International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705
Production of Haploids Plants from Anther Culture of Musa Paradisiaca cv. ‘Puttabale’ K. Girish Kumar1, V. Krishna2*, Venkatesh3, R. Shashikumar4, H. S. Arunodaya5 1, 2, 4, 5
Department of P. G. Studies and Research in Biotechnology, Bioscience Block, Kuvempu University, Jnana Sahyadri, Shankaraghatta‐577 451, Shivamogga (Dist.), Karnataka, India. 3
Department of Biochemistry Indian Institute of Science Bangalore. Karnataka, India * Corresponding author: V. Krishna
Abstract: Haploid plants were regenerated from the anther callus of banana Musa paradisiaca (AB) cv. Puttabale. The highest frequency of callus induction (90%) was observed at the concentration of 3mg/l 2, 4-D . After 20 days of incubation organization of embyroids were organised from the callus mass. Interaction of 4mg/l BAP and 0.4 mg/l IAA provoked shoot growth of the embryoids and well organised roots were developed at the concentration of 0.6 mg/l NAA and the media was agumented with 0.2% activated charcoal. Flow cytometry study was carried out to analyse the DNA content of the regenerated haploid plants. The results of the investigation reported the efficient production of haploid plants from the anther culture. Key words: Musa paradisica, puttabale, anther culture, haploid plants I. INTRODUCTION anana and plantain (Musa spp.) are the world’s major food crop for millions of people. Many breeders improved the quality by manipulating the ploidy of this crop (Pillay et al., 2003). But the conventional breeding method is laborious and took more generations for the development of haploid plants. The production of haploids through anther culture has many advantages since it does not imply the inbreeding depression caused by selfing and does not involve pollinator genome contamination. Many factors are involved and interact in the optimization of the anther culture protocol to produce haploid plants; therefore it is necessary to consider: pre‐treatment of anthers, genotype effects, the stage of pollen development, culture conditions and induction of embryos and regeneration of plants. Anther or microspore cultures have been found to be the most efficient techniques for obtaining a large number of haploid plants (De Buyser and Henry.,et al 1980). Haploid production is extremely important for crop improvement and eradication of pathogens (Brown and Thorpe et al., 1995) and dramatically improves the yield. (Badoni and Chauhan et al., 2010). Anther or microspore cultures has proved to be the most efficient techniques for obtaining a large number of haploid plants (Buyser and Henry et al., 1980) and valuable in plant breeding and genetics
B
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studies (Custódioa et al., 2005). The establishment of homozygous lines of new varieties is possible in a short period of time, hence provide a useful tool to accelerate plant breeding successions (Devaux and Li et al., 2001; Zhang et al., 2011). Haploids can obtained through natural parthenogenesis or androgenesis. Artificial induction of ovaries (Muren et al., 1989), ovules (Hansen et al., 1994), anthers (Foroughi et al., 1982), microspores (Kohler and Wenzel et al., 1985) is also a promising tool for haploid production. In the genus Musa, accurate determination of the ploidy by phenotypic traits, including stomatal size, density, and pollen size, and by chromosome counting is difficult and laborious, mainly due to strong genotypic influences (Hamill, 1992; Vandenhout et al., 1995; Van Duren et al., 1996; Dolezel, 2004). Musa paradisiaca cv. Puttabale (AB group) belongs to family Musacae and an indigenous banana cultivar of Karnataka, India. Previously we published the high frequency plant regeneration protocol from shoot tip, leaf and immature male floral explants. No reports available on haploid production from this plant. This paper deals with the in vitro haploid plant regeneration from anther culture of banana cultivar Puttabale. II. MATERIALS AND METHOD Explants Collection And Sterlization Banana cv puttubale inflorescence of appropriate stage was collected from farmyard of Shimoga district, Karnataka. The inflorescence were disinfected in 2% sodium hypochloride solution for 15 min, followed by 8-10 rinses in sterile distilled water.The immature male flowers are detached from bract, tepals aseptically. Male flowers contained five stamens with fully developed anthers. Anthers from the stamens was carefully isolated and transfered into pertriplates. Anthers with uninucleated microspore stage was determined using 1% aceto-carmine stain at this stage anthers were inoculated on to the callusing media. Plant Regeneration
