Nusantara Bioscience vol. 1, no. 2, July 2009

Adenium obesum photo by Abu Abdurrahman

| Nus Biosci | vol. 1 | no. 2 | pp. 53‐103 | July 2009 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

| Nus Biosci | vol. 1 | no. 2 | pp. 53‐103 | July 2009 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

FIRST PUBLISHED: 2009

ISSN: 2087-3948 (printed edition), 2087-3956 (electronic edition)

EDITORIAL BOARD: Abdulaziz M. Assaeed (King Saud University, Riyadh, Saudi Arabia), Alfiono (Sebelas Maret University, Surakarta), Edwi Mahajoeno (Sebelas Maret University, Surakarta), Ehsan Kamrani (Hormozgan University, IR Iran), Eko Handayanto (Brawijaya University, Malang), Endang Sutariningsih (Gadjah Mada University, Yogyakarta), Faturochman (Gadjah Mada University, Yogyakarta), Iwan Yahya (Sebelas Maret University, Surakarta), Jamaluddin (R.D. University, Jabalpur, India), Lien A. Sutasurya (Bandung Institute of Technology, Bandung), Magdy Ibrahim El-Bana (Suez Canal University, Al-Arish, Egypt), Mahendra K. Rai (Amravati University, India), Marsetyawan H.N. Ekandaru (Gadjah Mada University, Yogyakarta), Oemar Sri Hartanto (Sebelas Maret University, Surakarta), R. Wasito (Gadjah Mada University, Yogyakarta), Rugayah (Indonesian Institute of Science, Cibinong-Bogor), Sameer A. Masoud (Philadelphia University, Amman, Jordan), Supriyadi (Balitbiogen, Bogor), Sri Margana (Gadjah Mada University, Yogyakarta), Suranto (Sebelas Maret University, Surakarta), Sutarno (Sebelas Maret University, Surakarta), Sutiman B. Sumitro (Brawijaya University, Malang), Taufikurrahman (Bandung Institut of Technology, Bandung), Wayan T. Artama (Gadjah Mada University, Yogyakarta)

EDITOR-IN-CHIEF: Sugiyarto (sugiyarto_ys@yahoo.com)

EDITORIAL STAFF: Yansen M. Toha (lingkungan_global@yahoo.com), Ari Pitoyo (aripitoyo@yahoo.co.id)

MANAGING EDITORS: Ahmad Dwi Setyawan (unsjournals@gmail.com)

PUBLISHER: “Bioscience Community”, School of Graduates, Sebelas Maret University, Surakarta

ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbioscience@yahoo.com

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EXPERTISE OF THE EDITORIAL BOARD: AGRICULTURAL SCIENCES: Eko Handayanto (ehn_fp@brawijaya.ac.id), ANTHROPOLOGY: Sri Margana (margo15id@yahoo.com), APPLIED BIOLOGICAL SCIENCES: Suranto (surantouns@gmail.com), BIOCHEMISTRY: Wayan T. Artama (artama@ugm.ac.id), NATURAL PRODUCT BIOCHEMISTRY: MAHENDRA K. RAI, BIOPHYSICS AND COMPUTATIONAL BIOLOGY: Iwan Yahya (iyahya@uns.ac.id), CELL BIOLOGY: Sutiman B. Sumitro (sutiman@brawijaya.ac.id), DEVELOPMENTAL BIOLOGY: Lien A. Sutasurya (lien@bi.itb.ac.id), ECOLOGY: Magdy Ibrahim El-Bana (magdy.el-bana@ua.ac.be), ENVIRONMENTAL SCIENCES: Abdulaziz M. Assaeed (assaeed@ksu.edu.sa), EVOLUTION: Taufikurrahman (taufik@bi.itb.ac.id), GENETICS: Sutarno (nnsutarno@yahoo.com), IMMUNOLOGY: Marsetyawan H.N. Ekandaru (marsetyawanhnes@yahoo.com), MEDICAL SCIENCES: Alfiono (afieagp@yahoo.com), ANIMAL AND VETERINARY SCIENCES: R. Wasito (wasito@ugm.ac.id), MICROBIOLOGY: Endang Sutariningsih (annisah-endang@ugm.ac.id), NEUROSCIENCE: Oemar Sri Hartanto (oemarsrihartanto@yahoo.com), PHARMACOLOGY: Supriyadi (supriyadi@cbn.net.id), PHYSIOLOGY: Sameer A. Masoud (smasoud@philadelphia.edu), PLANT BIOLOGY: Rugayah (titikrugayah@yahoo.com), POPULATION BIOLOGY: Ehsan Kamrani (kamrani@hormozgan.ac.ir), PSYCHOLOGICAL AND COGNITIVE SCIENCES: Faturochman (fatur@cpps.or.id), SUSTAINABILITY SCIENCE: Jamaluddin (jamaluddin_123@hotmail.com), SYSTEMS BIOLOGY: Edwi Mahajoeno (edmasich@yahoo.com)

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 1-8 March 2009

Effect of VCO to leucocyte differential count, glucose levels and blood creatinine of hyperglycemic and ovalbumin sensitized Mus musculus Balb/c NOOR SOESANTI HANDAJANI1,3,♥, RUBEN DHARMAWAN2,3 ¹Department of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java, Indonesia. Tel./Fax.: +92-271-663375. ♥email: noor_handajani@yahoo.com. ²Faculty of Medicines, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia 3 Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 3 December 2008. Revision accepted: 24 February 2009.

Abstract. Handajani NS, Dharmawan R. 2009. Effect of VCO to leucocyte differential count, glucose levels and blood creatinine of hyperglycemic and ovalbumin sensitized Mus musculus Balb/c. Nusantara Bioscience 1: 1-8. Chemical medicines and insulin can decrease glucose blood level on hyperglycemic patients with macro vascular side effect. Diabetes and allergy incidences are influenced by quality and quantity of leucocytes. Lauric acid within VCO reports decreased glucose blood level of diabetes and some allergy incidents. The purpose of the study is to know the effect of VCO on glucose blood level, differential leucocytes count and creatinine blood level on hyperglycemic and normoglicemic ovalbumin sensitized mice. Forty five (45) male (mice) of Mus musculus Balb/c with average weight of 35 g are divided into 9 groups with 5 repetitions, those are 4 non alloxan groups and 5 alloxan induced hyperglycemic groups. On 22nd day to 36th day they are sensitize to ovalbumin as allergen. Blood sample was obtained by orbital vena using heparin as anti coagulant in order measuring glucose blood level by GOD method to 6 times, on 1st, 4th, 18th, 22nd, 32nd and 37th days, then are tested by ANOVA followed by DMRT 0.05. On 37th day, differential leucocytes are determined, blood level are counted, and then compared to normal value. The result of this study were that within differential leucocytes count of hyperglycemic mice, neutrophile percentage were much lower than the normal value (3.22%), and lymphocyte percentage were much higher than the normal value (94.54%). Consumed 0.003 mL/35 g VCO more 18 days decreased glucose blood level on hyperglycemic mice, decreased basophile percentage of ovalbumin sensitized mice, normalized neutrophile percentage no increased creatinine blood level.

Key words: VCO, hyperglycemic, allergy, leucocytes differential count, blood creatinine.

Abstrak. Handajani NS, Dharmawan R. 2009. Pengaruh VCO terhadap hitung jenis leukosit, kadar glukosa dan kreatinin darah Mus musculus Balb/c hiperglikemi dan tersensitisasi ovalbumin. Nusantara Bioscience 1: 1-8. Obat-obatan kimia dan insulin dapat menurunkan kadar glukosa darah pada pasien dengan efek samping hiperglikemi makro vaskular. Diabetes dan insiden alergi dipengaruhi kualitas dan kuantitas leukosit. Asam laurat dalam VCO dilaporkan menurunkan tingkat glukosa darah pada kejadian diabetes dan beberapa insiden alergi. Tujuan penelitian ini adalah mengetahui pengaruh VCO pada tingkat glukosa darah, diferensial leukosit dan kadar kreatinin pada mencit hiperglikemi dan normoglicemic tersensitisasi ovalbumin. Empat puluh lima (45) mencit Mus musculus Balb/c jantan dengan berat rata-rata 35 g dibagi menjadi 9 kelompok dengan 5 ulangan, yaitu 4 kelompok non aloksan dan 5 kelompok hiperglikemi yang diinduksi aloksan, Pada hari ke-22 sampai ke-36, mereka disensitisasi dengan ovalbumin sebagai penyebab alergi. Sampel darah diperoleh dari vena orbital menggunakan heparin sebagai anti koagulan, kadar glukosa darah diukur dengan metode GOD sebanyak 6 kali, pada hari ke-1, 4, 18, 22, 32 dan 37, kemudian diuji dengan ANAVA yang diikuti oleh DMRT 0,05 untk mengetahui tingkat perbedaan antar perlakuan. Pada hari ke-37, diferensial leukosit dan tingkat kreatinin darah ditentukan, lalu dibandingkan dengan nilai normal. Hasil penelitian menunjukkan bahwa dalam hitungan diferensial leukosit mencit hiperglikemi, persentase neutrofil jauh lebih rendah daripada nilai normal (3.22%), dan persentase limfosit jauh lebih tinggi daripada nilai normal (94.54%). Konsumsi 0.003 mL/35 g VCO lebih dari 18 hari menurunkan kadar glukosa darah pada mencit hiperglikemi, menurunkan persentase basophile pada mencit tersensitisasi ovalbumin, normalisasi persentase neutrophile tidak meningkatkan tingkat kreatinin darah.

Kata kunci: VCO, hiperglikemia, alergi, hitungan diferensial leukosit, kreatinin darah.

INTRODUCTION Diabetes mellitus is a disease of the pancreatic endocrine hormones, including insulin and glucagons. This was due to inadequate insulin activity so that the amount of insulin secretion is reduced (Suharmiati 2003) or a response decrease in peripheral tissue to insulin (insulin resistance) and a decrease in the ability of pancreatic beta cells to secrete insulin in response to glucose load. The

main manifestations include disorders of lipid metabolism, carbohydrate and protein which in turn stimulate the conditions of hyperglycemia. The condition of hyperglycemia will develop into diabetes mellitus with various forms of complications manifestation (Unger and Foster 1992; Nugroho 2006). According to the UKPDS (1999), although the sulfonylurea drugs and insulin can lower blood glucose levels, but it can cause macrovascular problems. Because glucose is very important in

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lymphocytes metabolism, the Diabetes can also cause characteristic changes in lymphocyte metabolism (Otton et al. 2002), which in turn can stimulate the apoptosis lymphocyte (Otton et al. 2004). Research of Alba-Loureiro et al. (2006) showed that diabetes also causes the characteristic on neutrophile function and metabolism of white rats.

Today, allergic is interpreted as immunologic reaction to antigens unreasonably or inappropriately in individuals who have previously sensitized by the antigen in question, so that the antigen is called an allergen. Ovalbumin, an allergenic protein that is found in white egg, can often cause allergic reactions in children (Endaryanto and Harsono 1996). Two processes that mark the occurrence of allergic process are exocytose of granule contents such as histamine and the induction of the formation of mediators that stimulate the formation of prostaglandins and leukotrienes that have a direct impact on the local tissue (Kresno 2001).

All this time, the use of VCO (Virgin Coconut Oil) for cases of diabetes and allergies are empirically much done, so the author wants to prove a reduction of blood glucose levels in hyperglycemia mice (Mus musculus Balb/c) with the treatment of VCO. Given the role of several types of leukocytes between the cases of diabetes and allergies, so in this study, it also should be noted that the VCO is potential for the vulnerability of normal and hyperglycemia mice on allergen exposure by viewing the leukocyte counts.

The consumption of nutritious substances that are expected to resolve a complaint needs to be watched out as the negative impacts may arise as a side effect. Therefore, it should be tested for the presence or absence of the rise of blood creatinine levels indicating the normality status of renal function physiologically, because the inability to excrete creatinine as a component of urine, so, in large numbers, it flows back into the bloodstream above its normal values.

Based on the background of the problems above, the purpose of this study are: (i) to examine the effect of VCO on blood glucose levels in mice (M. musculus) Balb/c hyperglycemia, (ii) to know the leukocyte counts of mice hyperglycemia, (iii) to investigate the VCO effect on leukocyte counts mice which are exposed to ovalbumin, (iv) the effect of VCO on leukocyte counts mice hyperglycemia which are exposed to ovalbumin, (v) the effect of VCO on levels of blood creatinine of mice in efforts to achieve normal glichemia conditions and protection against exposure to ovalbumin.

MATERIALS AND METHODS Time and place This research was conducted in October-November 2007. The Maintenance and the checking of blood sugar levels and creatinine in mice were conducted in LP3HPLPPT Gadjah Mada University, Yogyakarta.

Material VCO is obtained from PT Prima Solutions Medika

Jakarta, 45 mice (Mus musculus BaIb/c) which are 2-3 months old and male with an average body weight of 35 g are obtained from LP3HP-LPPT Gadjah Mada University, Yogyakarta.

Alloxan monohydrate (SIGMA), glibenclamide (Kalbe Farma), ovalbumin (SIGMA), pellets are used to feed the mice and tap water is as drink.

Procedures Before the treatment, 45 mice Balb/c which are 2-3 months old and male with an average body weight of 35 g are acclimated to suit with laboratory conditions, then they are divided into 9 groups. They are fed with pellets and gave drink with tap water, ad libitum. Then the following works are performed: Determination of dosage and injection of alloxan.

Alloxan dose given to monkeys (Macaca fascicularis) after being converted to white rats weighing 200 g is 35 mg/200 g BW (Widyastuti et al. 2005). For mice weighing 35 g is 35/200x35 mg = 6.125 mg/35 g BW. For injection, the alloxan is dissolved in 0.5 mL of distilled water.

Determination of dosage and the oral application of glibenclamide. The dose of glibenclamide in humans is 5 mg/day/70 kg BW (Tjay and Rahardja 2007), then converted in mice = 35/70000 x 5 mg = 0.00025 mg/day /35 g BW and for every application, it is dissolved with 0.5 CMC 1%.

Determination of dosage and the application of VCO. VCO is given to the human therapeutic dose is generally 3 tablespoons or 45 mL/day (Dayrit, 2000), when converted in mice, it is 35/70.000x45 = 0.00225 mL/day /35 kg BW. In this study, the doses are given in 2 variations, dose I = 0.002 mL/day and dose II = 0.003 mL/day.

Determination of blood glucose levels. Blood glucose level was determined with the GOD-PAP method (Enzymatic Photometric Test) which is the method of photometric measurement with spectrophotometer (Widowati et al. 1977). The measurements are taken 6 times during the study, day 1 (just before alloxan treatment), day 4, day 18, day 22, day 32 and day 37 (just before the sacrifice).

Determination of dose and way of allergen injection.

Determination of allergen dose and way of allergen injection refer to the work of Diding (2007) who managed to create mice with an average weight of 17.5 mg to have an allergy on its digestive system. This study used mice with an average weight of 35 mg, so every injection can be converted into 2X of the initial dose, with the protocol as follows: On day 22 sensitization of mice by injection of 0.30 mL ovalbumin out of 2.5 mg ovalbumin in 7.75 mL of Al(OH)3 in intra-peritoneal way. The re-sensitization is with the same solution and the same way on day 28. then, on day 32-34 consecutively the mice were re-sensitized by giving a solution of 0.30 mL ovalbumin of 2.5 mg ovalbumin in 2.5 mL of PBS orally.

Preparations of blood smear and determination of leukocyte count using Romanowski method. The bloods are dropped on the surface of glass objects then they are made into smears/blood film. Furthermore, they are airdried for a few minutes, and then they are dropped with methyl alcohol, and left untouched for 5 minutes.

HANDAJANI et al. – Effect of VCO on hyperglycemic mice

Furthermore, the preparations are lifted and placed in 1-2% of Giemsa dye solution for 30 minutes. Finally, the preparations are removed from the dye solution and washed with water, and carefully dried with a tissue, and then they are ready to be observed under microscope (Suntoro 1983).

After all form of leukocytes is observed under microscope with 10x100 magnification, with emersion oil paced on the surface of the smear preparations. Furthermore, the percentage of each type of leukocytes from 100 leukocytes found in every preparation is counted (Fox 1990).

Calculation of blood creatinine levels. Decrease in renal physiological function can be ascertained from the increased levels of blood creatinine. Blood creatinine levels were measured with a spectrophotometer of photometric system based on method of Daffe.

The experimental design This study used 45 mice (M. musculus Balb/c) which are divided into 9 groups. The blood glucose measurements were performed 6 times and then a Completely Randomized Design was carried out, using 9 kinds of treatments with five replications at each treatment, as follows: A. Treatment of normal controls: mice were given food and drink each day ad libitum without any treatment until the end of the study.

B. Treatment of non-alloxan, CMC and ovalbumin sensitization: on day 4 mice were given with 0.5 mL of 1% CMC, orally every day until the end of the study.

Ovalbumin sensitization was started on day 22-36.

C. Non alloxan treatment, dose I of VCO and ovalbumin sensitization: on day 4 mice were given orally with VCO 0.002 mL/35 g BW every day until the end of the study. Ovalbumin sensitization was started from day 22-36.

D. Treatment of non-alloxan, VCO dose II and ovalbumin sensitization: on day 4 mice were given orally with VCO 0.003 mL/35 g BW every day until the end of the study. Ovalbumin sensitization was started from day 22-36.

E. Treatment of hyperglycemia control: on day 1 mice were injected with alloxan 6.125 mg/35 g BW subcutaneous, then they were let to be fed and drank ad libitum without any treatment until the end of the study.

F. Treatment of alloxan, CMC and ovalbumin sensitization: on day 1 mice were injected subcutaneously with alloxan 6.125 mg/35 g BW, then on day 4-36 were given with 0.5 mL of 1% CMC. Ovalbumin sensitized was begun at day 22-36.

G. Treatment of alloxan, VCO dose I, ovalbumin sensitization: on day 1 mice were injected subcutaneously with 6.125 mg/35 g BW, then on days 4-36 were given with VCO 0.002 mL/35 g BW orally and ovalbumin sensitization began on day 22-36.

H. Treatment of alloxan, VCO dose II, ovalbumin sensitization: on day 1 mice were injected subcutaneously 6.125 mg/35 g BW, then on day 4-36 were given with 0.003 mL/35 g BW of VCO orally and ovalbumin desensitization began on day 22-36.

I. Treatment of alloxan, glibenclamide and ovalbumin sensitization: on day 1 mice were injected

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subcutaneously with alloxan 6.125 mg/35 g BW, then on day 4-36 was given orally with 0.0025 mg of glibenclamide in 1% CMC and ovalbumin sensitization began on day 22-36.

Data analysis Quantitative data of blood glucose levels were analyzed using the ANOVA (Analysis of Variance) and if there are significant differences, then it is followed by the DMRT test (Duncan's Multiple Range Test) at the significance level of 5% to determine the significance differences among the treatments. So the VCO effect in decreasing the blood glucose levels in hyperglycemic mice can be detected. Quantitative data of leukocyte differential were obtained by counting each type of leukocytes in each treatment group. And then, the data mean were compared with differential of normal leukocyte, according to Smith and Mangkoewidjojo (1988) and Jacoby and Fox (1984) to compare the leukocyte differential of mice in conditions of hyperglycemia, hyperglycemia that have been treated with the VCO, and hyperglycemia which is exposed to allergens, mice exposed to VCO which is then exposed to allergens and normal leukocyte differential in mice, so that can know the relationship between hyperglycemia, VCO and the development of allergy immunology. Quantitative data average blood creatinine levels of each treatment group is compared with the normal rate according to Jacoby and Fox (1984), so the effect of VCO treatments on the function status of renal physiology can be known.

RESULTS AND DISCUSSION Blood Glucose Levels (BGL) From the results, the state/condition of blood glucose levels data can be obtained as shown in Table 1 and Figure 1: the Mean of Blood Glucose Levels (BGL) of all treatment, measurement 1 to measurement 6, Figure 2: the Mean of BGL of non alloxan groups, measurements 1 to 6 and, Figure 3: Mean BGL of alloxan groups, measurement 1 to 6. The data obtained is analyzed by analysis of variance (ANOVA) one way and when there are significant difference, it is followed by Duncan test (DMRT) with significance level of 5% to determine the real differences among the treatments.

The first measurement of average blood glucose level on day 1 was started just before the injection of alloxan.

The mean blood glucose levels of 45 mice were classified into 9 treatment groups. Figure 1 shows that there are no significant differences of the blood glucose levels of all treatment groups from A to I. It means that between groups which were not given with alloxan and the group which were given with alloxan showed no significant difference, namely in a range of 61.40 to 71.80 mg/dl. This is because all the mice used were in normal-glychemia and relatively similar.

Figure 1 shows the measurement 2 (day 4), all treatment groups, which were given alloxan or not, had an increasing in blood glucose levels, although the non alloxan groups were not statistically significant compared

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Table 1. Changes in average blood glucose level groups of mice during treatment Blood glucose levels day by Treatment 1 (day-1) 2 (day-4) 3 (day-18) 4 (day-22) 5 (day-32) 6 (day-37) group mg/dl mg/dl % changes mg/dl % changes mg/dl % changes mg/dl % changes mg/dl % changes Non alloxan A 71,600a 96,600abc + 34,9 108,600a + 51,7 119,000ab + 66,2 137,000ab + 91,3 69,000a -3,6 a ab a a a B 61,400 79,200 +29 110,000 +79,2 82,000 +33,6 78,800 +28,3 92,800ab +51 66,400a -1,2 93,200a +38,7 75,200a +12 86,400a +28,6 101,600ab +51,2 C 67,200a D 61,400a 124,200abc + 102 87,800a + 43 113,000ab + 84 79,400a + 29,3 97,200ab +58,3 Alloxan 159,000c +128,4 157,800ab +126,7 160,800bcd +131 156,600ab +125 179,200c +157,5 E 69,600a a bc bc abc ab 142,000 +97,8 222,000 +209,2 128,000 +78,3 132,200 +84 150,200bc +109 F 71,800 G 67,200a 158,800c +136 338,000de +403 205,400d +205,7 220,200b +227,7 230,200c +242,6 122,800abc +90,7 299,800cd +365,5 168,200bcd +161,2 104,200a +61,8 200,200c +210,9 H 64,400a I 61,800a 298,800d +383,5 413,600d +413,6 177,000cd +186,4 162,200ab +162 191,200c +209,4 Note: The number followed the same superscript letter within a column indicate no significant difference between treatments (p> 0.05).

A: normal control group (non-alloxan, non-allergen), B: The treatment of non alloxan, CMC and allergens/ovalbumin, C: Treatment of non alloxan, VCO dose I and allergens/ovalbumin, D: Treatment of non alloxan, VCO II dose and allergen/ovalbumin, E: The control group alloxan/hyperglycemia (alloxan, non-allergen), F: The treatment of alloxan, CMC and allergens/ovalbumin, G: Treatment of alloxan, VCO dose and allergen/ovalbumin, H: Treatment of alloxan, VCO dose II and allergens/ovalbumin, I: Treatment alloxan, glibenclamide and allergens/ovalbumin.

A B C Figure 1. Blood Glucose Levels of 1 to 6 repetised measurement. A. All treatment, B. Non-alloxan measurement, C. Alloxan measurement. Note: Description of A to I are similar to Table 1.

to levels at the beginning of treatment. The increase in non alloxan groups allegedly was due to stress because of the treatment of grouping in a different cage from its usual cage. Stress conditions will stimulate the sympathetic nervous system for the adrenal medulla to secrete the epinephrine hormone which causes the increase in blood glucose levels (Guyton and Hall 2008). The same was experienced by mice in the groups treated with alloxan.

In the 3rd measurement (day 18), non alloxan groups has blood glucose levels that can be said to be stable, ie average maximum of about 100 mg/dl. The difference with the alloxan group is so contrast. This is because all of them had an increase in blood glucose levels significantly.

On day 1, shortly after the measurement of blood glucose levels, the groups were treated with alloxan.

Alloxan plays a role in pancreatic beta cells that produce insulin and selectively killing these cells (Suharmiati 2003; Guyton and Hall 2008). The destruction of pancreatic beta cells causes the quantity and or quality of insulin disturbing the smooth transport of glucose from the blood into the tissues; it causes the glucose pile up in the blood, causing an increase in levels (hyperglycemia).

The 4th measurements (day 32), the average of blood glucose level of groups of mice induced with alloxan had a

significant reduction. The same blood glucose levels found in group H (alloxan, VCO dose II, ovalbumin) and group I (alloxan, glibenclamide, ovalbumin), although it did not reach the previous level which was not significantly different from the level in the initial treatment, but giving a dose VCO 2 after 18 days gives the same effect with the provision of glibenclamide, which lowers blood glucose levels. According Tjay and Rahardja (2007), glibenclamide is one oral antidiabetic drug including sulfonylurea.

Sulfonylurea drugs play a role in stimulating beta cells of Langerhans islands, so that insulin secretion is increased, besides that it also can increase the sensitivity of beta cells for blood glucose levels through its influence on glucose transport proteins. There are indications that these drugs also improve the target organ of insulin and decrease absorption of insulin by the liver. While in group H (alloxan, VCO dose II, ovalbumin), a decline in blood glucose levels happened after the oral provision of VCO at a dose of 0.003 mL/35 g BW every day for 18 days. This was because of the VCO makes an improvement of metabolic activity. MCFAs in the VCO which particularly are monolauric can reduce protein catabolism in hypercatabolic status, and they can serve as a protein sparer which prevents oxidation of amino acids to produce

HANDAJANI et al. – Effect of VCO on hyperglycemic mice

energy, and can also provide a protein into the tissues. The supply of network protein stimulates the secretion of glucagon which activates adenil cyclase that will produce cyclic AMP, so the phosphorylation for the processes of normal cell metabolism is activated, such as the repairing of gland secretion and enzymes activity, hormones, and of their receptors (Guyton and Hall 2008). In this case, it is assessed that there was an increase in insulin sensitivity hormones and in tissues of insulin receptor, so the supply of glucose into the tissue was normal. The other insulin functions are also normal, so that high blood glucose levels could be lowered.

In group G (alloxan, VCO dose I, ovalbumin), the same mechanism with the group H (alloxan, VCO dose II, ovalbumin) occurred though the decrease was not as much as dose of VCO 2 group. The mechanism was assumed to be the same. In the normal group or the control solvent, decrease blood glucose levels occurred, although there was no substance that could act as healers, including group E (alloxan, non-therapeutic, non ovalbumin) and group F (alloxan, CMC, ovalbumin). This was due to the recovery levels of the hormone epinephrine in experimental mice as the response of the body to restore its normal conditions.

Therefore, 1% CMC solution was only used as a hydrophilic gel drug compounds that were nontoxic, were not digested and not absorbed by the individual (Tjay and Rahardja 2007), so it does not affect changes in blood glucose levels. Only the H group (alloxan, VCO dose II, and ovalbumin) is steadily declining blood glucose levels until day 32nd, but rose again on day 37th, after ovalbumin sensitization protocol is complete. This is because, despite improved metabolic activity with the VCO, due to the body's response to ovalbumin which acts as allergens, so that blood glucose levels which had increased because of damage to pancreatic beta cells selectively, was damaged again, although not fatal.

In groups of non alloxan, the rise of blood glucose levels that occurred is assumed because of stress. The decline did not happen simultaneously, it was because the response of the body to restore normal conditions of each group was also influenced by the initial conditions and the given therapeutic as well as the body's response to allergens given. Three non-alloxan groups except group A (non-alloxan, non-therapeutic, non ovalbumin) decreased maximally and reached levels which were not significantly different from day 32nd, Groups C and D which were given VCO had decreased first, due to the improvement of metabolic activity. In group A, a new decline occurred on day 32nd and continued to decline, it was because there was no influence in the body, including allergens.

Determination of leukocyte differential Data on the average percentage of each leukocyte type of all treatment groups is shown in Table 2. Data average percentage of each leukocyte type of each treatment group is compared to its normal standard of mice according to Smith and Mangkoewidjojo (1988) and according to Jacoby and Fox (1984).

The first measurement of average blood glucose level on day 1 was started just before the injection of alloxan.

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The mean blood glucose level of 45 mice was classified into 9 treatment groups. Figure 1 showed that there are no significant differences of the blood glucose levels of all treatment groups from A to I. It means that between groups which were not given with alloxan and the group which were given with alloxan showed no significant difference, namely in a range of 61.40 to 71.80 mg/dl. This is because all the mice used were in normal-glychemic and relatively similar.

