRWHG QXPEHU RI Ă€RZHU SHU LQĂ€RUHVFHQFH WKH VL]H RI WKH Ă€RZHUV ZDV QRW VLJQLÂżFDQWO\ LQĂ€XHQFHG ,Q FRQWUDVW WKH DSSOLFDWLRQ RI PJ /-1 CPPU not RQO\ LQFUHDVHG QXPEHU RI Ă€RZHU EXW DOVR HQODUJHG Ă€RZHU VL]H Flower width, with 10 mg L-1 &338 WUHDWPHQW VLJQLÂżFDQWO\ increased from 6.9 cm to 7.3 cm, compared to non-treated control samples (Table 1). )UHVK DQG GU\ ZHLJKWV RI LQĂ€RUHVFHQFH Fresh weights of the LQĂ€RUHVFHQFH VLJQLÂżFDQWO\ LQFUHDVHG IURP J WR g after the pseudobulbs were treated with 200-400 mg L-1 BA, or 5-10 mg L-1 CPPU, respectively (Fig. 1). In addition, the PD[LPXP GU\ ZHLJKW RI VLQJOH LQĂ€RUHVFHQFH ZDV REWDLQHG IURP pseudobulbs treated with 10 mg L-1 &338 7KH LQĂ€RUHVFHQFH dry weight increased from 2.8 to 3.4 g compared to non-treated control (Fig. 2). 3HUFHQWDJHV RI LQĂ€RUHVFHQFH REWDLQHG IURP GHIRUPHG Ă€RZHUV Foliar applications of BA concentrations (100, 200 and 400 mg L-1 IRU WKUHH WLPHV FDXVHG GHIRUPDWLRQ RI Ă€RZHU PRUSKRORJ\ ,Q DEQRUPDO Ă€RZHUV WKHUH ZHUH DGGLWLRQDO OLS OLNH RUJDQV SUHVHQW in the basal parts of columns (a fusion unit of orchids; male and IHPDOH UHSURGXFWLYH RUJDQV RU DW WKH SRVLWLRQ RI Ă€RZHU SROOHQ FDSV )LJ 7KH KLJKHVW SHUFHQWDJH RI LQĂ€RUHVFHQFH REWDLQHG LQ VXFK GHIRUPHG Ă€RZHUV ZDV IRXQG LQ WKH PJ /-1 BA treatment (33.3%), followed by the 200 mg L-1 BA treatment (26.7%) )LJ +RZHYHU WKHUH ZHUH QR GHIRUPHG Ă€RZHUV LQ DOO &338 treatments, and non-treated control specimen (Fig. 2).

Table 1. Effects of different BA or CPPU concentrations on flower characteristics of Dendrobium Sonia ‘Earsakul’ Cytokinins ,QĂ€RUHVFHQFH Flowers Treatment Concentration Number per Length Diameter Number per (mg L-1) pseudobulb (cm) (cm) LQĂ€RUHVFHQFH Control 0 1.1Âą0.1c 49.4Âą1.4b 0.57Âą0.02b 12.2Âą0.3c c b b BA 100 1.0Âą0.0 49.0Âą0.9 0.59Âą0.03 14.1Âą0.5a bc b ab 200 1.2Âą0.1 49.1Âą1.3 0.61Âą0.02 14.0Âą0.7a 400 1.3Âą0.1abc 49.1Âą1.0b 0.62Âą0.01ab 14.8Âą0.5a CPPU 1 1.3Âą0.1abc 52.6Âą0.7a 0.60Âą0.02ab 12.4Âą0.2c 5 1.4Âą0.2ab 53.1Âą0.8a 0.62Âą0.02ab 12.7Âą0.4bc a a a 10 1.7Âą0.1 55.1Âą1.2 0.66Âą0.02 13.8Âą0.2ab Values are meansÂą standard error. Mean separation within columns by least significant difference test at 3” 0.05

Width (cm) 6.9Âą0.1b 6.9Âą0.1b 6.8Âą0.1b 6.8Âą0.2b 7.0Âą0.1ab 7.0Âą0.1ab 7.3Âą0.1a

142

Effects of pre-harvested N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU) spraying on Dendrobium

Fig. 2. Percentage of inflorescence withdeformed flowers of the BA treatments. Values are meansÂą standard error. There were no deformed flowers in all CPPU treatments, and non-treated control.

Fig.1. Fresh weight (A) and dry weight (B) of inflorescence of D. Sonia ‘Earsakul’ after applying different BA or CPPU concentrations. Values are meansÂą standard error. Different letters indicate that the values are significantly different at the P<0.05 level.

'D\V WDNHQ IURP F\WRNLQLQ DSSOLFDWLRQ EHIRUH Ă€RZHU EXG initiation to harvesting of D. Sonia ‘Earsakul’: In commercial SURGXFWLRQ LQĂ€RUHVFHQFH RI D. Sonia ‘Earsakul’ are harvested ZKHQ REWDLQLQJ RSHQ Ă€RZHUV SHU LQĂ€RUHVFHQFH ,W ZDV IRXQG that the applications of different BA or CPPU concentrations had less effect upon harvesting time. It took 83.1 to 85.1 days DIWHU %$ RU &338 DSSOLFDWLRQ WR KDUYHVW Ă€RZHUV DQG WKHUH ZDV QR VLJQLÂżFDQW FKDQJH DV FRPSDUHG WR WKH QRQ WUHDWHG FRQWURO samples, which took 85.7 days to harvest (Fig. 3).

'LVFXVVLRQ In this study, we applied cytokinin-like compound CPPU to LPSURYH WKH FXW Ă€RZHU TXDOLW\ RI D. Sonia ‘Earsakul’ for the ÂżUVW WLPH LQ FRQWUDVW WR ZLGHO\ XVHG %$ 7KH UHVXOWV VKRZHG WKDW exogenous applications of CPPU (especially at 10 mg L-1) to the FXUUHQW SVHXGREXOEV EHIRUH Ă€RZHU EXG LQLWLDWLRQ VLJQLÂżFDQWO\ HQKDQFHG WKH QXPEHUV RI LQĂ€RUHVFHQFH SHU SODQW Ă€RZHU FRXQWV EDVHG XSRQ LQĂ€RUHVFHQFH DQG WKH OHQJWK RI LQĂ€RUHVFHQFH DQG VL]HV RI Ă€RZHU LQ Dendrobium orchids. Increasing numbers of LQĂ€RUHVFHQFH SHU SODQW PD\ EH D UHVXOW RI WKH UROHV RI F\WRNLQLQ LQ SURPRWLQJ FHOO GLIIHUHQWLDWLRQ GXULQJ WKH GHYHORSPHQW RI Ă€RUDO

Fig. 3. Days taken from BA or CPPU applications before flower bud initiation to harvesting of D. Sonia ‘Earsakul’. Values are meansÂą standard error. ns indicates not significant.

primodia. We found that pseudobulbs which had 10 mg L-1 CPPU WUHDWPHQW PRVWO\ SURGXFHG GRXEOH LQĂ€RUHVFHQFHV DW WKH WHUPLQDO ZKLOH RWKHU WUHDWPHQWV JHQHUDOO\ SURGXFHG D VLQJOH LQĂ€RUHVFHQFH (IIHFWV RI %$ DSSOLFDWLRQ LQ RUGHU WR SURPRWH Ă€RZHU PHULVWHP DFWLYLW\ DQG LQFUHDVHG QXPEHUV RI Ă€RZHU EXG LQLWLDWLRQ KDYH EHHQ previously reported in other orchid species: Dendrobium Jaquelyn Thomas ‘Uniwai Pricess’ (Sakai et al., 2000), Dendrobium Angel White (Nambiar et al., 2012), Doritaenopsis and Phalaenopsis orchids (Blanchard and Runkle, 2008). In this study, we found that %$ DSSOLFDWLRQ FRXOG SURPRWH KLJK QXPEHUV RI Ă€RZHU FRXQWV RI LQĂ€RUHVFHQFH \HW KDG IHZHU SURPRWLRQDO HIIHFWV XSRQ VWLPXODWLQJ QXPEHUV RI LQĂ€RUHVFHQFH SHU SODQW OHQJWK RI LQĂ€RUHVFHQFH DQG VL]H RI Ă€RZHU ,QFUHDVHG QXPEHUV RI Ă€RZHU FRXQW LQ LQĂ€RUHVFHQFH ZHUH IRXQG LQ both BA (100-400 mg L-1) and CPPU (10 mg L-1) treatments. In the Arabidopsis FN[ FN[ GRXEOH PXWDQW LQĂ€RUHVFHQFH FDUULHG KLJKHU QXPEHUV RI Ă€RZHUV WKDQ WKH ZLOG W\SH EHFDXVH WKH SODQWV KDG JHQHUDWHG KLJKHU QXPEHUV RI LQĂ€RUHVFHQFH PHULVWHPDWLF FHOOV GXULQJ Ă€RZHU GHYHORSPHQW 7KH FRQWHQW RI ELRORJLFDOO\ DFWLYH and inactive trans-zeatin-type cytokinins in this mutant was also

Effects of pre-harvested N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU) spraying on Dendrobium

143

Fig. 4. Flower morphology of D. Sonia ‘Earsakul’ treated with BA 0, 200 and 400 mg L-1 in Figure A, B and C, respectively. Normal flowers have only a lip (A). In abnormal flowers several additional lips are generated from the basal part of orchid columns (B, C). Scale = 1 cm.

dramatically higher than the wild-type (Bartrina et al., 2011). Therefore, there is a relationship between increased endogenous cytokinin levels and the production of higher numbers of inflorescence meristematic cells. The exogenous cytokinin application has been reported to promote increased endogenous cytokinin levels (Letham, 1994; Blanchard and Runkle, 2008). Length of inflorescence is a main criteria for the grading of cut orchids. CPPU at 10 mg L-1 had a dramatic effect upon H[WHQGLQJ WKH OHQJWK RI LQĂ€RUHVFHQFH LQ ZKLFK WKH &338 WUHDWHG LQĂ€RUHVFHQFH FRXOG EH FODVVLÂżHG DV H[WUD ORQJ JUDGHG Ă€RZHUV (more than 55 cm in length). The application of appropriate BA concentrations (150 or 200 mg L-1) was previously reported to LQFUHDVH WKH OHQJWK RI LQĂ€RUHVFHQFH LQ D. Angel White, when WKH SODQWV ZHUH VSUD\HG ZHHNO\ LQ WKH ÂżUVW PRQWK DQG HYHU\ WZR weeks in the subsequent months for a total of six months (Nambiar et al., 2012). However, foliar applications of BA or CPPU in this study were only performed at fortnight intervals for three times. We found that the BA application had less promotional effects XSRQ H[WHQGLQJ OHQJWKV RI LQĂ€RUHVFHQFH 'LIIHUHQW UHVSRQVHV WR exogenous BA applications to Dendrobium species may be due to different durations and frequencies of applications, genetic backgrounds and stages of the plants. Although applications of BA at 100, 200 or 400 mg L-1 had increased numbers of flowers per inflorescence, the flower VKDSHV ZHUH GHIRUPHG 7KH GHIRUPHG Ă€RZHUV KDG DGGLWLRQDO OLSV generated from the basal part of the column, or pollen cap (Fig. $EQRUPDO Ă€RZHUV PD\ EH GXH WR KLJK GRVDJHV RI %$ F\WRNLQLQ DSSOLFDWLRQV 7KH GHYHORSPHQW RI DEQRUPDOO\ IRUPHG Ă€RZHUV GXH to high concentrations of BA applications was formerly found in D. Jaquelyn Thomas ‘Uniwai Pricess’ (Sakai et al., 2000) and in Phalaenopsis orchids (Wu and Chang, 2009). &\WRNLQLQV DOVR SOD\ D UROH LQ UHJXODWLQJ WKH VL]H RI Ă€RZHU RUJDQV (Bartrina et al., 2011). Increasing endogenous cytokinins causes SURORQJHG SHULRGV RI FHOO GLYLVLRQ GXULQJ Ă€RZHU GHYHORSPHQW DQG UHVXOWV LQ LQFUHDVHG VL]HV DQG FHOO QXPEHUV RI Ă€RZHU RUJDQV in the CKX3 CKX5 mutants of Arabidopsis (Bartrina et al., 2011). We hypothesize that the exogenous application of CPPU in this study may enhance endogenous cytokinin levels and lead WR HQODUJHG VL]HV RI Ă€RZHU 7KHUHIRUH HQGRJHQRXV F\WRNLQLQ OHYHOV DQG LQĂ€RUHVFHQFH PHULVWHP DFWLYLW\ DIWHU H[RJHQRXV &338 application should be studied further. The effect of cytokinin

CPPU has been proven to increase fruit size and fresh weight in several fruit crops: kiwifruit (Patterson et al., 1993), and berries (Zabadal and Bukovac, 2006). In this study, we found CPPU as potential compound for promoting fresh weight of Dendrobium LQĂ€RUHVFHQFH ZKLFK FDQ LQFUHDVH IUHVK ZHLJKW RI LQĂ€RUHVFHQFH XOWLPDWHO\ WKH WRWDO SURGXFW volume for sale.

Acknowledgements We thank ‘Jitrakarn orchid farm’ in supporting with plant materials and growing facilities for this study. This research ZDV ÂżQDQFLDOO\ VXSSRUWHG E\ WKH 1DWLRQDO 5HVHDUFK &RXQFLO RI Thailand (NRCT).

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Letham, D.S.,1994. Cytokinins as phytohormones-sites of biosynthesis,translocation, and function of translocated cytokinin. In: Cytokinins: Chemistry, Activity, and Function, D.M.S. Mok and M.C. Mok (eds.). CRC Press. p. 57-73. Nambiar, N., C.S.Tee and M. Maziah, 2012. Effect of 6-Benzylaminopurine RQ ÀRZHULQJ RI D Dendrobium orchid. Aus J. Crop Sci., 6(2): 225-231. 3DQ % = DQG = ) ;X %HQ]\ODGHQLQH WUHDWPHQW VLJQL¿FDQWO\ increases the seed yield of the biofuel plant Jatropha curcas. J. Plant Growth Regul., 30(2): 166-174. Patterson, K.J., K.A. Mason and K.S. Gould, 1993. Effects of CPPU (N-(2-chloro-4-pyridyl)-N’-phenylurea) on fruit growth, maturity, and storage quality of kiwifruit. New Zealand J. Crop Hort. Sci., 21: 253-261. Sakai, W.S., C. Adams and G. Braun, 2000. Pseudobulb injected growth regulators as aids for year around production of Hawaiian Dendrobium RUFKLG FXWÀRZHUV Acta Hort., 541: 215-220. Sakakibara, H. 2006. Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol., 57: 431-449. Sim, G.E., C.S. Loh and C.J. Goh, 2007. High frequency early in vitro ÀRZHULQJ RI Dendrobium Madame Thong –In (Orchidaceae). Plant Cell Rep., 26: 383-393. Sugiyama, N. and Y.T. Yamaki, 1995. Effects of CPPU on fruit set and fruit growth in Japanese persimmon. Scientia Hort., 60: 337-343. Takei, K., H. Sakakibara, M. Taniguchi and T. Sugiyama, 2001. Nitrogendependent accumulation of cytokinins in root and the translocation to leaf: implication of cytokinin species that induces gene expression of maize response regulator. Plant Cell Physiol., 42: 85-93.

Tee, C.S., M. Maziah and C.S. Tan, 2008. Induction of in vitro ÀRZHULQJ in the orchid Dendrobium Sonia 17. Biol. Plant., 52(4): 723-726. Wingler, A., A. von Schaewen, R.C. Leegood, P.J. Lea and W.P. Quick, 1998. Regulation of leaf senescence by cytokinin, sugars, and light. Effects on NADH-dependent hydroxypyruvate reductase. Plant Physiol., 116: 329-335. Wu, P.H. and D.C.N. Chang, 2009. The use of N-6-benzyladenine to UHJXODWH ÀRZHULQJ RI Phalaenopsis orchids. HortTechnology, 19: 200-203. :X 3 + DQG ' & 1 &KDQJ &\WRNLQLQ WUHDWPHQW DQG ÀRZHU quality in Phalaenopsis orchids: Comparing N-6-benzyladenine, kinetin and 2-isopentenyl adenine. Afr. J. Biotechnol., 11: 1592-1596. Zhang, K., L. Diederich and C.L.P. John, 2005. The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be VDWLV¿HG E\ \HDVW &GF SURWHLQ W\URVLQH SKRVSKDWDVH LPSOLFDWLRQV for mechanisms of cytokinin response and plant development. Plant Physiol., 137: 308-316. Zabadal, T.J. and M.J. Bukovac, 2006. Effect of CPPU on fruit development of selected seedless and seeded grape cultivars. Hort. Sci., 41: 154-157. Submitted: November, 2014; Revised: March, 2015; Accepted: March, 2015

Journal

Journal of Applied Horticulture, 17(2): 145-150, 2015

Appl

Production, quality and aroma analysis of sapodilla (Manilkara achras (Mill) Fosb.) wine K. Ranjitha1*, C.K. Narayana1, T.K. Roy2 and A.P. John1 Division of Post Harvest Technology, 2Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Hessaraghatta Lake P.O., Bangalore-560089, India. * E-mail: ranjitha@iihr.ernet.in

1

Abstract Process was standardized for preparation of fermented beverage from sapodilla (Manilkara achras (Mill) Foseberg). The starter culture using yeast strain Saccharomyces cerevisiae UCD 522 fermented juice from two sapodilla varieties viz., Cricket Ball and Oval, to obtain wines with 10.1-11.2 % alcohol, 0.44- 0.58 % acidity, 3.6-3.9 pH, 0.26-0.28 % residual sugar, 300-645 mg/L phenolics and <0.09 % volatile acidity in six to nine days at 18 °C. Retention of peel while pulping improved the phenolics level; but reduced the VHQVRU\ TXDOLW\ RI ZLQH %HQWRQLWH GRVDJH DQG SHULRG UHTXLUHG IRU FODULÂżFDWLRQ ZDV RSWLPL]HG DV IRU GD\V DQG IRU 21 days for production of wine from peeled fruits of Cricket Ball and Oval varieties, respectively. Sensory evaluation of dry, sweet, DQG Ă€DYRUHG ZLQHV UHYHDOHG WKH SRWHQWLDO PDUNHW DFFHSWDELOLW\ RI WKH ZLQHV +HDG VSDFH YRODWLOH DQDO\VLV VKRZHG WKH SUHVHQFH RI QHZ RGRURXV FRPSRXQGV OLNH HVWHUV DQG VKRUW FKDLQ IDWW\ DFLGV GXULQJ YLQLÂżFDWLRQ RI VDSRGLOOD MXLFH 0HWKR[\ FRPSRXQGV DQG FDUERQ\O IUDFWLRQV ZHUH OHVV LQ WKH ÂżQLVKHG ZLQH FRPSDUHG WR QDWXUDO MXLFH Key words 6DSRGLOOD ZLQH \HDVW SKHQROLFV ZLQH FODULÂżFDWLRQ KHDG VSDFH YRODWLOHV

,QWURGXFWLRQ Sapodilla (Manilkara achras (Mill) Foseberg), also known as sapota, is a popular dessert fruit in tropical countries like Brazil, Guatemala and India with the centre of origin being in Mexico. The rapid increase in cultivation of this fruit crop is mainly due to its adaptability to wide range of conditions, low cost of production and high economic returns. Being climacteric, ripe fruits have shelf life ranging from 5-7 days and require quick disposal (Salunkhe and Desai, 1984). Ripe fruits of sapota are good source of digestible sugars (12-18%) and also contain 0.5 per cent SURWHLQ IDW FDUERK\GUDWHV ÂżEHU DQG DVK They are also rich source of minerals like calcium, phosphorus and iron. The Cricket Ball and Oval varieties contain several amino acids like alanine, aspartic acid, glutamine, glutamic acid, lysine, phenylalanine and threonine. Additionally Cricket Ball contains glycine, isoleucine, proline, citrulline, D-aminobutyric acid and Oval contain arginine and valine (Gopalan et al., 1981). Besides table purpose, ripe sapota fruits are also used for making value added products like intermediate moisture foods, beverages and bakery products (Relekar and Naik, 2012). Being sugar-rich, the fruit can be converted to fermented product like sapodilla wine. Impressive progress has been made in development of technologies for preparation of wines using fruits like mango, apple, pear, plum, pineapple, cashew-apple, banana, ber, strawberry, litchi etc. (Joshi and Attri, 2005). However, research work carried out on standardization of a suitable methodology for sapodilla wine is very limited and earlier work mainly focused RQ LQĂ€XHQFH RI IUXLW PDWXULW\ DQG SHFWLQDVH HQ]\PH RQ VDSRWD juice fermentation at room temperature (Pawar, 2009). Present paper describes the result of experiments on evaluation of popular sapodilla varieties for wine making, effect of fruit peel removal RQ ZLQH TXDOLW\ RSWLPL]DWLRQ RI FODULÂżFDWLRQ DJHQW SUHSDUDWLRQ

of diverse styles of sapodilla wines, and analysis of head space volatiles of sapodilla juice and dry wine.