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International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705 The callusing media consisted of MS media constituents agumented with 40 g/l sucrose, 8 g/l agar, 160 mg/l adenine sulfate, 100 mg/l tyrosine and the growth regulator 2, 4-D tested at the range of 1 to 6 mg/l. The pH was adjusted to 5.8 before autoclaving at 121ºC for 20 min. The cultures were incubated at 25 ± 2ºC with 12 hrs photoperiod and the callogenic frequency was evaluated after 30 days of incubation. Regenerated androgenic embryos like structures developed from callus were transferred to proliferation medium containing different conentrations of BAP (2 to 6 mg/l) and IAA( 0.2 to 0.6 mg/l) the media was agumented with 100mg/l of adanine sulphate and tyrosin. The well grown elongated shoots were aspectically transferred to the medium containg 0.25 to o.75mg/l NAA and without hormones fortified with 0.2% activated charcoal for the proper development of root system. The plantlets from culture bottles are moved from the laboratory later they are shifted to green house for primary hardening where they are first gently washed free of agar medium. The well rooted plants were washed with 0.2% of bovastin solution then followed by primary hardening in cocopeats, maintained in polythene house for a period of two weeks. During this period, 90-95 % humidity is maintained for the initial 6-8 days under diffused light, later they were maintained at 70 %, humidity and 26 °C Then transferred to green house for secondary hardening in a polythene bags containing red soil, sand and cattle manure in the ratio of 1:1:2 respectively. The regenarents grew to the height of 10 to 15 cm were transferred to field condition. Determination of Ploidy Level The flow cytometric technique was followed to determine the ploidy level of the anther-derived plants. Leaf pieces (1×1 cm) from in vitro plants derived from anther cultures were chopped with a razor blade in 600 μl buffer solution consisting of 45 mM MgCl2, 30 mM Tri-sodium citrate (Na3C6H5O7, 2H2O), 20 mM MOPS, 1% triton X100, 10 mM sodium-metabisulfite (Na2S2O5) (pH 7) to obtain a nuclei suspension. The whole sample was sieved through a 40-μm mesh nylon filter and stained with 6 μg ml-1 DAPI solution. A diploid parental plant was used as an internal standard. Nuclei analysis was carried out by using Partec CA II flow cytometer following the method of Assani et al., 2003. III. RESULTS AND DISCUSSION The anthers isolated aseptically were inoculated on to the callusing media (1 mg/l. 2,4D to 6 mg/l. 2,4D) showed callogenic response within a week of incubation. Prior to inoculation anthers were selected at the diod or tetraoid stage of meiosis (Fig A) Optimum callogenesis noticed at the concentration of 3 mg/l. 2,4D (Table.1). On callusing media anthers began to swell and the size increases to three folds. After ten days of incubation, callogenic mass noticed from the dorsal side while the anther wall cells turns black (Fig B). After 20 days of culture organization of embyronic nodules
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were appeared from the sprouted callus mass. The formation of androgenic embryroids from the protruded callus was similar to the findings of Assani et al., 2002. The interaction of BAP at higher levels (2.0 – 6.0 mg/l) and IAA at lower levels (0.2 ‐ 0.6 mg/l) provoked the growth and development of the enbryonic mass in to shoot buds. The mean number of shoots organized at each combination of BAP and IAA is shown in the (Table 1). The shoot multiplication was optimized at the concentration of 4 mg/l BAP and 0.4 mg/l IAA with a mean of 7.10±0.88 shoots per callus. shoot buds. The caulogenic mass with multiple shoots were subjected to colchecin treatment with a range of (0.025% and 0.05%). Then they were transferred to the development media consisted of 0.6 mg/l NAA and 0.2% activated charcoal. The well rooted shoots were maintained on the media for two to three cycles, then they were transferred to the rhizogenic media. The induction of roots depends on the composition of mineral nutrients and growth regulators. Shoot buds were transferred to MS with 0.6 mg/l NAA, 0.2% activated charcoal initiated roots from the base of microshoots ( Fig E), after three weeks of culture. The well rooted plantlets (Fig H) were hardened primarily in coco peats containing equal proportion of garden soil, sand, and cattle dung manure in polythene house for a period of two weeks (Fig I) and then they were subjected to secondary hardening in perforated polythene bags in green house (Fig J). The presence of diploid plants was also observed among the regenerants. While this could be a consequence of the regeneration of diploid anther tissues such as anther wall or connective tissue, in monocots the regeneration of somatic anther tissue has been very rarely reported. The other possibility is the spontaneous chromosome doubling in haploid cells. The possible factors leading to spontaneous doubled-haploid plants could be nuclear fusion in the early divisions of the microspores, endomitosis, endoreduplication or multipolar mitosis during the callus phase (Chen et al.1982; De Buyser and Henry1986). The polar transport of auxin is essential for the establishment of bilateral symmetry during embryogenesis in dicotyledonous and monocotyledonous species (Fisher,c, et al.1996). Androgenesis has been traditionally divided in two main stages, namely induction and regeneration. In the former one, somatic cells acquire embryogenic characteristics by means of a complete reorganization of the cellular state, including physiology, metabolism and gene expression (Feher, A. et al. 2002). In the present study also androgenic embryoids developed from the primary callogenic mass and upon subculture on the cytokinin supplemented media embryoids grew up in to plantlets. Many authors have reported that the developmental stage of anthers can drastically affect plant regeneration because microspores respond only at a specific development stage, which ranges from the tetrad, early and mid-uninucleate to the early binucleate stage. As such anther of the Lilium ×