Figure 1 shows the measurement 2 (day 4), all treatment groups, which were given alloxan or not, had an increasing in blood glucose levels, although the non alloxan groups were not statistically significant compared to levels at the beginning of treatment. The increase in non alloxan groups allegedly was due to stress because of the treatment of grouping in a different cage from its usual cage. Stress conditions stimulated the sympathetic nervous system for the adrenal medulla to secrete the epinephrine hormone which caused the increase in blood glucose levels (Guyton and Hall 2008). The same was experienced by mice in the groups treated with alloxan.

In the 3rd measurement (day 18), non alloxan groups had blood glucose levels that can be said to be stable, ie average maximum of about 100 mg/dl. The difference with the alloxan group is so contrast. This is because all of them had an increase in blood glucose levels significantly.

On day 1, shortly after the measurement of blood glucose levels, the groups were treated with alloxan.

Alloxan plays a role in pancreatic beta cells that produce insulin and selectively killing these cells (Suharmiati 2003; Guyton and Hall 2008). The destruction of pancreatic beta cells causes the quantity and or quality of insulin disturbing the smooth transport of glucose from the blood into the tissues; it causes the glucose pile up in the blood, causing an increase in levels (hyperglycemia).

The 4th measurements (day 32), the average of blood glucose level of groups of mice induced with alloxan had a significant reduction. The same blood glucose levels found in group H (alloxan, VCO dose II, ovalbumin) and group I (alloxan, glibenclamide, ovalbumin), although it does not reach the previous level which is not significantly different from the level in the initial treatment, but giving a dose VCO 2 after 18 days gives the same effect with the provision of glibenclamide, which lowers blood glucose levels. According Tjay and Rahardja (2007), glibenclamide is one oral antidiabetic drug including sulfonylurea.

Sulfonylurea drugs play a role in stimulating beta cells of Langerhans islands, so that insulin secretion is increased, besides that it also can increase the sensitivity of beta cells for blood glucose levels through its influence on glucose transport proteins. There are indications that these drugs also improve the target organ of insulin and decrease absorption of insulin by the liver. While in group H (alloxan, VCO dose II, ovalbumin), a decline in blood glucose levels happened after the oral provision of VCO at a dose of 0.003 mL/35 g BW every day for 18 days. This is because of the VCO makes an improvement of metabolic activity. MCFAs in the VCO which particularly are monolauric can reduce protein catabolism in hypercatabolic status, and they can serve as a protein sparer which

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prevents oxidation of amino acids to produce energy, and can also provide a protein into the tissues. The supply of network protein stimulates the secretion of glucagon which activates adenil cyclase that will produce cyclic AMP, so the phosphorylation for the processes of normal cell metabolism is activated, such as the repairing of gland secretion and enzymes activity, hormones, and of their receptors (Guyton and Hall 2008). In this case, it is assessed that there was an increase in insulin sensitivity hormones and in tissues of insulin receptor, so the supply of glucose into the tissue was normal. The other insulin functions were also normal, so that high blood glucose levels could lowered.

In group G (alloxan, VCO dose I, ovalbumin), the same mechanism with the group H (alloxan, VCO dose II, ovalbumin) occurred though the decrease was not as much as dose of VCO 2 group. The mechanism is assumed to be the same. In the normal group or the control solvent, decrease blood glucose levels occurred, although there was no substance that could act as healers, including group E (alloxan, non-therapeutic, non ovalbumin) and group F (alloxan, CMC, ovalbumin). This is due to the recovery levels of the hormone epinephrine in experimental mice as the response of the body to restore its normal conditions.

Therefore, 1% CMC solution was only used as a hydrophilic gel drug compounds that were nontoxic, were not digested and not absorbed by the individual (Tjay and Rahardja 2007), so it does not affect changes in blood glucose levels. Only the H group (alloxan, VCO dose II, and ovalbumin) is steadily declining blood glucose levels until day 32nd, but rose again on day 37th, after ovalbumin sensitization protocol is complete. This is because, despite improved metabolic activity with the VCO, due to the body's response to ovalbumin which acts as allergens, so that blood glucose levels which had increased because of damage to pancreatic beta cells selectively, was damaged again, although not fatal.

In groups of non alloxan, the rise of blood glucose levels that occurred is assumed because of stress. The decline did not happen simultaneously, it was because the response of the body to restore normal conditions of each group was also influenced by the initial conditions and the given therapeutic as well as the body's response to allergens given. Three non-alloxan groups except group A (non-alloxan, non-therapeutic, non ovalbumin) decreased maximally and reached levels which were not significantly different from day 32nd, Groups C and D which were given VCO had decreased first, due to the improvement of metabolic activity. In group A, a new decline occurred on day 32nd and continued to decline, it is because there is no influence in the body, including allergens.

The differential of leukocyte of group A as control (non-alloxan, non-therapeutic, non ovalbumin) was: 1.140% of neutrophils; 90.76% of lymphocytes, 1.52% of monocytes 0.22% of eosinophils and 3.00% of basophils.

All percentage of neutrophile of groups with treatment is less than the normal standard by Smith and Mangkoewidjojo (1988) and/or according to Jacoby and Fox (1984), except in group H (alloxan, VCO 0.003 mL/g BW, ovalbumin) which has a normal percentage.

Furthermore, all lymphocyte percentage of groups with treatment is high above normal standards according to Smith and Mangkoewidjojo (1988) and/or according to Jacoby and Fox (1984), except in group F which has a normal percentage. All the percentage of monocytes is normal, except in group D (non-alloxan, VCO 0.003 mL/g BW), most of the eosinophils percentage in groups with treatment is less than the normal percentage. Percentage of basophils is higher than normal standards, except for groups C, D, E, G, H, and I. From the above data, it is estimated that basically in animal studies, there has been immunological defense mechanism which is likely due to a viral infection. Because according to Burkitt et al. (1995), an increased number of lymphocytes is generally a marking of a viral infection.

Neutrophil percentage far below the percentage of normal, it also supports the allegation. According to Price and Wilson (2006), neutropenia can be caused by disorders of neutrophil formation or the formation of ineffective neutrophil due to hipoplastic or aplastic anemia caused by cytotoxic drugs, the presence of toxic substances and viral infections, starvation and replacement by normal bone marrow by malignant cells, as in leukemia. Since lymphocytes have a central role in all immunological defense mechanisms. The increasing percentage of basophiles is supposed due to the induction of sensitized lymphocytes to respond to antigens or environmental allergens that are less profitable. According to Price and Wilson (2006), basophiles and mast cells have a membrane receptor which is very typical for this segment of Fc&IgE produced by plasma cells as antigen/allergen environment.

Burkitt et al. (1995) also states that basophiles are responsible primarily to give an allergic reaction and antigen by releasing chemicals histamine that causes inflammation. According to the authors, the high percentage of basophiles in these groups is as a respond to environmental antigens that arise due to endogenous viruses that infected him. The percentage of neutrophil on group H (alloxan, VCO 0.003 mL/g BW, ovalbumin) occupies the normal range according to Smith and Mangkoewidjojo (1988) or by Jacoby and Fox (1984) In the study, mice groups were suspected of suffering from a viral infection, but with the consumption of VCO, the reduction of virus amount is assumed to occur because of the MCFAs role, mainly mono laurin, through its lipid membrane, so the percentage of its neutrophil is normal, and this will improve the immunologic status of these mice groups. These groups also had a normal percentage of basophils. Basophiles are primarily responsible for giving an allergic reaction and histamine release by antigen chemical that causes inflammation. Basophiles and mast cells have a membrane that is very specific for the segment of IgE and Fc which is generated by cells plasma as response to a number of environmental allergens. The exposure of allergens causes the formation of bridges between the adjacent IgE molecules that trigger evisceration of granule/degranulation appropriately. So the release of histamine and other vasoactive mediators is responsible for the occurrence of acute hypersensitivity reactions to allergic rhinitis (hay fever), some forms of

HANDAJANI et al. – Effect of VCO on hyperglycemic mice

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Tabel 2. The mean percentage of the types of leukocytes from all groups of Mus musculus (mice), day 37 (last day of the study) after treatment of a variety of healers and ovalbumin sensitization Treatment group

Neutrophils

Lymphocytes

Number of cells ( % ) Monocytes

Eosinophils Basophils Non alloxan A 1.140-90.760++ 1.520* 0.220* 3.000++ -+ * * B 4.460 88.920 0.920 0.520 3.760++ -++ * * C 1.580 95.580 1.920 0.200 0.480* D 2.600-93.580++ 4.580++ 0.0600.760* Alloxan E 3.22094.540++ 0.820* 0.260* 0.460* F 5.48082.120* 1.420* 0.1406.420++ G 0.860-95.460++ 1.180* 0.200* 0.060* * + * H 10.100 85.800 1.200 0.120 0.580* I 0.820-97.720++1.420* 0.1400.240* Standard normal J 12-30 55-85 1-12 0.2-4.0 K 6.7-37.2 63-75 0.7-2.6 0.9-3.8 0-1.5 Note: Description of A to I are similar to Table 1. J. The normal according to Smith dan Mangkoewidjojo (1988). K. J. The normal according to Jacoby and Fox (1984). +: Greater than normal according to Smith and Mangkoewidjojo (1988) / Jacoby and Fox (1984) or both. -: Smaller than normal according to Smith and Mangkoewidjojo (1988) / Jacoby and Fox (1984) or both. *: Fulfilling the normal range according to Smith and Mangkoewidjojo (1988) / Jacoby and Fox (1984) or both.

asthma, urticaria and anaphylactic shock. But, there are other incentives for the occurrence of IgE independent mast cell degranulation. Basophils also represents 15% of infiltrate cells in allergic dermatitis and skin alograf rejection, known as basophils skin hypersensitivity, induced by sensitized lymphocytes and is a kind of hypersensitivity with media of slow degranulation cells (Burkitt et al. 1995).

Because the MCFAs contained in the VCO are particularly monolaurin which can reduce protein catabolism in hiperkatabolik status and serve as a protein sparer which prevents oxidation of amino acids to produce energy, so it provides the supply the protein into tissue. The supply of tissue protein stimulates the secretion of glucagon which activates adenil cyclase, produces cyclic AMP; so, the phosphorylation for metabolism processes of cell is activated normally, such as gland secretion and activity of enzymes, hormones, or repairing their receptors (Guyton and Hall 2008). The repair of hormones activity is, among other, increasing the sensitivity of receptors that they will have the ability to actively induce immunological response toward the Th1-Th2 balance after being exposed to ovalbumin allergen. According to Endaryanto and Harsono (1996), allergic is a form of Th2 disease which its cure requires the patient to be on a balanced condition between Th1 and Th2. So if risk factors cannot be avoided for the prevention, active induction of immunological response toward Th1-Th2 balance needs to be strived.

According to Abbas and Lichtman (2004), allergy is a type of pathological reactions caused by the release of mediators from mast cells. Generally, the production of IgE antibody cells mast is in various tissues of the body. The situation is followed by inflammation in some individuals exposed by certain foreign antigens which have hit them before.

Basically, the immune response, whether specific or

nonspecific, is to act protectively. But the immune response can lead to bad consequences and even disease called hypersensitivity. In addition, according to Bratawidjaja (2004), levels of cAMP and cGMP in cells are one of the things that affect of degranulation of mast cell. The increase of cAMP prevents degranulation, while moderate increase in cGMP boosts the prevention. So normally the percentage of basophiles is in the groups protected by VCO, although they were sensitized with ovalbumin, they allegedly did not experience an allergic event, this was due to the prevention of degranulation of basophiles due to the increase in AMP cyclic caused by the provision of VCO. The role of monocytes and eosinophils are not needed specifically on this group, because this study allegedly has no relation to bacteria and the given ovalbumin gives no result in anaphylactic reaction. According to Burkitt et al.

(1995), the role of eosinophils are, among others, to destroy parasites, to respond to bacterial products and as complement components, especially a substance released by basophiles namely histamine and eosinophil chemotactic factor of anaphylaxis other than activated lymphocytes, the percentage of eosinophils in this group which was just below normal indicates high rates balance toward percentage of lymphocytes.

Measurements of blood creatinine levels The average data of blood creatinine levels of each group is presented in Table 3. The data is then compared with the standard normal creatinine levels in mice by Jacoby and Fox (1984). From the measurement results of 9 groups of mice, it was found that all group had a magnitude of blood creatinine levels between 0.320 to 0.5040 mg/dl which still meet the range of normal numbers by Jacoby and Fox (1984), namely 0.21 to 0.74 mg/dl.

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Table 3. Mean blood creatinine levels of all treated groups on day 37 (end of study) Treatment group Blood creatinine levels (mg/dl) Non alloxan A 0.3760 B 0.4080 C 0.4040 D 0.4200 Alloxan E 0.5040 F 0.4400 G 0.3200 H 0.4020 I 0.3900 Standard normal J 0.21-0.74 Note: Description of A to I are similar to Table 1. J. The normal according to Jacoby and Fox (1984).

Giving VCO with a dose of 0.002 mL/35 g BW and 0.0030 mL/35 g BW orally for 34 days (started on day 4th37th), which is intended to lower blood glucose levels for groups of hyperglycemia mice and to protect the body against allergic reactions for groups of normoglycemia mice or groups that hyperglycemia does not affect the increase in blood creatinine levels. The increased value of creatinine would indicate a decrease of excretion, for example due to the decrease of kidney function. Thus, plasma creatinine values which are still within the normal range after long-term consumption of this VCO do not damage the kidneys, thus it is safe for consumption even though for a long-term consumption. This is appropriate with Kabara’s opinion (1978) that after consumption, the high percentage of lauric acid contained in the VCO which is a medium chain saturated fat will come into the digestive tract and can be easily absorbed without enzymatic process, thus more quickly reach the bloodstream. Furthermore, through blood flow it is brought to liver (heart) and to other tissues directly into mitochondria without the need for carnitine, and then will be converted into energy (ATP), so it will not be accumulated as fat that can lead to problems of blood vessels, etc.. Lauric acid is the most important fatty acids in VCO which potentially build and maintain the immune system. According to him, coconut oil improve immune system, among others, by lowering the amount of H4 virus and increase the number of CD4 cell without the side effects that are concluded from various clinical tests, among others, the number of blood cells, liver function tests (ALT and AST), renal function tests (urea and creatinine), blood lipids (cholesterol, triglycerides, HDL) and body weight.

CONCLUSION Giving VCO (Virgin Coconut Oil), 0.003 mL per oral after 18 days can lower blood glucose levels of Mus musculus Balb/c with hyperglycemia. The leukocyte differential of mice with hyperglycemia in this study: 3.220% of neutrophil, lymphocytes: 94.540%, 0.820%, of monocytes, eosinophils: 0.260%, 0.460% of basophile,

which reflects the low percentage of neutrophil and the high percentage of lymphocytes. Giving VCO onto differential of mice leukocyte which are exposed to ovalbumin will normalize the percentage of basophils.

Giving VCO to differential of hyperglycemic mice leukocyte which are exposed to ovalbumin can normalize the percentage of basophils and neutrophils.

REFERENCES Abbas AK, Lichtman AH. 2004. Hypersensitivity disease, basic immunology. 2nd ed. Saunders. Philadelphia.

Alba-Loureiro TC, Hirabara SM, Mendonça JR, Curi R, Pithon-Curi TC. 2006. Diabetes causes marked changes in function and metabolism of rat neutrophils. J Endocrinol 188: 295-303 Bratawidjaja KG. 2004. Basic immunology. Faculty of Medicine, University of Indonesia. Jakarta. [Indonesia] Burkitt HG, Young B, Heath JW. 1995. Handbook and atlas of Wheater functional histology. In: Tambayong J, Melfiawati S (eds). EGC. Jakarta. [Indonesia] Caradang EV. 2005. Health benefits of virgin coconut oil explained.

Philippine Coconut Research and Development Foundation (PCRDF).

Manila.

Dayrit C. 2000. Coconut oil in health and desease’s potential, it’s and monolaurin’s potential as cure for HIV/AIDS. 36th Cocotech Meeting, Chennai India, July 25, 2000.

Diding HP. 2007. Effect of probiotics on cytokine profiles and expression of TOOL LIKE receptor of intestinal mucosa of mice Balb/c allergen model. Faculty of Medicine, Sebelas Maret University. Surakarta.

[Indonesia] Endaryanto A, Harsono A. 1996. The prospect of probiotics in allergy prevention through the active induction of immunological tolerance.

Faculty of Medicine, Airlangga University/RSUP Dr. Sutomo.

Surabaya. [Indonesia] Fife B. 2003. The healing miracles of coconut oil. Healthwise Pub. Colorado Springs.

Fox SI. 1990. A labolatory guide to human physiology (concept clinical applications). 4th ed. Macmilan. New York.

Guyton A, Hall JE. 2008. Textbook of medical physiology. 11st ed.

Elsevier. New York.

UK Prospective Diabetes Study (UKPDS) Group. 1999. Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complication in patient with type 2 diabetes. Lancet 352 (9131): 837-853 Hessel EM, Van Oosterhout AJ, Hofstra CL, De Bie JJ, Garssen J, Van Loveren H, Verheyen AK, Savelkoul HF, Nijkamp FP. 1995.

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice. Eur J Pharmacol 293 (4): 401-412.

Jacoby RO, Fox JG. 1984. Biology and desease of mice. Academic Press.

Orlando.

Kabara JJ. 1984. Antimicrobial agents derived from fatty acids. J Am Oil Chem Soc 61: 397-403.

Kresno SB. 2001. Imunologi: diagnosis dan prosedur laboratorium.

Faculty of Medicine, University of Indonesia. Jakarta. [Indonesia] Nugroho AE. 2000. Experimental animals with diabetes mellitus: pathology and mechanism of diabetogenic action. Biodiversitas 7 (4): 387-391. [Indonesia] Otton R, Mendonca JR, Curi R. 1999. Diabetes causes marked changes in lymphocyte metabolism. J Endocrinol 174: 55-61.

Otton R, Soriano FG, Verlengia R, Curi R. 2004. Diabetes induces apoptosis in lymphocytes. J Endocrinol 182: 145-156.

Price SA, Wilson LM. 2006. Pathophysiology: clinical concept of desease processes. 6th ed. Elsevier. New York.

Smith JB, Mangkoewidjojo S. 1988. Pemeliharaan, pembiakan dan penggunaan hewan percobaan di daerah tropis. UI-Press. Jakarta.

[Indonesia] Suharmiati. 2003. Tests for anti-diabetes mellitus bioactivity of medicinal plants. Cermin Dunia Kedokteran 140: 8-13. [Indonesia] Suntoro SH. 1983. Staining Method pewarnaan. Bhratara Karya Aksara.

Jakarta. [Indonesia] Tjay TH, Rahardja K. 2007. Essential medicines: efficacy, usage and side effects. 6th ed. Elex Media Komputindo. Jakarta. [Indonesia] Unger RH, Foster DW. 1992. Diabetes mellitus. In: Wilson JD, Foster DW. Endocrinology. W.B Sunders. London.

Widowati L, Dzulkarnain B, Sa’roni. 1997. Medicinal plants for diabetes mellitus. Cermin Dunia Kedokteran 116: 53-60. [Indonesia] Widyastuti SK, Ungerer T, Mansyoer I, Lelana RP. 2005. Long-tailed macaques (Macaca fascicularis) as a model of diabetes mellitus: effect of hyperglycemia on blood lipids, nitric oxide serum, and clinical behavior. J Veteriner 2 (2): 1-5. [Indonesia]

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 9-16 March 2009

Biomass, chlorophyll and nitrogen content of leaves of two chili pepper varieties (Capsicum annum) in different fertilization treatments SUHARJA1,♥, SUTARNO² ¹ SMA Negeri 1 Klaten. Jl. Merbabu 13, Gayamprit, Klaten Selatan, Klaten 57423, Central Java, Indonesia. Tel./Fax.: +62-272-321150 ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 28 October 2008. Revision accepted: 27 January 2009.

Abstract. Suharja, Sutarno. 2009. Biomass, chlorophyll and nitrogen content of leaves of two chili pepper varieties (Capsicum annum) in different fertilization treatments. Nusantara Bioscience 1: 9-16. This study aims to determine the influence of various fertilization treatments on biomass, chlorophyll and nitrogen content of leaves from two varieties of chili, Sakti (large chili) and Fantastic (curly chili). The study was conducted in the village of Gatak, Karangnongko sub-district, Klaten District, Central Java in September 2006 to March 2007. The study used a complete block design with two factorial of chili varieties and fertilizer treatment. Fertilization treatments includes no fertilizer (control) (P1); manure 2 kg/plant (P2), manure (1 kg/plant) + chemical fertilizer (ZA, SP-36, KCl = 2: 1: 1) + NPK (P3); and manure (1 kg/plant) + chemical fertilizer (SP-36: KCl = 1:1) + liquid organic fertilizer (P4). Chlorophyll content was measured refers to Harborne (1987), whereas leaf nitrogen concentration was measured with Kjeldahl method. Data were analyzed using ANOVA followed by DMRT. The results showed that on the Fantastic chili fertilizer treatment affected the biomass and chlorophyll a, but gave no effect on chlorophyll b, total chlorophyll and leaf nitrogen. On the curly chili fertilizer treatment effected plant fresh weight, chlorophyll a and total chlorophyll, but gave no effect on dry weight, fresh fruit weight, chlorophyll b and leaf nitrogen. It is, therefore, recommended to use the formulation of manure + chemical fertilizer (SP-36: KCl = 1: 1) + liquid organic fertilizer in the cultivation of chili.

Key words: biomass, chlorophyll, leaf nitrogen, chili, Capsicum annum, fertilizing.

Abstrak. Suharja, Sutarno. 2009. Biomassa, kandungan klorofil dan nitrogen daun dua varietas cabai (Capsicum annum) pada berbagai perlakuan pemupukan. Nusantara Bioscience 1: 9-16. Penelitian ini bertujuan untuk mengetahui pengaruh berbagai perlakuan pemupukan terhadap biomassa, kandungan klorofil dan nitrogen daun dari dua varietas cabai, Sakti (cabai besar) dan Fantastic (cabai keriting). Penelitian dilakukan di Desa Gatak, Kecamatan Karangnongko, Kabupaten Klaten, Jawa Tengah pada September 2006 sampai Maret 2007. Penelitian menggunakan rancangan blok lengkap dengan dua faktorial yaitu varietas cabai dan perlakuan pemupukan.

Perlakuan pemupukan meliputi tanpa pupuk (kontrol) (P1); pupuk kandang 2 kg/tanaman (P2), pupuk kandang (1 kg/tanaman) + pupuk kimia (ZA, SP-36, KCl = 2: 1: 1) + NPK (P3); dan pupuk kandang (1 kg/tanaman) + pupuk kimia (SP-36: KCl = 1:1) + pupuk organik cair (P4). Kadar klorofil diukur merujuk Harborne (1987), sedangkan kadar nitrogen daun diukur dengan metode Kjeldahl. Data dianalisis menggunakan Analisis Varians dilanjutkan DMRT. Hasil penelitian menunjukkan pada cabai Fantastic, perlakuan berbagai macam pemupukan berpengaruh terhadap biomassa dan klorofil a, namun tidak berpengaruh terhadap kandungan klorofil b, total klorofil dan nitrogen daun. Pada cabai Sakti perlakuan pemupukan berpengaruh terhadap bobot segar tanaman, kandungan klorofil a dan total klorofil, namun tidak berpengaruh terhadap bobot kering, bobot buah segar, kandungan klorofil b dan nitrogen daun. Oleh kerena itu direkomendasikan untuk menggunakan formulasi pupuk kandang + pupuk kimia (SP-36: KCl = 1: 1) + pupuk organik cair dalam budidaya cabai.

Kata kunci: biomasa, klorofil, nitrogen daun, cabai, Capsicum annum, pemupukan.

INTRODUCTION The demand of chili pepper (Capsicum annuum L) always increases with the increase of the number of food factory, family needs and various instant noodles, sauce, and chilly industry. Results of the 2007 chili crop was 6.30 tons/ha, which was lower than in 2006 6.51 tons/ha.

Meanwhile import of pepper in 2006 was 11,885,501 tons and the export of pepper in 2006 was 8,004,450 tons. This condition implies that the production of large chili per hectare needs to be developed. Therefore it is necessary to find cultivation technology which can increase growth and yield of chili (www.hortikultura.deptan.go.id 2008).

According to Nyakpa et al. (1988) the success of a farming business is largely determined by the growth and yield of cultivated plants. If the growth and yield is satisfactory then the business is said to be successful.

Allabi (2005), further states that peppers will give good results if the essential elements needs are met. To meet the needs of the essential elements, fertilization can be done.

The successful use of organic fertilizers in encouraging crop production is not doubted. The provision of organic fertilizer to increase growth and yield can give better yield than that of phosphorus.

Meanwhile Sadewa (2008) states that the type of fertilizer mixture (N: P: K = 8.31 g: 12.21 g: 8.81 g) can improve the growth of plant’s height, root’s length and

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chlorophyll content of three varieties of chili peppers at the vegetative phase. According to Chellemi and Lazarovits (2002) applying organic fertilizer 310 and 400 kg N/ha may cause a decrease in production of chili peppers and tomatoes, even providing 560 kg N/ha may lead to increase soil pH, NH3 and the total number of fungus/mushroom, which can be toxic and can cause death of the pepper.

Based on the researches of Sadewo (2008), Allabi (2005) and Chellemi and Lazarovits (2002), chili pepper plants require macro and micro nutrients for growing and increasing the production. These needs of nutrients can be met through the provision of organic fertilizer and inorganic fertilizer. Formulation of the appropriate types of fertilizer can affect the biomass, chlorophyll content and the nitrogen of the leaf. The thought that says organic fertilizers can increase agricultural production is not entirely correct. Only the appropriate formulations of fertilizer that can affect the growth and the yield of chili.

Therefore, research to find a formulation of organic and inorganic fertilizers and/or the combination of both fertilizers and its influence on the pepper plant needs to be done.

Big or large chili (C. annuum) has many varieties, among others: the long red chili (C. annuum var. longum (DC.) Sendt), round peppers (C. annuum var. Cerasiforme (Miller) Irish), sweet chili or bell peppers or paprika (C. annuum var. grossum) and green peppers (C. annuum var. annuum) and others. Curly chili is one of the varieties of long red chili (C. annuum var. longum (DC.) Sendt) (Pracaya 2000; Setiadi 1993).

Biomass is defined as the sum total of life at any given time and a certain area. Biomass can be expressed as the biomass volume, wet weight biomass, dry weight biomass and organo biomass (Michael 1994). Prawirohatmodjo et al. (2001) further state that biomass covers the entire body of a living creature, even when the body is only a branch or a leaf of a tree as long as still attached to the plants.

According to Salisbury and Ross (1995) fresh mass is determined by harvesting the whole plants or parts of the plant and weigh them quickly before too much water evaporates from the material. About 75% of plant biomass is produced several weeks before harvest time so that at that time the plant needs higher nutrient and it absorbs fertilizer more efficiently (Rubatzky and Yamaguchi 1995).

The measurement of biomass can also use dry mass. Dry mass measurement needs to be done, because of various problems arising from the content of the water so that the productivity of cultivated plants should also be measured by using the dry mass of the plants (Salisbury and Ross 1995). Dry weight biomass is measured to obtain the overall appearance of plant growth (Sitompul and Guritno 1995). Measurement of dry weight accumulation is an analogy to know the distribution pattern of assimilation from the source to the target (Gardner et al. 1991).

Chlorophyll is a magnesium-porphyryn attached to proteins (Nelson and Cox 2004). Chlorophyll is an important catalyst for photosynthesis found in thylakoid membranes as a green pigment in plant photosynthetic tissues, which is loosely bound to proteins but easily extracted into a solvent such as acetone and ether lipids (Harborne 1987). Taller plants have two types of

chlorophyll that are chlorophyll a and chlorophyll b.

Chlorophyll a is a complex compound of magnesium and porphyrin which has cyclopentanon ring (ring V). The four nitrogen atoms linked by ties of coordination with Mg2 + forming a firm planar compound. The hydrophobic side chain which is a terpenoid alcohol, or phytol, which are connected by ester bonds propionate group of ring IV. Chlorophyll b is the second chlorophyll found in plants (Wirahadikusumah 1985). The structure of chlorophyll b is different from chlorophyll a because chlorophyll a has a methyl catalyst, while chlorophyll b has an aldehyde group which is attached on the right top of the pyrrole ring (Harborne 1987). Chlorophyll b may derive from chlorophyll a, methyl groups are oxidized on its second ring and become the aldehyde group, or are possible for the porphyrin compounds which can be converted to chlorophyll a and b (Bonner and Varner 1965). Porphyrin in chlorophyll a is the precursor of chlorophyll both a and b (Mohr and Schopfer 1995).