Materials and methods Preprocessing operations: Two commercial varieties of sapodilla fruits viz., Oval and Cricket Ball grown in India were obtained from the orchards of Indian Institute of Horticulture Research, Bangalore. The mature fruits were harvested, washed, surface dried and stored in open plastic trays lined with paper till they reached optimum ripe stage. Fully ripe sapodilla fruits were subjected to pre-processing operations like washing, separation of seeds and milky latex portion and pulping. Pulping of peeled and unpeeled fruits were done in different experiments for checking the effect of peel retention on the quality of wine. Pulp was OLTXHÂżHG XVLQJ D FRPPHUFLDO JUDGH HQ]\PH 3HFWLQDVH &&0 3OXV (Biocon India Ltd) by adding at the rate of 5 mL/kg pulp followed by incubation at of 30 °C for 3 h. Juice was extracted by pressing this pulp in a muslin-cloth bag at the end of incubation period. Chemical composition of pulp and juice: The composition and quality parameters like total soluble solids, acidity, pH, total phenolics and ash were determined in all treatments as per the standard procedures (Ranganna, 1986) Fermentation of sapodilla juice: The expelled sapodilla juice was ameliorated by adjusting total soluble solids (T.S.S.) 22° Brix, and titratable acidity to 0.5 per cent by the addition of calculated amounts of cane sugar and tartaric acid. Dibasic ammonium phosphate (0.2g/L) was added as nitrogenous food for the yeast. Potassium metabisulphite (KMS) was added @ SSP IRU LQKLELWLQJ WKH JURZWK RI QDWXUDO PLFURĂ€RUD DQG facilitating the growth of pure yeast culture as well as to prevent browning reactions during fermentation and storage. Log phase culture of the yeast Saccharomyces cereviciae UCD 522 (2%

146

Production, quality and aroma analysis of sapodilla wine

v/v) with a population of 1012 cells/mL was inoculated to the ameliorated sapodilla juice and mixed well. The inoculated juice was fermented in BOD incubator at 18 °C till T.S.S. reduced to 0 o B. Total soluble solids (T.S.S.) was measured at regular intervals using Brix hydrometer. The completely fermented clear juice samples were separated from the sediment by siphoning, and were stored at 10Âą2 o& 7KH ERWWOHG ZLQHV ZHUH FODULÂżHG E\ DGGLWLRQ RI bentonite (0.04 - 0.08%), clear wine samples were further racked, bottled and plugged with cork stoppers. Analysis of wine: The biochemical parameters like pH, titratable acidity, volatile acidity, alcohol, phenolics and residual sugar were measured in the sapodilla wine as per the standard methods (Amerine and Ough, 1982). The clarity of the sapodilla wine was monitored by measuring the absorbance at 600 nm in UV-VIS spectrophotometer (Optima 300 plus). Preparation of different types of sapodilla wine: 7KH FODULÂżHG EDVH ZLQH REWDLQHG E\ IHUPHQWDWLRQ ZDV IRUWLÂżHG WR FRQWDLQ YDULHG Ă€DYRXU DQG VZHHWQHVV 7KH Ă€DYRXUHG ZLQH YHUPRXWK W\SH ZDV prepared by adding coarsely ground mixture of spices in the FODULÂżHG ZLQH 7KHVH ERWWOHV ZHUH NHSW DW “ ƒ& IRU ZHHNV DQG WKH VSLFHV ZHUH UHPRYHG E\ ÂżOWUDWLRQ 7KH VSLFHV XVHG IRU WKH SUHSDUDWLRQ RI Ă€DYRXUHG ZLQH ZHUH Âą DMZDLQ J / FLQQDPRQ (0.5 g/L), clove (0.3 g/L), coriander seeds (1 g/L), cardamom (0.20 g/L), nutmeg (0.50 g/L), cumin (0.20 g/L), anise (0.30 g/L), dried ginger (0.25 g/L), benzoin (0.05 g/L), black pepper (0.10 g/L), fenugreek seeds (0.10 g/L). Sweet wine was prepared by GLVVROYLQJ ÂżYH SHU FHQW VXFURVH LQ EDVH ZLQH Sensory evaluation of wine samples: Sensory appeal of the wines were judged on a nine point Hedonic scale (1=Dislike extremely, 2= Dislike moderately, 3= Dislike, 4=Dislike slightly, 5= neither like nor dislike, 6 = like slightly, 7= like moderately, 8= like much, 9= like extremely) Analysis of head space volatiles: The head space volatiles of the juice and dry wine from peeled fruits of sapodilla variety Cricket Ball was analyzed by SPME (GC-FID and GC-MS) method. Extraction process for head space volatiles of sapodilla fruit and wine was carried out as per the method suggested by Vermeir et al. (2009). Twenty milliliters of juice and wine sample were transferred to screw cap vials with silicon rubber septum and magnetic stirrer, to which sodium chloride was added. The SPME device (Supelco Inc. Bellefonte, PA, USA) FRDWHG ZLWK '9% &$5 3'06 Č?P KLJKO\ FURVV OLQNHG ÂżEHU ZDV FRQGLWLRQHG E\ LQVHUWLQJ LW LQWR WKH *& LQMHFWRU SRUW DW ƒ& IRU KUV 7KH FRQGLWLRQHG ÂżEHU ZDV WKHQ LQVHUWHG into the headspace under magnetic stirring for 90 min at 37 °C. Subsequently, the SPME device was introduced in the injector port for chromatographic analysis and was retained in the inlet for 5 min. The GC-FID analysis was performed on a Varian-3800 JDV FKURPDWRJUDSK V\VWHP ÂżWWHG ZLWK WKH '% FROXPQ KDYLQJ P ; PP ,' ZLWK Č?P ÂżOP WKLFNQHVV 7KH GHWHFWRU and injector temperature was 270 and 260 °C, respectively and the temperature of the column was raised from 50 °C to 200 °C with an increment of 3 °C /min with a holding time for 3 minutes, followed by a rise of 10 °C/min to 220 °C and maintaining the constant temperature for 8 minutes. The carrier gas was helium at D P/ PLQ Ă€RZ DQG VSOLW )RU WKH TXDOLWDWLYH LGHQWLÂżFDWLRQ of volatile substances and comparative variation of retention time

and index, the standard compounds viz., ethyl acetate, propanol, isobutanol, butanol, amyl alcohols, isoamyl acetate, pentanol, hexanol, 1-octene-3-ol, eugenol were co-chromatographed. 9RODWLOH FRPSRXQGV ZHUH LGHQWLÂżHG ZLWK D LRQ WUDS 9DULDQ GC-MS/MS mass selective detector using VF-5MS, 30 m X PP ,' ZLWK Č?P ÂżOP WKLFNQHVV FROXPQ 7KH PDVV spectrometer was operated in the external electron ionisation mode. The carrier gas used was helium @ 1 mL/min. The injector temperature was 250 °C; trap temperature was 220 °C, and the ion source-heating at 230 °C, transfer line temperature 250 °C, EI-mode was 70 eV, and the full scan-range 50-450 amu. The column temperature was programmed same as described above. The total volatile production was estimated by the sum of all GCFID peak areas in the chromatogram and individual compounds ZHUH TXDQWLÂżHG DV UHODWLYH SHUFHQW DUHD 9RODWLOH FRPSRXQGV ZHUH LGHQWLÂżHG E\ FRPSDULQJ WKH UHWHQWLRQ LQGH[ ZKLFK ZDV GHWHUPLQHG by using homologous series of n-alkanes (C5 to C32) as standard (Kovats, 1965) and comparing the spectra available with two spectral libraries using Wiley and NIST-2007.

5HVXOWV DQG GLVFXVVLRQ Rate of alcoholic fermentation and quality of wine is mainly LQĂ€XHQFHG E\ WKH FKHPLFDO FRPSRVLWLRQ RI MXLFH IHUPHQWHG 7KH carbohydrates, mainly sugars, act as substrates for fermentation by yeast for production of alcohol and carbon dioxide. Very high quantity of sugars leave higher proportion of non alcoholic residues in wine (Amerine and Ough, 1982). In the present study, juice obtained from two popular sapodilla varieties viz. Cricket Ball and Oval which were used for wine making were analyzed for the essential characteristics for wine making (Table 1). T.S.S. ranged from 19.8-24 °B, titratable acidity of the juice of Oval and Cricket Ball were 0.36 to 0.42 per cent, with pH ranging from 4.7 to 5.3. The ash content of pulp and juice of peeled Oval and Cricket Ball varieties ranged between 0.47 to 0.55 per cent. The amount of total phenolics was highest in juice of unpeeled Oval fruit (720 mg/L) and lowest in peeled Cricket Ball (496 mg/L). Sapodilla, commonly used as a dessert fruit is rich in sugars with a T.S.S. ranging from 21-28° Brix (Gopalan et al., 1981). Commercial standards suggest 0.5-0.6 per cent acidity and 3.5-4.0 pH in good quality grape wines (Amerine and Ough, 1982). Both the varieties recorded a lower acidity indicating the need of acidity amelioration which would also bring down the pH to optimum level. The fruit also possessed some quantity of SKHQROLFV ZKLFK FRXOG EULQJ LQ PLOG DVWULQJHQF\ WR WKH ÂżQLVKHG product on fermentation. Based on the above observations, it is suggested that sapodilla fruits are good raw materials for the production of wines. Effect of fruit peel removal on the quality of wine: Sapodilla peel is edible and has more nutritive value than pulp (Gopalan et al., 1981); and the use of unpeeled fruits for juicing can reduce the labor cost, thus lowering the cost of production. 0HFKDQL]DWLRQ RI SHHOLQJ LV GLIÂżFXOW GXH WR WKH VRIW WH[WXUH RI ripe fruits. Keeping this in view, juice extracted from peeled and unpeeled fruits were included in the study. Fermentation started faster in juice from peeled fruits of Cricket Ball variety and completed in six to seven days (Fig. 1). There was a delay in the initiation of fermentation in juice from Oval variety, and juice from unpeeled fruits. This observation suggests that Cricket Ball

Production, quality and aroma analysis of sapodilla wine

Based on these results, it is concluded that removal of skin prior to pulping is essential to get good quality wine from sapodilla fruits.

Peeled Oval

25

Unpeeled Oval

20

Unpeeled Cricket Ball Peeled Cricket Ball

o

Brix

15 10

5 0 0

2

4

6

8

147

10

Days

Fig. 1. Fermentation of juice from peeled and unpeeled sapodilla fruits

variety is suitable for fermentation as the risk of out growth of natural yeast is minimized and a faster completion of fermentation is achieved. The absence of fast initiation of fermentation results in stuck and sluggish fermentation due to the multiplication of WKH QDWXUDO ÀRUD %LVVRQ DQG %XW]NH 7KH FRPSRVLWLRQ and sensory score of wines made from the unpeeled and peeled fruit juice is given in Table 2. A marked increase in the phenolics, volatile acidity; as well as a low sensory score were noticed in wines from unpeeled fruits. High volatile acidity is a negative attribute of wine quality, and results mainly due to acetic acid formation. The probable reason of high volatile acidity would be the presence and subsequent metabolic activities of undesirable VXUIDFH PLFURÀRUD KDUERXUHG E\ WKH IUXLWV 7KH IUXLW VXUIDFHV DUH usually contaminated with undesirable acetic acid producing yeasts and bacteria and other spoilage micro organisms such as lactic acid bacteria and molds, which are acquired either from the ¿HOG RU GXULQJ KDUYHVWLQJ DQG IXUWKHU KDQGOLQJ RSHUDWLRQV )UD]LHU 8QGHVLUDEOH PLFUR ÀRUD DQG WKH LQKLELWRU\ FRPSRXQGV LQ the peel would have retarded the fermentation in starter culture.

6WDQGDUGLVDWLRQ RI FODUL¿FDWLRQ DJHQW GRVDJH The suspended particles originating from juice and yeast cause haziness in wine, which is an undesirable attribute. An attempt was made in the VWXG\ WR RSWLPL]H WKH GRVH RI FODUL¿FDWLRQ DJHQW EHQWRQLWH LQ sapodilla wine. Bentonite treatment did not alter the chemical composition of the base wine, but had a positive effect on color and clarity, which in turn could contribute to higher score for visual appearance and taste (Table 3 and 4). Bentonite dosage at WKH UDWH RI ZDV VXI¿FLHQW WR REWDLQ D FOHDU SURGXFW ZLWKLQ 14 days in Cricket Ball variety; while 0.08% and an incubation period of 21 days was required for obtaining clear wine in Oval variety. The observed difference in the clarification time is probably due to the difference in the pulp texture. Cricket Ball variety has coarse, gritty pulp and Oval is known to possess soft, mellow pulp. Bentonite is an inexpensive monmorrillanite FOD\ XVHG IRU JUDSH ZLQH FODUL¿FDWLRQ ,W FDQ UHPRYH KHDW VWDEOH proteins, unwanted metals like Cu, Fe, and Zn from the colloids in a dose dependant manner (Amerine et al., 1980). Composition and sensory quality of different types of sapodilla wine: Chemical composition of different types of sapodilla wine is presented in Table 5. Sweet wine possessed > 5% residual sugar, and the other types were fermented till dryness. The phenolic FRQWHQW LQ WKH ÀDYRXUHG ZLQH ZDV KLJK ZKLFK FRXOG EH GXH WR WKH H[WUDFWLRQ RI SKHQROLF FRPSRXQGV IURP WKH DGGHG ÀDYRXULQJ agents. Spices are rich in antioxidant compounds like vitamins, flavanoids, terpenoids, phytoestrogens etc (Suhaj, 2006). Different sapodilla wines were subjected to sensory evaluation to assess the acceptance among the consumers. The wines were golden yellow in color resembling white grape wine, possessed appealing aroma, and scored 6.6 to 7.9 for their overall acceptance on a nine point hedonic scale. Flavoured wines (vermouths) contain herbs and spices with medicinal properties and are more EHQH¿FLDO IRU KHDOWK 3DQHVDU et al., 2011). Ingredient herbs in the

Table 1. Proximate composition of juice from sapodilla varieties used for wine making Parameter TSS (°Brix) Total sugar (%) pH Titratable acidity (%) Phenolics (mg/L) Ash (%)

Cricket Ball Peeled 24.00Âą0.30 21.60Âą0.56 4.80Âą0.10 0.36Âą0.04 496Âą20 0.76Âą0.01

Oval Unpeeled 20.00Âą0.4 19.30Âą0.2 4.90Âą0.1 0.25Âą.03 670Âą24 0.89Âą0.1

Peeled 23.30Âą0.2 21.54Âą0.2 4.70Âą0.2 0.42Âą0.1 521Âą19 0.47Âą0.09

Unpeeled 19.80Âą0.3 17.41Âą0.29 4.70Âą0.2 0.21Âą0.05 720Âą25 0.55Âą0.06

Values given are mean of triplicates Âą standard deviation Table 2. Composition of wine prepared using juice obtained from peeled and unpeeled sapodilla fruits Parameters pH Total titratable acidity (%) Residual sugar (%) Total phenolics (mg/L) Alcohol (%) Volatile acidity (%) Sensory score

Cricket Ball Peeled 3.9Âą0.15 0.44Âą0.01 0.26Âą0.00 300Âą10.9 10.9Âą0.23 0.048Âą0.00 7.0Âą0.46

Values given are mean of triplicates Âą standard deviation

Oval Unpeeled 3.8Âą0.08 0.51Âą0.01 0.32Âą0.00 396Âą16.3 11.0Âą0.25 0.092Âą0.01 4.2Âą0.97

Peeled 3.6Âą0.00 0.57Âą0.02 0.28Âą0.00 402Âą19.3 10.1Âą0.50 0.036Âą0.00 6.7Âą0.31

Unpeeled 3.6Âą0.09 0.58Âą0.04 0.29Âą0.01 645Âą19.2 11.2Âą0.26 0.045Âą0.00 3.7Âą0.52

148

Production, quality and aroma analysis of sapodilla wine

Table 3. Effect of bentonite dosage on the clarity of sapodilla fruit wine Treatment Turbidity Visual appearance 14 days 21 days 14 days 21 days Cricket Ball Oval Cricket Ball Oval Cricket Ball Oval Cricket Ball Peeled, 0.08 % 0.022 0.063 0.015 0.061 + + ++ +++++++ Peeled, 0.04 % 0.025 0.106 0.020 0.100 ++++ ++-++++ Peeled, 0.02 % 0.042 0.106 0.035 0.101 ++-++-+++Peeled, 0% 0.044 0.124 0.043 0.120 ++-++-++-Unpeeled, 0.08% 0.065 0.058 0.064 0.052 ++++ +++++++ Unpeeled, 0.04 % 0.070 0.065 0.065 0.061 ++++ ++-++++ Unpeeled, 0.02 % 0.090 0.069 0.072 0.063 +++++-+++Unpeeled, 0% 0.200 0.085 0.198 0.080 +++++-+++++-- = slightly clear, +++- = moderately clear; ++++ = clear; * values given in Table are mean of triplicates Table 4. Effect of bentonite dosage on the composition of sapodilla fruit wine Treatment pH Acidity Residual sugar (%) (%)

Total phenolics (mg/L)

Alcohol (%)