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International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705 ‘Enchantment’, excised at the uninucleate microspores stage could form callus and regenerate bulblets (Niimi et al., 2001). Medium is another important factor affecting anther culture. Custódio et al. (2005) reported that the best medium for calli induction of carob tree was MS media supplemented with 2.3 μM 2, 4-D + 8.2 μM TDZ. Plant regeneration from anther of banana (Assani et al., 2003) was obtained in 4.4 μM BAP + 2.3 μM. While genotype or days in vitro are factors that favor the frequency of appearance of DNA polyploid plants or even DNA aneuploids (Tremblay et al. 1999; Endemann et al. 2001; Currais et al. 2013), Therefore, the changes observed in nuclear DNA content might be caused by the effect of growth regulators used in the culture media. We noticed that synthetic auxin 2, 4-D might be involved in embryoid formation from the microspore mass, because it was found to be a factor favouring changes in the genome of in vitro regenerated plants (May and Sink 1995; Jin et al. 2008; Clarindo et al. 2008). Additional assays would be useful to confirm the possible role of 2,4-D as a factor promoting the variations observed, as well as NAA or BAP, which were also mentioned as possible mutagenic agents (Lim and Loh 2003; Mishiba et al. 2006; Barow and Jovtchev 2007). The histogram developed from the result of cytometric analysis (Fig. 2 .A and B) showed a prominent peak of nuclei in PI fluorescent intensity in Gi l and G1 stages of interphase of the doubled diploid cells. The intensity of the nuclei in the interphase of doubled diploid plant and it is two folds as compared to haploid cells. Extensive fluorescent emissions at higher intensities are the indicative of populations of nuclei at increased ploidy levels. Flow cytometric analyses was performed by measuring the leaf DNA intensity of the in vitro regenerated plants. The report of Jin et al. (2008) suggested that detection of plants with lower nuclear DNA intensity value is due to the existence of aneuploid plants. This assumption is based on the fact that many of the changes that can be detected by FCM are associated with variations in the number of chromosomes (Clarindo et al. 2008; Currais et al. 2013). These variations might be enough to indicate the complete or partial loss of a chromosomes during doubling process (Pfosser et al. 1995; Roux et al. 2003).The flow cytometric studies enlightened many investigators to detect plants with values of nearly twice the nuclear DNA content, suggesting a possible in vitro polyploidization (Barow and Jovtchev 2007).
A
B
C
D
E
F
G
H
I
J
IV. CONCLUSION Anther culture is the classical method employed in the regeneration of haploid plants but in conventional breeding production of haloid plant require screening of many generations and it is difficult in pseudo fruit crops like banana. The results of the investigation reported here showed the efficient production haploid plants from the anther culture of Musa paradisica cv. Puttabale
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Fig. 1 High frequency regeneration of plantlets from anther explants of Musa paradisica cv. Puttabale. A. Uninuleate stage of anther. B. Development of anther callus. C and D. Androgenic embryos formed from the calli. E and F. Shoot growth of the embroids. G. Growth of haploid plants. H. Root induction from the haploid plants. I. Primary hardening plants. J. Secondary hardened haploid plants.
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International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705
Figure 2 Histograms of relative DNA content obtained after flow cytometric analysis of nuclei isolated from (A) leaf tissue of the diploid banana plant, and (B) leaf tissue of the haploid banana plant
Table 1 Effects of growth regulators on frequency of anther callogenesis and plant regeneration from Musa paradisiaca cv. Puttabale
Plant growth regulators mg/L 2, 4 -D mg/L
BAP mg/L
IAA mg/L
1 2 3 4 5 6 F Value
2 2 2 3 3 3 4 4 4 5 5 5 6 6 6
0.2 0.4 0.6 0.2 0.4 0.6 0.2 0.4 0.6 0.2 0.4 0.6 0.2 0.4 0.6
Frequency of callus formation per Number of multiple shoots per propagule explant Mean ± S.D % 16.66 36.66 90.00 56.66 43.33 33.33 -
0.90±0.74 1.60±0.70 1.30±0.95 2.00±0.47 3.00±0.67 2.10±0.57 3.20±1.14 7.10±0.88 4.40±1.71 4.00±1.15 5.50±0.97 4.60±1.35 3.30±1.16 5.40±1.58 4.20±1.03 27.3
The value of combination consisted of mean ± S.D. of 10 replicates. The F-value is significantly different when p< 0.05.
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