In plant tissues, nitrogen is a constituent component of many essential compounds such as proteins, amino acids, amides, nucleic acids, nucleotides, coenzymes (Loveless 1987), chlorophyll, cytosine, auxin (Lakitan 2007), and the main components of dry material derived from protoplasmic material plants (Salisbury and Ross 1992).

Plants absorb nitrogen element in the form of NO3- and NH4 + (Nyakpa et al. 1988).

Fertilizers are all materials provided for the ground to improve the physical, chemical and biological soil condition (Subagyo 1970). Fertilizer is a material that is given to soil, both organic and inorganic material, with objective to replace the loss of nutrients from the soil and to increase crop production (Sutejo 2002). Provision of different types of fertilizer can affect the growth and yield of plants. Yield and quality of paprika will be different depends on the different types of nitrogen fertilizer which is given to it, both PCU (polyolefinresin coated urea) and SCU (sulfur coated urea) (Guertal 2000).

Organic fertilizers have a role in influencing the physical properties, chemical and soil biological activity.

Organic fertilizers can improve soil physical characters through the formation of soil aggregate structure and a stable and closely related to water binding ability, water infiltration, reduce erosion, increase ion exchange capacity (CEC) and as a regulator of soil temperature, all affect the plant growth ( Kononova 1999; Foth 1990). Organic fertilizers contain nutrients that are needed by plant growth (Rauf 1995; Tandisau and Sariubang 1995). The use of organic extract (organic liquid fertilizer) with a concentration of 2-3 mL/L water can increase the yield of various crops, like peppers, tomatoes, and corn by about 25% (Sima 1999, 2005). Giving the organic extract with relatively short interval (7 days), can directly maintain the supply of nutrients and soil microbe vitamins that play a role in the decomposition process of soil organic matter and keep soil health (Diver 2001; Scheuerell 2004).

This study aims to know the influence of various fertilization treatments on biomass, chlorophyll content and leaf nitrogen in two varieties of chili pepper (Capsicum annuum L.), large chili and curly chili.

SUHARJA et al. – Effect of fertilization on two varieties of Capsicum annum

MATERIALS AND METHODS Time and place This research was conducted from September 2006 until March 2007 in the rice field in Gathak village, Karangnongko sub-district, Klaten District, Central Java.

The measurement of chlorophyll content and leaf nitrogen, was conducted at the Laboratory of Soil Science, Faculty of Agriculture, Sebelas Maret University, Surakarta.

Material Seeds of large chili from varieties of Fantastic and seed of curly chili varieties of Sakti which are from the same broodstock. It needs fertilizers such as NPK Mutiara, cattle manure, ZA, SP-36, KCl, and liquid organic fertilizer branded Batari Sri. According to PT. Batari Sri (2005), liquid organic fertilizer (LOF) Batari Sri is pure organic fertilizers that 97% are made from cattle urine and 3% of natural ingredients processed by fermentation to produce liquid fertilizer that do not contain zinc (Zn), copper (Cu) and lead (Pb).

Study design This research was a factorial with randomized complete block design (RCBD) with 2 factors: (i) variety (two levels) (ii) fertilization (four levels), with 3 replications and each replication consisted of 20 planted chili. Fertilization treatments included: P1 = no fertilizer as control; P2 = manure 2 kg/plant; P3 = manure (1 kg/plant) + chemical fertilizer (ZA, SP-36, KCl = 2: 1: 1) + NPK Mutiara; and P4 = manure (1 kg/plant) + chemical fertilizer (SP-36: KCl = 1:1) + liquid organic fertilizer Bathari Sri. Research parameter The parameters of this study are: biomass (plant fresh weight, dry weight of plants, and fruit fresh weight per plant), chlorophyll (chlorophyll a, chlorophyll b and total chlorophyll) and leaf nitrogen content. Measurement of research variables for parameters is done by taking three plants per block per treatment which was indicated and measured at the end of harvest.

Plant wet weight was measured by taking the entire plant and weighed. Plant dry weight was measured by drying wet plant in an oven for 48 hours (until constant weight is obtained). Wet weight of fruit was obtained in every harvest (from harvest 1 to finish). Total weight of fruit is obtained by summing the wet weight of fruit at each harvest. Weight of fruit per plant was calculated by counting the total weight of the fruit divided by the total number of plants per plot (20 plants). Plant chlorophyll content was measured with a spectrophotometer according to Harborne (1987). Plant leaf nitrogen content was measured by using the method of Sudarmaji et al. (1996).

Data analysis Data obtained from this study were analyzed by analysis of variance (ANOVA) and followed by DMRT (Duncan's multiple range test) using SPSS 10:05. In the analysis of variance, if F was greater than F table or a probability (sig) <0.05 hence H0 refused and H1 accepted.

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DMRT (Duncan) populations that had the same average grouped into one subset. In one subset the treatment was not different (Hanafi 2005; Pratista 2002).

RESULTS AND DISCUSSION Fertilization has long been known as a factor influencing the growth and yield of crops, including chili.

Fertilizing is an effort to provide the necessary elements of plant nutrient. Provision of nutrients affects the levels of organic and inorganic compounds of the plant (Rosmarkam and Yuwono 2002). Among the parameters that can be observed as a physiological phenomenon is fresh weight, dry weight, chlorophyll and nitrogen content of leaves (Marschner 1986).

Biomass Biomass in this study include the weight of wet and dry plants at the end of harvest and total wet weight of fruit per plant Plant fresh weight Fresh weight is the total weight of plants showing the results of metabolic activity. Fertilization can affect plant fresh weight as it provides nutrients from the soil. Fresh weight of pepper plants at different fertilizer treatment varied (Table 4). Fresh weight of the control group (P1) is equal to the weight of fresh manure treatment (P2), and not in one group with that treated with manure + chemical fertilizer + NPK (P3), and also that have different with that have treatment of chemical fertilizer + manure + liquid organic fertilizer (P4).

Table 1. Biomass plant two varieties of chili Varietas

Biomassa P1

P2

Plant fresh weight (g) Fantastic 228.93 a 496.01 ab Sakti 277.16 c 508.33 cd Plant dry weight (g) Fantastic 68.33b 111.11c Sakti 126.59a 155.41a Total fruit weight per plant (g) Fantastic 805.00 a 878.33 ab Sakti 530.83 c 546.67 c

P3

P4

602.11 b 607.56 d

665.65 b 656.52 d

139.67cd 354.28a

157 d 407.72a

770.83a 606.67 cd

990.93 b 705.83 d

Note: Number that are noted same on the same line means that they are not significantly different according to DMRT test at P = 0.05. P1: Control, P2: Manure (2 kg/plant), P3: Manure (1 kg/plant) + Chemical fertilizer (ZA: KCl: Sp-36 = 2: 1: 1) + NPK Mutiara, P4: manure + chemical fertilizer (KCl: SP-36 = 1: 1) + liquid organic fertilizer Bathari Sri.

Dry weight of plant Dry weight of plant of both varieties of peppers varies.

Various fertilization treatments significantly affect the increase in plant dry weight of Fantastic chili, but no significant effect on dry weight of Sakti chili (Table 5). The absence of influence of various fertilization treatments on

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the dry weight of chili is a powerful indication that fertilization has not been quite able to meet the nutrient requirements needed by chili. Fertilizer treatment on different varieties of chili show different effect. This is in line with the statement Kartasapoetra (1995) and Abdul Rahim and Jumiati (2007) that the plant will need a variety of nutrients for growth and development, it takes them different time.

Although the fertilizer treatment was not significantly different in the dry weight of Sakti, but it can be seen that there is a tendency that fertilizer treatment can improve the dry weight of both varieties of chili. This means there is a tendency of photosynthesis to increase, due to the addition of nutrients from the soil as a result of the fertilization process.

Total fruit weight per plant Total fruit weight per plant from both varieties of chili is different in various fertilizer treatments. The results showed that the fertilization treatment did not significantly affect fruit weight per plant in Sakti (Table 1). This gives an indication that the use of manure, chemical fertilizer and NPK (P3) is not always a solution to improve the yields.

Only the precise formulation that increases the weight of pepper. Meanwhile, fertilizer treatment significantly affected total fruit weight per plant in Fantastic (Table 1).

The presence of N, P, K fertilizer and organic matter deriving from chemical fertilizer and liquid organic fertilizer, make it possible to increase the weight of the chili. The combination of manure, chemical fertilizer and liquid organic fertilizer (P4) can be used to replace chemical fertilizer formulations that have been used by farmers in Klaten district.

Meanwhile in manure (1 kg/plant) + chemical fertilizer (ZA: KCl: SP-36 = 2: 1: 1) + Shake Mutiara NPK gave fruit weight per plant that was lower than the other treatments (Table 1). This can happen because the nitrogen in the treatment has exceeded the optimal point, thus causing partial breakaway of assimilated nitrogen as an amide, it just raises the nitrogen content of plants, and reduces the synthesis of carbohydrates (Rosmarkam and Yuwono 2005). Therefore, fresh weight of fruit that is formed is relatively lower than other treatments.

The increase in biomass of Sakti chili and the tendency of Fantastic chili biomass to increase show that the elements provided through fertilization function properly.

Results of correlation analysis shows that fresh weight is positively associated with plant dry weight, chlorophyll a, chlorophyll b and total chlorophyll of the two varieties of the chili. This means that if the fresh weight of plants increases then the dry weight will increase too, and so are the chlorophyll a, chlorophyll b and total chlorophyll leaves of both varieties of chili.

Organic fertilizers can provide soil organic matter that is very helpful in restoring fertility of physics, chemistry and biology of soil, because it is useful to bind soil particles through the process of soil aggregation.

Aggregation of soil can produce micro-pore space, so that the aeration in the soil becomes better and it creates optimum conditions for absorption of nutrients for plants

(Brady 1990). Influence of organic matter on soil chemical fertility, among others are kation and anion exchange capacity, the increase of soil microbial activity through decomposition and mineralization of organic matter (Suntoro 2002). Besides, organic material is also able to absorb and hold water (Juan et al. 2003), which in turn affects the accumulation of nutrients and products of metabolism that are stored in the fruit and seeds.

Meanwhile the chemical fertilizers progressively increase and complete nutrients (nitrogen, phosphorus, potassium, magnesium, sulfur) which are useful in increasing chili plant biomass. The nitrogen (from ZA and NPKMutiara) is capable of acting as a constituent of many essential compounds such as proteins, amino acids, amides, nucleic acids, nucleotides, coenzymes, and many compounds essential to metabolism, a constituent of chlorophyll, cytosine and auxine hormones and as main components of plant dry matter. Nitrogen will increase the green color of leaves, stem and leaf growth (Marschner 1986). Nitrogen is closely related to the synthesis of chlorophyll (Sallisbury and Ross 1992) and the synthesis of proteins and enzymes (Schaffer 1996). Rubisco enzyme acts as a catalyst in the fixation of CO2 that plants need for photosynthesis (Salisbury and Ross 1992; Schaffer 1996).

Therefore, increasing the nitrogen content of plants can affect the good photosynthesis through chlorophyll content and photosynthetic enzymes, thereby increasing the fotosintat (fresh weight, dry weight, and weight of chili pepper) is formed.

The phosphorus (from SP-36) is an important component of ATP constituent compounds that act as an energy source in the dark reactions of photosynthesis and the nucleoprotein, the genetic information system (DNA and RNA), cell membranes (phospholipids), and phosphoprotein. KCl Fertilization increases the availability and the absorbance of kalium, while the function of kalium in the chloroplast is to play role as a guard to keep a high pH. Kalium plays an important role in photosynthesis because it directly increases the growth and leaf area index, thus increases the assimilation of CO2 and increases translocation and assimilation of photosynthesis result (Suntoro 2002).

Sulphur (from SP-36) is needed by plants to form the amino acids cystine, cysteine and methionine. Besides, sulfur is also part of biotin, thiamine, co-enzyme A and glutationin (Marschner 1995). Sulfur also functions as an activator, cofactor or regulator of enzymes and plays a role in the process of plant physiology. Elemental sulfur is an important part of pherodoxyn, an iron and sulfur compound contained in the chloroplast and is involved in oxydoreduction reaction with electron transfer and also in the reduction of nitrate in the process of photosynthesis (Tisdalle et al. 1990).

Dolomite can increase chlorophyll because the supply of Mg from dolomite are able to increase the availability of soil Mg and Mg plant uptake (Suntoro 2002).

Artificial chemical fertilizers supplies certain nutrients in the form of highly concentrated inorganic compounds and easy to dissolve. Giving it repeatedly to plant can endanger the natural soil flora and fauna, bring in soil nutrient

SUHARJA et al. â&#x20AC;&#x201C; Effect of fertilization on two varieties of Capsicum annum

imbalances, cause water pollution, especially ground water.

Organic fertilizers supply various nutrients, especially in the form of a low concentration of organic compounds that are not easily soluble, so they do not cause nutrient imbalances in soil, even they can improve the nutrient balance. The supply of organic matter can nourish the life of the soil flora and fauna, which in turn will improve and maintain soil productivity (Nuryani and Sutanto 2002).

Significant increase in biomass, is a synergistic effect of organic extracts and chemical fertilizers in the soil. The supply of organic substrates through liquid organic fertilizer increases the activity of soil organisms that play a role in the decomposition of organic compounds and improve the physical fertility (soil structure, aggregation and aeration, increase water holding capacity) and increase nutrient availability (Reeves 1997). In such conditions the supply of oxygen which is required for respiration of soil organisms is available. Therefore, improvement of soil quality occurs in some phases and gives impact toward the increase Sakti chili biomass and tends to increase Fantastic chili biomass.

Chlorophyll content Chlorophyll a Fertilizer treatment gives effect on chlorophyll a content of two varieties of peppers (Table 2). Nutrients (nitrogen, phosphorus, magnesium, iron, manganese, potassium, calcium, sulfur) that accumulate in chemical fertilizer and organic fertilizer added to the manure treatment (1 kg/plant) + chemical fertilizer (ZA, SP-36, KCl = 2: 1: 1) + NPK Mutiara and manure (1 kg/plant) + chemical fertilizer (SP-36: KCl = 1:1) + liquid organic fertilizer can significantly increase the content of chlorophyll a in both varieties of chili. Chlorophyll a is in the leaves of both varieties of peppers varies. This is an indicator that the physiological response of these two varieties of chili against a given nutrient supply is different. In general, it is said that the supply of nutrients from fertilizer can increase chlorophyll a and both varieties of chili.

Table 2. Chlorophyll a, chlorophyll b, total chlorophyll at different fertilizer treatment given to two varieties of chili.

Varietas

P1

Kandungan klorofil (mg/L) P2 P3

Chlorophyll a (mg/L) Fantastic 2,52 a 5,99 ab Sakti 2,65 a 6,29 b Chlorophyll b (mg/L) Fantastic 1,66 a 2,80 a Sakti 1,68 a 3,23 ab Total chlorophyll (mg/L) Fantastic 4,17 a 8,78 ab Sakti 4,33 a 9,51 b

P4

6,99 b 7,48 bc

6,45 b 7,93 c

3,75 a 5,38 b

3,80 a 3,84 ab

10,72 b 13,31 b

10,25 b 11,31 b

Note: letters which are noted same on the same line means there were not significantly different according to DMRT test at P = 0.05. P1: Control, P2: Manure (2kg/tanaman), P3: Manure (1 kg/plant) Chemical Fertilizers (ZA: KCl: Sp-36 = 2: 1: 1) NPK Mutiara, P4: Fertilizer cage of chemical fertilizer (KCl: SP-36 = 1: 1) liquid organic fertilizer Bathari Sri

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Chlorophyll b Chlorophyll b functions as an antenna that gathers light and then transfer it to the reaction center. Reaction center is composed of chlorophyll a. Light energy is converted into chemical energy in the reaction center which can then be used for the reduction process in photosynthesis (Taiz and Zeiger 1991). Fertilizer treatment does not significantly affect chlorophyll b in two varieties of peppers (Table 2).

This likely occurs because most of the chlorophyll is still at the stage of chlorophyll a (chlorophyll a of both varieties are proven to be significantly influenced by different fertilizer treatment) and has not become chlorophyll b, as we know that chlorophyll a is the precursor of chlorophyll b (Robinson 1980). Although fertilization treatments do not significantly affect the chlorophyll b content in both varieties of chili, but there is a tendency that fertilization can increase the content of chlorophyll b both varieties of chili.

Total chlorophyll The use of manure (1 kg/plant) + chemical fertilizer (ZA, SP-36, KCl = 2: 1: 1) NPK Mutiara shows the highest total chlorophyll content for the two varieties of chili, because the artificial chemical fertilizers supply particular nutrients that are highly concentrated and easily soluble (N, P, K, Fe, Mg, S) that play a role in the formation of chlorophyll (Nuryani and Sutanto 2002). The control group had the lowest total chlorophyll content for both treatments, because there was no addition of nutrients from the outside, while the available nutrients in the soil were absorbed by the plant during the vegetative phase and early generative phase. Because of the low availability and low nutrient absorption then the formation of chlorophyll was disturbed. Therefore chili chlorophyll content of control group was relatively lower compared with other treatments.

The treatment of animal manure (1 kg/plant) + chemical fertilizer (SP-36: KCl = 1:1) liquid organic fertilizer is in the same group with the use of manure (1 kg/plant) of chemical fertilizer (ZA , SP-36, KCl = 2: 1: 1) NPK Mutiara.

Nutrients (nitrogen, magnesium, iron, manganese), which accumulates in the chemical fertilizer that is added through fertilization can increase the total chlorophyll content of leaves of Sakti chili (Table 2). This condition is possible to happen because the element of N, P, K, Mg, Fe, and S which are chlorophyll-forming element are available and can be absorbed by plants.

After the analysis of variant it is known that fertilizer treatment doesnâ&#x20AC;&#x2122;t significantly give effect on total chlorophyll of Fantastic chili, but significant effect on total chlorophyll of Sakti chili (Table 2). The existence of significant effect of fertilization on chlorophyll content indicates that the supply of nutrients (N, P, K, Mg, S) has a positive contribution to the process of leaf formation in Sakti chili. Meanwhile, Fantastic chili nutrient supply provided through fertilization has not been able to increase total leaf chlorophyll. This phenomenon indicates that the physiological response of both toward the fertilization is not the same, but there is a tendency that fertilization can increase the total chlorophyll content of leaves, and the manure and chemical fertilizer treatment (ZA: KCl: SP-36

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= 2: 1: 1) NPK Mutiara) is always higher than any other treatments (Table 2). The full content of nutrients in the fertilizer formulations is able to provide a stimulant for the increase in total chlorophyll content for both varieties of chili.

The addition of organic matter increases the leaf chlorophyll, and the increase will be higher if it is accompanied by adding the dolomite and KCl. KCl fertilization increases the availability and uptake of phosphorus, while the function of potassium in the chloroplast acts as a guard to keep a high pH. Giving dolomite can increase chlorophyll because the supply of Mg from dolomite are able to increase the availability of soil Mg and Mg plant absorption. Magnesium plays a very important role in the synthesis of chlorophyll (Suntoro 2002; Rahayu 2002; Santi 2002). Giving ZA, NPK Mutiara, organic fertilizer can increase the chlorophyll because it is able to provide a combination of nitrogen and magnesium which are known as elements that absolutely must be available for the formation of chlorophyll (Dwijoseputro 1986).

Nitrogen is closely related to the synthesis of chlorophyll (Salisbury and Ross 1992) and synthesis of proteins and enzymes (Schaffer 1996). Rubisco enzyme acts as a catalyst in CO3 fixation that plants need for photosynthesis (Salisbury and Ross 1992; Schaffer 1996).

Therefore, total nitrogen content in plants can influence the outcome of photosynthesis via the photosynthetic enzymes and chlorophyll formation. In plants, nitrogen initially is in the form of ammonia and subsequent ammonia has been changed into glutamic acid, catalyzed by the enzyme glutamine synthetase (Harborne 1987). Glutamic acid serves as the base material in the biosynthesis of amino acids and nucleic acids (Nyakpa et al. 1988). Robinson (1980) called glutamic acid as a precursor for the porphyrin ring in formation of chlorophyll.

Chlorophyll formation mechanism begins with the formation of α aminolevulinic acid (ALA) (Stryer 2002).

Formation of ALA through the stages of formation of glutamate through the glutamate-t-RNA, from glutamate and is converted into α ketoglut semialdehida then become the next-aldehyde with the enzyme transaminase or amino transferase enzyme ALA formed (Bonner and Varner 1965; Krogman 1979). Of the two molecules involves the enzyme ALA with dehydrated ALA will be formed porfobilinogen (PBG) containing pyrrole rings from four molecules of PBG to involve enzyme-ogen uroporfirin III. Decarboxylation changes uroporfirinogen III. Under aerobic conditions by involving kaproporfirinogen decarboxylase enzyme, kaproporfirinogen III will then be formed into proporfinogen IX. Oxidation toward proporfirinogen IX will result in proporfirin IX which does not have Mg, after joining Mg then Mg protoporfirin IX is formed. The addition of methyl groups on protoporfirin IX Mg with the help of esterase protoporfirin will form Mg porphyrin IX monomethyl ester. Next is the change of Mg porphyrin IX monomethyl ester into pro-chlorophyllide (Bonner and Varner 1965; Devlin 1983; Krogman 1979).

Change from prochlorophylide into chlorophyll takes place through the formation of proklorofilide holokrom

which binds to proteins that bind to ion 2H+, which will be given to the fourth ring which forms a holokrom proklorofilide, which can then be turned into chlorophyll a by releasing holokrom and apoprotein (Mohr and Schopfer 1995).

If chlorophyll a with the help of an enzyme that catalyzes the esterification compounds chlorophyllase phytol can be formed into chlorophyll a. Meanwhile leaf homogenate, and thylakoid supply and leaves that is protected from light can convert chlorophyll a to chlorophyll b. Therefore, chlorophyll a can become a precursor of chlorophyll b (Robinson 1980).

Results of correlation analysis shows that chlorophyll a has a positive relation with chlorophyll b and total leaf chlorophyll and is positively related to plant fresh weight.

An increased in chlorophyll a will increase the chlorophyll b, total chlorophyll and fresh weight of plant leaves. This can be easily understood because chlorophyll a is a precursor for chlorophyll b, while the chlorophyll a and b is the composition of total leaf chlorophyll, and also part of the plant fresh weight.

Leaf nitrogen content Results of the research shows that fertilizer treatments does not significantly influence the nitrogen content of leaves of two varieties of chili (Table 3). The results of this study supports the findings of the previous research which claims a combination of urea and organic fertilizer does not affect the nitrogen and chlorophyll content of hermada grass (Supriya and Soeharsono 2005). This is possible because in all treatment combinations, the plant minimum requirement for nitrogen has been met.

Therefore, although the nitrogen content provided through fertilization is high but the plants absorb only a certain number of plants as needed.

Table 3. Nitrogen content of leaves of two varieties of chillies on different fertilizer treatments.

Variety Fantastic Sakti

P1 3.64 a 3.92 b

Nitrogen content (mg/L) P2 P3 3.46 a 3.85 a 4.23 b 4.27 b

P4 4.12 a 4.25 b

Note: letters which are same on the same line means not significantly different according to DMRT test at P = 0.05. P1: Control, P2: Manure (2 kg/plant), P3: Manure (1 kg/plant) + Chemical fertilizer (ZA: KCl: Sp-36 = 2: 1: 1) + NPK Mutiara, P4 : manure + chemical fertilizer (KCl: SP-36 = 1: 1) + liquid organic fertilizer

Although the nitrogen content of leaves at different fertilizer treatment is not significant, but there is a tendency that the fertilizer treatment can increase the nitrogen content of leaves (Table 3). The tendency of leaf nitrogen content increase is a reflection of the increased nitrogen that can be absorbed by plants. The combination of ZA fertilizer, NPK Mutiara, and organic fertilizers cause an increase in leaf nitrogen content of plants.

Nitrogen that is available in the soil that can be absorbed by plant roots is in the form of nitrate ions and ammonium.

SUHARJA et al. â&#x20AC;&#x201C; Effect of fertilization on two varieties of Capsicum annum

Both forms of nitrogen is obtained as a result of organic material decomposition. Nitrates that are absorbed by the roots and transferred into the upper parts of the plant is a result the leaf transpiration process. Thus, nitrate assimilation in higher plants generally occurs in the leaves.

The first step is the reduction of nitrate into ammonia.

The second step, nitrite reaction which turn nitrite into nitrate that occurs to chlorophyll in the chloroplast. While the ammonia assimilation in most plants turn into glutamic acid. Glutamic acid serves as the base material in the biosynthesis of amino acids and nucleic acids (Harborne 1987; Nyakpa et al. 1988).

The tendency of increase in nitrogen content of plants can affect the photosynthesis either through chlorophyll content or photosynthetic enzyme. If the leaf nitrogen content increases, the fotosintat will increase, otherwise if the leaf nitrogen content decrease then fotosintat formed is also low. That's because the elements of nitrogen will increase the green color of leaves, support stem and leaf growth (Marschner 1986).

Nitrogen is closely related to the synthesis of chlorophyll (Sallisbury and Ross 1992) and synthesis of proteins and enzymes (Schaffer 1996). Rubisco enzyme acts as a catalyst in the fixation of CO2 that plants need for photosynthesis (Salisbury and Ross 1992; Schaffer 1996).

Meanwhile, Fantastic chili leaf nitrogen content, manure treatment (1 kg/plant) + chemical fertilizer (SP-36: KCl = 1:1) + liquid organic fertilizer of 4.12%, followed by the use of manure + chemical fertilizer (ZA, SP-36, KCl = 2: 1: 1) + NPK Mutiara (3.85%), followed by the control group (3.64%) and the lowest manure application (2 kg/plant ) amounted to 3.46% (Table 3).

The lower leaf nitrogen content in manure treatment than the control group at Fantastic chili, was caused by the nutrients in the manure treatment can not be absorbed optimally. This is related to the nature of the organic fertilizer that supplies a variety of nutrients, especially in the form of a low concentration of organic compounds that are not easily soluble (Nuryani and Sutanto 2002). The results of the research are consistent with research conducted by Salim (2006) which states that the type of soil management and organic fertilizer can not increase the total nitrogen, K-leaf and leaf nitrogen uptake.

CONCLUSION AND RECOMENDATION In Fantastic chili, a variety of fertilization treatment affects the biomass and chlorophyll a, but it does not affect chlorophyll b, total chlorophyll and plant leaf nitrogen. The treatment of various kinds of fertilizers to Sakti chili effects on plant fresh weight, chlorophyll a and total chlorophyll and has no effect on dry weight, weight of fresh fruit, chlorophyll b and the nitrogen content of leaves.

Researchers recommend to use the formulation of fertilizers with manure + chemical fertilizer (SP-36: KCl = 1: 1) + liquid organic fertilizer as a new alternative to chili cultivation which is more economical and environmental friendly.

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Tandisau P, Sariubang M. 1995. Manure and its relationship with soil fertility and cotton yield. J Ilmiah Penelitian Ternak Gowa. Edisi Khusus. 1-6. [Indonesia] Tisdale SL, Nelson WL, Beaton JD. 1985. Soil fertility and fertilizer.

Macmillan. New York.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp.: 17-22 March 2009

Effect of IAA and GA3 toward the growing and saponin content of purwaceng (Pimpinella alpina) 1

DASIYEM FATHONAH1,♥, SUGIYARTO2

SMA Negeri 1 Sapuran, Jl. Purworejo Km. 20 Sapuran, Wonosobo 56373, Jawa Tengah, Indonesia. Tel/Fax.: +92-286-611173, ♥email: sma1sapuran@yahoo.co.id ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 23 December 2008. Revision accepted: 4 February 2009.

Abstract. Fathonah D, Sugiyarto. 2009. Effect of IAA and GA3 toward the growing and saponin content of purwaceng (Pimpinella alpina). Nusantara Bioscience 1: 17-22. The aims of this research are to examine (i) the effect of IAA and GA3 in different concentrations to the growth of the plants and (ii) the saponin contained inside the P. alpina, leaves. The research was done in Sikunang Village, Kejajar Subdistrict, Wonosobo District, Central Java from July to November 2007. The experiment methods were used the Completely Random Design with two factors were used to analyze this experiment. First treatment gives IAA and GA3, second was done by giving different IAA and GA3 concentration. These experiments were repeated three times. Variables measured in this research were the growth of plant which is consisted of the number of leaves, their height, width, wet weight as well as dry weight. The chemical compound of the secondary metabolite in the form of leave saponin was employed. The result was analyzed by Analysis of Variance (ANOVA), then continued to Duncan Multiple Range Test in 5% level to analyze the real difference between those treatments. The result showed that giving IAA and GA3 differently affect the growth P. alpina. In variable of the height, the optimal wet weight and dry weight of the plant in GA3 treatment was 50 ppm; optimum number of leaves in GA3 treatment was 50 ppm where as the leave width in IAA treatment was 200 ppm and GA3 treatment was 75 ppm and optimum saponin treatment was IAA 200 ppm and GA3 25 ppm.