Oval ++++ +++++-++-++++ +++++-++--

Volatile Acidity (%)

a

b

a

b

a

b

a

b

a

b

a

b

Peeled, 0.08 %

3.86

3.6

0.51

0.57

0.28

0.282

396

402

11.0

10.1

0.092

0.036

Peeled, 0.04 %

3.93

3.6

0.48

0.60

0.28

0.283

398

410

10.9

10.0

0.096

0.048

Peeled, 0.02 %

3.89

3.6

0.49

0.58

0.28

0.285

400

450

10.8

10.0

0.04

0.054

Peeled, 0%

3.93

3.6

0.51

0.61

0.28

0.290

400

460

10.7

9.9

0.014

0.054

Unpeeled, 0.08%

3.84

3.60

0.40

0.58

0.28

0.281

300

645

11.0

11.2

0.090

0.054

Unpeeled, 0.04 %

3.83

3.6

0.42

0.60

0.28

0.281

310

645

10.9

11.0

0.084

0.042

Unpeeled, 0.02 %

3.89

3.60

0.42

0.60

0.28

0.283

310

650

11.1

10.9

0.09

0.048

Unpeeled, 0%

3.90

3.6

0.43

0.60

0.28

0.290

315

651

10.7

10.9

0.09

0.054

The values given in the table are mean of triplicates a: Cricket Ball, b: Oval Table 5. Composition of dry and flavoured sapodilla fruit wine Treatment

pH Dry

Alcohol (%v/v)

Residual sugar (%)

Phenolics (mg/L)

Volatile acidity (% acetic acid)

Sensory score

3.92 Âą .0

0.46 Âą .02

10.9 Âą 0.02

0.28 Âą 0.01

300 Âą 13.0

0.048 Âą 0.00

7.0 Âą.0.54

3.81 Âą 0.1

0.51 Âą 0.01

11.6 Âą 0.41

5.60 Âą 0.61

312 Âą 5.6

0.043 Âą 0.00

8.0 Âą 0.23

3.9 Âą 0..02

0.44 Âą 0.04

11.7 Âą 0.17

0.26 Âą 0.06

408 Âą 3.9

0.048 Âą 0.00

7.7 Âą 0.45

Dry

3.6 Âą 0.05

0.60 Âą 0.02

10.1 Âą 0.23

0.30 Âą 0.10

402 Âą 5.7

0.036 Âą 0.00

6.3 Âą 0.64

Sweet

3.68 Âą 0.07

0.51 Âą 0.06

10.9 Âą 0.34

5.40 Âą 0.54

392 Âą 6.7

0.035 Âą 0.00

7.2 Âą 0.49

Flavoured

3.6 Âą 0.04

0.57 Âą 0.04

11.9 Âą .25

0.33 Âą 0.01

532 Âą 12.5

0.035 Âą 0.00

7.5 Âą 0.47

Cricket Ball Sweet Flavoured Oval

Total acidity (% tartaric acid)

Values given are mean of triplicates Âą standard deviation

study included antimicrobial spices like clove, antipyretics such as black pepper and ginger; and carminative agents like cumin DQG DQLVH ,W LV OLNHO\ WKDW ÀDYRXUHG VDSRGLOOD ZLQHV DOVR SRVVHVV some medicinal properties. Head space volatile composition of juice and dry wine from sapodilla var. Cricket Ball: Though the catabolism of hexoses into ethanol is the primary objective of fermentation, this process also results in production of several volatile compounds, produced as by-products of metabolic processes of yeast. Volatile composition changes considerably during the conversion of juice to wine, suggesting the formation of new compounds during the process (Schreier, 1979; Ebeler, 2001). Relative abundance of head space volatiles of juice and wine is given in the Table 6. The proportion of various functional compounds in the expelled juice followed the pattern esters > methoxy compounds > alcohols > aldehydes and ketones > acids > hydrocarbons; while in wine, the order of abundance was esters > acids > alcohols > methoxy compounds > aldehydes and ketones. Esters formed the major part

in head spaces of both juice and wine with a higher proportion in wine. Methyl salicylate, 1-propyl ethanoate, 1-butyl butanoate, ethyl hexanoate, vinyl benzoate, benzene propyl acetate and ethyl hexadecanoate were the most predominant esters in juice. Earlier reports on sapodilla aroma analysis carried out by MacLeod and 'H7UDFRQLV DOVR LGHQWLÂżHG PHWK\O VDOLF\ODWH DQG PHWK\O benzoate as major esters contributing to sapodilla aroma. Esters like ethyl hexadecanoate, ethyl dodecanoate, ethyl benzoate, methyl- 9 - octadecenoate, ethyl tetradecanoate, ethyl - cis, cis-9, 12-octadecadienoate were predominant in sapodilla wine head space. Several acidic compounds are known to be produced or PRGLÂżHG GXULQJ WKH FRXUVH RI \HDVW IHUPHQWDWLRQ 7KHVH LQFOXGH volatile fatty acids of up to 12 carbons, primarily hexanoic, decanoic, and octanoic acids (Schreier, 1979). In the present study also, high amount of dodecanoic acid, pentadecanoic acid, 9hexadecenoic acid and their esters were found in higher amounts in wine. Benzyl alcohol was the major hydroxyl compound in head space of sapodilla juice, and was reduced to negligible quantity in wine head space. Cis -methyl isoeugenol and benzene compounds

Production, quality and aroma analysis of sapodilla wine

Table 6. Relative abundance of head space volatile compounds present in juice and wine from sapodilla variety Cricket Ball Name of the compound R.I. Relative area % Vinyl benzoate 1150 Juice Wine Ethyl benzoate 1170 Methyl salicylate 1198 Hydrocarbons Ethyl octanoate 1238 o-Xylene 852 0.152 N.D Ethyl salicylate 1270 Į 3LQHQH 938 0.074 0.019 Į 7HUSLQ\O DFHWDWH 1352 Į 3KHOODQGUHQH 1010 0.087 N.D Butyl benzoate 1352 Į 7HUSLQHQH 1025 0.082 N.D Benzenepropyl acetate 1373 Limonene 1029 0.078 N.D Ethyl 9-decenoate 1389 Ocimene 1039 0.069 0.018 Ethyl hydrocinnamate 1390 Naphthalene 1182 0.721 N.D Isoamyl benzoate 1415 Azulene 1310 N.D 0.424 Isoamyl octanoate 1450 Į &RSDHQH 1368 0.159 0.114 Methyl dodecanoate 1531 Į &DU\RSK\OOHQH 1462 0.094 0.188 Ethyl dodecanoate 1597 Calamenene 1521 0.177 N.D Isopentyl decanoate 1646 Cadinene 1532 0.077 N.D Methyl 2-methyltetradecanoate 1715 Total 1.770 0.764 Methyl pentadecanoate 1784 Alcohols Ethyl tetradecanoate 1793 1-Hexen-3-ol 769 0.067 N.D Methyl (9Z)-9-hexadecenoate 1885 3, 4-Dimethyl-3-hexanol 845 N.D 0.164 Methyl hexadecanoate 1890 2-Ethyl-2-hexanol 910 N.D 0.189 Diisobutyl azelaate 1938 1, 2-Pentanediol 926 1.512 N.D Ethyl hexadecanoate 1975 Benzyl Alcohol 1021 7.676 0.351 Isopropyl hexadecanoate 2014 2-methyl butan-2-ol 1023 0.105 0.127 Methyl 9-octadecenoate 2081 (Z)-5-Octen-1-ol 1052 0.420 N.D Ethyl heptadecanoate 2098 Veratrol 1151 0.250 N.D Methyl octadecanoate 2128 Lavandulol 1155 0.221 N.D Ethyl cis, cis-9, 12-octadecadienoate 2155 (E)-6-Nonen-1-ol 1167 0.718 N.D Ethyl cis-9-octadecenoate 2180 4-Terpineol 1181 0.275 N.D Ethyl octadecanoate 2195 3-Isopropenyl-2-methylcyclohexanol 1209 0.337 N.D Methyl eicosa-7, 10, 13-trienoate 2300 Į 0HWK\OEHQ]HQHHWKDQRO 1212 N.D 0.668 Total Benzenepropanol 1221 N.D 0.994 Aldehydes and ketones Nerol 1228 0.101 N.D Isovaleraldehyde 632 cis, trans-Nerolidol 1564 N.D 0.100 (E)-2-Hexenal 851 cis-(+)-Nerolidol 1537 N.D 0.227 3-methyl butanal 929 (Z, E)-3, 13-Octadecadien-1-1ol 2078 N.D 0.227 (E, E)-2, 4-Heptadienal 1016 Total 11.680 3.047 2-Nonanone 1090 CarboxylicAcids Benzenepropanal 1160 Benzoic acid 1191 N.D 1.196 Pulegone 1176 Dodecanoic acid 1566 N.D 2.408 Benzaldehyde 1497 Tetradecanoic acid 1760 0.011 1.363 (Z)-9, 17-Octadecadienal 1997 Pentadecanoic acid 1806 N.D 2.598 Total 9-Hexadecenoic acid 1898 0.054 2.236 Methoxy compounds Hexadecanoic acid 1961 0.070 0.017 p-Methylanisole 1026 14-Ethoxy-14-oxotetradecanoic acid 2135 N.D 0.040 Anethole 1287 Total 0.135 9.858 Methyleugenol 1337 Esters Eugenol 1384 Methyl propenoate 618 N.D 1.313 5-Allyl-2-methoxyphenol 1411 1-propyl ethanoate 712 3.784 0.094 cis-Methyl isoeugenol 1432 Ethyl 3-methylpropionate 809 0.599 N.D 1-Methoxy-4-(4-methyl-4-pentenyl)benzene 1459 Isoamyl acetate 872 2.535 0.052 Methylisoeugenol 1490 Methyl (2E)-4-hydroxy-2-butenoate 942 0.258 0.021 3, 4, 5-Trimethoxyallylbenzene 1554 1-Butyl butanoate 975 6.571 0.000 Total Ethyl hexanoate 996 4.383 0.080 1, 3-Dichlorobenzene 1002 Ethyl 3-furoate 1002 N.D 0.007 5-Chloro-1H-indole 1368 Hexyl ethanoate 1018 0.718 N.D Others Ethyl 2-furoate 1047 N.D 0.013 Methyl benzoate 1092 N.D 0.339 R.I. : Retention index; N.D.: Not detected

149

8.555 1.550 9.892 N.D 1.212 0.120 1.011 4.796 N.D N.D N.D N.D N.D N.D N.D N.D N.D N.D N.D N.D N.D 2.554 N.D N.D N.D N.D N.D N.D N.D N.D 48.539

N.D 13.447 N.D 1.378 N.D N.D 0.057 0.464 0.109 3.051 0.070 0.175 1.239 10.913 0.126 1.514 1.018 4.687 2.548 0.215 1.480 16.929 0.029 6.738 0.270 0.117 3.377 0.574 1.531 0.011 73.986

0.024 0.191 0.062 0.151 0.228 3.929 0.119 0.386 N.D 5.090

N.D N.D 0.075 N.D N.D N.D 0.036 0.420 0.647 1.179

0.081 0.391 0.040 0.037 0.981 12.961 10.992 0.081 N.D 25.565 0.110 N.D 0.110

N.D 0.948 0.857 0.574 0.261 0.771 N.D N.D 0.147 3.558 N.D 0.136 0.136

150

Production, quality and aroma analysis of sapodilla wine

were earlier reported as important components of sapodilla fruits (Shivashankar et al., 2007). Benzene propanal was the major aldehyde in sapodilla juice, while it was undetectable in wine. Similar was the observation with respect to cis-methyl isoeugenol and 1-methoxy- 4-(4-4, methyl-4-pentenyl) benzene. The present observation on proportion of carbonyl compounds in sapodilla juice and wine supports the earlier reports on vulnerability of carbonyl compounds to losses during fermentation (Kotsteridis and Baumes, 2000). Present study suggested the feasibility of a laboratory scale process for the preparation of high quality fermented beverage from sapodilla fruit. Fermentation of juice from peeled fruits of variety Cricket Ball using the yeast Saccharomyces cerevisiae UCD 522 for six to seven days followed by 0.04 % bentonite treatment for 14 days resulted in sapodilla wine; while, oval variety needed longer duration for fermentation and higher bentonite dosage or incubation period. It was also found that pulping and juice extraction from unpeeled fruits is not desirable as it could result lower consumer acceptability. All the three types viz GU\ VZHHW DQG ÀDYRXUHG ZLQHV ZHUH IRXQG WR KDYH high sensory appeal. Head space volatiles of sapodilla wine was very much different from unfermented juice, mainly due to formation of more esters and short chain fatty acids, as well as disappearance of carbonyl, methoxy, and hydroxyl compounds during fermentation. Most of these compounds are known to be highly aromatic, supporting the distinctiveness of the sapodilla wine from the unfermented juice.

Acknowledgements The authors thank Dr. A.S. Sidhu, Director, IIHR, Bangalore for the facilities provided for research and Mr. C. Lokesh for technical assistance.

References Amerine, M.A., H.W. Berg, R.E. Kunkee, C.S. Ough, V.L. Singleton and A.D. Webb, 1980. The Technology of Wine Making. 4th edition, AVI Publishing Company, Westport. Amerine, M.A. and C.S. Ough, 1982. Handbook of Methods For Analysis of Musts and Wines. 1st Edition, Wiley – Interscience Publications, New York. %LVVRQ / ) DQG & ( %XW]NH 'LDJQRVLV DQG UHFWL¿FDWLRQ RI sluggish and stuck fermentations. Amer. J. Enol. Vitic., 51(2): 168177.

Ebeler, S.E. 2001. Analytical chemistry: Unlocking the secrets of wine ÀDYRU Fd. Rev. Intl., 17: 45-64. Fraizer, W.C. and D.C.Westhoff, 1995. Food Microbiology. Tata McGraw Hill Publishing company Ltd, New Delhi, India. Gopalan, C.B.N., B.V.R. Ramasastri and S.C. Bala Subramaniam, 1981. Nutritive Value of Indian Food, National Instiute of Nutrition, Indian Council for Medical Research, Hyderabad. Joshi, V.K. and D. Attri, 2005. Panorama of wine research in India. J. Sci. Indu. Res., 64: 9-18. .RWVHULGLV < DQG 5 %DXPHV ,GHQWL¿FDWLRQ RI LPSDFW RGRUDQWV in bordeaux red grape juice, in the commercial yeast used for its fermentation, and in the produced wine. J. Agric. Fd Chem., 48: 400-406. Kovats, E. 1965. Gas chromatographic characterization of organic substances in the retention index system. Adv. Chrom., 1: 229-247. 0DF/HRG $ - DQG 8 'H7UDFRQLV 9RODWLOH ÀDYRXU FRPSRQHQWV of Sapodilla fruits. J. Agric. Fd. Chem., 30: 515-517. Panesar, P.S., V.K. Joshi, R. Panesar and G.S. Abrol, 2011. Vermouth: Technology of production and quality characteristics. In: Advances in Food and Nutrition Research. Vol.63 p. 255-280. R.S. Jckson (ed). Academic Press. Pawar, C.D. 2009. Standardisation of Wine Making Technology from Sapota (Manilkara achras (Mill) Forsberg. Ph.D. Diss., University of Agricultural Sciences, Dharwad. 2009. 140 pp. Ranganna, S. 1986. Handbook of Analysis and Quality Control for Fruits and Vegetable Products. Tata McGraw Hill Publishing Company Ltd., New Delhi. Relekar, P. and A. Naik, 2012. Value Added Products of Sapota (Manilkara achras (Mill) Foseberg : Standardization, Storage and Quality Analysis. Lambert Academic Publishing. Salunkhe, D.K. and B.B. Desai, 1984. Chemical control of loses. In: Post Harvest Technology of Fruits. Volume I : Boco Raton, F.L. (ed). CRC Press, Lodon. Shivashankara, K.S., T.K. Roy and Y. Selvaraj, 2007. Volatile components of two sapodilla (Achras sapota L.) cultivars. Ind. Perfumer, 51: 44-47. Schreier, P. 1979. Flavor Composition of Wines: A Review. CRC Crit. Rev. Fd Sci. Nut., 12: 59-111. Suhaj, M. 2006. Spices antioxidant isolation and their antiradical activity: A review. J. Fd. Comp. Anal., 19: 531-547. Vermeir, S., M.L.A.T.M. Hertog, K. Vankerschaver, R. Swennen, % 0 1LFRODL DQG - /DPPHUW\Q ,QVWUXPHQWDO EDVHG ÀDYRXU characterisation of banana fruit. Fd. Sci. Technol., 42: 1647-1653. Submitted: November, 2014; Revised: December, 2014; Accepted: December, 2014

Journal

Journal of Applied Horticulture, 17(2): 151-154, 2015

Appl

Effect of planting date on growth, development, aerial biomass SDUWLWLRQLQJ DQG Ă RZHU SURGXFWLYLW\ RI PDULJROG Tagetes erecta L.) cv. Siracole in Indo-gangetic plains of West Bengal Khumukcham Joshna* and P. Pal Department of Floriculture and Landscaping, Faculty of Horticulture, Bidhan Chandra Krishi Viswavidyalaya (BCKV) Mohanpur, Nadia-741252, West Bengal, India.*E-mail: joshna_kh@yahoo.com

Abstract 7KH LQYHVWLJDWLRQ ZDV FDUULHG RXW WR HYDOXDWH WKH JURZWK ÀRZHULQJ \LHOG DQG TXDOLW\ RI $IULFDQ PDULJROG FY 6LUDFROH DV LQÀXHQFHG by different planting dates. The crop planted on 9th June (T3) was found to have the highest plant height (96.93 cm). Maximum number of primary (5.3) and secondary (14.15) branches/plant, total fresh weight (502.00 g/plant), contribution by stem (385.00 g/plant) to the total fresh weight, higher dry (126.25 g/plant) matter accumulation and also the dry matter accumulation in stem per plant (98.00 g/plant) were found maximum with 12 April (T1 SODQWLQJ 7KH LQGLYLGXDO OHDI DUHD VT FP ZDV VLJQL¿FDQWO\ KLJKHU LQ WKH FURS planted in February (T11). It took minimum days (13.01 days) from visible bud to colour shown and bud emergence to full bloom GD\V PD[LPXP GLDPHWHU RI LQGLYLGXDO ÀRZHU FP ZHUH IRXQG ZLWK $SULO 71 SODQWLQJ +HDYLHVW ÀRZHU J ZDV recorded with October 12 (T7) planting. 16th May (T2 SODQWLQJ SURGXFHG PD[LPXP QXPEHU RI ÀRZHUV SHU SORW P2). Maximum carotene content was noted with 12th October (T7) planting. Crops planted between 50th MSW (T9) 2011 to 3rd MSW (T10) SURGXFHG YHU\ OHVV FURS ELRPDVV GU\ PDWWHU FRQWHQW DQG ÀRZHU \LHOG Key words: Carotene, Meteorological Standard Weeks, planting time, Tagetes erecta L. cv. Siracole.