Key words: Pimpinella alpina, IAA, GA, growth, saponin, Dieng.

Abstrak. Fathonah D, Sugiyarto. 2009. Pengaruh IAA dan GA3 terhadap pertumbuhan dan kandungan saponin purwaceng (Pimpinella alpina). Nusantara Bioscience 1: 17-22. Tujuan penelitian ini adalah untuk mengkaji (i) pengaruh IAA dan GA3 dengan konsentrasi yang berbeda untuk pertumbuhan tanaman dan (ii) saponin yang terkandung di dalam daun Pimpinella alpina. Penelitian dilakukan di Desa Sikunang, Kecamatan Kejajar, Kabupaten Wonosobo, Jawa Tengah pada Juli-November 2007. Metode percobaan menggunakan Rancangan Acak Lengkap dengan dua faktor digunakan untuk menganalisis percobaan ini. Pertama memberikan perlakuan IAA dan GA3, kedua memberikan perlakuan IAA dan GA3 dengan konsentrasi berbeda. Percobaan diulang tiga kali. Variabel yang diukur adalah pertumbuhan tanaman yang terdiri dari jumlah daun, tinggi tanaman, lebar daun, berat basah maupun berat kering; serta senyawa kimia metabolit sekunder dalam bentuk saponin. Hasil penelitian ditelaah dengan Analisis Varian (ANAVA), kemudian dilanjutkan ke Uji Jarak Berganda Duncan pada tingkat 5% untuk mengetahui perbedaan nyata antara perlakuan. Hasil penelitian menunjukkan bahwa pemberian IAA dan GA3 yang berbeda mempengaruhi pertumbuhan P. alpina. Pada variabel tinggi, berat basah dan berat kering tanaman perlakuan GA3 yang optimal adalah 50 ppm, jumlah daun optimal dalam perlakuan GA3 adalah 50 ppm dimana lebar daun optimal pada perlakuan IAA adalah 200 ppm dan pada perlakuan GA3 adalah 75 ppm, sedangkan kadar saponin optimal adalah perlakuan IAA 200 ppm dan GA3 25 ppm.

Kata kunci: Pimpinella alpina, IAA, GA, pertumbuhan, saponin, Dieng.

INTRODUCTION Lately, traditional medicinal plants become popular and are wanted by modern society (the city) because it is believed that the effects of traditional medicines are relatively small when compared to modern medicines. But one of the weaknesses of traditional medicine is not much information about their chemical constituents and compounds which is responsible for biological activity.

Traditional medicine is the medicine where the ingredients are derived from nature either from plant, animal or mineral materials (MoH 1981). Purwaceng or purwoceng (Pimpinella alpina Molk.; previously named Pimpinella pruatjan Molk.) is one of the plants which has a property as

a traditional medicine that its natural existence is already scarce in Dieng Plateau, its natural habitat, along with the loss of protected forest in the region as a result of the uncontrolled forest encroachment activities by the local community. In Wonosobo District, purwaceng is naturally found only in the Villages of Sikunang, Siterus and Dieng, District of Kejajar. Even according Rahardjo (2003) and Shaheed et al. (2004) these plants exist only on a very narrow area of cultivation in Sikunang Village, no longer found in their natural habitat. Basically, this plant can grow in any area in the Dieng Plateau and planted anytime during the dry season even though it does not rain for a long time because it needs no watering as much as in potato cultivation. The morphology of P. alpina illustrated

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in Figure 1.

Figure 1. Morphology of P. alpina

Purwoceng as medicinal plants contains active compounds which give the effect of warmth on the body and increase the emotion. This crude drug has been known as a sexual desire arousing (aphrodisiac) and urine laxative drug (diuretic) (Astuti 2005). Purwaceng contains major phytochemical groups of alkaloids, polyphenols, flavonoids and saponins. Bergapten, isobergapten, and sphondin belong to the group of furanokumarin (Sidik et al. 1975), also coumarins, saponins, sterols, alkaloids, and several kinds of sugar compounds ( oligosaccharide) (Caropeboka and Lopez 1975), stigmasterol (Suzery et al. (2004), bergapten, marmesin, 4-hydroxy coumarin, umbeliferon, and psoralen (Hernani and Rostiana (2004).

Saponins have many roles, on healthy plants it functions as an anti-fungal (Zehavi et al. 1993; Bowyer et al. 1995) and anti-virus (Wu et al. 2007). Saponins also have significant anti-microbial effects (Papadopoulou et al.

1999). These molecules also act to overcome a heart attack.

Some saponins are also known as active cure against virus attacks (Zao et al. 2008). Commercially, saponins are used to inhibit tumor cell growth and to lower blood cholesterol (Ridker 2005). Low cholesterol on blood serum of East African people, who consume food products of animal which have many fat and cholesterol, because it is counterbalanced by eating the herb-rich with saponins (Oleszek and Marston 2000; Davidson 2004).

By looking at the potential of saponin which is so much in helping the body's physiologic functions, it would require further research on the chemical content of saponins in Purwaceng plants. Because purwaceng are plants that have low flexibility in terms of adaptation, it is necessary to the cultivation of purwaceng crops by manipulating the environment in order to obtain optimal results. Optimal environmental factors are expected to increase purwaceng growth and chemical content of secondary metabolites, so that purwaceng production is expected to increase economic value for people who cultivate it.

According to Salisbury and Ross (1995), high light intensity increases karotinoid content and nitrogen content, resulting in leaf surface becomes more open, but on the other hand, a very high light intensity can reduce leaf chlorophyll content. Some of the knowledge required in the cultivation technique is related to factors of light, knowledge of plants, spacing and the use of crops cover.

In addition for its fast-growing, Purwaceng is managed to contain higher secondary metabolites, one with the application of growth regulators. Growth regulator (PGR), known as plant hormones (phytohormones) is the "regulator" produced by the plant itself and at low levels, it regulates plant biochemical, physiological and morphological processes. Therefore, the effort to improve crop yields of purwaceng is necessary with the use of PGR, so it is expected that it will be more optimal for growing Purwaceng as well as increasing the content of chemical compounds. This study examines how the effect of IAA and GA3 on the growing and the saponin content of plant leaves of Purwaceng and how to influence the interaction of IAA and GA3 on growing and the saponin content of plant leaves of Purwaceng.

MATERIALS AND METHODS Plant material Materials used in this study are obtained from Purwaceng plants from Dieng Plateau on the border of Wonosobo and Banjarnegara Districts, Central Java Province.

Procedures Experiments are conducted using Completely Randomized Design (CRD), the first factor in the form of IAA and GA3 and the second factor is the concentration of IAA and GA3 which are performed differently for three repetitions including the number of leaves, plant’s height, leaf’s area, fresh weight, dry weight, while the secondary metabolites of saponin content of leaves and analyzed using analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) at 5% test level to find out the real differences among the treatments.

Table 1. The experimental design Concentration of IAA (A) 0 ppm (A0) 100 ppm (A1) 200 ppm (A2) 300 ppm (A3)

0 ppm (B 0) A0B0 A1B0 A2B0 A3B0

Concentration of GA3 (B) 50 ppm 25 ppm (B 2) (B 1) A0B1 A0B2 A1B1 A1B2 A2B1 A2B2 A3B2 A3B1

75 ppm (B 3) A0B3 A1B3 A2B3 A3B3

Seeding. Seeding is started from the manufacture of growing media and site preparation on seedling plots of land measuring 100 cm x 400 cm plus compost with a ratio of 3: 1 under protective cover/shelter. Seeding process is started by selecting a good seed that is not broken and at the same size. Sowing seeds is in the morning and in the

FATHONAH et al. – Effect of IAA and GA3 on growth and saponin content of purwoceng

early of sowing seeding, the watering is done once in 2 days until the plants are 6 weeks old and ready to be moved into polybags.

Planting. Planting is done when seeds are 6 week old on the planting medium i.e poly bag measuring 9 cm x 15 cm and filled with soil and compost with a ratio of 3: 1. At this stage watering is done every 3 days.

Treatment. Treatment begins after the age of 4 weeks of planting. Treatment includes: spraying IAA and GA3 in combination with appropriate design of experiments on plants with 10 weeks of age for 8 weeks with 8 times spraying for 1week in the morning at 09.00 am. Treatments include: controlling plants, spraying of IAA concentration of 0 ppm, 100 ppm 200 ppm and 300 ppm at different polybags, spraying of GA3 concentration of 25 ppm, 50 ppm, 75 ppm, also at different polybag. Each spraying is done as much as 5 ml with 5 times spraying with the same pressure.

Observations. The variables measured in the observations included leaf number obtained by counting all the existing leaves on the plant, plant’s height measured from the base of the stem (leaf midrib) to the tallest part of plants, the leaf was calculated with the method grafimetri (Sitompul and Guritno 1995) which is formulated as follows: LD = Wr x

LD Wr Wt Lk

LK Wt

= leaf’s area = weight of paper leaf replica = total weight of paper = total area of paper

The next variable is the wet weight of plants that are weighed with an analytical balance, dry weight of plants which is calculated by, first, the plants are harvested and immediately measured and placed in a paper bag and then roasted at a temperature of 60°C for 5 days to achieve a constant dry weight, then weighed by analytical scales.

Then the leaf saponin content analysis is conducted after harvest (plants are 2 months old) with UVspectrophotometric method with following steps, first, the extraction phase, the dry leaves are crushed with a mortar till they become powder, then 0.1 grams of powder of them are extracted with 10 milliliters of ethanol 70% above the water steam bath with a temperature of 80° C for 15 minutes, then, the phase of making a standard curve that is made by Merck Saponin standard solution with concentration 2.5, 5.0, 7.5, 10 ppm and then the absorbance is measured using UV-Vis spectrophotometer at a wavelength of 365 nm in order to obtain a standard curve of saponin (Stahl 1985). The phase of counting saponin content of the leaves, extracted leaves are counted their saponin levels using UV-Vis spectrophotometer based on the standard curve of saponin Merck. The levels obtained then is converted into mg/g dry weight of leaves (Suskendriyati et al. 2004) with the following formula: S=

Saponin content of sample x volume of dilution Sample weigh of leaves

S = concentration of saponin

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RESULTS AND DISCUSSION Plant growth According to Abidin, plant growth regulator or hormone is believed to regulate plant physiological processes (1994), because hormones can affect protein synthesis and regulation of enzyme activity. An increase in protein synthesis as raw material constituent enzymes in plant metabolic processes would enhance growth. This process can enhance the future growth can increase the biosynthesis of secondary metabolites (Taiz and Zeiger 2006).

Plant growth is influenced by several factors, including external and internal factors. Internal factors that influence the growth including auxin (IAA) and gibberellin (GA3).

Some effects of hormones on plant cell growth were as follows: stimulating effect on the growth hormone was highly inhibited by the actinomyosin D antibiotic. This substance use its influence on the cell with a very precise manner i.e. by binding DNA nucleus and prevent the two DNA bands to split up so that DNA cannot be used as a mold for manufacturing of, whether additional DNA molecules or RNA molecules. Without additional new RNA, protein synthesis by the cells freezes quickly (Kimball 1991). This understanding can be used as a base on exogenous hormone use with a certain concentration to stimulate or inhibit growth. Gene activity begins with the transcription of DNA into mRNA. mRNA comes out from the nucleus to the cytosol and is translated at the ribosomes, resulting a protein synthesis. Protein synthesis forms new enzymes and activates certain enzymes that affect metabolism. A series of metabolic processes will affect plant growth (Salisbury and Ross 1995).

Growing variable in the study includes number of leaves, leaf’s area, plant’s height, wet weight, dry weight and saponin content. The Treatment with various concentrations of IAA in this study is given to 3-month-old purwaceng plants until they reach young harvest time, namely at age of 5 months. The results are shown in Table 2.

Number of leaves Based on Table 2, it is known that the treatment will give real effect at a concentration of IAA 0 ppm GA3 50 ppm. For the overall provision of IAA, GA3, or a combination of IAA and GA3 will increase the number of leaves, except at a concentration of IAA 300 ppm GA3 0 ppm. It is possible because the provision of growth hormone IAA 300 ppm would hamper growth. The experiment of Noggle and Fritz (1983) show that exogenous IAA plays a role in inhibiting the mother leaves bone, and then the inhibition of mother leaves bone formation will also inhibit the formation of the leaf itself.

Leaf’s area Leaf is one of the growth parameters that can be observed due to environmental changes. A change in the leaf is very sensitive to environmental changes. Leaf growth is closely associated with water availability in the environment. The development of the leaves is very

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Table 2. The plant growth of P. alpina with the IAA and GA3 treatment Concentration of GA3 (ppm) 25 50 75

Concentration of IAA (ppm) Number of leaves 0 100 200 300

7.67a 9.67de 8.67cd 7.00ab

9,00cd 9,00cd 9,00cd 8,33bc

10,33e 8,67cd 9,67de 8,33bc

8,67cd 8,00bc 9,67de 8,00bc

Plant leaf’s area 0 100 200 300

24.20be 24.80bf 25.40df 19.00a

21,60bc 25,20cf 22,40bd 22,00bc

27,60f 27,60f 24,60bf 26,40ef

26,40eg 26,60ef 31,40g 22,40bd

Plant’s height 0 100 200 300

12.00ab 15.17dc 16.67c 10.83a

14,33cd 12,17de 15,33d 11,83a

20,50e 15,83d 13,83ce 11,67ab

15,00de 11, 83ab 16,00de 12,67ac

Plant’s fresh weight 0 2.57a 2.92ac 100 2.77ab 200 2.63a 300

4,12gh 3,60ef 2,77ab 3,25ee

5,82i 4,27h 3,35de 3,26ce

3,88fg 2,93ac 3,91fg 3,10bd

Plant’s dry weight 0 100 200 300

0.38ab 0.49dc 0.41bc 0.35a

0,53eg 0,55fh 0,62i 0,40ac

0,74j 0,57gi 0,42bc 0,43bc

0,51e 0,52eg 0,59hi 0,45cd

Saponin content of plant 0 9.59e 9.28d 100 9.05c 200 8.93b 300

8,92bc 8,54a 12,00l 11,53k

8,92bc 11,16j 10,83i 10,85i

8,78b 10,68h 10,54g 10,38f

0

sensitive to environmental changes and also to growth hormone either exogenous or endogenous. The following table shows the results of studies of leaf’s area of P. alpina plant on different IAA and GA3 treatment. By providing a combination of IAA and GA3, it is expected to give positive effect on changes in leaf’s area. Table 2 notes that the treatment combination of IAA and GA3 has a significant influence on leaf’s area. The highest leaf’s area is at the treatment of IAA 200 ppm GA3 75 ppm, so the capacity indicates that it is the best response of the optimal growth of leaf’s area compared to other treatments. At the treatment of IAA 300 ppm GA3 25 ppm, the leaves area are, in average, at least number, this shows that to this treatment the response of plant for exogenous hormones has the character of inhibiting the growth of leaves area.

Plant’s height Plant’s height is very sensitive to water availability in the soil. Plant’s height is the parameter most frequently observed to measure the influence of the environment (Sitompul and Guritno 1995). Treatment of plant’s cells

with auxin causes an increase not only in the synthesis of RNA but also in protein synthesis. At first, the application of synthetic gibberellin to plant cells leads to the explosion of RNA synthesis which is then followed by a synthesis of various hydrolytic enzymes (Kimball 1999). These activities encourage good growth processes of roots, stems and leaves. The following table is the results of research on plant’s height of P. alpina on different IAA and GA3 treatment. From Table 4, it is noted that the combination treatment of IAA and GA3 has a significant influence on plant’s height. The average plant’s height is seen in combination IAA 0 ppm GA3 50 ppm. This shows that this combination is best treatment for variable height. By giving IAA 300 ppm GA3 0 ppm, the lowest plant’s height is created, this is possible that in such combinations there are negative feedback so that the plant suffered intrauterine growth retardation.

Plant fresh weight All synthetic plant hormones or compounds that have physiological and biochemical properties similar to plant hormones are plant growth regulators (plant growth regulator substances). Plant hormones and plant growth regulator in general encourage the growth and development occurs. The effect of the plant growth regulator (PGR) depends on plant species, the PGR site of action on plants, plant growth stage and concentration of PGR. One PGR does not work alone in influencing the growth and development of plants. In general, equilibrium of concentration of some PGR will control the growth and development of plants (Kusumo 1989). It is noted in Table 2 that the treatment combination of IAA and GA3 have a significant influence on fresh weight of plants. The lowest fresh weight is obtained in the combination treatment of IAA 0 ppm GA3 0 ppm. The highest fresh weight occurrs in the treatment of IAA 0 ppm GA3 50 ppm. This means that the plant growth regulator provided at that concentration affects optimally on the growth of almost all aspects of growing except the leaf’s area growth.

Dry weight of plant Plant’s dry weight depends on the speed capability of cells to divide, enlarged and elongated. The speed of cell activity can be influenced by growth of hormones such as auxin and cytokinin endogenous. The addition of some exogenous growth hormone is expected to accelerate the growth process. Auxin affects stem length increment, growth, differentiation and branching roots. While the provision of gibberellins promotes bud development, stem elongation and leaf growth, influencing growth and differentiation are also roots, plant dry weight of P. alpina.

Table 2 notes that the treatment combination of IAA and GA3 have a significant influence on plant dry weight. The highest plant dry weight was obtained at treatment combinations of IAA 0 ppm GA3 50 ppm, this indicates that the combination is optimal growth. The lowest dry weight contained in the IAA treatment 300 ppm GA3 0 ppm. This shows that the combination treatment does not occur in an optimal growth due to metabolic disorders.

FATHONAH et al. – Effect of IAA and GA3 on growth and saponin content of purwoceng

Saponin content in leaf Secondary metabolites in plant cells accumulate in different amounts. Secondary metabolism contributes to survival, one of which was in self defense (Manito 1992).

Saponin is one class of terpenoid secondary metabolites which are synthesized through the acid path mevalonate of respiration. From Table 2, it is noted that the combination treatment of IAA and GA3 has a significant influence on levels of leaf saponins. The highest saponin content present in treatment of IAA 200 ppm GA3 25 ppm and saponin content of the lowest in the treatment of IAA 100 ppm GA3 25 ppm.

Discussion The results of this study show giving ZPT on the various treatments had significant effect on growth and saponin content of plant P. alpina. The following table calculates the average dry weight and saponin content of each crop in each treatment (Table 3).

The effect of IAA IAA at a concentration of 100-200 ppm affects various growth parameters including leaf number, plant’s height, leaf’s area and plant fresh weight. Giving IAA at low concentrations of 100 ppm gives a real difference to the number of leaves formed. The number of leaves is strongly influenced by genetic factors (Goldworthy and Fisher 1992). In this experiment, 300 ppm IAA treatment produces the least number of leaves, although no significant difference with control plants. The higher concentration of IAA is given, the fewer leaves are formed.

Table 3. Average dry weight calculations and saponin content of each crop in each treatment Treatment Saponin Saponin content Dry weigh content (mg/g) of each plant (ppm) IAA 0 + GA3 0 0.35 9.59 3.36 IAA 100 + GA3 0 0.49 9.28 4.55** IAA 200 + GA3 0 0.41 9.05 3.71 IAA 300 + GA3 0 0.38 8.93 3.39 IAA 0 + GA3 25 0.53 8.92 4.23 IAA 0 + GA3 50 0.74 8.85 6.55 ** IAA 0 + GA3 75 0.51 8.79 4.48 IAA 100 + GA3 25 0.55 8.54 4.70 IAA 200 + GA3 25 0.62 12.00 7.44** IAA 300 + GA3 25 0.40 11.53 4.61 IAA 100 + GA3 50 0.57 11.16 6.36 IAA 200 + GA3 50 0.42 10.82 4.55 IAA 300 + GA350 0.43 10.85 4.67 IAA 100 + GA3 75 0.52 10.68 5.55 IAA 200 + GA3 75 0.59 10.54 6.22 IAA 300 + GA3 75 0.45 10.38 4.67

Based on the research it is known that the treatment will give real effect on leaf’s area at a concentration of 200 ppm IAA, although not significantly different from a concentration of 100 ppm. In the provision of the 300 ppm IAA, the plant’s height is low, although not significantly different from control plants. The provision of IAA which

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is not optimal will inhibit the growth of the plant itself (Hopkins 1999). Provision of 200 ppm IAA shows the highest number of leaves, although not significantly different from 100 ppm IAA and control, while the provision of 300 ppm IAA significantly different with the control. Giving the IAA will increase leaf’s area formed.

IAA triggers the formation of mesophyll tissue so leaf’s area which is formed also increases. The provision of the concentration of 100 ppm IAA gives the highest fresh weight, although not significantly different from the concentration of 200 ppm IAA treatment. Giving IAA 300 ppm gives no significant difference in wet weight. IAA plays a role in cell elongation, especially in the vertical direction. Elongation will be followed by cell enlargement and increased wet weight. Increased wet weight is mainly due to higher water uptake by these cells (Noggle and Fritz 1983).

Giving IAA concentration of 100 ppm gives a lower weight control, while the provision of 300 ppm IAA not significantly different from the control. Growth associated with increasing volume and number of cells, the formation of protoplasm, and in subsequent, weight of the dry weight.

Drying aims to stop the cellular metabolism of these materials (Sitompul and Guritno 1995). Provision of IAA in plants P. alpina has the effect of accelerating growth at a concentration of 100-200 ppm, but not so with the content of saponin. The content of saponin in this study is best in the condition without treatment and then followed at a concentration of 100, 200 and 300 ppm.

The effect of GA3 The results of this study show that the growth of P. alpina is strongly influenced by the provision of GA3. The provision of GA3 within 8 weeks had an impact on the process of growth. From the observed parameters, the provision of 50 ppm GA3 had optimal growing the number of leaves, plant’s height, leaf’s area, fresh weight, dry weight and this effect is inversely proportional to the saponin content. The higher concentration of GA3 given, the saponin levels produced decreases. The decrease occurred in the treatment of 75 ppm is possible because the concentration gives a negative feedback effect on the growth of the primary plant.

Taiz and Zeiger (2006) explains that the provision of high GA3 will cause a decrease transcription of GA20 oxides which is a major target in the regulation of feedback. If transcription of these compounds decreased, there will be biosynthesis hindrance of GA3 which will cause activity of GA3 to decrease. The results are consistent with reports of Chairani (1988) that the application of concentration of 50 ppm GA3 gives good effect in increasing the biomass of leaves of Mentha piperita plants.

At a concentration of 50 ppm, leaf dry weight is 56% higher than the controls and is different from the results of research Khristyana et al. (2005) that the concentration of 75 ppm GA3 treatment in Plantago major shows significant difference with control.

Results analysis of variance of plant P. alpina shows that GA3 provide significant different levels of saponins at level of 5% in the test. Based on researched data, the

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highest levels of saponin was in the control and the levels decreased along with the increasing concentrations of GA3.

GA3 affects nucleic acid metabolism that play a role in protein synthesis and regulate the activity of enzymes for plant growth. Increased protein synthesis as a raw material constituent enzymes in plant metabolic processes may increase the biosynthesis of secondary metabolites, including saponins at a later time (Martin 1998).

Providing a combination of IAA and GA3 on various treatments significantly affects the growth and saponin content of plant P. alpina. The results of this study show the growth of P. alpina is strongly influenced by a combination of IAA and GA3 treatment. The provision of IAA and GA3 within 8 weeks affects on the growth process which is shown on its dry weight at the treatment of IAA 200 ppm GA3 25 ppm, although no significant difference in the treatment this. In general, the combination of IAA and GA3 treatment that will increase the growth can be seen from the increasing of wet weight and dry weight. The treatment combination of IAA and GA3 enables the influence of IAA and GA3 to be optimal since the IAA is required for maximum effect of GA3 work. The research shows a combination of IAA 200 ppm GA3 25 ppm produced the highest saponin content. It means that the treatment combinations gave a real influence on the growth and on saponin content of P. alpina plant on the concentration of IAA 200 ppm GA3 25 ppm.

CONCLUSION The provision of different plant growth regulator (PGR) affects the growth of P. alpina and at the variables of plant’s height, leaf’s number, fresh weight, dry weight which was optimal at GA3 50 ppm treatment, the optimal leaf’s area growth is at the treatment of IAA 200 GA3 75 ppm, while the saponin content is optimal at the treatment of IAA 200 GA3 25 ppm. The provision of different plant growth regulator affects the leaf saponin content of P. alpina. In a single treatment, saponin content is lower than control plants, whereas the treatment combination of IAA 200 GA3 25 ppm increases leaf saponin content of 12 mg/g.

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Penerbit ITB. Bandung. [Indonesia] Suskendriyati H, Solichatun, Setyawan AD. 2004. Growth and saponin production Talinum paniculatum Gaertn callus cultures. with a variety of carbon sources.. Biosmart 6 (1): 19-23. [Indonesia] Suzery M, Cahyono B, Ngadiwiyana, Nurhasnawati H. 2004. Stigmasterol compounds from Pimpinella alpina Molk. Suplemen 39 (1): 39-41.

[Indonesia] Syahid SF, Rostiana O, Rohmah M. 2004. Effect of NAA and IBA on rooting purwoceng (Pimpinella alpina Molk.) in vitro. Indonesian Biopharmaca Excibition and Conference. Yogyakarta, 14-15 Juli 2004. [Indonesia] Taiz L, Zeiger E. 2006. Plant physiology. 4th ed. Sinauer. Sunderland.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 23-30 March 2009

Body weight and statistic vital of Texel sheep in Wonosobo District by giving the ramie hay as an additional woof AGUS KUNTJORO1,♥, SUTARNO², OKID PARAMA ASTIRIN² ¹ Office of Animal Husbandry and Fisheries of Wonosobo District, Jl. Mayjend Bambang Sugeng Km. 1 Wonosobo 56319, Central Java, Indonesia; Tel./Fax.: +92-286-321470, ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 3 March 2008. Revision accepted: 30 June 2008.

Abstract. Kuntjoro A, Sutarno, Astirin OP. 2009. Body weight and statistic vital of Texel sheep in Wonosobo District by giving the ramie hay as an additional woof. Nusantara Bioscience 1: 23-30. This research is aimed to observe the body weight and statistic vital measurement of 50 Texel sheep. Sheep are classified into five treatments of giving woof P0 (giving tree greenish woof without concentrate), P1 (giving greenish woof and concentrate without adding the ramie hay/0%) concentrate), P2 (giving greenish woof and concentrate by adding 10%) ramie hay), P3 (giving greenish woof and concentrate by adding 20%) ramie hay), P4 (giving greenish woof and concentrate by adding 30%) ramie hay), every treatment was repeated 10 times. The result shows that even it can’t yet replace the concentrate function, but adding ramie hay as much as 10%), 20%) and 30%) on sheep woof can increase the body weight’s growth respectively 186.67 g/day, 153.34 g/day dan 103.34 g/day. The addition of ramie hay 10%), 20%) and 30%) can increase the addition of statistic vital’s measurement on breast of sheep livestock 1.20 cm); 0.95 cm) and 0.90 cm); the addition of statistic vital measurement on the body length of sheep livestock 0.05 cm); 1.00 cm) and 0.75 cm) and also the addition of breast width is 1.50 cm); 0.15 cm) and 0.3 cm). Meanwhile the addition of ramie hay on livestock woof can only increase the addition of statistic vital mesurement on breast at giving 30%) as big as 0.15 cm). It is needed to know further on giving ramie hay by concentration comparasion of hay of different leaf and stem.

Key words: ramie hay, body weight, statistic vital, Texel sheep.

Abstrak. Kuntjoro A, Sutarno, Astirin OP. 2009. Bobot badan dan statistik vital domba Texel di Kabupaten Wonosobo dengan pemberian limbah rami sebagai pakan tambahan. Nusantara Bioscience 1: 23-30. Penelitian ini bertujuan untuk mengamati bobot badan dan pengukuran statistik vital dari 50 domba Texel. Domba dikelompokkan ke dalam lima perlakuan pemberian pakan, yaitu P0 (pakan hijauan, tanpa konsentrat), P1 (pakan hijauan dan konsentrat tanpa limbah rami/0%), P2 (pakan hijauan dan konsentrat dengan menambahkan 10% limbah rami), P3 (pakan hijauan dan konsentrat dengan menambahkan 20% limbah rami), P4 (pakan hijauan dan konsentrat dengan menambahkan 30% limbah rami), setiap perlakuan diulang 10 kali. Hasil penelitian menunjukkan bahwa meskipun belum dapat menggantikan fungsi konsentrat, penambahan limbah rami sebanyak 10%, 20% dan 30% pada pakan dapat meningkatkan pertumbuhan bobot tubuh domba masing-masing sebesar 186,67 g/hari, 153,34 g/hari dan 103,34 g/hari; juga meningkatkan statistik vital pada dalam dada domba sebesar 1,20 cm, 0,95 cm dan 0,90 cm; panjang tubuh 0,05 cm, 1,00 cm dan 0,75 cm; dalam dada 1,50 cm, 0,15 cm dan 0,3 cm. Sementara itu penambahan limbah rami pada pakan ternak hanya dapat meningkatkan penambahan ukuran statistik vital dalam dada pada pemberian rami 30% sebesar 0,15 cm. Perlu kajian lebih lanjut tentang pemberian limbah rami dengan berbagai konsentrasi limbah daun dan batang yang berbeda.

Kata kunci: sampah goni, berat badan, statistik vital, domba Texel.

INTRODUCTION In Wonosobo district a kind of sheep, namely Texel, has been limitedly domesticated. This Texel, or locally named dombos, is a superior type of sheep which produces meat and wool with a fairly good quality. With the support of the potential areas available in Wonosobo District, it is easy to develop the farm of Texel sheep (Ovis aries) with excellent results, with relatively rapid growth and with the weight of the adult males can reach 90-100 kg, and adult females can reach 50-70 kg. With that consideration, many people raise them in their farm (Livestock Office of Wonosobo District, 2001, 2007).

Besides, Wonosobo is a producer of ramie plants (Boehmeria nivea (Linn.) Gaudich.), which are used as raw material for textiles (Brink dan Escobin 2003; Escobin 2005). Ramie (Boehmeria nivea Gaudich) is a typical textile raw material in China (Bally 1957) and used for Chinese burial shrouds over 2,000 years and also used in mummy cloths in Egypt about 3000-5000 years BC. It is used for the production of textiles and ropes because it is extremely absorbent, dries quickly, dyes fairly easy, resists shrinkage, and is unusually tolerant with bacteria, mildew and insect attacks (Wang et al. 2007). Ramie has long been used in Indonesia, the kingdom of Majapahit mentioned ramie among the goods brought from China (Yoshimoto 1988). Rami is very important. Many research have bbeen

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done to increase production by phytohormones (Prayudhiani 2000; Wang et al. 2007), microorganisms (Mayerni 2004), in vitro propagation (Wang et al. 2008), and even genetic engineering (Dusi et al. 1993).

Currently, ramie hay is just thrown away or used as fertilizer in Wonosobo. While ramie has the necessary nutrients that cattles need, ramie leaf hay has a relatively high protein content (24%), so that when they are mixed as animal feed ingredients, they can increase the efficiency for the food costs (Agrina Prima 2006). Ramie plants are suitable to be planted in Indonesia at the ideal height of 400-1500 m asl (above sea level), with daily temperature 20-27oC and rainfall > 140 mm/month evenly throughout the year, open-structured soils such as clay, sand lightly with a pH of 5.6 to 6.5 by ages productive 6-8 years, harvested 5-6 times a year (Dempsey 1975; Sudiro 2004).

This plant has a drought tolerant cultivar (Liu et al. 2005).

Fiber productivity are highest in the highlands (> 700 m asl.), 2.5 to 3.0 tons/ha/year, followed by mainland medium (400-700 m asl.), 2.0 to 2.5 tons/ha/year, and lowlands (400 m asl.), 1.5 to 2.0 tons/ha/year (Setyo-Budi et al. 2005).

Intensive cultivation of ramie in Bogor, can produce up to 4.5 tons/ha/year, and crop yields have increased in the following year, in order to obtain an average production of 5.0 tons/ha/year, with leaf and stems production ratio of 45% and 55%.

Ramie leaves contain protein, fat and high fiber, so as to improve the nutritional value of feed when used as a concentrate (Sastrosupadi 2004). Provision of ramie on a small cattle no significant negative effect, but granting ramie above 30 kg/day in dairy cows resulted in wet eczema disease in the legs (Lahiya 1984). Dinh et al.

(2007) states that ramie good fresh whole plants or parts of it have leaf crude protein (> 21% dry weight and ash (1922% dry weight) is high, drying can reduce the crude protein content. The coefficient of digestibility of organic matter, crude protein and fresh leaves of ramie fiber, respectively, 78.5, 80.9 and 82.6%, while the dried leaves, respectively, 63.1, 60.6, and 76.1% and in all their fresh crops respectively are 66.1, 75.9, and 62.5%, so that ramie has a high nutritional value for ruminants.

Animal feeding is one factor that is very strategic for the success of poultry farms, because it contributes Âą 70% of the total production cost (BPTP 2000). Potential agricultural waste can be used as concentrate feedstuffs.

Waste is always associated with low prices, but there are some things to consider in its utilization of continuity, availability, nutrient content and the possibility of limiting factors such as anti-nutritional substances and whether or not the material is processed before it can be used as feedstuffs (Mathius and Sinurat 2004 .)

Physiologically, sheep require roughage in their feed, especially coming from forage such as fresh grass, hay, silage or hay and grain mixtures containing minerals and vitamins. Twigs, branches from trees and shrubs can be used as food additives for the sheep beside fresh grass and forage (Hanafi 2004). Sheep food is in the form of food concentrates and forages. Greenery is good forage that has more fiber content of 18% which is a natural food for ruminants, either in the form of grasses consisting of grass

and grass field as well as the form of superior legume.

Concentrate is a kind of food with a high protein content and low crude fiber content (Sofyan and Lili 2000).

For growth, production, reproduction and basic living, animals need nutrients. Fattening aims to produce a high and efficient weight gain and to produce a high-quality carcass, then it is needed the food that contains high nutrition, because livestock production would increase if the nutrient content of food increased (Tillman et al. 1991).

Concentrates can stimulate the growth of beneficial bacteria in the rumen forage digestion that in turn increases the body weight (Dirdjopranoto et al. 2007).

Protein is essential for life because these substances are active protoplasm in all living cells (Anggorodi 1990).

Protein plays an important role in the process of growth, production and reproduction. According to Tillman et al.

(1991) growth is generally expressed by the measurement of weight gain, carried out by weighing repeatedly and checking the growth of body weight every day, every week or every other time. Growth stages which have fast and slow stages. The fast phase occurs at the time of puberty and the slow phase occurs when the animals have reached adult ages. Meanwhile, according Sugeng (2000) to assess the outer form of the animals, the measurements of certain parts such as body length, width and in the chest, chest circumference and height are carried out.

This study is aimed to determine weight gain and measurement of vital statistics including chest circumference, body length, high gumba, in the chest and chest width on the sheep of Texel after the addition of ramie hay on the additional food.

MATERIALS AND METHODS Time and place The experiment was conducted in the farmland belonging to Bina Tani group at Tegalgot Village, Kepil Subdistrict, Wonosobo District, Central Java. The experiment was conducted from September until October 2007. Preparation of concentrate food was carried out in the Fooder Factory Mill belonging to Bina Tani, Wonosobo. Proximate analysis of food was carried out at the Laboratory of Animal Nutrition and Feed, Department of Animal Science, Faculty of Agriculture, Sebelas Maret University, Surakarta.

Materials The sheep used in this research was cross-fertilization between Texel sheep and cattle belonging to a group of local sheep of Bina Tani, Tegalgot Village, Kepil Subdistrict, Wonosobo District, with the age of 80-10 months, consisting of 50 animals with the weight of 14-25 kg.

Cage used as research material is the one owned by farmers of Bina Tani group with the size of 40 cm x 120 cm consisting of 50 cages.

Forage was obtained by Bina Tani group members by finding the form of forage grass and Leguminous that are available around the Village area Tegalgot, District Kepil,

KUNTJORO et al. – Effect of hemp on body weight and statistic vital of texel sheep

with each group member brought a basket weighing about 30 kg.

Concentrates were obtained from the forage factory owned by Bina Tani group under the technical supervision of the Faculty of Animal Science, Gadjah Mada University, Yogyakarta, and Office of Animal Husbandry and Fisheries of Wonosobo District.

The ramie in the form of leaves and stems (45%: 55%) in dry condition (moisture content 10-15%), was obtained from the farmers' ramie and ramie-processing factory of PT. Prima Agrina, Wonosobo.

Experimental design The research design used a completely randomized design with 4 treatments in the form of food concentrates and the addition of ramie hay (0%, 10%, 20%, 30%), as control animals given only greenery. Each treatment and control was carried out with 10 replications for observation time 0 week, 2 weeks and 4 weeks.

Procedures Food making The addition of ramie hay in the concentrate is mixed homogeneously using a machine (mixer) of the factory by mixing the following: P0 = forage alone.

P1 = concentrate without the addition of ramie hay; P2 = 90% concentrate plus 10% ramie hay; P3 = 80% concentrate plus 20% ramie hay P4 = 70% concentrate plus 30% ramie hay To know the nutrient content of the food concentrate for each treatment (addition of ramie hay 0%, 10%, 20% and 30%) we conducted the proximate analysis at the Laboratory of Animal Nutrition and Feed, Faculty of Agriculture, Sebelas Maret University, Surakarta.

Feeding The five groups of animals (P0, P1, P2, P3, and P4) were given forage in sufficient quantities as needed (± 20% of body weight) or about 4 kg/day and the feeding was done twice a day morning and afternoon. Providing additional food and concentrates were given to the groups of animals (P1, P2, P3, and P4) in sufficient quantities as needed (± 2.5% of body weight) or 500 g/day and administered twice a day, given before the greenery was given.

Weighing the body’s weight Weighing done with scales (dacin) by placing animals in ramie sacks that have been specially made. Weighing is done three times, namely before the start of treatment (0 weeks), after the research had been carried out for 2 weeks and at the end of the study (4 weeks).

Measurement of vital statistics Measurements made with dipsticks and metline. The chest line was measured by using the metline encircling the chest at the back of the shoulder. The length of the torso was measured by using the dipstick which was the distance

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between the front edge of the shoulder joint and the filter bone. The height of gumba was measured with dipstick of the highest part of gumba to the ground following the perpendicular line. The inner line of the chest is measured by using a dipstick by drawing a vertical line between the edge of the back and the chest. The width of the chest is measured by using a dipstick by drawing a horizontal line between the outer edge of the left and right shoulder joints.

Data analysis The data obtained was analyzed by using the analysis of variance (ANOVA) one way with the observed variables of body weight and vital statistics of the sheep, then the further tests with LSD were conducted.

RESULTS AND DISCUSSION Nutritional content of the food Test results proximate of nutrient content of the food given in this trial are presented in Table 1.

Table 1. Nutrient content of the foods given in the research.

Treatment

Composition

Crude protein

Crude fiber

P1 P2 P3 P4

without ramie added by 10% ramie added by 20% ramie added by 30% ramie

13.84% 13.14% 12.64% 11.02%

11.61% 13.95% 14.61% 15.29%

Protein is essential for life, because these substances are active protoplasm in all living cells (Anggorodi 1990).

Protein has an important role in the process of growth, production and reproduction. The protein content of ramie leaves is 24-26%, 5-6% for fat; 25-30% for Phosphorus, 56% for calcium, 36-46% for carotene (Sudiro 2004).

Protein has an important role in the process of growth, production and reproduction. The needs of the rude protein for the sheep range from 12.5 to 14.4% from the rations (BPTP Ungaran 2000), while according Anggorodi (1990) protein content of leguminosa ranges from 14.3 to 17.4% and the one of grass ranges from 4.3-10.3%, so the feeding should be mixed with legume and grass or other supplementary food.

High fiber content can inhibit the growth of the animals. According to Hanafi (2004) twigs, branches from trees and shrubs can be used as food additives other than fresh grass and other greenery. However, the selection of the food ingredients should consider possible limiting factors such as nutrition and the anti nutrient material whether or not it is processed before it can be used as food (Mathius and Sinurat 2004). According to Sutardi (1980), the lignin content of the greenery is closely related to the benefits of fodder. If the rate is high, then the coefficient of food digestibility related to the benefits of forage is low.

Texel sheep body weight In this study, the initial data is the result of the initial weight of the animals gained by weighing all the sheep to

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above the ones treated with additional food for fattening sheep given with ramie hay carried out by The Office of Animal Husbandry and Fisheries of Wonosobo District in 2005 which only gained 71 g/head/days. This situation can occur because the sheep which is used in the research the result of cross-fertilization of the Texel sheep species for the fattening project. Standard average weight gain for the pure species of Texel sheep is 300 g/day, with the test results on individual growth varies between 250 and 540 g/day (Anon 2000). According to Sodiq and Abidin (2002) the highest daily growth could be gained with the weight gain of 0.3 kg per day. Average daily weight gain that can be achieved with intensive maintenance is 0.2 kg per day.

In the efforts of cattle fattening, growth is an important goal. Excess food from basic living needs will be used to increase the body weight. The cattle’s weight gain reflects the extent of the benefits of food given to the cattle (Hanafi 2004). After multiple comparison tests between the means using the LSD method, it is showed that the significance difference in the control group with each treatment was only found in P1 and P2. This is proved from the results of the test for each mean of which all have significance under 0.05 (P, 0.05). Thus it indicates that the treatment of giving the 10% and 20% of ramie hay has significant impact (P <0.05) on weight gain for the Texel sheep. Besides, according to Gabbi et al. (2004) the increase in the supply of ramie hay to fattening White New Zealand rabbits has a negative response on the productive performance of animals, when those already receive high levels of fiber on the diet. According to de Toledo et al. (2008) the combination of ramie and alfalfa hays, as main fiber source ingredient in the diets of rabbits, caused a positive synergic effect and improve growth performance.

determine the initial weight of each treatment. The initial weight of Texel sheep throughout the study group had a range of 14-25 kg with an average body weight of 20 kg.

Texel sheep with body weight of the observation period of 2 weeks after treatment had a range of 16-28 kg with an average body weight of 23.48 kg. Texel sheep’s body weight of the observation period of 4 weeks after treatment had a range of 18-29 kg with an average body weight of 25.86 kg (Figure 1).

The highest average initial body weight (0 weeks) is in the group given with the addition of ramie hay by 30% which was P4 (21.3 kg), and then flowed by P0 and P3 (which had a weight of 20 kg), and P1 (19,6 kg). The results of weighing the average body weight on the second week obtained the highest body weight which was P4 (23,6 kg), followed by P1 (23,5 kg), P2 and P3 (22,9 kg), and then the lowest was P0. The result of weighing the average body weight after 4 weeks was that highest body weight was gained by P1 (26,4 kg), then consecutively followed

by P2 (24,7 kg), P3 (24,6 kg) and P4 (24,4 kg) and the lowest achieved by P0 (22,4 kg).

The body weight Texel sheep on all treatments shows different increase in the body weight. The highest weight was gained by P1 (213,34 g/head/day), followed by P2 (186,67 g/head/day), P3 (153,34 g/head/day), and P4 (103,34 g/head/day), while the lowest was P0 (96,67 g/head/day). The highest percentage of the increase in body weight was achieved by P1 (32%), followed by P2 (29,32%), P3 (23%), while the lowest was P4 (14,55%). It is below the average daily gain control group which was 18.37% (Figure 2).

From Figure 2, it can be concluded that the Texel sheep which has been researched are still below the standard weight gain of the pure species of Texel sheep, but already

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Figure 1. The average frequency distribution measurements of Texel sheep according to the treatment period. A. body weight of sheep, B. chest circumference, C. body length, D. high gumbai, E. inner chest, F. chest width.

KUNTJORO et al. – Effect of hemp on body weight and statistic vital of texel sheep

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Figure 2. The average percentage increasement in Texel sheep on each treatment. A. body weight of sheep, B. chest circumference, C. body length, D. high gumbai, E. inner chest, F. chest width. P0: ♦ , P1: ■ , P2: ▲ , P3: X , P4: *.

The data of body weight increase in Figure 2 shows that the greater the addition of ramie hay means the more negative impact on the weight gain. This situation is caused by a decrease in protein content of the food given with the additional ramie hay, even though the food given is already in accordance with the needs of the sheep.

Vital statistics of the Texel sheep Chest circumference The average chest circumference of Texel sheep at the beginning, middle and end of treatment is presented in Figure 1. The highest average of chest circumference of Texel sheep at the beginning of treatment (0 weeks) is at P4 (65.6 cm), then at P3 (64.8 cm), P1 (64.6 cm), and P0 (59.45 cm), while the lowest is at P2 (56.75 cm). The measurement results on average chest circumference after two weeks of treatment shows the highest chest circumference is at P4 (66 cm), followed by P3 (65.15 cm), P1 (64.8 cm) and P0 (59.85 cm), and the lowest average of chest circumference was obtained at P2 (57.45 cm). The measurement results on average chest circumference after four weeks of treatment achieved the highest chest circumference at P4 (66.5 cm), followed by P1 (65.85 cm), P3 (65.75 cm), P0 (59.7 cm) and the lowest at P2 (57.95 cm).

Chest circumference of Texel sheep on all treatments shows a different increment. The highest chest circumference is reached at P1 (1.25 cm), followed by P2 (1.2 cm), P3 (0.95 cm), and P4 (0.9 cm), whereas the

lowest is at P0 (0.40 cm). The highest increment percentage of chest circumference is achieved in P1 (1.25%), followed by P2 (1.2%), P3 (0.95%), P4 (0.9%), while the lowest is at P0 (0.4 %) (Figure 2).

The results of Anova further test with LSD show that the addition of ramie hay 10%, 20% and 30% can increase the vital statistics growth of each Texel sheep chest circumference of 1.20 cm, 0.95 cm and 0.90 cm. This situation is caused by degradation of feed protein and increment of feed crude fiber due to the addition of ramie hay. The addition of ramie hay in cattle feed lowers the percentage of protein content of feed and increases feed crude fiber, where protein plays an important role in the process of growth, production and reproduction.

Body length The average body length of Texel sheep at the beginning, middle and end of treatment is presented in Figure 1. The highest average body length at the beginning of treatment (0 weeks) is found in P4 (67.8 cm), then in P0 (63.9 cm), in P3 (53.8 cm), while the lowest is in P1 and P2 (respectively, 52.4 cm). The result of measuring the average body length on two weeks after treatment obtain the highest body length is on P4 (68.15 cm), followed by P0 (64.05 cm), P3 (54.6 cm), P2 (53.2 cm) and the lowest is at P1 (53 cm). The measurement results of average body length four weeks after treatment show the highest body length is P4 (68.15 cm), then P0 (64.1 cm), P3 (54.6 cm), P1 (53.55 cm), and the lowest is P2 (53.45 cm).

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The average body length of Texel sheep on all treatments shows different increment. The highest average length increment of sheep is at P1 (1.15 cm), followed by P2 (1.05 cm), then P3 (0.8 cm) and P4 (0.35 cm), while the lowest is at P0 (0.2 cm). The highest increment percentage on body length is achieved in P1 (2.19%), then P2 (2%), P3 (1.49%), and P4 (0.52%), while the lowest is in P0 (0.31%) (Figure 2).

The results of Anova further test with LSD show that the addition of ramie hay of 10%, 20% and 30% in the feed can increase the vital statistics addition of body length, consecutively, of 1.05 cm, 0.80 cm and 0.35 cm. The decrease condition is caused by the degradation of feed protein and crude fiber increment on feed due to the addition of ramie hay.

Wither’s height The wither’s average height of Texel sheep at the beginning (0 weeks), middle (2 weeks) and end of treatment (4 weeks) is presented in Figure 1. At the beginning (0 week), the highest average of withers height is found in P1 (56.4 cm), then P4 (56 cm), P0 and P1 (respectively, 55.7 cm), whereas the lowest is in P3 (54.8 cm). The result of withers average height measurement two weeks after treatment shows that the highest withers height is at P4 (56.5 cm), followed by P1 (56.4 cm), then P0 (55.9 cm), P2 (55.7 cm), and the lowest is at P3 (55.2 cm). The result of withers average height measurement four weeks after treatment shows that the highest withers height is at P4 (56.75 cm), followed by P1 (56.4 cm), P0 (56.05 cm), P3 (55.8 cm) and a the lowest is at P2 (55.75 cm).

The withers average height of Texel sheep on all treatments shows different increment (Figure 2). The highest increment of withers height of sheep is at P3 (1 cm), followed by P4 (0.75 cm), P0 (0.35 cm), and P2 (0.05 cm), whereas P1 did not experience any withers height increment. The highest percentage of withers height increment is achieved in P3 (1.83%), followed by P4 (1.34%), P0 (0.63%), and P2 (0.09%), whereas P1 does not increased at all.

The Anova further test results with LSD shows that the addition of ramie hay of 10%, 20% and 30% in the food can increase the vital statistics addition of withers height of each treatments by, consecutively, 0.05 cm, 1.00 cm and 0.75 cm. The increase which is relatively limited is due to the reduced protein content of food and the increase of crude fiber as a result of ramie hay additions.

Inner part of chest The average of inner part of chest of Texel sheep at the beginning (0 weeks), middle (2 weeks) and end time of treatment (4 weeks) is presented in Figure 1. Measurement of the average inner part of chest at the beginning of treatment (0 weeks) are highest at P3 (25.3 cm), then P2 (23.3 cm), P4 (21.5 cm), P1 (20.5 cm), while the lowest was in P0 (19.8 cm). Results of average measurement of inner part of chest two weeks after the treatment shows the highest is on P3 (25.3 cm), followed by P2 (23.3 cm), P4 (21.6 cm), P1 (20.5 cm), and the lowest at P0 (19.8 cm).

Results of average measurement of inner part of chest four

weeks after treatment shows the highest achievement is at P3 (25.3 cm), followed by P2 (23.3 cm), P4 (21.65 cm), P1 (21.15 cm), and the lowest is at P0 (19.85 cm) (Figure 2).

The highest average addition of inner part of chest is reached at P1 (0.65 cm), followed by P4 (0.15 cm) and P0 (0.05 cm), while P2 and P3 does not experience any increment. The highest percentage increment of inner part of chest is achieved in P1 (3.17%), followed by P4 (0.69%), P0 (0.25%), while P2 and P3 not increased (Figure 2).

The Anova further test results with LSD show that the addition of 30% ramie hay in feed can increase the vital statistics addition of inner part of chest (0.15 cm), whereas other treatments give no significant effect. This situation is caused by the degradation of feed protein and crude fiber increment on feed due to the addition of ramie hay.

Chest width The average chest width of Texel sheep at the beginning of treatment (0 weeks), middle of treatment (2 weeks) and end of treatment (4 weeks) is presented in Figure 1. The measurement of the average width of the chest at the beginning of treatment (0 weeks) shows the highest number is found in P4 (18.35 cm), and P3 (16.9 cm), P0 (15.95 cm) and P2 (15.8 cm), while the lowest is at P1 (15.15 cm). Chest width average measurement results on two weeks after treatment show the highest chest width is at P4 (18.45 cm), followed by P3 (17.05 cm), P2 (16.8 cm), P0 (16.2 cm), and the lowest is at P1 (15.9 cm). Chest width average measurement results after four weeks of treatment show the highest chest width is at P4 (18.65 cm), then P2 (17.3 cm), P3 (17.05 cm), P1 (16.5 cm), and the lowest is at P0 (16.3 cm).

The average chest width of Texel sheep in each treatment represents different increments (Figure 2). The highest increment of sheep chest width is at P2 (1.5 cm), followed by P1 (1.35 cm), P0 (0.35 cm), P4 (0.3 cm), while the lowest is in P3 (0.15 cm). The highest percentage of chest width increment is achieved by P2 (9.49%), followed

by P1 (8.91%), P0 (2.19%) and P4 (1.63%), while the lowest is in P3 (0.89 %). Considering the vital statistics data of Texel sheep, it appears that the size of vital statistics of research sheep is still below the class D standard measure of vital statistics on Texel sheep with 2.5 years of age i.e. the chest circumference is 80 cm, withers height is 65 cm and 65 cm of body length (Office of Animal Husbandry and Fisheries of Wonosobo District 2007).

The Anova further test results with LSD shows that the addition of ramie hay of 10%), 20%) and 30%) in the food can increase the vital statistics addition of chest width, each for 1.50 cm); 0.15 cm) and 0.30 cm). This situation is caused by degradation of feed protein and increment of crude fiber in the feed due to the addition of ramie hay.

After the data analysis with one-way Anova is performed to all the calculation results obtained in all treatment, it is obtained the value of statistical test, i.e. Fobs > Fα, thereby H0A is rejected, while H1A is accepted. Thus, the results of these tests show that there are significant differences among all treatments on body weight and size

KUNTJORO et al. – Effect of hemp on body weight and statistic vital of texel sheep

of the vital statistics including chest circumference, body length, withers height, inner part of chest and chest circumference of Texel sheep on each treatment. Anova further test results with LSD show that the addition of ramie hay will increase body weight and size of the statistics vital.

Economic calculation The result of observation toward the addition of ramie hay in the food given to the Texel sheep shows the economic cost of food as presented in Table 2.

Economically, though not yet to replace concentrate food, but the maintenance of Texel sheep with the addition of ramie hay by 10%, 20% and 30% can reduce the costs of the food from Rp 330,000, - to Rp 319,000, -; Rp 309,000, - and IDR 298 000, - and gained the profit per head of for each sheep Rp 52,050, -; Rp 38,100, - and Rp 16,650, -.

While the maintenance of sheep by feeding them with concentrates without any additional food of ramie hay gained a benefit of Rp 63,000, - and the sheep fed only with greenery alone without giving concentrates gained a profit of Rp 28,500, -. This situation is caused by the cost of feed control group, P1 (without hay of ramie), P2 (the addition of ramie hay 10%), P3 (ramie hay 20%) and P4 (30% ramie hay) respectively IDR 150,000.00/month; Rp 330,000.00/month; IDR 319 500.00/month; IDR 309,000.00/month and Rp 298,500.00/month. The cost of feed is treated with grass price of Rp 3,000.00/buckets (for 6 heads), concentrates IDR 1,200.00/kg and ramie hay price IDR 500.00/kg and livestock prices Rp 15,000/kg body weight.

Tabel 2. Economic analysis of sheep feed costs by treatment.

Treatment P0 P1 P2 P3 P4

Added Prices body weight feed (IDR) (kg) 150,000 29 330,000 64 319,500 56 309,000 46 298,500 31

Price sheep (IDR) 435,000 960,000 840,000 690,000 465,000

Advantages (IDR) 285,000 630,000 520,500 381,000 166,500

CONCLUSION The highest weight gain of sheep was achieved by the group with the additional food of ramie hay respectively 0%, 10%, 20% and 30%. Though not yet able to replace the function of the concentrate, but the addition of ramie hay as much as 10%, 20% and 30% in food increases the weight gain of sheep for each percentage 186.67 g/head/day, 153.34 g/head/day and 103 , 34 g/head/day. The increased size of the vital statistics of chest line, body length and chest width is achieved by the additional of ramie hay in the cattle food as much as 0%, 10% and 20%, while the highest body height was reached by the group with the additional food of 20% and 30% ramie hay, and the highest chest width was achieved by the group with the additional food of 0% and 30% ramie hay.

29

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Increasing levels of ramie (Boehmeria nivea) hay on the diets of fattening rabbits. Proceedings of 8th World Rabbit Congress, September 7-10, 2004, Puebla, Mexico Hanafi ND. 2004. Silage treatment and ammoniation palm leaves as a raw material feed sheep. Animal Production Program, Faculty of Agriculture, University of North Sumatra. Medan. [Indonesia] Lahiya A. 1984. About versatile ramie plant (Boehmeria nivea).

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 31-37 March 2009

Protein expression on Cr resistant microorganism using electrophoresis method UMI FATMAWATI1,♥, SURANTO², SAJIDAN1,² ¹Biology Education Program, Department of Mathematics and Natural Sciences Education, Faculty of Teacher Training and Education Science, Sebelas Maret University Surakarta 57 126, Central Java, Indonesia ²Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 19 Augustus 2008. Revision accepted: 17 November 2008.