,QWURGXFWLRQ Marigold (Tagetes erecta) is one of the most important commercially grown flower crops in India. Besides, orange FRORXUHG PDULJROG ÀRZHUV DUH LPSRUWDQW VRXUFH RI FDURWHQRLG pigments which is mainly used in poultry feed industry. In recent years carotenoid has represented a good alternative for the pharmaceutical and food industries and especially for the human health. It prevents different diseases, such as cancer, mascular degradation and cataracts (Arvayo et al., 2013 ). The crop is known to respond by day length and temperature. Time of planting D QRQ PRQHWDU\ LQSXW SOD\V D VLJQL¿FDQW UROH LQ LPSURYLQJ the yield of many crops and governs the crop phenological GHYHORSPHQW DQG WRWDO ELRPDVV SURGXFWLRQ DORQJ ZLWK HI¿FLHQW conversion of biomass in to economic yield (Khichar and Niwas, 2006). Moreover, meagre information is available about the response of African marigold cv. Siracole to different planting time. Keeping this in view, a study was undertaken to evaluate WKH JURZWK ÀRZHULQJ \LHOG DQG TXDOLW\ RI $IULFDQ PDULJROG FY 6LUDFROH DV LQÀXHQFHG E\ GLIIHUHQW SODQWLQJ GDWHV

Materials and methods The study was conducted during April to March (2011 to 2012) at Horticultural Research Station, Mondouri, BCKV, Nadia, (23.5 0N latitude; 89 0E Longitude) at about 8.75 m above mean sea level under irrigated condition. The site experiences a mean annual temperature of 26.15 0C. Rainy season accounts for 35 % of the total rainfall and is associated with low sunshine hours.The treatments comprised of twelve planting dates viz., T1 - 12 April, T2 - 16 May, T3 - 9 June, T4-12 July, T5- 9 August, T6-12 September, T7-12 October, T8-12 November, T9-15 December, T10-20 January,

T11- 20 February and T12 -13 March, corresponding to15, 20, 23, 28, 32, 37, 41, 46, 50, 3, 8 and 11 meteorological standard weeks (MSW) and were tested in a randomized block design with four replications. The experimental plot size was laid with an area of 6.4 m2 (3.2 m × 2 m). A basal dose of 23.5 q mustard oil cake ha-1, 100 kg P2O5 ha-1 and 100 kg K2O ha-1 was applied. Well rooted cuttings of 21-25 days old with more or less uniform growth and vigour were planted at 40 × 20 cm and adopted uniform agronomical practices for all treatments. Top dressing (50 kg N ha-1 as urea) was given 30 days after planting. Soluble fertilizers (NPK- 19:19:19) @ 1.5 gm/L of water were sprayed on every 15 days intervals. Plants were pinched 30 days after planting to encourage axillary branches. Periodical observations were taken RQ YHJHWDWLYH SDUDPHWHUV 5HSURGXFWLYH DQG ÀRZHULQJ SDUDPHWHUV were recorded at one day interval. The observations recorded on plant height, number of branches, individual leaf area, and dry matter accumulation in leaves, stem and roots were recorded 90 days after planting (DAP). Flower petals were collected randomly on 10 days interval from each treatment and the total carotene content (Swain and Hill, 1959) was estimated and expressed in mg/g. Data on various characters studied during the course of investigation were statistically analysed. The meteorological data of the cropping period and harvesting week are given in Fig. 1.

5HVXOWV DQG GLVFXVVLRQ Vegetative attributes: Data presented in Table 1 revealed that SODQWLQJ WLPH RI PDULJROG FY 6LUDFROH VLJQLÂżFDQWO\ LQĂ€XHQFHG the plant height, number of primary and secondary branches, leaf area, and weight of plant parts i.e. fresh and dry weight of plants. 3ODQW KHLJKW FP ZDV VLJQLÂżFDQWO\ KLJKHU LQ thJune (T3) transplanting as compared to other planting dates. It may be due to

3ODQWLQJ GDWH LQĂ€XHQFH RQ JURZWK GHYHORSPHQW DHULDO ELRPDVV RI PDULJROG

152

Fig. 1. Meteorological data of the growing period

the fact that 23rd MSW planted crop grew under high temperature range (average day 32.56 to 24.840C and night 31.08 to 14.150C) and long day condition, consequently registered longer duration (82 days) of vegetative phase, enhanced the linear growth of the SODQW 7KLV LV LQ FRQIRUPLW\ ZLWK WKH ÂżQGLQJV RI 1DLU et al. (1985) and Yulian et al. (1995). The crop planted on 20th January (T10) showed minimum plant height (14.10cm). During the growth phase plants exposed to low temperature range (average DT 20.2 to 29.4 0C and NT 7.6 to 14.5 0C) and short day condition. Low temperature can result in poor growth. Another cause may be the severe blight infestation. The number of both primary (5.3) and secondary (14.15) branches/plant, total fresh weight (502.00 g/plant) and contribution by stem (385.00 g/plant) to the total fresh weight was maximum with 12 April (T1) planting. The increase in fresh weight of plant may be due to formation of more

number of primary and secondary branches per plant in T. erecta cv. Siracole. The crop planted on 12th November (T8) recorded VLJQLÂżFDQWO\ ORZHU QXPEHU RI EUDQFKHV DQG WRWDO IUHVK ZHLJKW (38.25 g/plant, compared to others but remained statistically at par with 15th December (T9) and 20th January planting (T10). At '$3 WKH LQGLYLGXDO OHDI DUHD VT FP ZHUH VLJQLÂżFDQWO\ higher in the crop planted in February (T11) followed by June (T3) planting (4.35sq cm). The crop which were planted on 15th December (T9) (1.06 sq cm), 20th January (T10) (1.14 sq cm) and 12th November (T8) (1.67 sq cm) remained at par with each other EXW UHFRUGHG VLJQLÂżFDQWO\ ORZHU OHDI DUHD WKDQ RWKHUV ZKLFK might be accumulated lesser number of degree days and resulted in poor growth. The accumulation of crop biomass ultimately depends on the interception of solar radiation by leaf canopy and

Table 1. Effect of planting dates on vegetative parameters of marigold Treatment Plant Number of branches/ Leaf area Fresh weight g/plant height plant (cm2) (90 DAP) (cm) Primary Secondary 90 DAP Leaf Stem Root T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 LSD(0.05)

72.75 91.10 96.93 91.05 74.10 56.50 39.25 18.00 19.85 14.10 74.15 70.60 4.72

5.3 3.5 3.25 2.65 2.85 2.7 2.4 1.6 1.75 1.7 3.25 3.3 0.67

14.15 12.60 10.60 8.85 9.80 10.90 7.10 3.70 4.45 4.00 6.35 7.30 1.30

3.88 3.75 4.35 2.67 2.90 2.93 2.84 1.67 1.06 1.14 4.73 3.83 0.84

79.25 73.50 105.25 60.50 17.25 23.75 17.50 10.00 12.00 19.00 29.00 75.75 5.11

385.00 319.00 195.25 172.75 103.00 71.25 35.25 25.00 25.00 30.25 36.75 340.00 19.81

37.75 51.75 54.25 20.75 19.00 5.25 5.75 3.25 2.25 1.50 8.75 30.25 7.67

Total fresh weight (g/plant) 502.00 444.25 354.75 254.00 139.25 100.25 58.50 38.25 39.25 50.75 74.50 446.00 27.58

Dry weight g/plant (90 DAP) Leaf Stem Root

Total dry weight (g/plant)

20.00 19.75 20.25 11.75 6.25 9.75 4.42 1.75 1.50 5.25 9.00 20.25 1.87

126.25 125.00 75.75 56.25 38.50 31.00 15.42 5.25 4.75 13.42 25.00 123.25 3.02

98.00 93.25 41.75 40.25 28.25 19.25 9.25 3.25 3.00 7.92 14.25 95.00 2.43

8.25 12.00 13.75 4.25 4.00 2.00 1.75 0.25 0.25 0.25 1.75 8.00 1.58

3ODQWLQJ GDWH LQÀXHQFH RQ JURZWK GHYHORSPHQW DHULDO ELRPDVV RI PDULJROG on active photosynthesis by the individual leaves. Dry matter accumulation in different plant parts of T. erecta cv. Siracole viz OHDYHV VWHP DQG URRWV DQG WRWDO ELRPDVV ZDV VLJQL¿FDQWO\ LQÀXHQFHG E\ SODQWLQJ GDWHV 7KH FURS SODQWHG RQ $SULO 71) UHFRUGHG VLJQL¿FDQWO\ KLJKHU GU\ PDWWHU DFFXPXODWLRQ g/plant) as compared to others but remained at par with May 16 (T2) and March 13 (T12). Whereas, December 15th (T9) planted FURS UHFRUGHG VLJQL¿FDQWO\ ORZHU GU\ PDWWHU DFFXPXODWLRQ 7DEOH 1). The total energy available to any crop is never completely converted to dry matter under even most favourable conditions. (I¿FLHQF\ RI FRQYHUVLRQ RI KHDW HQHUJ\ LQWR GU\ PDWWHU GHSHQGV upon genetic factors, sowing time and crop type (Hundal et al., 2004). Dry matter accumulation in stem per plant was VLJQL¿FDQWO\ KLJKHVW LQ $SULO J SODQW IROORZHG E\ March (95.00 g/plant) and16 May (93.25 g/plant), respectively. This may be attributed to the fact that lesser dry matter was accumulated in different plant parts of T. erecta cv. Siracole planted beyond Oct 12 to Dec 15. Willits and Bailey (1999) observed increased plant weight with increasing temperature of both heat sensitive and heat tolerant chrysanthemum. The crops planted at 46th MSW (T8) partitioned more towards leaf (86.89%) followed by 47th MSW (82.62%).The marigold planted on 3rd MSW (T10) partitioned more towards stem (71.66%) and least towards root (1.99%). The crop planted on April 12 (T1) recorded VLJQL¿FDQWO\ KLJKHU IUHVK J SODQW DQG GU\ J plant) matter accumulation as compared to all other treatments. Whereas, December 15th (T9 SODQWHG FURS UHFRUGHG VLJQL¿FDQWO\ lower dry matter accumulation. Reproductive attributes: The crop transplanted on June 10 (T3) LQLWLDWHG ÀRZHU EXGV RQ GD\V DIWHU SODQWLQJ '$3 ZKHUHDV only 33 days were required for those planted on September12 (T6) followed by T1 (34 days). It may be due to shortening of WKH YHJHWDWLYH SKDVH DQG SHULRG EHWZHHQ EXGGLQJ DQG ÀRZHULQJ is curtailed due to minimum DIF, which might have shortened WKH SKHQRSKDVH GXUDWLRQ RI ODWH WUDQVSODQWHG FURS 6LJQL¿FDQW YDULDWLRQ ZDV QRWHG LQ GD\V WDNHQ IURP ÀRZHU EXG HPHUJHQFH WR FRORXU VKRZQ DQG ÀRZHU EXG HPHUJHQFH WR IXOO EORRP XQGHU different planting dates (Table 2). Crops planted on 12 thApril (T1) took minimum days (13.01 days) from visible bud to colour

shown and bud emergence to full bloom (20.16 days) than other dates of transplanting but remained at par with the crop planted on13 March (T 12). During this period plants were exposed to favourable high temperature regime (35/ 25.4 0C). Crops planted on 12 November (T8) took more days from visible bud to colour shown and bud emergence to full bloom (42.77 days). Low temperature (22/9.2 0C) during vegetative phase delayed the visibility of colour. Karlsson et al. (1989) and Wilkins et al. (1990) concluded that low night temperatures of 5 0C or 13 0C KDG GHOD\LQJ DIIHFWV RQ ÀRZHULQJ RI FKU\VDQWKHPXP 'LDPHWHU DQG ZHLJKW RI LQGLYLGXDO ÀRZHU DOVR YDULHG VLJQL¿FDQWO\ GXH to different planting dates. Maximum diameter of individual ÀRZHU FP ZDV QRWHG ZLWK $SULO 71) planting whereas November 12 (T8) planted crop produced small size (0.62 cm) ÀRZHUV FRPSDUHG WR RWKHU SODQWLQJ GDWHV +LJKHU DWPRVSKHULF temperature during vegetative stage and lower diurnal variation and bright sun shine (BSS) hours during reproductive stage lead WR JUHDWHU ÀRZHU GLDPHWHU +HDYLHVW ÀRZHU J ZDV UHFRUGHG with October12 (T7) planting. Individual flower weight was favoured by lower atmospheric temperature. Among the different planting dates, 16th May (T2) planting produced maximum number RI ÀRZHUV SHU SORW P2 ,Q WHUPV RI ÀRZHU ZHLJKW maximum yield was recorded with 12th Sept (T6) planting. This may be due to availability of lower atmospheric temperature. This allows the plant to photosynthesize (build up) and respire (break down) during an optimum daytime temperature, and to curtail the rate of respiration during a cooler night. Yield potential of a crop is resultant effects of growth, development and qualitative performance of the plant in a particular agro-climatic condition. Crop growth and yield are the results of the interaction of weather that prevail during the crop growth period and genetic FRQVWLWXWLRQ RI WKH FURS SODQWV &DURWHQH FRQWHQW LQ WKH ÀRUHWV YDULHG VLJQL¿FDQWO\ GXH WR GLIIHUHQW SODQWLQJ GDWHV 0D[LPXP carotene content was noted with 12 th October (T7) planting. Carotene content showed higher values when the plants received lower atmospheric temperature. The study clearly indicated that planting dates and weather variables like rainfall, temperature and bright sunshine hours had SURIRXQG LQÀXHQFH RQ JURZWK DQG GHYHORSPHQW RI T. erecta cv.

Table 2. Effect of planting dates on reproductive parameters of marigold Treatment Days to Day Vegetative Reproductive Total Individual colouration from bud stage stage duration of ÀRZHU from bud emergence to (days) (days) FURS LQ ¿HOG weight emergence full bloom (days) (g/plot) T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 LSD(0.05)

13.01 15.16 15.37 15.81 19.43 20.33 20.89 33.19 28.63 29.41 15.59 13.97 2.06

20.15 22.86 22.54 22.89 26.95 28.86 30.56 42.77 37.80 39.09 23.76 21.37 2.07

52.00 70.00 82.00 72.00 59.00 48.00 46.00 60.00 64.00 63.00 52.00 54.00 2.09

78.00 88.00 78.00 68.00 68.00 68.00 46.00 48.00 38.00 58.00 58.00 59.00 2.54

130.00 158.00 160.00 140.00 127.00 116.00 92.00 108.00 102.00 121.00 110.00 113.00 2.93

153

2.18 1.24 1.80 1.74 1.68 2.46 2.55 1.14 0.57 0.33 1.19 1.16 0.24

Individual ÀRZHU diameter (cm)

Number of ÀRZHUV

Weight of ÀRZHUV (g/plot)

3.99 3.23 3.65 3.48 3.45 3.47 3.79 0.62 0.63 0.83 3.69 3.77 0.07

3369.24 7434.67 5552.68 6810.51 6325.58 5027.16 3319.66 255.10 209.10 667.03 2188.74 965.57 794.14

7329.25 9164.00 10015.00 11851.75 10572.75 12312.75 8445.00 289.75 102.00 214.50 2587.50 1117.25 1280.25

Total carotene contents (mg/g) 0.31 0.84 1.35 1.48 1.81 1.80 2.68 1.20 1.00 1.24 1.01 0.74 0.78

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References Arvayo, E.H., I.M. Fernandez, P.G. Moroyoqui, J.L. Cervantes and R.P. 5DPLUH] &DURWHQRLGV H[WUDFWLRQ DQG TXDQWLÂżFDWLRQ D UHYLHZ Annals Methods, 5: 2916-2924. Hundal, S.S., P. Kaur, S.D.S. Malikpuri and J. Joy, 2004. Prediction of growth and yield of pearl millet using agro-climatic conditions. J. Agromet., 6: 166-170. Karlsson, M.G., R.D. Heins, J.E. Erwin and R.D. Berghage, 1989. Development rate during four phases of chrysanthemum growth as determined by preceding and prevailing temperatures. J. Amer. soc. HortScience, 114(2): 234-240. .KLFKDU 0 / DQG 5 1LZDV 0LFURFOLPDWLF SURÂżOHV XQGHU GLIIHUHQW sowing environments in wheat. J. Agromet., 8: 201-209.

Nair, S.R., K.G. Kumar and K.K. Santha, 1985. Effect of planting time DQG VSDFLQJ RQ JURZWK ÀRZHU SURGXFWLRQ DQG VHHG \LHOG LQ PDULJROG Orissa J. Hort., 13: 14-20. Swain, T. and W.E. Hill, 1959. Standard Methods of Biochemical Analysis, Thimmaih (Ed), Kalyani Publishers, Ludhiana, p.304-305. Wilkins, H.F., W.E. Healy and K.L. Grueber, 1990. Temperature regime DW YDULRXV VWDJHV RI SURGXFWLRQ LQÀXHQFHV JURZWK DQG ÀRZHULQJ of 'HQGUDQWKHPD JUDQGLÀRUXP. J. Amer. Soc. Hort. Sci., 115(5): 732-736. Willits, D.H. and D.A. Bailey, 1999. Night cooling as a means of improving warm weather chrysanthemum production: the effect RI QLJKW WHPSHUDWXUH RQ JURZWK DQG ÀRZHU GHYHORSPHQW LQ PXPV Amer. Soc. Agr. Eng., 944072: 12. Yulian, F.Y. and N. Okuda, 1995. Effects of day length on growth, budding and branching of garland chrysanthemum (Chrysanthemum coronarium L.). Tech. Bul. of Faculty of Agr. Kagawa Univ., 47(1): 7-13. Submitted: May, 2014; Revised: December, 2014; Accepted: December, 2014

Journal

Journal of Applied Horticulture, 17(2): 155-159, 2015

Appl

'LYHUVLW\ RI EHH IRUDJLQJ Ă RUD DQG Ă RUDO FDOHQGDU RI 3DLWKDQ taluka of Aurangabad district (Maharashtra), India Bhalchandra Waykar and R.K. Baviskar* Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431004 (Maharashtra), India. *E-mail: rkbaviskar@gmail.com

Abstract 7KH VWXG\ ZDV FRQGXFWHG DW 3DLWKDQ WDOXND RI $XUDQJDEDG GLVWULFW GXULQJ 2FWREHU ¹6HSWHPEHU WR LGHQWLI\ H[LVWLQJ EHH ÀRUD DQG WR GHWHUPLQH KRQH\ ÀRZ DQG GHDUWK SHULRG WR GHYHORS WKH ÀRUDO FDOHQGDU 7KH ÀRZHULQJ SODQWV ZHUH YLVLWHG DQG REVHUYHG IRU WKH presence of honey bees and their foraging activities. Plants were reported as bee foraging species when at least three honey bees had YLVLWHG WKH ÀRZHUV ZLWKLQ WKH SHULRG RI PLQXWHV 7KH UHVXOW UHYHDOHG WKDW SODQW VSHFLHV ZHUH XVHIXO WR KRQH\EHHV DV VRXUFH RI IRRG RXW RI ZKLFK ZHUH ZLOG DQG ZHUH DJUR KRUWLFXOWXUDO SODQWV 7KH LGHQWL¿HG ÀRUD ZDV IXUWKHU JURXSHG LQWR QHFWDU SROOHQ DQG ERWK nectar and pollen supplying plants. Out of 41 wild bee plant species, 17 were nectar producing, 4 were pollen producing and 20 were both nectar and pollen producing. Results also revealed that out of 22 agriculture bee plant species, 6 were nectar producing, 5 were SROOHQ SURGXFLQJ DQG ZHUH ERWK QHFWDU DQG SROOHQ SURGXFLQJ 0LG 2FWREHU WR PLG 'HFHPEHU ZDV LGHQWL¿HG DV KRQH\ ÀRZ SHULRG RI WKH \HDU KDYLQJ QXPEHU RI ÀRZHULQJ SODQWV 0LG 0D\ WR PLG $XJXVW ZDV WKH FULWLFDO GHDUWK SHULRG ZLWK IHZ ÀRZHULQJ SODQWV %DVHG RQ WKH DYDLODELOLW\ RI ÀRUD PDMRU FKDUDFWHULVWLFV RI WKHVH SODQW VSHFLHV XWLOLW\ VWDWXV DQG ÀRZHULQJ GXUDWLRQ WKH EHH ÀRUDO FDOHQGDU ZDV GHYHORSHG IRU 3DLWKDQ WDOXND RI $XUDQJDEDG GLVWULFW 7KH UHVXOW LQGLFDWHG WKDW WKH DUHD KDV ULFK EHH ÀRUD DQG LV VXLWDEOH IRU FRPPHUFLDO bee keeping. Paithan taluka has four honey bee species, viz., Apis dorsata, A. cerana indica, $ ÀRUHD and A. mellifera. Among these, $ ÀRUHD and A. dorsata were dominant bee species, whereas A. mellifera was introduced species and only few colonies of A. cerana indica were observed. Key words: %HH ÀRUD ÀRUDO FDOHQGDU KRQH\ ÀRZ SHULRG GHDUWK SHULRG Apis dorsata, A. cerana indica, $ ÀRUHD, A. mellifera.

,QWURGXFWLRQ Beekeeping is agro-horticultural and forest based industry and LW LV RI JUHDW LPSRUWDQFH WR IDUPHUV IRU SROOLQDWLRQ EHQH¿W %\ investing limited expenses, less land requirement, beekeeping can be practiced to obtain maximum subsidiary income through honey, beeswax and other bee products with increased agricultural output. The demand of bee keeping has been increased tremendously in world. Success of beekeeping depends upon many factors, among which abundant availability of bee ÀRUD ZLWKLQ WKH VXUURXQGLQJ DUHD RI DQ DSLDU\ LV PRVW LPSRUWDQW (Akratanakal, 1987; Crane, 1990; Singh, 2005). There are three W\SHV RI EHH ÀRUD SODQWV WKDW RQO\ VXSSO\ QHFWDU SODQWV WKDW RQO\ supply pollen, and plants that provide both (Crane et al., 1989; Allen et al., 1998; Bhattacharya, 2004; Waykar et al., 2014). 7KH KRQH\ ÀRZ SHULRG DQG GHDUWK SHULRG YDULHV IURP RQH ORFDWLRQ WR DQRWKHU DQG ZLWK DOWLWXGHV 7KH ÀRZHULQJ SODQWV RI VHYHUDO plant families blossom at different time interval of the year (Free, 1970). Depending upon the soil type, climatic factors and the habitat of the vegetation, the time of the blooming may change for even the same nectar plant (Rodinov and Shabanshov, 1986; $EURO 7KH H[WHQVLYH NQRZOHGJH RQ ÀRZHU W\SH ÀRZHULQJ GXUDWLRQ PDLQ EORRPLQJ WLPH GHQVLW\ DQG TXDOLW\ RI EHH ÀRUD LQ D UHJLRQ DUH SUHUHTXLVLWHV IRU HQKDQFLQJ WKH HI¿FLHQF\ RI beekeeping industry and successful beekeeping (Kumar et al., 2013). Such information enable beekeepers to utilize them at the maximum level, so that they can harvest a good yield of honey and other bee products in addition to effective pollination which enable higher crop yields.

$ ÀRUDO FDOHQGDU IRU EHHNHHSLQJ LV D WLPH WDEOH WKDW LQGLFDWHV the approximate date and duration of the blossoming period of the important honey and pollen plants in the area. Preparation RI D ÀRUDO FDOHQGDU IRU DQ\ VSHFL¿F DUHD UHTXLUHV WKH FRPSOHWH observations of the seasonal changes in the vegetation patterns and/or agro ecosystems of the area (Yadav and Kaushik, 6XFK NQRZOHGJH RQ WKH EHH ÀRUD KHOSV LQ WKH HIIHFWLYH PDQDJHPHQW RI EHH FRORQLHV GXULQJ WKH KRQH\ ÀRZ SHULRG DQG dearth period. 7KH VWXG\ DLPHG RQ LGHQWL¿FDWLRQ RI SODQW VSHFLHV XVHIXO WR KRQH\ bees as source of food and critical dearth period for effective management of bee colonies.

Materials and methods Study area: Geographically, Paithan taluka of Aurangabad district is located at 19029’ N 750 26’ E. The average altitude of this area is 458 meter above sea level. The farmers cultivate PDMRU FURSV VXQÀRZHUV PXVWDUG VXJDUFDQH FRWWRQ EDMDUD wheat and jawar), pulses and vegetables in the area. The study area is also known for cultivation of horticultural crops. Paithan taluka of Aurangabad district has been greatly endowed with these resources and is one which has not been explored so far for beekeeping. ,GHQWL¿FDWLRQ RI EHH ÀRUD Field data was collected through regular visits to the study sites, during October 2012-September 2013 regularly. Each study visit served as pseudo replicates for the site and all observations were made between 0700-1800 hours

156

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in winter and monsoon season and 0700-1830 hours in summer season. The study included observation of bee’s activities on ÀRZHUV RI GLIIHUHQW SODQW VSHFLHV :KHQHYHU EHHV ZHUH IRXQG RQ WKH ÀRZHUV RI VXFK SODQWV WKHLU IRUDJLQJ EHKDYLRU ZDV REVHUYHG for a period of 10 minutes. If the success of any foraging attempt was ascertained, the plant was scored as bee foraging species after DW OHDVW WKUHH KRQH\EHHV YLVLWHG WKH ÀRZHUV VLPXOWDQHRXVO\ RU within the observation period (10 minutes). The observation on nectar and pollen source was based on activities performed by honeybees on different flowers. Honeybees with their activity of extending their proboscis into the ÀRZHUV DUH FRQVLGHUHG DV QHFWDU VRXUFH DQG EHHV FDUU\LQJ SROOHQ LQ their pollen basket were considered as pollen source. Honeybees ZLWK WKHLU DFWLYLW\ RI H[WHQGLQJ WKHLU SURERVFLV LQWR WKH ÀRZHUV DQG also collecting pollen on their hind legs were determined as nectar and pollen yielding plants (Bista and Shivakoti, 2001). Samples RI SODQWV WKDW FRXOG QRW EH LGHQWL¿HG LQ WKH ¿HOG ZHUH FROOHFWHG and saved in herbarium sheets in specimen box. All collected VDPSOHV ZHUH LGHQWL¿HG LQ WKH 'HSDUWPHQW RI %RWDQ\ 'U % $ Marathwada University, Aurangabad by taxonomist and then compared with the published reports (Shrestha, 1998; Sivaram, 2001; Waykar et al., 2014) for their probable use by honeybees. $ FRPSOHWH FKURQRORJLFDO UHFRUG RI ÀRZHULQJ SHULRGV RI WKH plants species was made during the surveys. The data was UHFRUGHG DQG FRPSLOHG LQWR DQQXDO ÀRUDO FDOHQGDU WR SUHSDUH KRQH\ ÀRZ DQG GHDUWK SHULRG

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The bee colony efficiency and its development as well as production of honey, beeswax and other bee products depend on quality and quantity of pollen and nectar obtained from bee forage plants (Keller et al., 2005; Brodschneider et al., 2010). The nectar acts as source of honey and provides heat and energy for bees and pollen provides the protein, vitamins, fatty substance and other nutrients to bees (Fluri and Bogdanov, 1987). Therefore, a GLUHFW FRQVHTXHQFH RI QXWULWLRQDO GH¿FLHQF\ SROOHQ VKRUWDJH LV a decrease in the colony population (Keller et al., 2005). %HH ÀRUDO FDOHQGDU During the survey, a complete chronological UHFRUG RI ÀRZHULQJ SHULRGV RI WKH SODQW VSHFLHV ZDV PDGH DQG REWDLQHG GDWD ZDV FRPSLOHG LQWR DQQXDO ÀRUDO FDOHQGDU DQG GDWD is presented in Tables 1 and 2 and Figs. 1 and 2. Amongst total 41 ZLOG SODQWV LGHQWL¿HG DV EHH SODQWV ZHUH EORRPLQJ LQ VXPPHU season, 18 in winter season and 21 in monsoon season. Out of 22

The bee-flora consists of mostly ornamental, medicinal,

Abitulon pannosum Alternanthera paronochyoides Amaranthus dubius Amaranthus polygonoides Amaranthus spinosus Asystasia dalzelliana Azadirachta indica Caesalpinia bonduc Canavalia gladiate Celosia argentea Chenopodium album Cleoserrata speciosa Cuphea micropetala Cyanotis cristata Delonix regia Eleusine indica Euphorbia hirta Gliricidia sepium Guizotia abyssinica Hygrophilla schulii Hyptis suaveolens Ipomoea obscura Lagascea mollis Lantana camara Launaea procumbens Maytenus emarginata Medicago sativa Melilotus indica Mimosa hamata Pentas lanceolate Paracalyx scariosa Parthenium hysterophorus Plectranthus mollis Pongamia pinnata Senna ostusifolia Sonchus asper Spinaceae oleraceae Tamarindus indica Tridax procumbens Vernonia cinerea Ziziphus jujuba

Flowring period (months)

The survey on the flowering plants with special reference to beekeeping importance was carried out during the study period October 2012–September 2013 and obtained data was summarized in Table 1 and 2 and Figs. 1 and 2. The results revealed that 63 plant species 14 were useful for beekeeping as source of food, out of 12 which 41 were wild and 22 were agro-horticultural plants, 10 which were well distributed and commonly found in the 8 study area. The identified flora was further grouped 6 into nectar, pollen and both nectar and pollen producing 4 plants (Table 1 and 2), out of 41 wild bee plant species 17 2 were nectar producing, 4 were pollen producing and 20 plant 0 species were both nectar and pollen producing. Results also revealed that out of 22 agrohorticulture bee plant species 6 were nectar producing, 5 were pollen producing and 11 were both nectar and pollen producing.

vegetables, horticultural and other commercially important plants OLNH VSLFHV SXOVHV FHUHDOV RLO \LHOGLQJ ¿EUH DQG IRGGHU FURSV etc. Four species of weeds viz., Alternanthera paronochyoides St. Hil, Lantana camara L. Hil, Parthenium hysterophorus L. and Tridax procumbens, ZLWK WKH ÀRZHULQJ SHULRG RI and 7 months, respectively, four wild plants, the Azardirachta indica A. Juss., Pongamia pinnata L. Pierre. Tamarindus indica L. and Delonix regia +RRO 5DI ZLWK WKH ÀRZHULQJ SHULRG RI DQG PRQWKV UHVSHFWLYHO\ DQG ¿YH DJUR KRUWLFXOWXUDO FURSV viz., Citrus aurantium L., Cajanus cajan L. Mill sp., Moringa oleifera Lamk., Mangifera indica L., and Triticum aestivum L., ZLWK WKH ÀRZHULQJ SHULRG RI DQG PRQWKV UHVSHFWLYHO\ ZHUH GRPLQDQW LQ WKH ¿HOG DUHD 7KHVH SODQW VSHFLHV VHUYHG DV WKH excellent sources of pollen and nectar in the study area. In dearth period when agro-horticultural plants were not in blooming stage, ZHHGV DQG ZLOG ÀRZHULQJ SODQWV ZHUH REVHUYHG DV DOWHUQDWH IRRG source for honeybees.

Fig. 1. Flowering duration of wild bee plants recorded during October 2012-September 2013

'LYHUVLW\ RI EHH IRUDJLQJ ÀRUD DQG ÀRUDO FDOHQGDU RI 3DLWKDQ WDOXND RI $XUDQJDEDG GLVWULFW ,QGLD Table 1. The wild bee flora and floral calendar of Paithan taluka during October 2012–September 2013 Botanical name Family Flowering period. Nectar Malvaceae Abitulon pannosum Forst. f. Schlecht. Amaranthaceae Alternanthera paronochyoides St. Hil. Amaranthaceae Amaranthus dubius Mart. Ex. Thell. Amaranthaceae Amaranthus polygonoides L. Amaranthaceae Amaranthus spinosus L. Acanthaceae Asystasia dalzelliana Meliaceae Azadirachta indica A Juss. Caesalpiniaceae Caesalpinia bonduc L. Roxb. Fabaceae Canavalia gladiate Jacq. DC. Amaranthaceae Celosia argentea L. var. argentea Chenopodiaceae Chenopodium album L. Cleomaceae Cleoserrata speciosa Raf. Iltis Lythraceae Cuphea micropetala L Commalinaceae Cyanotis cristata L. D. Don. Fabaceae Delonix regia Hook Raf. Poaceae Eleusine indica L. Gaertn. Euphorbiaceae Euphorbia hirta L. Fabaceae Gliricidia sepium Jacq. Kunth ex Steud. Asteraceae Guizotia abyssinica L. f. Cass. Hygrophilla schulii Buch-Ham. M.R. and S.M. Almeida. Acanthaceae Lamiaceae Hyptis suaveolens L. Poit. Convolvulaceae Ipomoea obscura L. Ker-Gawl. Forma obscura Asteraceae Lagascea mollis Cav. Verbenaceae Lantana camara L. Asteraceae Launaea procumbens Roxb. Ramayya and Rajgopal. Celastraceae Maytenus emarginata Willd. Ding Hou. Fabaceae Medicago sativa L. Fabaceae Melilotus indica L. All. Mimosaceae Mimosa hamata Willd. Rubiaceae Pentas lanceolate )URVVN 'HÀHUV. Fabaceae Paracalyx scariosa Roxb. Ali. Asteraceae Parthenium hysterophorus L. Lamiaceae Plectranthus mollis Ait. Spreng. Fabaceae Pongamia pinnata L. Pierre. Caesalpiniaceae Senna ostusifolia L. Irwin and Barneby Asteraceae Sonchus asper L. Hill. Chenopodiaceae Spinaceae oleraceae L. Legiminosae Tamarindus indica L. Asteraceae Tridax procumbens L. Asteraceae Vernonia cinerea L. Less. Rhamnaceae Ziziphus jujuba Mill.

January-June February-April. November-March June-October April-August January-December April-June February-April January-December April-August March-June January-November August-November July-October April-June July-September. January-December February-June July-October September-May August-October October-March August-March January-April, July-September September-December March-July June-August August-February August-February January-December November-February January-December August-December May-September May-November July-October June-September May-July January-December January-March July-October

N N N N N N N N N N N N N N N N N -

157

Bee forage value Pollen Nectar+Pollen NP P NP P NP NP NP NP NP NP NP P NP NP NP NP NP NP NP P NP NP NP NP

N – Nectar yielding plant. P – Pollen yielding plant. NP – Nectar and pollen yielding plants

DJUR KRUWLFXOWXUDO SODQWV LGHQWL¿HG DV EHH SODQWV SODQW VSHFLHV bloom in summer season, 9 in winter season and 11 in monsoon VHDVRQ 7KH GDWD ZDV DOVR XVHG WR SUHSDUH KRQH\ ÀRZ DQG GHDUWK period of the region. The knowledge of blooming seasons and variation in plant species having different blooming season is important for sustainable management of bee colonies and for good honey harvest. The flowering duration of any given region helps in migratory beekeeping practice. +RQH\ ÀRZ DQG GHDUWK SHULRG For Paithan taluka of Aurangabad GLVWULFW WKH KRQH\ ÀRZ DQG GHDUWK SHULRG ZDV GHWHUPLQHG DQG DUH summarized in Table 1. The peak periods of honeybee foraging DFWLYLW\ KRQH\ ÀRZ SHULRG ZDV UHFRUGHG GXULQJ PLG 2FWREHU WR mid-December of winter season of the year. During the season, abundant bee floral plants were found blossoming. During honey flow period mid-October to mid-December of winter season, 15 wild plant species were recorded as source of food

for honeybees. Out of 15 plants, 6 plants species viz Asystasia dalzelliana Santapau, Azadirachta indica A Juss, Canavalia gladiate Jacq. DC., Melilotus indica L. All., Pentas lanceolate )URVVN 'HÀHUV DQG Tamarindus indica L., were nectar producing, two plant species viz., Amaranthus dubius Mart. Ex. Thell. and Plectranthus mollis Ait. Spreng, were pollen producing and 7 plant species viz., Delonix regia Hook Raf., Euphorbia hirta L., Launaea procumbens Roxb., Mimosa hamata Willd., Paracalyx scariosa Roxb. Ali., Parthenium hysterophorus L. and Tridax procumbens L. were both nectar and pollen producing. During the same period 7 agro-horticultural plants were blooming viz., Citrus aurantifolia Christm. Sw., Citrus aurantium L., Coriandrum sativum L., Cucumis sativus L., Cucurbita pepo L., Lagenaria siceraria Molina Standl., and Moringa oleifera Lamk., out which 1 agro-horticultural plant was pollen producing and remaining ZHUH ERWK QHFWDU DQG SROOHQ SURGXFLQJ 2WKHU EHH ÀRUD RI WKH UHJLRQ VXSSRUWHG KRQH\ SURGXFWLRQ 7KH ÀRZHULQJ SODQWV RI DQ area having good value as bee pasture are necessary to maintain

158

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Table 2. The agro-horticultural bee flora and floral calendar of Paithan taluka during October 2012–September 2013 Botanical name Allium cepa L. Brassica juncea L. Czern. Et cross. Cajanus cajan L. Millsp. Carthamus tinctorius L. Cicer arietinum L. Citrullus lanatus Thunb. Matsum. &Nakai Citrus aurantifolia Christm. Sw. Citrus aurantium L. Coriandrum sativum L. Cucumis melo L. Cucumis sativus L. Cucurbita pepo L. Cyamopsis dentata N.E.Br. Torre Helianthus annuus L. Lagenaria siceraria Molina Standl. Mangifera indica L. Moringa oleifera Lamk. Pisum sativum L. Punica granatum L. Rosa damascene Mill. Solanum melongena L. Triticum aestivum L.

Family

Flowering period.

Alliaceae Brassicaceae Fabaceae Asteraceae Fabaceae Cucurbitaceae Rutaceae Rutaceae Apiaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Leguminosae Asteraceae Cucurbitaceae Anacardiaceae Moringaceae Fabaceae Punicaceae Rosaceae Solanaceae Poaceae

Nectar N N N N N N

June– August July-August July-September May-July December – March. July – August October – January, July – September March- November January – December March – May August– October August– October June- August July – September October – February January-April November – February August- September March – June February-April January to March, June to July. Febrauary – April.

Bee forage value Pollen Nectar+Pollen P P NP NP NP P P NP NP NP NP NP NP NP NP P -

N – Nectar yielding plant. P – Pollen yielding plant. NP – Nectar and pollen yielding plants

2011; Kumar et al., 2013 and Waykar et al., 2014).

bee colonies. Honeybees visited these plants extensively for honey production and colony multiplication.

7KH SUHVHQFH RI QXPEHU RI GLYHUVL¿HG EHH ÀRUDO VSHFLHV LQ WKH area suggests that the study area is undoubtedly suitable for commercial beekeeping. Zamarlicki (1984) reported that the knowledge of honey plants is the most important factor in bee management and that the survival of honey bees is related to the abundance of bee plants. The success of bee plants in a given area including botanical and palynological aspects provides information on beekeeping potential (Sharma, 1972).

Flowring period (months)

7KH PLG 0D\ WR PLG $XJXVW SHULRG ZDV LGHQWL¿HG DV WKH GHDUWK period for honey bee at Paithan taluka of Aurangabad district. Based on the climatic conditions, the dearth period of study area may be divided into two periods. The mid-May to mid-June was critical dearth period with high temperature (over 390C), scarcity RI ZDWHU IRU ÀRZHULQJ SODQWV DQG ZDV XQIDYRUDEOH IRU KRQH\EHH foraging. The few of wild plants like, Abitulon pannosum (Forst. f.) Schlecht. Azadirachta indica A Juss. Chenopodium album L. Delonix regia Hook Raf., and Gliricidia sepium Jacq. Kunth 14 ex Steud, and agricultural plants like, Carthamus tinctorius L., Citrus aurantium L., and Punica 12 granatum L. were blossomed during the season.