Abstract. Fatmawati U, Suranto, Sajidan. 2009. Protein expression on Cr resistant microorganism using electrophoresis method.

Nusantara Bioscience 1: 31-37. Hexavalent chromium (Cr(VI)) is known as toxic heavy metals, so the need is reduced to Cr(III) is much less toxicity. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are resistant Cr(VI) microorganism and have ability to reduce Cr(VI). The aim of this research is to know ability of microorganism to reduce Cr(VI) and to know protein band pattern between Cr(VI) resistant microorganism and non resistant microorganism which inoculated on LB broth. SDS-PAGE was used to indentify protein expression. While, Cr(VI) concentration was identified by 1.5 diphenylcarbazide method. The quantitative data was analyzed by two factorial ANOVA that continued with DMRT at 1% level test.

The qualitative data i.e. protein expression analyzed by relative mobility (Rf). The results showed that the ability of microorganisms to reduce Cr(VI) at initial concentration of 0.5 ppm, 1 ppm, 5 ppm and 10 ppm may vary, the average percentage of the ability of each microorganism in reducing Cr(VI) is P. putida (65%) > S. cerevisiae (64.45%) >. P. aeruginosa (60.73%) > Pantoea sp. (50.22%) > K. pneumoniae (47.82%) > without microorganisms (34.25%). The adding microorganisms have significantly influenced toward reduction of Cr(VI). The SDS-PAGE shows that protein expression between resistant and not resistant microorganisms are no different, but resistant microorganisms have more protein (protein band is thicker).

Key words: Cr heavy metal, microorganism, protein, electrophoresis.

Abstrak. Fatmawati U, Suranto, Sajidan. 2009. Ekspresi protein pada mikroorganisme resisten Cr dengan metode elektroforesis.

Nusantara Bioscience 1: 31-37. Krom heksavalen (Cr(VI)) dikenal sebagai logam berat beracun, sehingga perlu direduksi menjadi Cr(III) yang lebih rendah toksisitasnya. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. dan Saccharomyces cerevisiae adalah mikroorganisme resisten dan mampu mereduksi Cr(VI). Tujuan penelitian ini adalah mengetahui kemampuan mikroorganisme dalam mengurangi Cr(VI) dan mengetahui pola pita protein antara mikroorganisme resisten Cr(VI) dan mikroorganisme tidak resisten yang diinokulasi pada medium kaldu LB. SDS-PAGE digunakan untuk mengetahui ekspresi protein, sementara konsentrasi Cr(VI) diidentifikasi dengan metode 1,5 difenilkarbazid. Data kuantitatif dianalisis dengan ANAVA dua faktorial dilanjutkan dengan uji jarak berganda Duncan pada taraf 1%. Data kualitatif yaitu ekspresi protein dianalisis dengan mobilitas relatif (Rf). Hasil penelitian menunjukkan bahwa kemampuan mikroorganisme dalam mereduksi Cr(VI) pada konsentrasi awal 0.5 ppm, 1 ppm, 5 ppm dan 10 ppm berbeda-beda, persentase rata-rata kemampuan masing-masing mikroorganisme dalam mereduksi Cr(VI) adalah: P. putida (65%) > S. cerevisiae (64,45%) > P. aeruginosa (60,73%) > Pantoea sp. (50,22%) > K. pneumoniae (47,82%) > tanpa mikroorganisme (34,25%). Penambahan mikroorganisme secara nyata mempengaruhi reduksi Cr(VI). SDS-PAGE menunjukkan bahwa ekspresi protein antara mikroorganisme resisten dan tidak resisten tidak berbeda, tetapi mikroorganisme resisten memiliki lebih banyak protein (pita protein lebih tebal).

Kata kunci: logam berat Cr, mikroorganisme, protein, elektroforesis.

INTRODUCTION Chromium (Cr) as one of heavy metal contaminants can potentially become a pollutant as a result of coloring fabrics in the textile industry, paints, leather tanning, metal plating, batteries or industrial chromium (Ackerley et al.

2004). Through the food chains chromium can be deposited in a living body part which at a certain size it can cause toxicity (Mulyani 2004). Generally chromium in open nature is in the valence of 3 (Cr3+) and valence 6 (Cr 6 +).

Cr6+ is more toxic than Cr3+. The toxicity of Cr6+ is due to its high solubility and mobility in the environment (Palar

1994; Lowe et al. 2002; Uprati et al. 2003; Rahman et al.

2007). If it enters into cells, then it can cause DNA structural damage or further it can generate mutations (Larashati 2004).

Several microorganisms species such as Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are microorganisms that have resistance to heavy metal contamination and the ability to reduce Cr(VI) to Cr(III).

Research conducted by Krauter and Krauter (2002) concludes that S. cerevisiae is able to reduce Cr(VI) at 100% of initial 1.89 ppm concentration at an optimum pH

32

1 (1): 31-37, March 2009

of 6.5-7. While in acidic condition (pH = 2.1) the corps of S. cerevisiae is still able to reduce the level of Cr(VI) by 70%. Jianlong et al. (2003) tested the tolerance level of S. cerevisiae at a concentration of Cr(VI) 5 uM and it was found that it did not affect microbial growth, while at the concentration of Cr(VI) 15 uM it inhibited microbial growth by 30%.

Ganguli and Tripathi (2002) reports that the bacteria P. aruginosa is able to reduce Cr(VI) by 96% of the initial concentration of Cr(VI) at 10 ppm. But the bacteria P aeruginosa also has a limited ability to reduce the concentration of 50 ppm which is only reduced by 16%. P. putida is a bacterium that is resistant to Cr and Cd so that it can be used in reducing Cr in a certain medium (Timothy et al. 1989; Lowe et al. 2002; Ackerley et al. 2004; Rahman et al. 2007). Bacteria P. putida can reduce Cr(VI) with a speed of 6 ppb min-1 which was previously tested on gelatin containing Cr (IV) (Lowe et al. 2002). K. pneumoniae which was inoculated in BHI medium is capable of reducing Cr(VI) by 27% (Mardiyono 2005).

Obraztsova et al. (2002) suggested that the reduction of Cr(VI) 150 ppm by Pantoea sp was optimum with the addition of sulfate (SO4-2) which took 20 hours.

Knowing the potential for some microorganisms such as P aeruginosa, P. putida, K. pneumoniae, Pantoea sp. and S. cerevisiae in reducing heavy metal Cr, then a research was conducted on the ability of these microorganisms to reduce Cr(VI) in liquid media containing heavy metal Cr, to know the genetic changes in microorganisms through the expression of protein bands by polyacrylamide gel electrophoresis detection in SDSPAGE. Electrophoresis is the ideal analysis to purify the protein component of the mixture sample by the adding the medium that can bind proteins during electrophoresis process. The best method in the purification of proteins by electrophoresis is by using polyacrylamide gel electrophoresis (PAGE). Polyacrylamide gel is a solution of acrylamide and bisakrilamid (Davis and Heywood 1963; Hames and Rickwood 1990; Matsudaira 1993).

The aim of this research was to know ability of microorganism to reduce Cr(VI) and to know protein band pattern between Cr(VI) resistant microorganism and non resistant microorganism which inoculated on LB broth.

MATERIALS AND METHODS The microorganism used. Microorganisms such as K. pneumoniae, P. aeruginosa, P. putida, Pantoea sp., and S. cerevisiae are used in testing the reduction of Cr(VI), and the expression pattern of protein bands is obtained from the University of Gadjah Mada University, Yogyakarta.

Bacterial isolates are multiplied in LB liquid medium with the composition of each isolate was 100 mL of 1 g Tryptone, 0.5 g yeast extract, and 0.5 g NaCl, and for the proliferation of molds liquid PDA (potato dextrose agar) medium was used.

Preparation of liquid media which contains heavy metal Cr. A total of 0.1414 g K2Cr2O7 was reconstituted

with distilled water in a 1 liter measuring flask and diluted until it reached mark boundaries to obtain solution concentrations of 0.05 mg/mL to prepare supply of standard Cr. Then chromium standard solution was made by diluting 1 mL of Cr stock solution into 100 mL liquid medium, until we obtained liquid media containing concentrations of heavy metals Cr 0.5 ppm. For concentrations of 1 ppm, 5 ppm and 10 ppm Cr preparations was done by diluting standard solution into a liquid medium (LB or PDA) as much as 100 mL. Counting the number of microorganism cells. To determine the ability of living microorganisms to survive in media containing heavy metals, then the number of cells inoculated in microorganisms was calculated in liquid media with Cr(VI) 0 ppm and 10 ppm for 16 hours. The growing culture was diluted several times by 10-5 and 10-6.

The result of dilution was grown on 100 µL of solid LB media, and then it was incubated again for 16 hours at 37ºC. Colonies that were formed then were calculated by colony counter and the number of microorganisms cells was also counted in units of cells/mL (Hadioetomo 1993).

Cultures which grew on both Cr(VI) were resistant microorganisms, while the cultures which only grew on the control were not resistant microorganisms.

Inoculation of microorganisms in liquid LB media.

To determine the ability to reduce Cr(VI), each microorganism was taken with an ose needle and was grown in erlenmeyer containing liquid LB media (LuriaBertani media) with Cr(VI) 0 ppm, 0.5 ppm, 1 ppm, 5 ppm and 10 ppm concentration. Each species of microorganism was grown in 100 mL of LB liquid medium on five different initial concentrations of Cr(VI) above and then they were incubated in an incubator with a temperature of 30-36ºC for 16 hours.

Hexavalent chromium test. A total of 50 mL liquid bacterial culture medium containing chromium was put into Eppendorf tubes and centrifuged at 3000 rpm for 30 minutes, the supernatant was collected and filtered with Whatman filter paper of 0.2 μm and then the heavy metal content was analyzed (Lowe et al. 2002). The solution was then neutralized by adding H2SO4 (1+1) or NH4OH, and then it was added with 1 mL of H2SO4 (1+1) and 0.3 mL of 85% H3PO4. The solution was then quantitatively transferred into 100 mL measuring flask and 2 mL diphenylcarbazide solution was added, diluted to mark boundaries and whipped until it was well mixed. After 5-10 minutes, it was measured with UV-VIS spectrophotometer with a wavelength of 540 nm.

Preparation of standard solution of chromium.

Standard supply chromium solvent as much as 20-20 mL was taken with pipette (2, 4, 6, 8, and 10, and so on in stages) into several pieces of measuring 100 mL flask. Put 25 mL of distilled water into the other measuring flask a blank. Into each measuring flask, add 1 mL of H2SO4 (1 +1), 0.3 mL 85% H3PO4 and 2 mL of solution diphenylcarbazide, then diluted until it gets the boundary mark and whipped until blended, allowed to stand for 5-10 minutes. Define the absorption in the wavelength of 540 nm and calibration curve is made then, calculate the chromium content in mg/L for the calibration curve.

FATMAWATI et al. â&#x20AC;&#x201C; Protein expression of Cr resistant microorganisms

SDS-PAGE electrophoresis. A total of 5 mL bacterial culture was poured into 1.5 mL an eppendorf tube and the cells was sedimented by centrifuging in for 5 minutes at speed of 13,000 rpm. The cell sediment then was cleaned from liquid LB medium by removing its supernatant. The pellet cell which settled then was suspended with Phosphate Buffered Saline Solution (PBS) twice, then it was again disentrifused back, and the PBS supernatant was discarded. One mL of PBS was added into the pellets. To break the cells sonication was used for 30 seconds for 4 times. Redisentrifuse it and take the supernatan for running by adding sample buffer (4:1/v: v). Before being placed in wells, the mixture of sample and sample buffer was boiled in boiling water for 2 minutes, then put it in ice for + 5 minutes, after which the samples are ready for running. The gel that has been formed (discontinuous gel 10% and 3% stacking gel), was transferred into the electrophoresis tank (Hames and Rickwood 1990).

Furthermore, electrophoresis tank was filled with running buffer (0.19 M Glycine, 10 mL SDS 10%, and 0.0248 M Tris in 1 L) until it is full. As much as 10 ÂľL of mikropippete of mixture of the sample and the sample buffer was poured into the electrophoresis wells carefully Then close the tank lid and set the voltage (100V, 90 minutes). To identify the molecular weight of proteins, use protein markers that have a molecular weight range 21211.3 kDa. Comassie Blue staining. Solution was made with the composition of 1 g Comassie Blue coloring that was dissolved in 1 L of destaining solution (100 mL acetic acid, 400 mL of methanol, then diluted with the addition of distilled water until it reaches the volume of 1 L) (Hames and Rickwood 1990). After running, the gel was soaked in dye solution Comassie blue for 12 hours, then it was washed with destaining for 3-4 times for 2 hours until the protein band pattern was formed.

Data analysis. Differences in the ability of each microorganism to reduce the heavy metals Cr(VI) at each concentration were analyzed by analysis of variance (ANOVA) followed by further test of Duncan Multiple Range Test (DMRT). The differences in protein expression between the microorganisms were described descriptively.

RESULTS AND DISCUSSION The growth of microorganisms in media containing Cr Preliminary test results in the form of counting the number of colonies, or Colony Form Unit (CFU) of five species of microorganisms which were grown in an gel medium containing 10 ppm Cr(VI) indicated that the five species of microorganisms were able to live in a media containing Cr(VI) ( Table 1). The highest number of cells contained in S. cerevisiae was 460x106 cells/mL at a concentration of 0 ppm Cr(VI), while the concentration of 10 ppm Cr(VI) S. cerevisiae cells also produce most of 317x106 cells/mL. The number was obtained from the calculation of the average number of colonies of two types of dilution: 10-5 and 10-6. The gel medium that was used

33

to grow S. cerevisiaea was Potato Dextrosa Agar (PDA), because S. cerevisiae is a type of yeast that can ferment and it needs a lot of substrate fermentation of glucose that will be converted into ethanol. The ability of S. cerevisiae to survive in an gel medium containing Cr(VI) indicates that this microorganisms are resistant or tolerant to Cr heavy metals (Jianlong et al. 2003; Mulyani 2004; Gao et al.

2006). P. aeruginosa also has the ability to live in a media containing heavy metal Cr(VI) 10 ppm, where it is proven by the growth of colonies on LB agar media containing Cr(VI) 10 ppm with an average cell amount of 287x106 cells/mL. Other microorganisms also have the ability to live in a media containing heavy metal Cr(VI) 10 ppm. The fewest number of cells observed on Pantoea sp. with the number of cells of 177x106 cells/mL. Table 1. The number of cells inoculated microorganisms on solid LB medium with the addition of Cr(VI) 0 ppm and 10 ppm.

Species P. aeruginosa P. putida

Average number of cells (sel/mL) 0 ppm 10 ppm 6 370x10 287x106 352x106

230x106

34.6%

6

6

20.7% 30.3%

K. pneumoniae

303x10

240x10

Pantoea sp.

254x106

177x106

6

6

S. cerevisiae

Percentage decrease in the number of cells 22.4%

460x10

317x10

31%

Five species of microorganisms in general have a decrease in the number of cells in media containing heavy metal Cr(VI). The lowest decrease in the number of cells is present in K. pneumoniae by 20.7%, while the highest decrease is in P. putida at 34.5%. The decrease in number of cells of inoculated microorganisms in media containing heavy metal Cr(VI) shows that these microorganisms select the tolerant variant toward the heavy metals. Based on Table 1, the decrease in the number of cells of microorganisms after being inoculated into the gel medium with the addition 10 ppm of Cr(VI), generally as much as 20-30%, a decline that is not too extreme and indicates that the five species of microorganisms are resistant to the environment containing metal weight of Cr(VI).

The microorganisms that are capable of living in media containing Cr(VI) can also serve as a reductor of heavy metal Cr(VI) to Cr(III). Several studies have proven that the presence of Cr(VI) at levels of 0-50 ppm in the microorganisms cells does not interfere cell growth microorganisms (Jianlong et al. 2003; Gao et al. 2006; Rahman et al. 2007) because besides growth, the microorganisms will make side product of H2S. The increase in the number of cells of microorganisms will increase the speed of H2S production that will accelerate the reduction of Cr(VI). H2S which is produced by the bacteria will react with chromium to form chromium sulfides that are not stable in solution and will more quickly be deposited to form Cr (OH)3 namely Cr with a valence of three who has a lower toxicity of Cr valence six.

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Reduction of Cr(VI) in liquid media Based on the result of the preliminary research it is proven that the five species of microorganisms are capable of living in an gel medium containing heavy metal Cr(VI) then test was conducted to determine the changes in levels of Cr(VI) before and after treatment. The treatment was done with five species of microorganisms namely: K. pneumoniae, Pantoea sp., P. aeruginosa, P. putida, and S. cerevisiae which were inoculated in LB liquid media containing Cr(VI) 0.5 ppm, 1 ppm, 5 ppm and 10 ppm with the initial inoculum concentration of 1% (Mardiyono 2005) and was incubated for 16 hour at room temperature on a shaker. Decrease percentage in Cr(VI) by five species of microorganisms at different concentrations are shown in Figure 1.

pH 6.5-7, while at acidic pH its bioremoval capabilities are less effective.

One of the benefits for Saccharomyces as an agent of biosorbtion of Cr(VI) is that its character is not pathogenic compared to other bacterial pathogens such as P. aeruginosa and K. pneumoniae. S. cerevisiae also has the ability to reduce other types of heavy metals such as Mo, Co, Ca, Zn, Sr, Hg and Cu in water (Krauter and Krauter 2002; Mulyani 2004).

Pseudomonas putida has the second largest percentage in reducing Cr(VI) that is 66.8% in the initial concentration of Cr(VI) initial 5 ppm. The lowest ability is bacteria K. pneumoniae at 20.4% on the initial concentration of 0.5 ppm. In the treatment without addition of microorganisms there was also a decrease as much as 31%. Several other studies also use bacterium P. putida to reduce Cr(VI) into Cr(III); among others are Ackerley et al. (2004), Lowe et al. (2002), Timothy et al. (1989), and Rahman et al. (2007).

The results shows that P. putida is able to reduce Cr(VI).

The highest initial concentration of Cr(VI) that was used in this study is 10 ppm. At this concentration the microorganisms still can grow and reproduce, it is seen from the turbidity of liquid LB medium inoculated with five species of microorganisms. Logically, with higher survival ability, certainly there must be more microorganisms that live so as to increase the ability to reduce Cr(VI) into Cr(III) in the environment where Cr(VI) is also greater. The highest percentage in decreasing Cr(VI) occurrs at concentration of 1 ppm and 5 ppm.

Figure 1. Percentage decrease in Cr(VI) by five species of microorganisms. Note: K = without microorganisms or control, 1 = K. pneumoniae, 2 = Pantoea sp., 3 = P. aeruginosa, 4 = P. putida, 5 = S. cerevisiae.

Cr(VI) on five species of microorganisms Based on Figure 2 we can see the order of the ability of microorganisms in reducing Cr(VI). The highest ability is in bacteria P. putida with an average percentage of reduction as much as 65%, while the lowest is bacteria K. pneumoniae with an average percentage reduction of 47.8%. The sequence of comparison of the ability to reduce Cr(VI) among the microorganisms are as follows: P. putida (65%)> S. cerevisiae (64.45%)> P. aeruginosa (60.73%)> Pantoea sp. (50.22%)> K. pneumoniae (47.82%)> without microorganisms (34.25%).

The analysis results of variance calculations of the influence of initial concentration of heavy metals Cr(VI) and species of microorganisms toward the decrease in Cr(VI) is shown in Table 2. Based on the calculation of two-factorial ANOVA where the main factor is the species of bacteria and the subplot factor is the concentration, it can be concluded that the species of microorganism have real effects on decreasing the concentration of Cr(VI) at level 1%. Initial concentration of Cr(VI) has real effect on decreasing the concentration of Cr(VI) at level 1%, and the interaction of species of microorganisms with initial concentration of Cr(VI) has significant effect on decreasing the concentration of Cr(VI) at level 1%.

From the fact above, there are significant variations of effect in the species of microorganisms and the initial concentration of heavy metals Cr(VI) to the decline of heavy metal concentrations of Cr(VI). The results of further test namely the DMRT to the decline of heavy metal concentrations of Cr(VI) are shown in Table 3.

Reduction ability of Cr(VI) by the five species of microorganisms is tested on LB liquid medium with a solution of 1 ppm Cr(VI). On visual observation there is a difference between liquid LB media that are not inoculated microorganisms and LB liquid medium which are inoculated with five species of microorganisms, and that the liquid media which are inoculated by microorganisms look more cloudy. This indicates that there are growth and proliferation of cells in the media. In addition to the growth activity, some microorganisms also have the ability to reduce Cr(VI) (Suzuki et al. 1992; Ganguli and Tripathi 2002; Krauter and Krauter 2002; Jianlong et al. 2003; Mulyani 2004; Upreti et al. 2004; Mardiyono 2005).

Saccharomyces cerevisiae has the highest ability to reduce among other microorganisms. The decrease percentage in S. cerevisiae is as much as 87% at the initial concentration of 5 ppm. At the initial concentration of this 5 ppm the lowest percentage decline occurred in K. pneumoniae that is as much as 56.7%. In the treatment without bacteria, decrease also happens by 42.5%. Several other studies state that Saccharomyces is a microorganism that has the highest effectiveness in reducing Cr(VI). Krauter and Krauter (2002) states that S. cerevisiae can reduce Cr(VI) 100% at

FATMAWATI et al. â&#x20AC;&#x201C; Protein expression of Cr resistant microorganisms

35

a valence of three who has a lower toxicity of Cr with a valence of six.

Meanwhile, according to F table Degrees of Sum of Middle F Suhendrayatna (2001) reduction of Source of diversity freedom squares Squares Calculate 5% 1% Cr(VI) to Cr(III) by microorganisms Replication 2 108.258 54.129 called bioremoval has two kinds of Microorganisms (A) 5 8626.752 1752.350 50.858 3.330* 5.640 mechanisms, namely passive and Error (a) 10 339.249 33.925 active. Passive absorption is known Initial conc. of Cr (VI) (B) 3 8988.810 2996.270 101.644 2.860* 4.380 as biosorbsi. This process occurs AxB 15 1350.517 90.034 3.054 1.960* 2.580 when heavy metal ions bind to the Error (b) 36 1061.207 29.478 General 71 20474.792 cell wall in two different ways, Note: kk (a) = 10.631%; kk (b) = 9.910%; * = significant difference at level 1% namely (i) ion exchange of monovalent and divalent ions which like Na, Mg, and Ca on the cell wall Table 3. Further test results significantly different from Duncan's to the decline of Cr (VI) was replaced by heavy metal ions, concentrations. and (ii) complex formation between heavy metal ions with functional Initial concentrations of Cr (VI) Species of microorganisms groups such as carbonyl, amino, 0.5 ppm 1 ppm 5 ppm 10 ppm e e e e thiol, hydroxyl, phosphate, hydroxyl, Without microorganisms 18.8 43.06 42.52 31.83 carboxyl located on the cell wall.

K. pneumoniae 20.46e 59.82abcd 56.72cd 54.42abcd Biosorbsion process can occur Pantoea sp 38.16abcd 62.59abcd 64.00bcd 59.94abcd 68.95ab 67.32bc 60.14abc P. aeruginosa 48.12ab back and forth and quickly. The P. putida 48.16a 69.50a 75.63ab 60.65a process of alternating bond of heavy 65.4abc 87.00a 66.87ab S. cerevisiae 44.81abc metal ions on the surface of these Note: numbers followed the same letter showed no significant difference in the level of cells can occur in both dead cells and DMRT 1%. living cells from a biomass. The biosorbtion process can also be more The presence of Cr(VI) in the environment can interfere effective at certain pH and the presence of other ions in the the organism but also can result in selection of resistant medium in which heavy metals can be deposited as salt bacteria. Compounds of Cr(VI) are really more dangerous which is not dissolved. Heavy metal absorption can also than the Cr(III) due to its high solubility in water, rapid occur actively, which occurs in various types of living permeability and subsequent interaction with intracellular cells. This mechanism simultaneously occurs in line with proteins and nucleic acids (Upreti et al. 2004). the consumption of metal ions for the growth of Microorganisms can develop resistance mechanisms to microorganisms or the accumulation of intracellular heavy select the next resistant variants. metal ions. Heavy metals can also be deposited in the metabolism and excretion process. This process depends on 70 the energy contained in it and the sensitivity of different parameters such as pH, temperature, ionic strength, and 60 light. This process can also be inhibited by low 50 temperatures, lack of energy sources and inhibition of cell 40 metabolism (Suhendrayatna 2001).

30 Protein expression in microorganisms resistant Cr 20 in Figure 3 shows the results of running the pattern of 10 protein bands on SDS-PAGE of the five species of 0 microorganisms namely K. pneumoniae, Pantoea sp., P. M0 M1 M2 M3 M4 M5 aeruginosa, P. putida, and S. cerevisiae, each sample of which is extracted from the cells of microorganisms that Jenis mikroorganisme are grown in LB liquid culture with concentrations of Figure 2. The average reduction capability. Note: M0 = without Cr(VI) 0 ppm and 10 ppm. This two kinds of concentration microorganisms (control), M1 = K. pneumoniae, M2 = Pantoea are chosen to distinguish between populations of sp., M3 = P. aeruginosa, M4 = P. putida, M5 = S. cerevisiae microorganisms that are resistant and that is not resistant.

Protein markers can be used to identify the molecular As noted previously, according to Rahman et al. (2007) weight of a mixture of polypeptides (Hames and Rickwood reduction of Cr(VI) happens because not only of growth, 1990). In this study, markers of protein that are used have a but also the fact that microorganism produces byproducts molecular weight range 212-11.3 kDa. From the results of in the form of H2S. The increasing number of cells of electrophoresis there is a number of protein bands which microorganisms will increase the speed of H2S production have different thicknesses. The protein that has a more that will accelerate the reduction of Cr(VI). H2S that is thickness and greater color intensity than any other proteins produced by the bacteria will react with chromium to form and is always there in every variety is called major protein chromium sulfides which are not stable in solution and will (Wijaya and Rahman 2005). more quickly deposited to form Cr (OH)3 which is Cr with Penurunan (%)

Table 2. Results of analysis of variance the variation of initial concentration of Cr(VI) and species of microorganisms to the decrease of Cr(VI).

Â

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1 (1): 31-37, March 2009

212 kDa 155 kDa 130 kDa 92,5 kDa

63,4 kDa

14,4 kDa M1a

M1b

M2a M2b M3a M3b

M4a

M4b M5a M5b M

Figure 3. Protein expression in five species of microorganisms are grown on LB liquid medium with Cr(VI) 0 ppm and 10 ppm. Note: M = marker proteins, M1a, M1b = K. pneumoniae, M2A, M2b = Pantoea sp., M3a, M3b = P. aeruginosa, M4A, M4b = P. putida, M5a, M5b = S. cerevisiae. a = 0 b = 10 ppm ppm

In the electrophoresis results, there are several major proteins on the species of P. aeruginosa (M3) and P. putida (M4). This major protein molecular weight ranges from 148.7 kDa, 121.6 kDa, and 105 kDa. The two major proteins of these type have same color intensity and thickness because this two microorganisms belong to the same genus namely Pseudomonas. Bacteria K. pneumoniae (M1) has several protein bands, but because proteins from the sample that is running is only a few, then the formation of bands on polyacrylamide gel is still less than optimal.

From Figure 3 we can note that there are three major protein bands of resistant microorganisms and not resistant microorganisms with the molecular weight of 121.6 kDa, 14.4 and 14 kDa, 3 kDa. Generally, the pattern of protein bands from two samples that running do not have a dramatic difference, because they are actually of one type.

Protein running of S. cerevisiae (M5) shows the same banding pattern among the microorganisms that are resistant and which are not resistant. These microorganisms have three pairs of major proteins with molecular weight of 212 kDa, 188.5 kDa, 14.5 kDa, and 14.4 kDa. In addition, there are some bands with less color intensity, due to less protein concentration. The lower the location of protein bands are, the smaller the molecular weight. This happens because the low molecular weight have a greater speed to migrate in the matrix medium polyakrilamid.

The reason of why electrophoresis was used in this study is because it has a very important role in the process of separating biological molecules, especially proteins.

This method does not affect the structure of biopolymers, as well as very sensitive to the difference in charge and in small molecular weight (Bachrudin 1999). Proteins that run in a medium that contain electric field causes the charged compounds will move in the solution as a result of the nature of the opposite polarity, so that the mobility of a

molecule is a function of shape, molecule size, and large content type.