Triticum aestivum

Solanum melongena

Rosa damascene

Pisum sativum

Punica granatum

Moringa oleifera

Mangifera indica

Helianthus anus

Lagenaria siceraria

Cucurbita pepo

Cyamopsis dentata

Cucumis melo

Cucumis sativus

Citrus aurantium

Coriandrum sativum

Citrus aurantifolia

Cicer arietinum

Citrullus lanatus

Cajanus cajan

Carthamus tinctorius

Allium cepa

Brassica juncea

The period of early monsoon i.e. from mid10 June to mid-August was critical dearth period because of unfavorable environmental condition 8 for foraging. Though relatively more flowers bloomed during rainy season, but due to heavy 6 and continuous rain fall, bee foraging activity was limited. The few wild plants like, Amaranthus 4 polygonoides L., Cyanotis cristata L. D. Don., Eleusine indica L. Gaertn., Lantana camara L., 2 Medicago sativa L., Spinaceae oleraceae L., and Ziziphus jujuba Mill and agricultural plants like, 0 Allium cepa L., Brassica juncea L. Czern. Et cross., Cajanus cajan L. Millsp., Citrullus lanatus Thunb. Matsum. & Nakai, Citrus aurantifolia Christm. Sw., Cyamopsis dentata N.E.Br. Torre and Solanum melongena L. were blossomed during the season. These minor sources are utilized by bees during the time of scarcity of food (Dalio, 2012). Similar studies have also been carried out by some Fig. 2. Flowering duration of agro-horticultural bee plants recorded during October investigators (Singh, 2005; Adhikari and Ranabhat, 2012-September 2013

'LYHUVLW\ RI EHH IRUDJLQJ ÀRUD DQG ÀRUDO FDOHQGDU RI 3DLWKDQ WDOXND RI $XUDQJDEDG GLVWULFW ,QGLD Beekeeping practice is very much useful for enhancing the quality and quantity of various agricultural crops. Sahli and Conner, (2007) reported that bee pollination increase the crop yield in a kind of mutualistic relationships. The economically important bee plants provide substantial quantity of pollen and nectar for bees during different months of the year. According to Thakur (2012), in India, about 80 percent or more of the crop plants were dependant on insect pollination. At different locations of Paithan taluka of Aurangabad district, four honey bee species, viz., A. dorsata, A. cerana indica; A. ÀRUHD and A. mellifera were reported. Among these four species $ ÀRUHD and A. dorsata were dominant bee species, whereas A. mellifera was introduced species and only few colonies of A. cerana indica were observed. The results revealed that 63 plant species were useful to honey bees as source of food, out of which 41 were wild and 22 were agro-horticultural plants. Mid-October to mid-December (winter VHDVRQ ZDV KRQH\ ÀRZ SHULRG DQG PLG 0D\ WR PLG $XJXVW ODWH summer and early monsoon season) was critical dearth period at Paithan taluka. The results also shows that the area has large number of plants producing nectar and both pollen and nectar than pollen producing. Paithan taluka has four honey bee species, viz., A. dorsata, A. cerana indica, $ ÀRUHD and A. mellifera. Among these $ ÀRUHD and A. dorsata are dominant bee species, whereas A. mellifera is introduced species and only few colonies of A. cerana indica were observed. Based on the study and available ÀRUD 3DLWKDQ WDOXND FDQ EH VXLWDEOH WR LQLWLDWH VXVWDLQDEOH DQG commercial beekeeping. However attention must be given to PDLQWDLQ WKH H[LVWLQJ EHH ÀRUD DQG PXOWLSOLFDWLRQ RI PXOWLSXUSRVH plant species in order to make it sustainable. In addition, there LV D QHHG WR SURYLGH DUWL¿FLDO IRRG WR EHHV GXULQJ WKH UDLQ\ DQG summer months.

Acknowledgements The authors are thankful to Prof. A.S. Dhabe, Department of Botany, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad for extending the help for identification and authentication of bee plants.

References Abrol, D.P. 2013. Asiatic Honey bee Apis cerana: Biodiversity Conservation and Agricultural production. Springer Dordrecht Heidelberg, London New York. $GKLNDUL 6 DQG 1 % 5DQDEKDW %HH ÀRUD LQ PLG KLOOV RI &HQWUDO Nepal. Botanica Orientalis. Journal Plant Science, 8: 45-56. Akratanakal, P., 1987. Beekeeping in Asia. FAO, United Nations. Allen, W. G., B. Peter, R. Bitner, A. Burquezs, S.L. Buchmann, J. Cane, P. A. Cox, V. Dalton, P. Feinsinger, M. Ingram D. Inouge, E.E. Jones, K. Kennedy, P. Kevan, H. Koopowitz, R. Medellin, M.S. Medellin and G.P. Nabnam, 1998. The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crop yields. Conserv. Biology, 12: 8-17.

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Bhattacharya, A. 2004. Flower visitor and fruit set of Anacardium occidentole. Annales Botanici. Fennici., 41: 385-392. %LVWD 6 DQG & 3 6KLYDNRWL +RQH\ EHH ÀRUD DW .DEUH Dolakha District. Nepal Agriculture Research Journal, 4-5: 18-25. Brodschneider, R. and K. Crailsheim, 2010. Nutrition and health in honeybees. Apidologie, 41: 278-294. Crane, E., 1990. Bees and Beekeeping: Science Practice and World Resource. Henemann News, Hally court Jordan Hill OX28Ej. Crane, P.R., E.M. Friis and K.R. Pedersen, 1989. Reproductive structure and function in Cretaceous Chloranthaceae. Plant Systematics and Evolution, 165: 211-226. Dalio, J.S. 2012. Cannabis sativa-An important subsistence pollen source for Apis mellifera. IOSR Journal of Pharmacy and Biological Sciences, 1: 1-3. Fluri, P. and S. Bogdanov, 1987. Age dependence of fat body protein in summer and winter bees (Apis mellifera). In: Chemistry and Biology of Social Insects, Eder, J; Rembold, H. (eds) Verlag J Peperny; Munic, Germany, 170-171. Free, J.B. 1970. Insect Pollination of Crops. Academic press, London, 544. Keller, I., P. Fluri and A. Imdorf, 2005. Pollen nutrition and colony development in honey bees: Part II. Bee World, 86(1): 3-10. Kumar, R., G.S. Rajput, R.C. Mishra and O.P. Agrawal, 2013. A study on assessment of duration of dearth period for Honey bees in Haryana, India. Munis Entomology Zoology, 8(1): 434-437. Rodinov, V.V. and Shabanshov, 1986. The Fascinating World of Bees. Mir Publishers, Moscow (Russia). 6DKOL + ) DQG - . &RQQHU 9LVLWDWLRQ HIIHFWLYHQHVV DQG HI¿FLHQF\ of 15 genera of visitors to wild radish, Raphanus raphanistrum (Brassicaceae). American Journal Botany, 94: 203-209. 6KDUPD 0 6WXGLHV LQ WKH ÀRZHU RI Datura stramonium Linn. in relation to bee-botany. Journal Palynology, 8: 17-21. Shrestha, K.1998. Dictionary of Nepalese Plant Names. Mandala Book Point, Kathmandu, Nepal. Singh, S.T. 2005. Bee plant diversity in Southern Peninsular India, Ph.D. thesis, submitted to University of Pune, India. 6LYDUDP 9 +RQH\ EHH ÀRUD DQG EHHNHHSLQJ LQ .DUQDWDND 6WDWH India. Proceedings of the 37th International Apicultural Congress, Apimondia, Durban, South Africa. 28 October -1 November 2001. Thakur, M. 2012. Bees as Pollinators – Biodiversity and Conservation. International Research Journal Agricultural Science Soil Science, 2(1): 1-7. Waykar, B., R.K. Baviskar and T.B. Nikam, 2014. Diversity of QHFWDULIHURXV DQG SROOHQLIHURXV EHH ÀRUD DW $QMDQHUL DQG 'XJDUZDGL hills of Western Ghats of Nasik district (M.S.) India, Journal of Entomology and Zoology Studies, 2(4): 244-249. Yadav, S. and H.D. Kaushik, 2012. Pollination syndrome in relation to insect pollinators. Advances in bio-ecology and management of insect pollinators of crops. Centre of advance faculty training department of entomology, Hisar Haryana, 204-210. Zamarlicki, C.C. 1984. Evaluation of honeybee plants in Burma – A case study. Proceedings of the FAO (UN) expert committee, 57-76. Submitted: December, 2014; Revised: March, 2015; Accepted: March, 2015

Journal

Journal of Applied Horticulture, 17(2): 160-164, 2015

Appl

'HYHORSPHQW RI DQ HIÀFLHQW in vitro regeneration protocol IRU ÀJ Ficus carica L.) S.S. Dhage, V.P. Chimote*, B.D. Pawar, A.A. Kale, S.V. Pawar and A.S. Jadhav State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri-413722, Maharashtra, India. *E-mail: vivekchimote@rediffmail.com

Abstract 7KH SUHVHQW LQYHVWLJDWLRQ ZDV XQGHUWDNHQ WR GHYHORS DQ HIÂżFLHQW in vitro UHJHQHUDWLRQ SURWRFRO LQ IRXU ÂżJ FXOWLYDUV viz., Poona Fig, Brown Turkey, Conadria and Deanna. Highest shoot tip establishment was observed in Deanna (100 %), followed by Conadria (79.2 %) and Brown Turkey (76.7 %) on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine (BAP), 0.5 mg/L gibberellic acid (GA3). Establishment of shoot tips was very poor in cultivar Poona Fig (11.7-13.3 %). Further inoculation of shoots on MS medium VXSSOHPHQWHG ZLWK PJ / LQGROH EXW\ULF DFLG ,%$ UHVXOWHG LQ ERWK PXOWLSOH VKRRWLQJ DV ZHOO DV URRWLQJ 6LJQLÂżFDQW QXPEHU RI newly formed shoots were observed in Conadria (4.7) and Deanna (3.8) as against in Brown Turkey (1) and Poona Fig (0.6). Highest root induction was observed in Conadria (73.3 %), followed by Deanna (52.2 %), Brown Turkey (26.7 %) and Poona Fig (24.4 %). 7KHVH UHVXOWV FRQÂżUPHG WKDW WKH VKRRW EXG HVWDEOLVKPHQW DQG PXOWLSOH VKRRW LQGXFWLRQ LQ ÂżJ LV KLJKO\ JHQRW\SH VSHFLÂżF $V WKH UHVSRQVH of popular cultivar Poona Fig to shoot tip culture was very poor, tender leaf explants were further used for regeneration study. Optimum regeneration was observed using MS medium supplemented with 4.0 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) for callusing; 7 PJ / WKLGLD]XURQ 7'= DQG PJ / ÄŽ QDSKWKDOHQH DFHWLF DFLG 1$$ IRU VKRRWLQJ DQG PJ / ,%$ IRU URRWLQJ Key words: )LJ UHJHQHUDWLRQ JHQRW\SH VSHFLÂżF VKRRW WLS FXOWXUH PXOWLSOH VKRRWLQJ URRWLQJ

,QWURGXFWLRQ 7KH FRPPRQ ÂżJ Ficus carica L.) is well known for its nutritive value and are consumed both as fresh and process dried. Figs are ULFK VRXUFH RI FUXGH ÂżEUH PLQHUDOV YLWDPLQ . DQG DQWLR[LGDQWV compounds (Vallejo et al., 2012). It is native to Southwest Asia and the Mediterranean region (from Afghanistan to Portugal). :RUOGZLGH ÂżJ LV ZLGHO\ FXOWLYDWHG ZLWK DQ DUHD RI ODNK KD and production of 10.93 lakh tonnes harvested in 2012. However despite of huge demand both cultivation area (5500 ha) and production (19,000 tonnes) is very low in India (FAOSTAT, ([RWLF FXOWLYDUV GR QRW ÂżQG IDYRU ZLWK ,QGLDQ IDUPHUV DV WKH\ DUH PRUH SURQH WR QHPDWRGH 7KH VHHGV RI ÂżJ DUH QRQ YLDEOH KHQFH WKH ÂżJV DUH SURSDJDWHG WKURXJK YHJHWDWLYH DSSURDFKHV However, these propagation approaches are slow and limited. Development of clonal propagation methods have numerous potential applications e.g., plant transformation, germplasm conservation, synthetic seeds and mutation breeding (Ji et al., 2011). Micropropagation has been successfully employed for rapid multiplication of genetically identical and superior quality planting material in many fruit crops. In vitro propagation in ÂżJ VHUYHV WKH SXUSRVH RI PDVV VFDOH SURGXFWLRQ RI KLJK TXDOLW\ planting material (Rout et al., 2006). In vitro UHJHQHUDWLRQ LQ ÂżJ using various explants such as shoot tips (Murithi et al., 1982; Haelterman and Docampo, 1994; Gella et al., 1998; Hepaksoy and Aksoy, 2006), nodal explants (Fraguas et al., 2004), leaves (Kim et al., 2007; Dhage et al., 2012; Soliman et al., 2010) and apical buds (Kumar et al., 1998; Gella et al., 1998) has been reported. 'HYHORSPHQW RI DQ HIÂżFLHQW UHJHQHUDWLRQ SURWRFRO LV HVVHQWLDO for successful in vitro SURSDJDWLRQ DQG WUDQVIRUPDWLRQ LQ ÂżJ Fig is recalcitrant in its production of adventitious shoots (Kim et al., 2007) and factors affecting shoot proliferation have not

been optimized (Fraguas et al., 2004). Thus, the objective of our study was to optimize in vitro UHJHQHUDWLRQ SURWRFRO LQ ÂżJ XVLQJ shoot tips and leaves.

Materials and methods Preparation of explants: $[LOODU\ VKRRW WLSV RI IRXU ¿J FXOWLYDUV viz., Poona Fig, Brown Turkey, Conadria and Deanna as well as tender leaves of Poona Fig were collected in morning hours from healthy mature plants from experimental orchard of the All India Coordinated Research Project Arid Zone Fruits, Mahatma Phule Krishi Vidyapeeth, Rahuri. These explants were sterilized with 0.1, 0.2, 0.3 % (w/v) mercuric chloride (HgCl2) or 4 % (w/v) sodium hypochlorite (NaClO) solution for 3, 5, 7, 9 min. They ZHUH IXUWKHU ULQVHG ¿YH WLPHV ZLWK VWHULOH GLVWLOOHG ZDWHU Shoot tip culture establishment: Initially shoot tip culture was attempted in cv. Poona Fig. However, very poor bud breaking was observed. In order to check whether initial poor results LQ FY 3RRQD )LJ ZDV GXH WR JHQRW\SH VSHFL¿F UHVSRQVH WKUHH other fig cultivars i.e. Brown Turkey, Conadria and Deanna were further included in the present study. Sterilized shoot tips were inoculated on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) supplemented with 2.5 or 3.5 mg/L 6-benzylaminopurine (BAP), 0.5 mg/L gibberellic acid (GA3), 100 mg/L ascorbic acid and 150 mg/L citric acid. They were initially kept in dark for a week and then incubated at 25 °C temperature with 16 h light period and 8 h dark period. Multiple shoot induction and rooting: Individual shoots from shoot tip induction medium were further transferred to MS medium with either of 1/2/3/4 mg/L of indole-3-butyric acid (IBA), 100 mg/L ascorbic acid, 150 mg/L citric acid and 0.2 % activated charcoal for further induction of shoots and roots.

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161

Table 1. Effect of growth regulators on in vitro shoot tip establishment Genotype

Treatment

Poona Fig

Brown Turkey

Conadria

Deanna

EM1

Establishment (%) 13.3 Âą0.70 a

Days to bud break/ sprouting 6.7 Âą0.02 a

Days to leaf emergence 7.6Âą0.03

Shoots with more than one leaf (%) 18.3 Âą0.61 a

Multiple shoot (%) 0.00

EM2

11.7 Âą0.65 a

7.0Âą0.02

7.9Âą0.05

20.8 Âą0.59 a

0.00

EM1

76.7Âą1.15

6.5Âą0.02

7. 5Âą0.03

90.8Âą0.85

0.00

EM2

53.3Âą0.48

6.7 Âą0.03 a

7.3 Âą0.04 a

52.5Âą0.83

0.00

EM1

79.2Âą0.59

6.1 Âą0.01 b

7.1Âą0.04

EM2

87.5Âą1.26

bc

EM1

100

EM2

62.5Âą0.50

6.2 Âą0.03

95.0Âą1.96

52.50

a

60.8Âą0.49

39.17

7.3 Âą0.02 a

94.2Âą0.99

41.67

a

54.2Âą0.48

60.00

7.2 Âą0.02

6.2Âą0.03 c 6.4Âą0.03

7.3 Âą0.03

2.08 0.07 0.103 2.75 CD at 5% EM1: MS + 2.5 mg/L BAP+ 0.5 mg/L GA3 + 100 mg/L ascorbic acid +150 mg/L citric acid EM2: MS + 3.5 mg/L BAP+ 0.5 mg/L GA3 + 100 mg/L ascorbic acid +150 mg/L citric acid *All values are means ÂąSE. Mean values in each column/row followed by the same lower-case letter(s) are not significantly different (P < 0.05) by the FCRD test.acid EM2: MS + 3.5 mg/L BAP+ 0.5 mg/L GA3 + 100 mg/L ascorbic acid +150 mg/L citric acid *All values are means ÂąSE. Mean values in each column/row followed by the same lower-case letter(s) are not significantly different (P < 0.05) by the FCRD test.

In vitro regeneration using leaf explants in cv. Poona Fig: Tender leaves were cut across the midrib and placed with adaxial surface up for callusing on MS medium supplemented with six different hormonal combinations (Table 3) along with 100 mg/L ascorbic acid and 150 mg/L citric acid. They were initially kept in dark for a week and then incubated at 25 °C with 16 h light period and 8 h dark period. All treatments of regeneration experiments had three replicates with 25 explants in each replication. Nine weeks after culture, the calli were transferred to shooting medium i.e. MS medium with ten different hormonal combinations (Table 4). Subculturing of cultured material was done after every 4 weeks. Shoots were transferred to MS medium with 1.0 mg/L IBA for rooting. Plantlets thus produced were transferred to pots containing coco peat and farmyard manure (2:1), and irrigated with water at regular intervals. They were initially covered

with plastic bags for a week and then kept in polycarbonated polyhouse.

5HVXOWV DQG GLVFXVVLRQ Preparation of explant: Contamination of axillary shoot tip and OHDI H[SODQWV ZDV PDMRU SUREOHP LQ ÂżJ GXULQJ HVWDEOLVKPHQW RI in vitro culture. In all the genotypes tested 0.2 % HgCl2 for 7 min was found to be optimum for shoot tip sterilization. Though 0.2 % HgCl2 for 9 min gave highest sterilization but percent explant establishment was low. Decrease in concentration of disinfectant and duration of treatment resulted in high percentage of contamination while increase in concentration leads to browning of shoot tip. Sterilization with 0.1 % HgCl2 for 7 min was found to be optimum for leaf explants of Poona Fig. Higher HgCl 2

Table 2. Response of fig genotypes to multiple shoots and root induction Genotype Poona Fig

Brown Turkey

Conadria

Deanna

CD at 5%

Treatment RM1 RM2 RM3 RM4 RM1 RM2 RM3 RM4 RM1 RM2 RM3 RM4 RM1 RM2 RM3 RM4

Rooting (%) 24.4Âą0.75a 0c 0c 0c 26.7Âą1.26b 0c 0c 0c 73.3Âą1.23 30Âą1.19 25.6Âą0.75ab 0c 52.2Âą0.63 0c 0c 0c 1.82

Shoot length (mm) 23.0Âą0.04a 14.0Âą0.04 23.0Âą0.03a 19.1Âą0.04 15.1Âą0.04 10.1Âą0.09 22.0Âą0.02 18.0Âą0.04 47.5Âą0.04 37.6Âą0.04 37.8Âą0.03 50.2 Âą0.06 30.0Âą0.05 42.7Âą0.06 35.3Âą0.03 25.0Âą0.02 0.13

Number of leaves/ shoot 1.7Âą0.02a 1.0Âą0.02c 2.2Âą0.02 1.8 Âą0.02a 1.8 Âą0.03a 1.0 Âą0.03c 2.3Âą0.03 1.8 Âą0.02a 8.1Âą0.09 6.5Âą0.04 5.7Âą0.04b 5.7 Âą0.04b 6.3Âą0.04 9.0Âą0.04 7.5Âą0.04 4.7Âą0.04 0.10

Newly formed shoots (#) 0.6Âą0.04b 0 0 0.5Âą0.02b 0.8Âą0.04 0 1.0Âą0.04 0 4.7Âą0.07 3.3Âą0.02 3.5Âą0.03 2.7Âą0.04 2.5Âą0.04 3.2Âą0.02 c 3.8Âą0.03 3.3Âą0.02 c 0.09

RM1: MS + 1 mg/L IBA, RM2: MS + 2 mg/L IBA, RM3: MS + 3 mg/L IBA, RM4: MS + 4 mg/L IBA *All values are means ÂąSE. Mean values in each column/row followed by the same lower-case letter(s) are not significantly different (P < 0.05) by the FCRD test.

162

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concentrations (0.2-0.4 %) and 4 % NaClO proved to be more toxic leading to browning and death of leaf explants.

(0-1.0 additional shoots/explant). Multiple shoots may be likely due to carry over effect of previous medium.

Shoot tip culture establishment: Browning due to oxidation of phenolic compounds, released from the cut ends of the explants is a major problem during in vitro cultures of woody plant species. In preliminary experiments it was observed that browning of explants hindered explant establishment in cultivar Poona Fig. The shoot tips that survived showed poor growth in terms of shoot elongation and leaf emergence. There was high-browning in both shoot buds and leaf tissues. Therefore in the present study antioxidants such as ascorbic acid and citric acid were used in all medium to reduce browning. Soliman et al. (2010) used different antioxidants such as polyvinyl-pyrrolidone, citric acid and ascorbic acids to control browning.

*HQRW\SH VSHFLÂżF UHVSRQVH WR ,%$ WUHDWPHQW ZDV REVHUYHG LQ terms of shoot length after 30 days of culture. Higher shoot length was observed in Conadria (37.6-50.2 mm) and Deanna (25.0-42.7 mm), as compared to Poona Fig (14.0-23.0 mm) and Brown Turkey (10.1-22.0 mm). Average number of leaves / shoots after 30 days of culture, was highest in Deanna (9.0) at 2 mg/L IBA followed by Conadria (8.1) at 1 mg/L IBA concentration. Even in other IBA treatments, high leaf bearing was observed in Conadria (5.7-6.5) and Deanna (4.7-7.5). However leaf bearing was very poor in Poona Fig (1.0-2.2) and Brown Turkey (1.0-2.3), with best treatment being 3 mg/L IBA in both cases.

During shoot tip culture, it was observed that establishment of H[SODQWV ZDV VLJQLÂżFDQWO\ LQĂ€XHQFHG E\ JHQRW\SH DQG JURZWK regulator used (Table 1). Highest shoot tip establishment was observed on MS medium supplemented with 2.5 mg/L BAP and 0.5 mg/L GA3. However in Conadria optimum shoot tip establishment was observed on MS medium supplemented with 3.5 mg/L BAP and 0.5 mg/L GA3. Establishment was higher in three genotypes i.e. Deanna (62.5-100 %), Conadria (79.2-87.5 %) and Brown Turkey (53.3-76.7 %). However, response to shoot tip establishment was very poor in Poona Fig (11.7-13.3 %). BAP KDYH EHHQ UHSRUWHG WR EH XVHIXO LQ ÂżJ VKRRW HVWDEOLVKPHQW DQG proliferation (Kumar et al., 1998; Kim et al., 2007). Mustafa and Taha (2012) reported enhanced shoot multiplication and callus IRUPDWLRQ IURP VKRRW WLS H[SODQWV RI GLIIHUHQW ÂżJ FXOWLYDUV XVLQJ 2.5 mg/L BAP. Hepaksoy and Aksoy (2006) used combination of BAP, GA3 and IBA for in vitro shoot tip culture as well as in PXOWLSOLFDWLRQ PHGLXP LQ ÂżJ *HQRW\SH VSHFLÂżF UHVSRQVH GXULQJ in vitro FXOWXUH LQ ÂżJ KDV DOVR EHHQ UHSRUWHG HDUOLHU +HSDNVR\ DQG Aksoy, 2006; Kim et al., 2007; Dhage et al., 2012). Number of leaves per shoot also varied with genotype and culture medium used (Table 1). Cultivar Poona Fig showed poor leaf bearing with only 17.7 % shoots having multiple leaves as compared to other three genotypes (52.5-95 %). There was not much effect of genotype and growth regulator on days to bud sprouting (6.1-7.0 days) and leaf emergence (7.1-7.9 days). Earliest bud sprouting (6.1 days) and leaf emergence (7.1 days) was observed in Conadria genotype. Callus formation at the base was observed in Brown Turkey and Poona Fig after 12-16 days. Multiple shoot formation was observed only in Deanna (41.7 and 60.0 %) and Conadria (52.5 and 39.17%) at BAP concentrations of 2.5 mg/L and 3.5 mg/L, respectively with GA3 (0.5 mg/L). However, no multiple shooting was observed in genotype Poona Fig and Brown Turkey in either treatment. Shoot tip culture studies clearly indicated that Poona Fig was very poor in response to initial in vitro morphogenesis as compared to other genotypes. Multiple shoot induction and rooting: Individual shoots obtained from shoot tip culture were transferred to rooting medium containing four different IBA combinations (1-4 mg/L). However, multiples shoots were observed in addition to rooting. 5HVSRQVH WR VKRRW PXOWLSOLFDWLRQ ZDV KLJKO\ JHQRW\SH VSHFLÂżF (Table 2). Higher new shoot induction was observed in Conadria (2.7-4.7 additional shoots/explant) and Deanna (2.5-3.8 additional shoots/explant). Negligible new shoot induction was observed in Poona Fig (0-0.6 additional shoots/explant) and Brown Turkey

Highest rooting percentage was observed in MS medium supplemented with 1 mg/L IBA in all genotypes i.e. Conadria (73.3 %), followed by Deanna (52.2 %), Brown Turkey (26.7 %) and Poona Fig (24.4 %) (Table 3). However, in Brown Turkey shoots exhibited only aerial rooting. Cultivar Conadria also showed significant rooting at 2 mg/L and 3 mg/L IBA concentrations. However, there was almost no rooting in rest of genotype-IBA treatment combinations. Earlier, there are reports RI URRWLQJ LQ ¿J DW YDU\LQJ OHYHOV RI ,%$ i.e. 0.5 mg/L IBA and NAA (Danial et al., 2014); 1 mg/L IBA (Yakushiji et al., 2003; Soliman et al., 2010); 2 mg/L IBA (Kumar et al., 1998) and hormone free medium (Yakushiji et al., 2003; Kim et al., 2007). In vitro regeneration using leaf explants in cv. Poona Fig: Poona Fig is very popular local cultivar in western India due to its JRRG ÀDYRU HDVLO\ UHPRYDEOH VNLQ DQG VRIW VHHGV +RZHYHU WKLV variety showed very poor response to shoot tip culture therefore OHDI H[SODQWV IURP ¿HOG ZHUH XVHG IRU UHJHQHUDWLRQ VWXG\ 0RVW of the reports published are based on use of leaf explants derived IURP VKRRW WLS FXOWXUH LQ ¿J <DNXVKLML et al., 2003; Soliman et al., 2010; Dhage et al., 2012). Browning was observed in all media, of which least browning (18.7-28.0 %) was observed on medium containing only 2,4-dichlorophenoxy acetic acid (2,4-D), while highest incidence of browning (58.7-68.0 %) was observed on both combinations RI %$3 DQG Ď QDSKWKDOHQH DFHWLF DFLG 1$$ 'D\V UHTXLUHG IRU callus treatments also varied with treatments, ranging from 35.345.7 days. Late callus formation (48.3-49.0 days) was observed on medium containing combinations of kinetin and 2,4-D. Among all treatments, calli induction frequency varied from 21.3 % to 89.3 % (Table 3). Highest (89.3 %) and earliest callus Table 3. Callus formation in cv. Poona Fig after 60 days of inoculation Hormonal concentration (mg/L) BAP NAA 8.0 8.0 10 .0 10.0 2,4-D Kinetin 2.0 0 2.0 0.2 4.0 0 4.0 0.4 LSD (P=0.05)

Callusing (%)

Browning Days to callus (%) initiation

21.3Âą0.92 60.0Âą1.64

68.0Âą1.4 58.7Âą0.77

45.7Âą0.88 40.3Âą0.88 a

66.7 Âą1.32 28.0Âą1.5 41.7 Âą0.33 a 48.0Âą1.64 40.0Âą1.4 49.0Âą0.58 b 89.3Âą2.38 18.7Âą0.99 35.3Âą0.33 41.3Âą0.77 41.3Âą0.77 48.3 Âą0.33 b 4.75 3.60 1.87 *All values are means ÂąSE. Mean values in each column/row followed by the same lower-case letter(s) are not significantly different (P < 0.05) by the CRD test.

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163

induction was observed when leaf explants were cultured on MS medium supplemented with 4.0 mg/L 2,4-D. Next highest calli formation (66.7 %) was observed on medium containing 2.0 mg/L 2,4-D. This is as expected since it is well known that 2,4-D serves as a callusing agent at higher concentrations. Addition of NLQHWLQ UHVXOWHG LQ VLJQLÂżFDQW UHGXFWLRQ RI FDOOXVLQJ DQG LQFUHDVH in browning at both 2,4-D concentrations. This result contradicts earlier reports of Soliman et al. (2010) of higher callus formation (86%) obtained on MS medium supplemented with 2 mg/L 2,4-D and 0.2 mg/L kinetin. Sixty per cent callusing was observed on MS medium supplemented with 10.0 mg/L BAP and 10.0 mg/L NAA. Lowest callus formation (21.33 %) was observed on medium supplemented with 8.0 mg/L BAP and 8.0 mg/L NAA. Previously, Soliman et al. (2010) reported 73 and 68% callusing, respectively on MS medium with 10.0 mg/L BAP and NAA each and 8.0 mg/L BAP and NAA each. For shoot induction ten different medium were used, out of which VKRRW LQGXFWLRQ ZDV REVHUYHG RQO\ LQ ÂżYH RI WKHP WKUHH RI ZKLFK were supplemented with thidiazuron (TDZ) and NAA (Table 4). These three NAA and TDZ combinations showed earliest shooting (26.0-35.7 days); longest shoot length (19.2-22.3 mm); most shoots/ callus (2.4-2.8). Highest shoot induction (82.7 %) was observed in MS medium supplemented with 7.0 mg/L TDZ DQG PJ / 1$$ 7'= KDV EHHQ UHSRUWHG WR EH HIÂżFLHQW LQ stimulating adventitious shoot production in several recalcitrant ZRRG\ SODQWV LQFOXGLQJ ÂżJ(Huetteman and Preece, 1993). Soliman et al. (2010) reported optimum indirect shooting on medium comprising 7 mg/L TDZ in combination with 0.25 mg/L NAA. Shooting was also observed in MS medium supplemented with 8.0 mg/L N6-[2-Isopentyl] adenine (2iP) + TDZ 2.0 mg/L and 8.0 mg/L BAP + 2.0 mg/L kinetin. Later treatment showed delayed shooting (44 days), poor shoot growth (12.2 mm) and lowest multiples (1.3). High browning was observed in the treatments that failed to induce shoot. After shooting for 4-5 weeks regenerated shoots were rooted on full-strength MS medium with 1 mg/L IBA and acclimatized into pots containing cocopeat and manure in polycarbonate polyhouse. ,Q VXPPDU\ KLJKO\ HIÂżFLHQW VKRRW PXOWLSOLFDWLRQ DQG UHJHQHUDWLRQ Table 4. Details of shoot induction from calli of cv. Poona Fig Hormonal Percent concentration shoot ( mg/L) induction NAA TDZ 0.25 7.0 82.7Âą0.99 0.50 7.0 64.0Âą1.38 0.25 8.0 25.3Âą2.31 a 2iP TDZ 8.0 2.0 45.3Âą2.03 10.0 2.0 0.0 20.0 2.0 0.0 2iP 30.0 0.0 BAP Kinetin 8.0 2.0 22.7Âą0.92a 8.0 4.0 0.0 10.0 2.0 0.0 3.39 LSD (P=0.05)

Days to shoot induction

Shoot length (mm)

Number of shoots/ explant

30.7Âą0.88 20.2Âą0.09 2.8Âą0.15 26.0Âą0.58 22.3Âą0.09 2.5Âą0.09a 35.7Âą0.88 19.2Âą0.09 2.4Âą0.06 a 41.0Âą0.58 21.6Âą0.09 0.0 0.0 0.0 0.0 0.0

0.0

44.0Âą0.58 12.2Âą0.12 0.0 0.0 0.0 0.0 2.25 0.30

2.0Âą0.06 0.0 0.0 0.0 1.3Âą0.06 0.0 0.0 0.28

Fig. 1. In vitro regeneration in fig. (A) Shoot tip establishment on MS+2 mg/L BAP + 0.5 mg/L GA3 in Conadria; (B) Multiple shoot induction MS + 1 mg/L IBA in Conadria; (C) Rooting; (D) Aerial rooting in cv. Brown Turkey:; (E): Callus initiation from cv. Poona Fig leaf explant on 4 mg/L 2,4-D; (F): Shoot induction from the callus on MS + 0.25 mg/L NAA + 7 mg/L TDZ in cv. Poona Fig.

protocol was developed. Shoot bud breaking at 2.5-3.5 mg/L BAP + 0.5 mg/L GA3 and further inoculation at 1.0 mg/L IBA resulted in multiple shooting as well as rooting. Best regeneration was observed after callusing from leaf explants on 4.0 mg/L 2, 4 D followed by subsequent shooting on 7 mg/L TDZ and 0.25 mg/L NAA. The present investigation may be helpful for commercial PLFURSURSDJDWLRQ RI GLIIHUHQW YDULHWLHV RI ÂżJ

Acknowledgements The authors are grateful to authorities of Mahatma Phule Krishi Vidyapeeth, Rahuri for providing necessary facilities to undertake WKLV VWXG\ +HOS UHQGHUHG E\ 'U 9 6 6XSH 2IÂżFHU ,QFKDUJH All India Coordinated Research Project on Arid Zone Fruits, Mahatma Phule Krishi Vidyapeeth, Rahuri, India during this study is also gratefully acknowledged.

164

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References Danial, G.H., D.A. Ibrahim, S.A. Brkat and B.M. Khalil, 2014. Multiple VKRRWV SURGXFWLRQ IURP VKRRW WLSV RI ÂżJ WUHH Ficus carica L.) and callus induction from leaf segments. Intl. J. Pure Appl. Sci. Tech., 20(1): 117-124. Dhage, S.S., B.D. Pawar, V.P. Chimote, A.S. Jadhav and A.A. Kale, 2012. In vitro FDOOXV LQGXFWLRQ DQG SODQWOHW UHJHQHUDWLRQ LQ ÂżJ Ficus carica L.). J. Cell Tiss. Res., 12: 3395-3400. FAOSTAT, 2014. http://faostat3.fao.org/browse/Q/QC/E Fraguas, C.B., M. Pasqual, L.F. Dutra and J.O. Cazetta, 2004. 0LFURSURSDJDWLRQ RI ÂżJ Ficus carica L.) ‘roxo de valinhos’ plants. In vitro Cell. Dev. Biol. Plant, 40: 471-474. Gella, R., J.A. Marin, M.L. Corrales and F. Toribio, 1998. Elimination RI ÂżJ PRVDLF IURP ÂżJ VKRRW WLS FXOWXUHV E\ WKHUPRWKHUDS\ Acta Hort., 480: 173-177. Haelterman, R.M. and D.M. Docampo, 1994. In vitro propagation of PRVDLF IUHH ÂżJ Ficus carica L.) cultivars, using thermotherapy and shoot tip cultures. Revista de Investigaciones Agropecuarias, 25(3): 15-22. Hepaksoy, S. and U. Aksoy, 2006. Propagation of Ficus carica L. clones by in vitro culture. Biol. Plant., 50: 433-436. Huetteman, C.A. and J.E. Preece, 1993. Thidiazuron: A potent cytokinin for woody plant tissue culture. Plant Cell Tiss. Org. Cult., 33: 105119. Ji, A., X. Geng, Z. Yan, H. Yang and G. Wu, 2011. Advances in somatic embryogenesis research of horticultural plants. Amer. J. Plant Sci., 2: 727-732.