The use of SDS and merkaptoetanol accompanied by heating will break three-dimensional structure of proteins, particularly the disulfide bonds into polypeptides sub units individually. SDS also wraps the chain of proteins that are not bound by the same negative charge to form SDSprotein complex. SDS-protein complexes have an identical charge density and move on the gel based on the size of the protein (Wijaya and Rohman 2005). Therefore, the greater SDS-protein complexes have slower mobility than the smaller SDS-protein complexes.

Methods for extracting proteins in microorganisms are done with Phosphate Buffer Saline Solution (PBS) solution followed by breaking the cell by using sonification (cellbreaking equipment using sound waves that produce high frequency). The purpose of the cell breaking is to provide opportunities to extract the protein that will be purified.

The cells that have been broken must be preserved from the influence of oxygen, because oxygen can cause the protein to be inactive, denatured, and compact (Bachrudin 1999).

CONCLUSION The species of microorganisms Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Pantoea sp., and Saccharomyces cerevisiae have a very significant effect on the percentage reduction of Cr(VI). The variation of initial concentration of Cr(VI) also has a very significant effect on the percentage reduction of Cr(VI) and interaction species of microorganism with initial concentration of Cr(VI) which have significant effect on decreasing the concentration of Cr(VI). Microoganisms ability in reducing Cr(VI) to Cr(III) can be sorted as follows: P. putida (65%)> S. cerevisiae (64.45%)> P. aeruginosa (60.73%)>

FATMAWATI et al. â&#x20AC;&#x201C; Protein expression of Cr resistant microorganisms

Pantoea sp. (50.22%)> K. pneumoniae (47.82%)> without microorganisms (34.25%). Expression of proteins that are formed on each of the microorganisms that are resistant and are not resistant have almost the same pattern of protein bands.

REFERENCES Ackerley DF, Gonzales CF, Park CH, Blake R Keyhan M, Martin A. 2004. Chromat reducing properties of soluble flavoprotein from Pseudomonas putida and Escherichia coli. Appl Environ Biol 70 (2): 873-882.

Bachrudin Z. 1999. Laboratory manual: isolation, identification, and coloring protein. Inter University Center of Bioteknologi, Gadjah mada University. Yogyakarta. [Indonesia] Davis PH, VH Heywood. 1963. Basic methods in molecular biology. 2nd ed. Appleton & Lange. Conecticut.

Ganguli A, Tripathi A. 2002. Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa A2Chr in two bioreactors. Appl Microbiol Biotechnol 58: 416-420.

Gao J, Zhang Y, Ntoni J, Begonia MFT, Lee KS, Hicks L, Hwang WW, Hwang H-M. 2006. Effects of selected by-products of an acid hydrolyzate on cell growth and ethanol fermentation by Saccharomyces cerevisiae. J Mississippi Acad Sci. October 01, 2006 www.accessmylibrary.com/article-1G1-156274382/effects-selectedproducts-acid.html Hadioetomo RS. 1993. Microbiology basis in practice. UI Press. Jakarta.

[Indonesia] Hames BD, Rickwood D. 1990. Gel electrophoresis of proteins: a practical approach. Oxford University Press. London.

Jianlong W, Zeyu M, Xuan Z. 2003. Response of Saccharomyces cerevisiae to chromium stess. Process Biochem 39 (10): 1231-1235.

Krauter PAW, Krauter GW. 2002. Water treatment process and system for metals removal using Saccharomyces cerevisiae. The Regents of the

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University of California, United States Patent and Trademark Office Granted Patent. www.uspto.gov/patft/index.html Larashati S. 2004. Reduction of chromium (Cr) in vitro by mixed culture of bacteria isolated from landfill leachate (landfill). [Thesis]. Bandung Institute of Technology. Bandung. [Indonesia] Lowe KL, Fliflet RE, Ly T, Little BJ, Jones-Meehan J. 2002. Chromium tolerant microbial communities from the Chesapeake Bay watershed.

Virginia J Sci 53(3): 142-155.

Mardiyono. 2005. Reduction of Cr(VI) textile industrial waste by a bacterium Pseudomonas aeruginosa, Ecsherichia coli, and Klebsiella pneumoniae. Thesis. Environmental Science, School of Graduates, Sebelas Maret University. Surakarta. [Indonesia] Matsudaira P. 1993. A practical guide to protein and peptide purification for microsequencing. 2nd ed. Academic Press. San Fransisco, CA. Mulyani B. 2004. Variation analysis of biomass of Saccharomyces cerevisiae to chromium metal uptake. Sains dan Matematika 2 (4): 19. [Indonesia] Obraztsova AY, Francis CA, Tebo BM. 2002. Sulfur disproportionation by the facultative anaerob Pantoea aglomerans sp-1 as a mechanisme for chromium (VI) reduction. Geomicrobil J 19: 121-132 Palar H. 1994. Analisis variasi biomassa Saccharomyces cerevisiae terhadap serapan logam krom. Rineka Cipta. Jakarta. [Indonesia] Rahman MU, Gul S, UlHaq MZ. 2007. Reduction of chromium (VI) by locally isolated Pseudomonas sp. C171. Turkey J Biol 31: 161-166 Suhendrayatna. 2001. Bioremoval of heavy metals using microorganisms: a literature review. Biotechnology Seminar,Sinergy Forum-Institute of Technology. Tokyo Suzuki T, Miyata N, Horitsu H, Kawai K, Takamizawa K, Tai Y, Okazaki M. 1992. NAD(P)H dependent chromium (VI) reductase of Pseudomonas ambigua G-1:a Cr(V) intermediated is formed during the reductin of Cr(VI) to Cr(III). J Bacteriol 174 (16): 5340-5345 Timotius KH, Widianarko B, Laksmani S. 1989. Interaction between bacteria and heavy metals. Scientific Seminar on the Soil Ecology and Ecotoxicology. Faculty of Biology, Satya Wacana Christian University. Salatiga. [Indonesia] Upreti RK, Srivastha R, Chaturvedi UC. 2004. Gut microflora, toxic metal: chromium as a model. Indian J Med Res 119: 49-59.

Wijaya SKS, Rahman L. 2005. Fractionation and characterization of the major proteins of soybean seeds. Fakulty of Mathematics and Natural Sciences, University of Jember. Jember. [Indonesia]

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 38-42 March 2009

Characterization of white grubs (Melolonthidae: Coleoptera) at salak pondoh agroecosystem in Mount Merapi based on isozymic banding patterns SRI WARDANI1,♥, SUGIYARTO1,2 ¹Department of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java, Indonesia. Tel./Fax.: +92-271-663375. ♥email: noor_handajani@yahoo.com. ²Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 5 March 2008. Revision accepted: 24 May 2008.

Abstract. Wardani S, Sugiyarto. 2009. Characterization of white grubs (Melolonthidae: Coleoptera) at salak pondoh agroecosystem in Mount Merapi based on isozymic banding patterns. Nusantara Bioscience 1: 38-42. The aim of this research is to know the characteristics of white grubs (Melolonthidae: Coleoptera) based on isozyme banding patterns. This research was conducted at Sleman, Yogyakarta and Magelang-Central Java for the morphological purposes. The sample was taken from 5 places with different height in wich 5 samples were taken from each location. The method used in this research was polyacrylamide gel electrophoresis (PAGE) using the vertical type. The enzyme system used in this research were peroxidase and esterase to detect the isozyme banding patterns. The results showed that there was a variation in isozyme banding patterns of white grubs (Melolonthidae: Coleoptera) at salak pondoh agroecosystem in Mount Merapi’s slope (peroxidase in station II and IV while esterase in station III and V). It’s mean that genetic variation on white grubs population at salak pondoh agroecosystem in Mount Merapi’s slope was found. The environmental condition also contributed to the influence of the appear of isozyme banding pattern’s variation because each location had a different condition.

Key words: white grub, isozyme banding patterns, electrophoresis, Mount Merapi, salak pondoh.

Abstrak. Wardani S, Sugiyarto. 2009. Karakterisasi lundi putih (Melolonthidae: Coleoptera) pada agroekosistem salak pondoh di Gunung Merapi berdasarkan pola pita isozim. Nusantara Bioscience 1: 38-42. Tujuan penelitian ini adalah mengetahui karakteristik lundi putih (Melolonthidae: Coleoptera) didasarkan pada pola pita isozim. Penelitian morfologi dilakukan di Sleman, Yogyakarta dan Magelang, Jawa Tengah. Sampel diambil dari lima tempat dengan ketinggian yang berbeda dimana lima sampel diambil dari setiap lokasi. Metode yang digunakan dalam penelitian ini adalah elektroforesis gel poliakrilamida (PAGE) menggunakan jenis vertikal.

Sistem enzim yang digunakan adalah peroksidase dan esterase untuk mendeteksi pola pita isozim. Hasil penelitian menunjukkan bahwa terdapat variasi pola pita isozim lundi putih (Melolonthidae: Coleoptera) pada agroekosistem salak pondoh di lereng Gunung Merapi (peroksidase di stasiun II dan IV sedangkan esterase di stasiun III dan V). Hal ini menunjukkan bahwa terdapat variasi genetik pada populasi lundi putih pada agroekosistem salak pondoh di lereng Gunung Merapi. Kondisi lingkungan juga berpengaruh terhadap munculnya variasi pola pita isozim karena setiap lokasi memiliki kondisi lingkungan yang berbeda.

Kata kunci: lundi putih, pola pita isozim, elektroforesis, Gunung Merapi, salak pondoh.

INTRODUCTION Salak pondoh (Salacca zalacca (Gaert) Voss) is one type of fruit that is loved by the people of Indonesia. The area around the slope of Mount Merapi, particularly in Sleman district, Yogyakarta and Magelang regency, Central Java is one of the salak cropping center, especially salak pondoh (Kusumo et al. 1995; Suskendriyati et al.

2000). The pests often attack salak pondoh plants in Sleman, Yogyakarta is the white grub. White grub is the name of a group of insect larvae (Family Melolonthidae, order Coleoptera) that have a body shape like the letter "C" or "scarabaeid". It has cream or white body, reddish brown head with cutters mouth type, three pairs of leg right at the back of the head. The body length ranges from 2-6 cm, body diameter ranged from 0.5 to 1.5 cm. In some areas of Java, it is also known by the name "uret" or "embug"

(Sugiyarto 2000; Sugiyarto et al. 2002; Kompas 4/10/2003). These pests attack sporadically that resulted in crop damage of salak pondoh widespreadly.

Pracaya (1999) states that at the beginning, these pests only eat humus and other debris, but after a little bigger, they eat the roots of plants that are still alive, sometimes even eat salak tree in the soil so that it can cause plant to die. In the adult phase, in the form of beetles, these pests eat the leaves of plants but the damage is not so visible. In area of Texas and other sub-tropical regions, the white grub of Phyllophaga crinita species is known as the pest of grassland and various kinds of ornamental plants with huge losses (Crocker et al. 1999; Drees and Jackman 1999).

White grub is also the pest of main auxiliary crops that attack the area of sengon-based agroforestry in Jatirejo, Kediri District and we have not found how to control them (Sugiyarto 2004).

WARDANI et al. – White grubs at salak agroecosystem in Mount Merapi

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MATERIALS AND METHODS Field research White grub (Melolonthidae: Coleoptera) were taken from the agroecosystem of salak pondoh on the slopes of Mount Merapi, precisely in Sleman regency, Yogyakarta and Magelang regency, Central Java. Samples were taken at 5 stations with different altitude, namely: station I: 484 m asl (Turi, Sleman), station II: 545 m asl (Srumbung, Magelang), station III: 620 m asl (Srumbung, Magelang), station IV: 751 m asl (Turi, Sleman), and station V: 820 m asl (Pakem, Sleman). Five samples were taken from each station and are well-treated in order to make them stay alive until the isozyme banding pattern analysis is done.

The measurement of environmental factors includes air temperature, soil temperature, soil pH, soil moisture and Soil Organic Matter (SOM).

Figure 1. Morphology of white grub an insect larvae of family Melolonthidae, order Coleoptera.

So far, the white grub pest control efforts have been made through various approaches such as physical and chemical approaches, but the results are not satisfactory. In order to develop a biological control, the key to success is the presence of complete information on the characteristics of the specimen. Until now there has been no complete information about the characteristics of white grub, and therefore the pest needs to be characterized.

Characterization of the morphological approach has several weaknesses, including the appearance of the characters are often influenced by environmental factors. But the main weakness of this morphological approach, according to Delluchi et al. (1989) and Suskendriyati et al (2000), is character recognition at the level of sub-species, especially with the presence of twin species or sibling species.

One alternative way that can be used to characterize the white grub is through isozyme. Isozymes are some enzymes that have different chemical structures but catalyze the same reaction. Isozyme has several advantages, including: can be used to identify the properties that are not visible in morphology (Mariani 2002), can be applied to determine the genetic structure of intra-and inter-population (Fitriyah 2002), and many samples can be analyzed in relatively short time (Hadiati and Sukmadjaja 2002). The purpose of this study is to determine the characteristics of white grub (Melolonthidae: Coleoptera) on salak pondoh agroecosystem on the slopes of Mount Merapi on the basis of isozyme banding pattern.

Isozyme analysis Isozyme banding pattern analysis was performed by polyacrylamide gel electrophoresis (PAGE). The preparation of buffers and stock solutions follows the method of Suranto (1991, 2001, 2002).

Making a buffer. Tank buffer (borax buffer) was made by dissolving the borax acid of 14.4 g and 31.5 g of borax in distilled water until it reaches the volume of 2 liters.

Extraction buffer is made by dissolving 0.018 g of cysteine, 0.021 g of ascorbic acid, and 5 g of sucrose in 20 mL of buffer tank with pH 8.4.

Preparation of stock solutions. A stock solution was prepared by dissolving 4.5 g of tris and 0.51 g of citric acid in 500 mL aquabidest. B stock solution was prepared by dissolving 30 g of acrylamide, combined with 0.80 g of N,N'-methylene-bis-Acrylamide (bisacrylamide) into 100 mL aquabidest.

Preparation of gel. Gels prepared according to the method of Suranto (1991) with modification, namely by mixing 2.5 mL of stock solution B and 5 mL of stock solution A, then added with 0.02 mL of N,N,N',N'tetramethyl-ethylenediamine (TEMED) and mixed them carefully. For gel polymerization, it was added by 30 mL of ammonium persulphate (APS).

Extraction and sample preparation. Digestive organ was extracted using extraction buffer with a ratio of 1:3, in μg for samples and μl for buffer extraction. Organs are crushed using a mortar on top of ice crystals flake. Samples that have been destroyed then centrifuged at 8500 rpm for 3 minutes. Supernatant is taken as many as 7 μL for peroxidase staining and 15 μL for esterase staining.

Electrophoresis device used for the analysis of isozyme banding pattern is a BIO-RAD Mini Protean 3 Cell vertical type, made in USA. Staining. The staining in this study used two enzyme systems, namely peroxidase and esterase. For peroxidase staining, a total of 0.0125 g of O-dianisidin dissolved in 2.5 mL of acetone and then added by 50 mL of acetate buffer pH 4.5 and 2 drops of hydrogen peroxide. While for esterase staining a total of 0.0125 g of α-naphthyl acetate was dissolved in 2.5 mL of acetone, then added with 50 mL

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of 0.2 M phosphate buffer pH 6.5 and 0.0125 g of fast Blue BB salt.

Data analysis Band formed was drawn in the shape of zimogram.

Data obtained by calculating the value of Rf, which is the ratio of migration distance of band to the migration distance of loading dye. Data were analyzed based on whether the tape appeared on the gel and the thickness of thin band formed.

RESULTS AND DISCUSSION Environmental factors All environmental parameters measured can affect physiological processes (metabolism) of white grub. There are variations on the five environmental parameters measured at five observation stations (Table 1).

Table 1. Environmental factors of the research site.

Air temp. Soil temp. Soil GWT SOM (oC) (oC) pH (%) (%) I: 484 m asl 30.9 27.3 6.72 12.77 5.52 II: 545 m asl 29.7 25.8 5.44 19.24 3.61 III: 620 m asl 32.3 27.2 6.76 20.16 5.72 IV: 751 m asl 27.8 24.4 6.98 5.14 3.45 V: 820 m asl 26.8 29.3 7 7.95 6.14 Note: I&IV: Turi, Sleman, II&III: Srumbung, Magelang, V: Pakem, Sleman, SOM: Soil Organic Matter, GWT: Ground Water Levels.

Station

The temperature tends to decrease as the height increases (from station I to V). Soil temperature plays a key role in soil environment. Insects have a certain temperature range where he can live. Outside the temperature range, the insects will die from the cold or heat. Temperature effect is clearly visible in the process of insect physiology. At a certain temperature, insect activity is high, but at other temperatures, it will be reduced (down). In general, the minimum temperature is 15°C, the optimum temperature is 25°C and 45°C for maximum temperature (Jumar 2000).

The five stations showed a normal pH range, ie close to pH 7, except for station II, which has the lowest soil acidity (pH 5.44). For land animals, soil pH also influences. Living things can run these processes with a good life if in the range of their optimum pH. The existence of extreme pH can affect the survival of the organism. The soil also contained water needed by plant roots and soil organisms to survive. Soil water content will affect the soil moisture.

The higher soil water content, soil moisture will also be higher. Soil organic matter is a source of food (energy source) for the major white grub so that its availability is needed.

By considering the vegetation that makes up each research station, the station I is salak pondoh agrotourism, so the plants that dominate this place is the salak pondoh.

Station II is salak pondoh garden that has been treated with water treatment in anticipation of a white grub pests, therefore the soil moisture content is high enough. Beside

salak pondoh plants, cassava and bananas crops are planted nearby, as intercropping plants to distract the white grub not to eat the plant roots of salak pondoh. Station III is pure salak garden and is not planted by the other plants. Station IV represents salak pondoh garden planted with crops intercropping, as well as station II. Other vegetation found in this station is a cassava, banana, coconut, distance, mahogany, and weed is pretty much found. Station V had the most different field conditions among other observation stations because the soil tends to dry and sandy.

White grub (Melolonthidae: Coleoptera) Among the group of insects, beetles (Coleoptera) are the largest group since they set about 40% of all insect species and consist of not less than 250 thousand species (Pracaya 1999). White grub is included in Melolonthidae family of Coleoptera order (Chu 1992). Borror (1992) also include a white grub into a Scarabaeidae family. Larvae (uret) included in this family often damages the roots of plants and when they grow up, the adults will eat the leaves, but the damage is not as bad as its larvae stage.

White larva lives in the soil and takes ± 7 months before it becomes a cocoon.

Of the five study sites, i.e. the stations I to V which are determined based on the gradation of heights, it can be found a white grub that has similar morphological features.

The morphological features including: a body shape like the letter 'C', head reddish brown, 3 pairs of legs just behind the head, and 3 segments of thorax (the first segment is spiracles). Spiracles serve as the exit point of O2 and CO2, it also is as the evaporation of H2O (Jumar 2000). The abdomen has 10 segments, 8 segments have spiracles on the lateral body, while the last 2 segments do not have spiracle and serve as a place to store the rest of digestion so the color is darker (black) and there is an anus.

The body is yellowish-white color, the body length ranges from 2-6 cm, the diameter ranges from 0.5 to 1.5 cm.

Based on morphology, white grub found in the study area is included in the genus of Phyllophaga, and has species name of Phyllophaga javana Brsk and the other name is Holotrichia javana Brsk (Pracaya 1999). Local name for this insect species is ampal. P. javana larvae is multiphytophagous, if soil organic matter content is high, this larvae is more as saprophagous, but if the soil conditions is in shortages of organic material, the larvae eats the roots of plants so it causes crop damage.

Isozyme banding pattern Isozyme is the enzyme which has different chemical structures but catalyze the same reaction. The difference form of an enzyme molecule can serve as the basis of chemical separation, such as by electrophoresis, which produces tapes with a range of different migration.

Peroxidase isozyme Peroxidase (PER) enzyme is categorized in the group of oxydo-reductase. The reactions that occur in the peroxidase staining were: 2 H2O2 Æ 2 H2O + O2

WARDANI et al. â&#x20AC;&#x201C; White grubs at salak agroecosystem in Mount Merapi

Peroxidase catalyzes the H2O2 into H2O and O2. The existence of peroxidase can be easily detected because of high activity and stability and it can use a number of substrates as hydrogen donors (Cahyarini 2004).

Figure 1. Zimogram peroxidase isozyme electrophoresis results of white grub in agroecosystem salak pondoh on the slopes of Mount Merapi. Description: 1.2 = sample station I; 3.4 = sample station II; 5.6 = sample station III; 7.8 = sample station IV; 9.10 V = sample station

From zimogram of peroxidase isozyme electrophoresis results, it is known that the isozyme peroxidase produce 12 bands based on the relative motion of the enzyme (Rf). Of the twelve bands, four bands always appear or are found in all individuals from station I to station V. The four bands are located at a distance migration 1, 1.5, 5 and 9 mm from the slot or at Rf 0.017, 0.026, 0.086 and 0.155. The second tape is faintly visible or thin, which suggests that small molecular weight enzyme. The four bands can be used as a characteristic pattern of peroxidase isozyme bands on white grub. Besides the four major bands that appear, the individuals from station II has a band that is not present in other individuals of the five stations. The band is located at 3.5 mm (Rf 0.060) and at 4 mm from the slot (Rf 0.069).

Figure 1 shows the band of individual No. 3 and No. 4.

Quantitatively, the two bands are thin. Besides the individuals of station II, individuals from station IV also shows the anomaly with the advent of tape on the migration distance of 15 and 18 mm or at Rf 0.259 and 0.310 (in Figure 1, it is shown by individual No. 8), and 38, 41, 45 and 52 mm or at Rf 0.655, 0.707, 0.776 and 0.896 (in Figure 1 is shown by individual No. 7). The emergence of bands in individuals from station IV which is absent in individuals from other stations also shows a variation pattern of peroxidase isozyme bands on white grub.

Based on the results above, it can be explained that the individuals from stations I, III and V have the same banding pattern. While individuals from stations II and IV show the diversity of isozyme banding pattern so that it can be assumed that there are genetic differences that encode the enzyme. According Cahyarini (2004), the difference in migration distance bands is a manifestation of differences in content and form of the enzyme molecule. Rahayu et al.

(2006) adds that the enzyme or protein can be used to show variation both qualitatively and quantitatively. These variations are result from the role of the gene that directs

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the formation of the enzyme in question, therefore the variation of enzyme can describe the gene variation.

In terms of vegetation, station II and IV are prepared not only by salak pondoh plants, but there are other plants that make up the ecosystem. While the stations I, III, and V is pure salak pondoh plantations, in other word, there is no other plant that dominates. It is possible that differences in vegetation gives the effect of the variation of isozyme banding pattern of white grub, considering that every living being will try to maintain its survival in case of environmental changes.

Band thickness can basically be divided into two, namely a thick band and a thin band. A thin band or vaguely indicates that the isozyme content or concentration is small.

Esterase isozyme Esterase enzyme included in the reaction hidrolase class specific chemical bond is determined by adding the element of water (Salisbury and Ross 1992). Esterase is a hydrolytic enzyme that functions to withhold simple esters in organic acids, inorganic acids and alcohols and phenols that have a low molecular weight and soluble (Subronto 1989 in Setianto 2001).

Figure 2. Zimogram esterase isozyme electrophoresis results of white grub in agroecosystem salak pondoh on the slopes of Mount Merapi. Description: 1.2 = sample station I; 3.4 = sample station II; 5.6 = sample station III; 7.8 = sample station IV; 9.10 V = sample station

From Figure 2, it is known that esterase isozymes in white grub produce 7 bands. The first band, the second band and the third band appear on all individuals from the five stations, with each migration distance is 1.5, 2 and 6 mm from the slot or at Rf 0.026, 0.034 and 0.103. The three of these bands are unique because they are always found in all individuals from the five stations that show uniformity in banding pattern. The specialties are in individuals from station III and V, because beside having a third band mentioned above, they also have a band on the migration distances of 10 and 14 mm (Rf 0.172 and 0.241).

In addition, band at a distance of 35 and 38 mm (Rf 0.603 and 0.655) are found only in individuals from station V. Based on the comparison of bands that appear at the five stations, it appears that individuals from station I, II, and IV have the same number of bands and the same banding pattern. Meanwhile, individuals from station III and V

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shows the variation of esterase isozyme banding pattern.

Band esterase isozyme in the gel appears to be brown with almost the same intensity.

Based on the results of electrophoresis of either peroxidase staining or esterase staining, the diversity of isozyme banding pattern is more likely to belong to the qualitative diversity, namely the presence or absence of bands on the gel. The thickness of the tape which is a quantitative nature is mostly the same. According to Setianto (2001), qualitative properties are preferred because they relate to the presence of a particular band at a particular distance migration that reflects the presence or absence of amino acids making up the enzyme that is a product of the gene itself.

From those facts, it can be explained that the peroxidase and esterase isozyme can show the variation of isozyme banding pattern on a white grub. The variation of isozyme banding pattern shown by the distance of migration of different bands indicates different forms or different chemical structures (conformation), and it can be presumed that the genes that encode enzymes are not the same. In addition, environmental factors also affect the appearance of this isozyme banding pattern variation considering that the sampling sites have variation of environmental conditions. Salisbury and Ross (1992) states that if there are environmental factors that change, the most active isozyme in those environments would carry out its functions and help the organism to survive.

CONCLUSION Based on research results obtained, it can be concluded that the white grub (Melolonthidae: Coleoptera) on salak pondoh agroecosystem on the slopes of Mount Merapi showed good variation of isozyme banding pattern of peroxidase isozyme (in individuals from station II and IV) and esterase (in individuals from station III and V). Thus there is genetic variation among populations of white grub (Melolonthidae: Coleoptera) in agroecosystem salak pondoh on the slopes of Mount Merapi. Environmental factors also affect the appearance of this isozyme banding pattern variation considering the sampling sites have environmental conditions variation.

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Cahyarini RD. 2004. Identifikasi keragaman genetik beberapa varietas lokal kedelai di Jawa berdasarkan analisis isozim. Tesis. Program Pascasarjana Universitas Sebelas Maret, Surakarta.

Chu HF. 1992. How to know immature insects. WMC Brown. Dubuque.

Crocker RL, Nailon WT, Matis JH, Woodruff RE. 1999. Temporal pattern of ovipositional readiness in spring species of Phyllophaga (Coleoptera: Scarabaeidae) in North Central Texas. Ann Entomol Soc Am 92 (1): 47-52.

Delluchi VD, Rosen D, Schlinger EI. 1989. Relation of systematic and biological control. In: Huffaker CB, Messenger PS (eds) Theory and practice of biological control. University of Indonesia Press. Jakarta.

Drees BM, Jackman J. 1999. Field guide to Texas insect. Gulf Publishing.

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Fitriyah F. 2002. Comparative study of isozyme banding pattern and morphology of green leafhoppers (Nephotettix virescens) from West Java, Central Java and East Java. [Thesis S1]. Faculty of Agriculture, University Eleven March. Surakarta. [Indonesia] Hadiati S, Sukmadjaja D. 2002. The diversity of some accessions of pineapple based on isozyme banding pattern analysis. J Bioteknol Pertanian 7 (20): 62-70. [Indonesia] Jumar. 2000. Agricultural entomology. Rineka Cipta, Jakarta. [Indonesia] Kompas. 2003. Salak pondoh plant threatened by white grubs pests in Sleman. 10 April 2003. [Indonesia] Kusumo S, Farid AB, Sulihanti S, Yusri K, Suhardjo, Sudaryono T. 1995.

Salak production technology. Research and Development Center for Horticulture. Research and Development Agency, Department of Agriculture. Jakarta. [Indonesia] Mariani Y. 2002. Study on isozyme variation of a few colonies of green leafhoppers (Nephotettix virescens) as a vector of rice tungro disease.

[Thesis S1]. Faculty of Agriculture, Sebelas Maret University.

Surakarta. [Indonesia] Pracaya. 1999. Pests and plant diseases. Penebar Swadaya. Jakarta.

[Indonesia] Rahayu S, Sumitro SB, Susilawati T, Soemarno. 2006. Analysis of isoenzymes to study the genetic variation of Bali cattle in Bali Province. Berk Penel Hayati 12: 1-5. [Indonesia] Salisbury FB, Ross CW. 1992. Plant physiology. Vol. 2. Penerbit ITB. Bandung. [Indonesia] Setianto A. 2001. Characterization of a large orange (Citrus grandis (L.) Obsbeck) in Jepon and Jiken Subdistrict, Blora District based on isozyme and morphological of fruit. [Thesis S1]. Faculty of Agriculture, Sebelas Maret University. Surakarta. [Indonesia] Subronto 1989. Isolation and properties of isozymes of palm leaf Deli Dura. [Thesis]. Bogor Agricultural University. Bogor. [Indonesia] Sugiyarto, Sugito Y, Handayanto E, Agustina L. 2002. Effect of land use systems on soil macroinvertebrate diversity in RPH Jatirejo, Kediri, East Java. BioSMART 4 (2): 66-69. [Indonesia] Sugiyarto. 2000. Diversity of soil macrofauna in different sengon age stands at RPH Jatirejo, Kediri District. Biodiversity 1 (2): 47-54.