Kim, K.M., M.Y. Kim, P.Y. Yun, T. Chandrasekhar, H.Y. Lee and P.S. Song, 2007. Production of multiple shoots and plant regeneration IURP OHDI VHJPHQWV RI ÂżJ WUHH Ficus carica L.). J. Plant Biol., 50(4): 440-446. Kumar, V., A. Radha and S.K. Chitta, 1998. In vitro plant regeneration of ÂżJ Ficus carica L. cv. Gular) using apical buds from mature trees. Plant Cell Rep., 17: 717-720. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15: 473-497. Murithi, L.M., T.S. Rangan and B.H. Waite, 1982. In vitro propagation RI ÂżJ WKURXJK VKRRW WLS FXOWXUH HortScience, 17: 86-87. 0XVWDID 1 6 DQG 5 $ 7DKD ,QĂ€XHQFH RI SODQW JURZWK UHJXODWRUV and subculturing on in vitro PXOWLSOLFDWLRQ RI VRPH ÂżJ Ficus carica) cultivars. J. Appl. Sci. Res., 8(8): 4038-4044. Rout, G.R., A. Mohapatra and M.S. Jain, 2006. Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects. Biotechnol. Adv., 24: 531-560. 6ROLPDQ + , 0 *DEU DQG 1 $EGDOODK (IÂżFLHQW WUDQVIRUPDWLRQ DQG UHJHQHUDWLRQ RI ÂżJ Ficus carica L.) via somatic embryogenesis. GM Crops, 1: 47-58. Vallejo, F., J.G. Marin and F.A. Tomas-Barberan, 2012. Phenolic FRPSRXQG FRQWHQW RI IUHVK DQG GULHG ÂżJV Ficus carica L.). Food Chem., 130: 485-492. Yakushiji, H., N. Mase and Y. Sato, 2003. Adventitious bud formation DQG SODQWOHW UHJHQHUDWLRQ IURP OHDYHV RI ÂżJ Ficus carica L.). J. Hort. Sci. Biotech., 78: 874-878. Submitted: October, 2014; Revised: December, 2014; Accepted: January, 2015

Journal

Journal of Applied Horticulture, 17(2): 165-168, 2015

Appl

Effect of packaging in extending shelf life of fresh curry leaves Dawn C.P. Ambrose*, S.J.K. Annamalai and Ravindra Naik Central Institute of Agricultural Engineering, Regional Centre, Coimbatore-3, Tamil Nadu, India. *E-mail: dawncp@yahoo.com

Abstract Curry leaf, which is a leafy spice, used in Asian culinary has limited shelf life. Investigation was carried out to extend the shelf life of fresh curry leaf by prepackaging in different packaging materials i.e., polyethylene bags of 38 and 75 micron thickness, polypropylene bags of 20 and 38 micron thickness and stored under ambient (30r2qC) and refrigerated (5 r1qC) conditions. It was found that prepackaging fresh and stripped curry leaf in polypropylene bag of 20 micron thickness with 0.1 % vent area of 5 mm diameter vent could prolong the keeping quality for 4 days under ambient storage. Also under refrigerated condition, under the same packaging treatment, the sample kept well for a period of 16 days in polyethylene bag of 75 micron thickness. Key words: Curry leaf, prepackaging, color scores, physiological weight loss, volatile oil

,QWURGXFWLRQ Curry leaf (Murraya koenigii) or curry patte (Hindi) are derived from a handsome, aromatic more or less deciduous shrub or small tree, found almost throughout India including Andaman Islands XS WR DQ DOWLWXGH RI PHWHUV ,W KDV EHHQ XVHG DV ÀDYRXULQJ agent in Indian food since time immemorial. It is cultivated on a commercial scale in large areas in Tamil Nadu, Andhra Pradesh and Karnataka. The leaves have 66.3% moisture, 6.1 SURWHLQ FDUERK\GUDWH ¿EUH PJ SKRVSKRUXV 0.93 mg iron and 7.56 mg E- carotene per 100 g (Shankaracharya DQG 1DWDUDMDQ &XUU\ OHDYHV SURYLGH KHDOWK EHQH¿WV E\ SURYLGLQJ WKH PXFK QHHGHG GLHWDU\ ¿EHUV VHYHUDO HVVHQWLDOV minerals and vitamins to the human diet. The antioxidant and anticarcinogenic effect of curry leaves have been studied and it has been reported that the curry leaves have a high potential as a reducer of the toxicity of carcinogens (Khanum et al., 2001). Among the horticultural products, leafy vegetables are one that leads to spoilage rapidly. There is change in developmental, structural, physiological and biochemical processes leading to senescence of the leaves. The most obvious symptoms of VHQHVFHQFH LQ WKH OHDYHV DUH ORVV RI IUHVK ZHLJKW VKULYHOLQJ ÀDYRU changes etc. (Halvey and Mayak, 1981). One of the symptoms of senescence in harvested leafy vegetables is loss of greenness with the degradation of chlorophyll (Yamauchi and Watada, 1991). Loaiza and Cantwell (1997) stated that fresh coriander with very good visual quality was maintained for 18 to 22 days at 0 qC, 12 to 14 days at 5 qC, 7 to 8 days at 7.5 qC and only 4 to 5 days at 10 qC. Curry leaf is one of the most widely traded leafy spices all over the world. The leaves have good export potential besides internal consumption (Lathan Kumar et al., 2003). The leaves are available throughout the year. Fresh curry leaves rapidly lose their moisture and get wilted. They are highly perishable and cannot be retained fresh for more than a day. Extending the shelf life of fresh curry leaf will improve its export market owing to the international demand. Also prepackaging of fresh leaves

helps in easy transportation and handling of the produce. Hence an attempt has been made to extend the shelf life of fresh curry leaves by prepackaging in different packaging materials under different storage conditions.

Materials and methods Raw material procurement and preparation: Curry leaves of the local cultivar, Senkaambu with pink petiole were obtained from the Horticultural Farm of TNAU, Coimbatore for the study. The leaves were harvested during the early morning hours. Fresh curry leaves were washed and air-dried to remove the surface PRLVWXUH 7KH\ ZHUH VWULSSHG IRU WKH OHDĂ€HWV /HDYHV UHYHDOLQJ abnormalities like yellowing, pest damage were eliminated. Prepackaging of fresh curry leaf: Prepackaging of fresh curry leaf was carried out in bags having perforations and bags without perforations. The number of holes for providing different ventilation levels, 0.1, 0.2 and 0.3 % vent were decided based on the total area of the bag and number of holes. The prepackaging treatments were as follows: Non-perforated bag – (T1), perforated bag having 5 mm diameter holes and 0.1 % vent – (T2), 0.2 % vent– (T3), 0.3 % vent– (T4), Perforated bag having 1 mm diameter holes and 0.1 % vent – (T5), 0.2 % vent – (T6) and 0.3 % vent – (T7) The experiments were carried out in three replications. For each replication, 50 g of fresh curry leaves was used. The samples were packed in different packaging materials viz., polyethylene bags of 38 and 75-micron thickness, polypropylene bags of 20 and 38 micron thickness having a size of 22x15 cm. They were then sealed and kept for storage under ambient (30Âą2°C) and refrigerated conditions (5 Âą1°C) to evaluate their shelf life. Statistical analysis: An ANOVA procedure for the statistical analysis of the results was done by using the “AgResâ€? software. 6LJQLÂżFDQFH ZDV WHVWHG DW P= 0.05.

166

Effect of packaging in extending the shelf life of fresh curry leaves

5HVXOWV DQG GLVFXVVLRQ Effect of packaging treatment on the shelf life of fresh curry leaf at ambient storage: Quality characteristics of fresh culinary herbs include a fresh appearance, uniformity of leaf size, form DQG FRORU FKDUDFWHULVWLF DURPD DQG ÀDYRU DQG D ODFN RI GHIHFWV such as decay or yellowing (Cantwell and Reid, 1993). Curry leaves, kept under various packaging treatments were evaluated for their shelf life using the color scale ranging from 5 to 1 (dark green and fresh to light green with >20% browning). The VDPSOHV SDFNHG LQ QRQ SHUIRUDWHG EDJV RI YDULRXV SDFNDJLQJ ¿OPV showed accelerated browning and had a poor shelf life compared WR WKRVH SDFNHG LQ SHUIRUDWHG EDJV 7KLV ZDV VLPLODU WR WKH ¿QGLQJV on storage of brinjals reported by Talukder et al. (2003) where brinjals became rotten in non perforated polyethylene bag but remained fresh for six days in perforated polyethylene bags. +HQFH SHUIRUDWLQJ ¿OP SDFNDJHV ZDV IRXQG WR EH QHFHVVDU\ IRU adequate oxygen to prevent anaerobic respiration and to avoid CO2 injury thereby extending the shelf life. Five mm diameter holes covering a ventilation area of 0.1% gave better results in terms of freshness retention during storage both at ambient and refrigerated conditions, compared to other pre-treatments. Higher Table 1. Color scores of fresh curry leaf packed in various packaging films during storage at ambient condition Packaging Packaging materials Days of storage treatment 0 2 4 6 Non perforated 20 micron Polypropylene 5 2 38 micron Polypropylene 5 3 1 38 micron Polyethylene 5 2 75 micron Polyethylene 5 3 5 mm diameter. 20 micron Polypropylene 5 5 5 4 Hole, 0.1% vent 38 micron Polypropylene 5 5 4 3 38 micron Polyethylene 5 5 4 2 75 micron Polyethylene 5 4 5 5 mm diameter. 20 micron Polypropylene 5 5 4 2 Hole, 0.2% vent 38 micron Polypropylene 5 4 2 38 micron Polyethylene 5 4 2 75 micron Polyethylene 5 4 3 5 mm diameter. 20 micron Polypropylene 5 5 3 2 Hole, 0.3% vent 38 micron Polypropylene 5 4 1 38 micron Polyethylene 5 3 2 75 micron Polyethylene 5 4 2 1 mm diameter. 20 micron Polypropylene 5 5 5 5 Hole, 0.1% vent 38 micron Polypropylene 5 5 5 4 38 micron Polyethylene 5 5 4 3 75 micron Polyethylene 5 5 5 5 1 mm diameter. 20 micron Polypropylene 5 5 4 2 Hole, 0.2% vent 38 micron Polypropylene 5 5 3 38 micron Polyethylene 5 4 3 75 micron Polyethylene 5 4 2 1 mm diameter. 20 micron Polypropylene 5 4 2 Hole, 0.3% vent 38 micron Polypropylene 5 4 38 micron Polyethylene 5 4 2 75 micron Polyethylene 5 4 2 LSD=0.055 (P=0.05) Color scale (5 to 1 scale): 5= dark green; 4= bright green; 3= light green with yellowing or browning affecting <5% of leaf area; 2= light green with noticeable yellowing or browning; 1= light green with >20% yellowing or browning

percentage of vent area resulted in poor quality of the product both in the case of 5 mm and 1 mm diameter holes. Under ambient storage (30r2qC), curry leaves could retain their freshness up to four days in polypropylene bags of 20 and 38 micron thickness. The shelf life of fresh curry leaf in polyethylene bags of 38 and 75 micron thickness was found to be four and three days, respectively. It was noted that polypropylene bags gave better result than polyethylene bags under ambient condition (30r2qC). This may be due to the reason that at ambient condition, the heat of respiration is more, which results in condensation of moisture inside the bags due to respiration. Since, polyethylene bags have high barrier property, the built up heat inside package leads to accelerated browning and decay. Based on the visual necrotic symptoms seen, the color score was arrived at (Table 1). Statistical analysis revealed that among the different treatments, there ZDV VLJQLÂżFDQW GLIIHUHQFH LQ WKH FRORU VFRUHV RI WKH VDPSOHV LQ perforated and non perforated bags. The color score of the samples in non perforated bags was poorer than rest of the treatments. Among the different packaging treatments, samples stored in 5 mm diameter hole perforations at 0.1% vent gave good results. Similarly, among the different packaging materials used, samples packed in 20 micron polypropylene gave best results compared to other materials. Effect of packaging treatment on the shelf life of fresh curry leaf at refrigerated storage: Under refrigerated storage (5 r1qC), the samples retained their freshness more than those kept under ambient condition. From the color scores of samples kept under refrigerated condition (5 r1qC) (Table 2), it could be seen that samples kept in 75 micron PE bag gave better results than other samples. The samples in 75 micron PE bags could retain their freshness up to 16 days, which is an indication of their better shelf life. However, the shelf life of samples kept in polypropylene bags was lesser than those in polyethylene bags under refrigerated conditions. This may be due to the reason that under refrigerated condition (5 r1qC); the metabolic activity of any living material is slowed down. In other words, respiration rate is slow under such conditions. Hence, the samples retain their freshness for a long time than at ambient condition. In case of polyethylene bags, because of their barrier property, there is no migration of moisture towards inside or outside of the bag. Packing of fresh herbs in polyethylene lined cartons reduced water loss and prevented wilting stored at 6 ÂşC for 5 days (Aharoni et al., 1989). However, in polypropylene bags the moisture condensed over the surface during storage penetrates into the bag thereby accelerating its spoilage. Statistical analysis of the samples showed that there was a significant difference among the treatments and packaging material during storage. Among the various treatments, samples packed in non perforated bags gave poor results compared to rest of the packaging treatments and hence not found suitable. Among the various packaging materials used, samples stored in 75 micron polyethylene bags gave best results at all treatments. However among the treatments, 5mm diameter hole at 0.1 % vent gave the highest color score for 75 micron thickness polyethylene bags. Effect of storage on the physiological weight loss of curry leaf: The physiological loss in weight of the samples packaged LQ YDULRXV SDFNDJLQJ ÂżOPV OLNH SRO\HWK\OHQH DQG SRO\SURS\OHQH during storage under ambient (30r1qC) and refrigerated condition (5 r1qC) was recorded daily.

Effect of packaging in extending the shelf life of fresh curry leaves

167

Table 2. Color scores of fresh curry leaf packed in various packaging films during storage at refrigerated condition Packaging treatment Packaging materials Days of storage 0 2 4 6 8 10 Non perforated 20 micron Polypropylene 5 4 38 micron Polypropylene 5 4 38 micron Polyethylene 5 4 75 micron Polyethylene 5 5 3 5 mm diameter. Hole, 0.1% vent 20 micron Polypropylene 5 5 5 4 2 38 micron Polypropylene 5 5 5 5 4 3 38 micron Polyethylene 5 5 4 3 75 micron Polyethylene 5 5 5 5 5 5 5 mm diameter. Hole, 0.2% vent 20 micron Polypropylene 5 5 4 2 38 micron Polypropylene 5 5 5 4 38 micron Polyethylene 5 5 4 3 3 75 micron Polyethylene 5 5 5 5 5 5 5 mm diameter. Hole, 0.3% vent 20 micron Polypropylene 5 5 3 2 38 micron Polypropylene 5 5 4 3 38 micron Polyethylene 5 5 3 75 micron Polyethylene 5 5 5 5 4 4 1 mm diameter. Hole, 0.1% vent 20 micron Polypropylene 5 5 5 5 4 2 38 micron Polypropylene 5 5 5 4 4 3 38 micron Polyethylene 5 5 4 3 75 micron Polyethylene 5 5 5 5 5 5 1 mm diameter. Hole, 0.2% vent 20 micron Polypropylene 5 5 5 4 3 38 micron Polypropylene 5 5 4 3 38 micron Polyethylene 5 5 4 3 75 micron Polyethylene 5 5 5 5 5 5 1 mm diameter. Hole, 0.3% vent 20 micron Polypropylene 5 5 3 2 38 micron Polypropylene 5 5 4 3 38 micron Polyethylene 5 5 5 2 LSD=0.023 (P=0.05)

Non perforated

1 mm diameter hole, 0.1% vent

5 mm diameter hole, 0.1% vent

1 mm diameter hole, 0.2% vent

5 mm diameter hole, 0.2% vent 5 mm diameter hole, 0.3% vent

1 mm diameter hole, 0.3% vent

16

16 5 3 3 4 -

18 3 3 3 -

At 0.1 % vent level, physiological weight loss was minimum for 1 mm diameter and 5 mm diameter holes, compared to other vent levels under ambient and refrigerated storage. Non perforated 5 mm diameter hole, 0.1% vent 5 mm diameter hole, 0.2% vent 5 mm diameter hole, 0.3% vent

1 mm diameter hole, 0.1% vent 1 mm diameter hole, 0.2% vent 1 mm diameter hole, 0.3% vent

16

12

12

8

4

0

14 5 4 3 4 4 -

It could be seen that the PWL encountered during the period of storage was at a slow rate at refrigerated condition (Fig. 2). In the cold storage, PWL is lower due to reduced metabolic activity (respiration rate) and lower difference of relative humidity between the fruit surface and the storage environment. Nasrin et al. (2008) reported that tomatoes stored in perforated polyethylene bags at refrigerated storage encountered minimum weight loss.

Physiological weight loss (%)

Physiological weight loss (%)

The physiological weight loss (PWL) of the samples packed in 20 micron thickness at various levels of ventilation and vent diameter DW DPELHQW FRQGLWLRQ LV SUHVHQWHG LQ )LJ )URP WKH ÂżJXUH LW could be seen that the trend of percent weight loss was increasing with the advancement of storage period. The physiological weight loss was more in the case of perforated samples than the nonperforated one. There was a gradual rise in the weight loss of the samples during the period of storage. Vent holes of 5mm diameter, covering 0.1% vent area and 1 mm diameter hole covering 0.1% vent area encountered less weight loss during storage compared to other ventilation levels.

12 5 5 4 5 5 -

0

1

2

3 Days

4

5

Fig.1. Physiological weight loss of fresh curry leaves in 20 micron polypropylene at ambient storage

8

4

0

0

2

4

6

8

10

12

14

16

18

Days

Fig. 2. Physiological weight loss of curry leaf in 75 micron polyethylene in refrigerated storage

168

Effect of packaging in extending the shelf life of fresh curry leaves

Effect of storage on the volatile oil content of the prepackaged curry leaf samples: Curry leaf stored in 20 micron thickness polypropylene at 5 mm diameter perforation, 0.1 % vent gave better results in terms of shelf life of the produce at ambient storage. Similarly, the samples in 75 micron thickness polyethylene at 5 mm diameter perforation, 0.1 % vent under refrigerated condition IRU VKHOI OLIH 7KH YRODWLOH RLO FRQWHQW ZKLFK GHFLGHV WKH ÀDYRU characteristic of curry leaf, was estimated periodically for the optimized sample both under ambient (30r2 qC) and refrigerated conditions. There was no change in the volatile oil content at 4 days ambient storage. However there was only negligible change in the volatile oil at the 15 days of storage under refrigerated conditions (Table 3). The study revealed that the shelf life of fresh curry leaf is influenced by prepackaging material and its thickness. Prepackaging fresh and stripped curry leaf in polypropylene bag Table 3. Changes in volatile oil content of fresh curry leaf during storage Packaging treatment 20 micron polypropylene, 5 mm diameter hole, 0.1% vent

Days of storage

Volatile oil (%)

Ambient 0 2

0.60 0.60

4 75 micron polyethylene, 5 mm diameter hole, 0.1% vent

0.60 Refrigerated

0 5 10 15

0.60 0.60 0.60 0.56

of 20 micron thickness with 0.1 % vent area of 5 mm diameter vent could prolong the keeping quality for 4 days under ambient storage. Under refrigerated condition, the sample kept well for a period of 16 days in polyethylene bag of 75 micron thickness.

References $KDURQL 1 $ 5HXYHQL DQG 2 'YLU 0RGL¿HG DWPRVSKHUHV LQ ¿OP SDFNDJHV GHOD\ VHQHVFHQFH DQG GHFD\ RI IUHVK KHUEV Acta Hort., 258: 255-262. Cantwell, M.I. and M.S. Reid, 1993. Postharvest physiology and handling of fresh culinary herbs. J. Herbs, Spices, Medicinal Plants, 1: 93-127. Halevy, A.H. and S. Mayak. 1981. Senescence and post harvest SK\VLRORJ\ RI FXW ÀRZHUV 3DUW ,, Hort. Rev., 3: 59-143. Loaiza, Julio and Maria Cantwell, 1997. Postharvest physiology and quality of cilantro (Coriandrum sativum L.). Food Science, 32: 104-107. Khanum, F., .K.R. Anilakumar, K.K.R. Sudarshana and K.R. Viswanathan, 2001. Anticarcinogenic effects of curry leaves in dimethylhydrazine – treated rats. Plant Foods Hum. Nutr., 55: 347-355. Lathan Kumar, K.J., Kakoli Dassharma and A. Mohandas, 2003. Curry leaf – an inevitable spice of Indian cuisine. Spice India, August, 8-9. Nasrin T.A.A., M.M. Molla, M. Alamigir Hossaen, M.S. Alam and L. Yasmin, 2008. Effect of postharvest treatments on shelf life and quality of tomato. Bangladesh J. Agril. Res., 33: 579-585. Shankaracharya, N.B. and C.P. Natarajan. 1971. Leafy spices – Chemical composition and uses. Indian Food Packer, 25: 29-40. Talukder, S., K.M. Khalequzzaman, S.M.K.E. Khua and Md. Sham-UdDun, 2003. Prepackaging, storage losses and physiological changes RI IUHVK EULQMDO DV LQÀXHQFHG E\ SRVW KDUYHVW WUHDWPHQWV Journal of Biological Sciences, 3: 474-477. Yamauchi, Naoki and Alley E. Watada, 1991. Regulated chlorophyll degradation in Spinach leaves during storage. J. Amer. Soc. Hort. Sci., 116(1): 58-62. Submitted: October, 2014; Revised: January, 2015; Accepted: March, 2015

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