[Indonesia] Sugiyarto. 2004. Macroinvertebrate diversity between the soil and crop productivity in sengon agroforestry systems. [Dissertation].

Postgraduate Program, Brawijaya University. Malang. [Indonesia] Suranto. 1991. Studies of population variation in species of Ranunculus.

[Thesis]. Department of Plant Science, University of Tasmania.

Hobart, TAS. Suranto. 2001. Isozyme studies on the morphological variation of Ranunculus nanus populations. Agrivita 23 (2): 139-146.

Suranto. 2002. Cluster analysis of Ranunculus species. Biodiversitas 3 (1): 201-206.

Suskendriyati HA, Wijayati, Nurhidayah, Cahyuningdari D. 2000.

Morphological studies and phylogenetic relationship of salak pondoh varieties (Salacca zalacca (Gaert.) Voss.) in the highlands of Sleman.

Biodiversitas 1 (1): 26-31. [Indonesia]

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 1, No. 1, Pp. 43-52 March 2009

Review: Effect of global warming on plant evolution and diversity; lessons from the past and its potential recurrence in the future AHMAD DWI SETYAWAN♥ ¹Department of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java, Indonesia. Tel./Fax.: +92-271-663375. ♥email: volatileoils@gmail.com Manuscript received: 11 November 2009. Revision accepted: 4 February 2010.

Abstract. Setyawan AD. 2009. Effect of global warming on plant evolution and diversity; lessons from the past and its potential recurrence in the future. Nusantara Bioscience 1: 43-52. Lessons from the past shows that global warming and glaciation is a natural cycle of repeated, the trigger factor is not always the same, but global warming is always accompanied by elevated levels of CO2 and greenhouse gases in the atmosphere which cause the other rising global temperatures. Present and destruction of various plants and other living makhluh continue to happen from time to time. Every era has its own life form, as a mirror of global environmental conditions at the time. Biodiversity is not always the same between one period of global warming are with the next global warming, or one period of glaciation that one with the next glaciation, although new breeds always show traces the evolution of his ancestors. Man is one of the agents of global warming that began with the development of agricultural systems since 8000 years ago. The impact of climate change due to global warming should continue to be wary of. Based on past experience, global warming is always followed by mass extinctions, but various forms of life will still survive even though its shape is almost certainly not the same as before. Living organisms can survive it will evolve into new taxa that are different from its parental taxa. Humans who were present at that time probably were not a men who are present at this time, given Homo sapiens may have been extinct for not being able to adapt or otherwise has evolved into a new man who may no longer shows characteristics of human wisdom.

Key words: global warming, evolution, diversity, new species.

Abstrak. Setyawan AD. 2009. Pengaruh pemanasan global terhadap evolusi dan keanekaragaman tumbuhan; pelajaran dari masa lalu dan kemungkinan terulangnya kembali di masa depan. Nusantara Bioscience 1: 43-52. Pelajaran dari masa lalu menunjukkan bahwa pemanasan global dan glasiasi merupakan siklus alamiah yang terus berulang; faktor pemicunya tidak selalu sama, namun pemanasan global selalu disertai peningkatan kadar CO2 dan gas-gas rumah kaca lainnya di atmosfer yang menyebabkan meningkatnya suhu bumi.

Hadir dan musnahnya berbagai tumbuhan dan makhluh hidup lainnya terus terjadi dari waktu ke waktu. Setiap jaman memiliki bentuk kehidupannya sendiri-sendiri, sebagai cermin kondisi lingkungan global pada saat itu. Keanekaragaman hayati tidak selalu sama antara masa pemanasan global yang satu dengan masa pemanasan global berikutnya; atau dari masa glasiasi yang satu dengan glasiasi berikutnya, meskipun keturunan-keturunan baru selalu menunjukkan jejak evolusi dari nenek moyangnya. Manusia merupakan salah satu agen pemanasan global yang dimulai dengan dikembangkannya sistem pertanian sejak 8000 tahun yang lalu. Dampak perubahan iklim akibat pemanasan global perlu terus diwaspadai. Berdasarkan pengalaman di masa lalu, pemanasan global selalu diikuti kepunahan massal, namun berbagai bentuk kehidupan tetap akan bertahan meskipun bentuknya hampir pasti tidak sama dengan yang ada sebelumnya. Makhluk hidup yang dapat bertahan akan berevolusi menjadi taksa baru yang berbeda dengan taksa tetuanya. Manusia yang hadir pada saat itu barangkali bukanlah manusia yang hadir saat ini, mengingat boleh jadi Homo sapiens telah punah karena tidak mampu beradaptasi atau sebaliknya telah berevolusi menjadi manusia baru yang barangkali tidak lagi menunjukkan ciri-ciri manusia bijaksana.

Kata kunci: pemanasan global, evolusi, keanekaragaman, jenis baru.

INTRODUCTION Climate change is a study that has long been a topic in biology. The effect of climate change on the lives of living things has long been rooted in biological studies, long before the politicians pay attention to their impact on the environment. The study of the climate influence on changes in the distribution of living things has been done in Europe since England started the industrial revolution (around 1765). Grinnell (1917) describes, in detail, the influence of climate on the spread of many species, followed by Andrewartha and Birch (1954) and MacArthur

(1972). These observations include various species of birds, butterflies, insects, herbs, and trees. Research which is mostly done is the change of the distribution of birds (Gudmundsson 1951; Harris 1964; Kalela 1949, 1952; Salomonsen 1948; Williamson 1975; Thomas and Lennon 1999), of butterflies (Ford 1945; Parmesan et al 1999; Parmesan 2002, 2003) , and of insects (Uvarov 1931; Dobzhansky 1943, 1947; Dennis 1993; Bale et al. 2002) to the north due to warmer summers and winters are less harsh, while the plants are relatively more limited and generally involves the spread of plants around Arctic (Sturm et al. 2001; Stirling 2002; Smol et al. 2005) and

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tropical mountains (Pounds et al. 1999, 2005; Wilson et al.

2005). At present, the study of the impact of climate change on biota includes, among others, the single impact of extreme weather on ecosystems, changes in distribution and abundance, phenology, physiology, morphology, genetics, and behavior (Roy and Sparks 2000; Stefanescu et al. 2003; Root and Hughes 2005, Parmesan 2006).

Climate change, either global warming or glaciations, is a natural cycle that continues to occur since the formation of the earth. Since the Precambrian period (600 million years ago; mya), there has been at least twice of global warming and three times of glaciations in large scale. These changes are caused by many factors, including: volcanic activity (Fischer 1984), falling of celestial body (Hildebrand et al. 1991), the separation of ancient continental (tectonic) (Strecker et al. 2007) and others. These changes are marked with the extreme and wide change of temperature and gas composition, and directly impact the lives, so the diversity of living things change continuously from time to time, either in the form of extinction due to failure in adaptation or the appearance of new taxa as the response to the evolution of these changes (Fishcer 1984).

Global warming is the result of an imbalance between the amounts of solar radiation energy received and released by the Earth, as it is restrained by the gases which lie between the earth's surface and the stratosphere, so the Earth's surface temperature rises. Greenhouse gases include water vapor, CO2, CH4, N2O, CFCs, aerosols, etc.

(Ramaswamy et al. 1992, 2001). These gases can be the result of natural events or human activities (anthropogenic).

Agricultural and industrial activities are the main source of anthropogenic global warming. Agriculture has donated greenhouse gases of CO2 and CH4 for thousands of years, because the conversion of forests to agricultural land on a large scale and the discovery of wet rice fields techniques (Yagi et al. 2000; Komiya et al. 2010). Industry donates gases of CO2 and other greenhouse gases as the effect fossil fuel use, ie coal and oil industries (IPCC 2001; Iijima et al. 2010).

This literature review has the aim to explain the connection of global warming with adaptation and evolution of plants and its effect on diversity and classification of plants. Nowadays, the study in this field is relatively still limited, although climate change is an old phenomenon in the study of biology, the issues discussed are generally only associated with the impact on agriculture, health, and responses at the ecosystem level, particularly changes on biota and the environment due to an extreme single incident, such as El Nino of Southern Oscillation (ENSO) and North Atlantic Oscillation (NAO).

Therefore, studies that connecting global warming with evolution and classification, especially in plants, need to be done.

GLASS HOUSE GAS AND GLOBAL WARMING Greenhouse gases. Greenhouse gases are the gases forming atmosphere, either naturally or anthropogenic, that

are able to absorb and re-emit the infrared radiation at specific wavelengths, where the radiation is emitted by the Earth's surface, atmosphere and clouds. The main greenhouse gases in the atmosphere are aqueous vapor (H2O), carbon dioxide (CO2), nitrogen oxide (N2O), methane (CH4) and ozone (O3). Aqueous vapor and CO2 contribute about 95% of the greenhouse effect; and the rest, about 5%, is contributed mainly by O3, CH4, N2O and chlorofluorocarbons (CFCs). Aqueous vapor is the most abundant gases in the troposphere, but because the amount is relatively fixed and short residence of time, then the impact on global warming is negligible (Table 1).

International agreements have been made to regulate greenhouse gas emissions. Montreal Protocol regulates the emissions of greenhouse gases which are entirely due to human activities, such as halocarbon, chlorine and bromine. The Kyoto Protocol regulates greenhouse gas emissions of CO2, N2O, and CH4, hexafluoride sulfur (SF6), hydrofluorocarbons (HFCs) and perfluorocarbons (PFCs) (IPCC 2001).

Greenhouse effect. Greenhouse gas is the main cause of the greenhouse effect, namely global warming as the effect of the rising of the earth’s surface temperature.

Greenhouse gases absorb infrared radiation emitted by the earth, atmosphere, and clouds. The main source of energy is solar radiation that is emitted in all directions.

Greenhouse gases can absorb heat between the earth's surface and troposphere ("natural greenhouse effect") (Figure 1). Radiation greatly affects the temperature of the atmosphere at an altitude where it is emitted. In the troposphere, temperatures generally decrease with increasing altitude. Infrared radiation emitted at altitude with temperature of about -19°C, while the earth's surface is maintained at a higher temperature, approximately +14°C. the increasing levels of greenhouse gases lead to the increasing of impermeability of infrared radiation into the atmosphere, therefore the radiation has begun to occur at a higher altitude with a temperature lower than -19°C, resulting in increasing of infrared radiation followed by the rising of temperatures greater than +14°C ("enhanced effect of greenhouse"). This is generally a result of anthropogenic activities. The theory of the greenhouse effect was first proposed by Arrhenius in 1896. He estimated that the surface temperature will increase due to multiplying of CO2 in the atmosphere (IPCC 2001; Loaiciga et al. 1996).

HUMAN ROLE IN GLOBAL WARMING Pre industry. Agriculture has started 11,000 years ago in the fertile area of the eastern Mediterranean, later followed by Chinese and American Indians. This activity grew very rapidly, about 2000 years ago all the major food crop has been cultivated. Agricultural activities have began donating CO2 greenhouse gases since about 8000 years ago, with the conversion of natural forest to agricultural land on a large scale in Europe and China. The remains of the burned or decomposing trees release CO2 into the atmosphere (Naik et al. 2007). Other greenhouse gases, CH4, is produced by the agriculture supporting activities

SETYAWAN – Global warming effect on the plant evolution and diversity Table 1. Concentration and resident time of some greenhouse gases (Loaiciga et al. 1996).

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and CH4, but also produces various kinds of other Greenhouse gases greenhouse gases as set forth a Parameter H2 O CO2 CH4 CFC-11 CFC-12 N2O O3 in the Montreal Protocol and (ppm) (ppm) (ppm) (ppt) (ppt) (ppb) (ppb) Kyoto Protocol. In addition, it b Early industrialization (1750-1800) 3000 280 0,8 0 0 285 1-15 is believed that in the future it 1990 3000 353 1,72 280 484 310 10-100 c will still be found and Resident time 10-15 50-100 10 65 years 130 years 150 n.a. produced greenhouse gases days years years years which are new, including Note: ppm = part per million, ppb = part per billion, ppt = part per trillion; a = < 12 kn, b = various types of aerosols approximate value, c = ozone produced continuously in the stratosphere via photolysis, n.a. = not applicable. which are difficult to quantify scientifically (Hansen et al.

1998; 2006).

CO2 gas is the largest contributor of greenhouse gases. Since the industrial revolution, its levels in the atmosphere have increased up to 83 ppm, from 280 ppm (in 1800) to 363 ppm (in 1990) (Table 1.). If the level of CO2 emissions in 1990 is not reduced, then the levels in 2100 will nearly be doubled from pre-industrial levels. The main sources of CO2 are coal and petroleum. Coal remains a potential source of CO2 in the future, because the world’s needs tend to rise higher than oil or gas (Loaiciga et al. 1996).

Figure 1. Average annual energy balance of the earth. Solar radiation entering Earth's atmosphere Another source is the change 2 2 on average per year is 342 W/m , which were 107 W/m directly reflected into space by clouds, 2 of land use in tropical area atmospheric and earth surface. The remaining 235 W/m , mostly absorbed by the earth's surface and natural resources either in (168 W/m2) and a small portion absorbed by the atmosphere (67 W/m2). The earth surface the ocean, biosphere, and radiates energy back into the atmosphere in the form of infrared radiation (24 W/m2), heat (78 land. Therefore, the reduction W/m2), water vapor and direct lost through the atmospheric "window". Finally, by 235 W/m2 of radiation emitted back to space through the atmosphere (165 W/m2), clouds (30 W/m2) and the of fossil fuel consumption, atmospheric "window" (40 W/m2) (Kiehl and Trenberth 1997). which continuously grew 1.2% per year since 1975, needs to be done. CO2 gas is such as animal husbandry and burning of land for hunting, the largest contributor of greenhouse gases up to now, but the CH4 in large numbers began to be donated about although recent studies of some NASA researchers give 5,000 years ago, since the discovery of wet fields opinion about a large number of the contribution of nontechniques in southern China. This technique led to the CO2 gases in global warming (Hansen and Sato 2001). The decomposition of organic materials by anaerobic bacteria United States is the largest contributor to CO2 and up to and produce CH4 (Zhang et al. 2008). This technique has now is not willing to implement the Kyoto Protocol. spread to Southeast Asia and India since 3000 years ago.

Mitigation scenario. Contribution of greenhouse Its use was more widespread with the discovery of gases due to anthropogenic activities can have a positive or techniques of terraced rice fields in the hills of Southeast negative effect on global climate. Agriculture is the most Asia since 1,000 years ago. Therefore, long before the influencing factor during interglaciations climate. During industrial period, Europe and Asia has contributed to the pre-industrial, agricultural activities increased the greenhouse gas (Ruddiman 2005). temperature of the earth about 0.8°C, so the earth is warm Industrial period. The industrial revolution in England enough for occupancy; because since 8,000 years ago began in 1765 with the discovery of steam engine with coal glaciations cycles have begun which is marked by the fuel. This machine contributes the releasing of CO2 and emergence of ice domes in northern Canada. On the other carbon black (aerosols) in the air, but an increase rate of hand, the rising temperatures about 0.6°C after the CO2 emissions to the air on a large scale began with the industrial revolution has raised the fears of global warming, discovery of gasoline engine (1876) and diesel (1893) of where the increase of about 0.5°C occurred in the past three petroleum fuel. Industrial activity not only produces CO2 decades (Figure 2) (Hansen et al. 2000; Ruddiman 2005).

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Figure 2. Effect of greenhouse gases from human activities that counteract glaciation began about 5,000 years ago. Early agricultural activity in pre-industrial produce greenhouse gases to offset the cooling of the earth's natural tendency (shaded), by heating the earth nearly around 0.8oC. Effects of early global warming (a) obscured when compared with the 0.6oC warming (b) measured in the late 17th century due to rapid industrialization (black). After running out of fossil fuels and increases as the peak temperature of greenhouse gases, the Earth will cool down towards the next glaciation, which has been delayed for thousands of years (Ruddiman 2005).

Figure 2. The estimated strength of the factors forming the climate between the years 1850-2000 (Hansen and Sato 2001).

earth's temperature will be equal to the period of midPliocene (2.75 mya), when the earth is around 2°C warmer than current temperature and sea level is 25 m higher than today. Studies in Greenland show that the temperature changes abruptly with an annual average of 5-8°C for 3 years happened in ancient times.

Therefore it is necessary to attempt to limit the rate of global warming.

The IPCC has several scenarios to inhibit the rate of global warming, including A1F1 scenario, A1B scenario, and B1 scenario. In the scenario of 2°C increase temperature, if emissions of CO2 could be maintained at current levels, and the technology to reduce or capture emissions of CO2 is found in the second quarter of this century, then in 2050 it is estimated that the temperature rise is only to 0.5°C, and in 2100, it can be stabilized again at the level as the beginning of the industrial revolution. To optimize this plan, the alternative scenario can also be made to keep the climate strength for the next 50 years is at 1 W/m2 or less and to keep the global warming of 0.75° C or less (Figure 3) (Hansen and Sato 2004).

IMPACT OF GLOBAL WARMING AT ANCIENT TIMES ON BIOLOGICAL EXTINCTION Life in ancient times, from the Precambrian period until now, shows the presence Figure 3. The growth rate of greenhouse gases (5-year mean) that affect climate change (18502000), and mitigation scenarios of global warming in the coming century (Hansen and Sato of genetics radiation, genetic 2004). innovation, and mass extinction of taxa. Evolution is triggered by changes in At this time, the trend of temperature rise due to conditions of nature, where the periodic climate changes greenhouse gases is 0.15 ± 0.05°C per decade, which is from cold conditions (glaciation) to hot conditions (global slightly lower than the scenario of "business as usual" warming). The factors that trigger climate change are not namely CO2 that is added at 1% per year. If this growth always the same. Fischer (1984) shows the correlation of occurs continuously till the next century, then in 2100 the various factors that cause the biology crisis and affect the

SETYAWAN â&#x20AC;&#x201C; Global warming effect on the plant evolution and diversity

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Paleozoic. Paleozoic era is started and ended with global warming, in the middle of it, there was an interspersing of glaciations in the short time.

When approaching to the era change of Precambrian to Cambrian, there were the major changes including the breakup of Rodinia continent and very extensive glaciations and also the Cambrian explosion, namely the soft tissue biomineralization of various organisms produce vertebrate animals, so their tracks appear in the fossil. In the Cambrian period, the levels of O2 and CO2 in the atmosphere were respectively about 0.2 and 20 times of today's level, thus Figure 4. Periodic cycles of changing environmental conditions on the Precambrian period until they greatly affected the now that indicate the relationship between volcanism, sea level, climatic conditions, and the respiration and the extinction of living things (Fischer 1984). distribution of living things.

In the era of mid-early Cambrian and early Ordovician, the lack of O2 due to natural disasters in the ocean caused a massive extinction of trilobites (Barnes 1999). In boundary period of Ordovician and Silurian, it happened 200 million years global warming and it interspersed with cooling for 10 million years.

This cooling occurred because of the decrease of CO2 due to the separation of Antarctica from Gondwana. In the Ordovician era, CO2 levels are estimated to be about 16 times today, while the O2 content of about 15-20% of current time due to the spread Figure 5. Geologic time scale; Proterozoic, Archaean, and Priscoan (Hadean) commonly known of marsh plants producing as the Precambrian period (Microsoft Encarta 2003). coal.

Levels of both significantly affect the average surface temperature, evolution, namely the volcanism, the climate change, and the extinction of living things (Figure 4). The primeval as well as CH4 and aqueous vapor content. Climate in the experience prove that the change of CO2 and O2 levels late Paleozoic era was more diverse and complex. In the greatly affect the biological extinction. Earth's CO2 gas is limit of Permo-Carboniferous era, glaciations occurred produced naturally through volcanic and tectonic activity, during 60 Ma, because the decrease in CO2 due to the the release of hydrates gas and oxidation of organic formation of Pangea. In the Permian era, the continent of material. The O2 gas continued to be multiplied by the Pangea was very dry and dusty, had limited vegetation phytoplankton since 3.5 Ga) (Barnes 1999). To facilitate cover, but in some raised parts of the edge of the the understanding of time scales in ancient times, Figure 5 continental, the rainfall was very abundant (Barnes 1999).

In Permo-Triassic boundary, there occurred global presents the geological time scale. warming because the release of CO2 on a large scales due

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to the eruption of volcano in Siberia. Global warming caused a mass extinction of about 85-95% species on earth, so it takes 5 million years in the early Triassic to recover from this devastation. Extinction is also driven by the occurrence of gas hydrate release and the oxidation of organic matter that also increase levels of CO2 in the atmosphere, and the existence of ecological instability due to changes in habitat and annual climate fluctuations (Erwin 1993; Barnes 1999).

Mesozoic. In the Mesozoic era, the global warming happened because of the opening of sea water circulation from the equator to the poles, the formation of a broad shallow seas and warm and also the volcanic activity. In the early Jurassic period, the temperature range 5-10°C higher than at present. In the Cretaceous period, the temperature is getting hotter, where the level of CO2 is almost 4 times today. At the end of the Mesozoic era, on the Cretaceous-Tertiary era boundary, an asteroid crashed in Chicxulub, Yucatan Peninsula, Mexico which cause climate change, acid rain and a big fire, resulting in mass extinction of dinosaurs, ammonites, and other biota. This great extinction gave adequate space for the development of new plants, Angiosperms, which its genetic innovation began to emerge in the early Cretaceous era (Prinn and Fegley 1987; Barnes 1999; Beerling et al. 2002).

Cenozoic. In the Cenozoic era, there was global warming that is followed by glaciations and by slow global warming until the temperature was as today. Global warming in the Paleocene-Eocene era boundary was caused by the release of hydrate gas. At that time, the earth's temperature was 2-4°C higher than the current temperature, and it caused mass extinction of benthos foraminifera, the plankton turned into calcareous, and the mammals began to emerge on mainland. Glaciations at the Eocene-Oligocene boundary were due to the decreasing levels of CO2 in the atmosphere which were as the result of the formation of alpine region (Himalayas, Alps, Andes, and the Cordillera), and also the intermixture of warm and cold sea water circulation due to the opening of Drake Passage (between South America and Antarctica) and the closing of Panama isthmus. This led to the extinction of foraminifera plankton.

In the late of Neogene, the global cooling was widespread happened due to the continued freezing of Arctic region.

The glaciations continued in the early Quaternary Holocene due to the melting of ice on a large scale in North America, besides the influence of the cycle of the sun's orbit (Barnes 1999; Thompson et al. 2006).

ADAPTATION AND EVOLUTION OF PLANT DUE TO GLOBAL WARMING Increased levels of CO2, CH4 and other greenhouse gases in the atmosphere have an impact on climate change, among others: the increase of average global temperatures, the changing of rainfall patterns, and the increased frequency and intensity of extreme weather. This will affect the living things in the distribution, phenology, physiology, morphology, genetics, and behavior. Species

that are able to adapt in the long run, likely will experience the evolution and speciation to form new species.

Changes in distribution and abundance. Global warming can also lead to invasion of species from tropical and sub tropical to temperate areas or from low land to high land. On Galindez Island, Antarctica, global warming led to increase the rates of germination and seedling resistance of Deschampsia antarctica, so that between 1964-1990 its number increased rapidly from 500 to 12,030 individuals. In some places in the Alps, the observation in 1992-1993 shows that for decades biodiversity increased rapidly by 70% due to upward colonization. In Quelccaya, Andes, the decrease of ice dome causes the return of Distichia muscoides which was present in the region around 11,000 - 6,000 years ago when the temperature is 1.5-2.0°C warmer than today (Hughes 2000; IPCC 2001; Parmesan 2006; Thompson et al . 2006).

Changes in phenology. The time which promotes inflorescence determines the success of Angiosperms’s reproduction. The phenology of plant species may change due to changes in rainfall and temperature. In the forest areas that are sensitive to the early arrival of rains in the spring, the vegetation changes will occur in line with changes in rainfall patterns. In North America, for many decades, the earlier time of inflorescence of Syringa vulgaris and Lonicera spp. (L. tatarica and L. korolkowii) has been gone forward in average of 2 and 3.8 days per decade. On the other hand, studies in Lapland, Sweden showed that temperature increases do not affect phenology of Saxifraga oppositifolia and Ranunculus nivalis, although a large number of other species are affected (IPCC 2001; Molau et al. 2005; Parmesan 2006).

Changes in physiology and morphology. The increase in temperature and CO2 concentration directly affects photosynthesis, growth and productivity of plants.

In temperate areas, increased levels of CO2 can be observed from the annual ring width line of cambium and wood biomass. Global warming since the mid-19th century led to the biomass of trees in temperate regions increased rapidly, for example, on Pinus aristata and Populus spp., Even in Populus spp. increased up to 33%. On the other hand, since the 1950's the average of wood biomass of tropical plants has begun to decline. Increased levels of CO2 affect the density of stomata. Current plants have fewer stomata than herbarium specimens of the same species that were collected about 200 years ago, because the number of stomata openings is less, CO2 for photosynthesis needs are met (Hughes 2000; IPCC 2001; Gielen and Ceulemans 2001).

Genetic differences and behavioral changes. In some species of plants, the success of fruit and seed formation is greatly influenced by the temperature at the reproduction period. Two cultivars of Prunus avium L., where one is able in adapting to the cold temperature and the other to warm temperatures show that rising temperatures reduce pollen germination of both, but increases the pollen tube growth. The mikrogamet ability of both in reaching the base of the stylus is different. At a temperature of 20°C, both mikrogamet populations are relatively similar, but at a temperature of 30°C, the cold

SETYAWAN – Global warming effect on the plant evolution and diversity

cultivars mikrogamet populations decrease and at a temperature of 10°C, cultivars decrease slightly warm.

Different genotypes respond differently to temperature during the reproductive period, in which the plants need time to adapt to changes in temperature (IPCC 2001; Hedhly et al. 2004).

PLANT DIVERSITY AND CLASSIFICATION CHANGES DUE TO GLOBAL WARMING Taxa extinction due to global warming. The past time has proven that global warming is one of the main factors causing extinctions, and evolution should be done by taxa to survive. In future, climate change is also expected to be the main factor of taxa extinction. Modeling study that includes 1,103 species of animals and plants in an area of 20% of the earth's land surface shows that in 2050 approximately 15-37% of species will extinct if global warming continues at current pace. Global warming causes climate homogenization in large areas and causing loss of habitat with a special climate (niche), therefore, it increases the invasion of alien species and wipes out many native species (Thomas 2004).

The formation of new taxa of Angiosperms. Global warming causing the extinction of taxa, on the other hand, encourages the development of new taxa through evolutionary process. Global warming in the Mesozoic era, which began in the early Jurassic to the temperature of 510°C higher than at this time and ended by the asteroid falls at the Cretaceous-Tertiary boundary, has caused the extinction of most of the gymnosperms which dominate the earth in those days, and allowed the development of new taxa of Angiosperms and its users, mammals. Early ancestor of Angiosperms has been present since 142 mya, but most has just started attending in the early Cretaceous period, especially in the Aptian era (122-125 mya). Most of

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Angiosperms which persisted up to now comes from the upper Albian era (110-113 mya). Figure 6 shows the possibility of evolution of Angiosperms and their close relatives (Doyle and Donoghue 1988).

In the Cenomanian stage, Angiosperms diversity began to increase. In the Turonian and Senonian stage, Angiosperms is more abundant than ferns and gymnosperms. A number of modern Angiosperms familia from Cenomanian stage has the form of leaves and fruits very similar to modern taxa. In the Maastrichtian stage, a number of modern genera and families began to attend, such as Nypa (Arecaceae), Ctenolophon (Linaceae), Proteaceae, Myrtaceae, Ilex (Aquifoliaceae), Poaceae, Sapotaceae, Nothofagus (Fagaceae), or Sarcococca Pachysandra (Buxaceae), Ascurinu (Chloranthaceae), Anacolasia (Olacaceae), Alnus (Retulaceae), Guarea (Meliaceae), and Symplocos (Symplocaceae). Some of the modern family is expected to appear on Turonian stage (90-100 mya) and some orders are appeared on Cenomanian stage, some earlier. In the Paleocene period, there appeared Alyxia (Apocynaceae), Betula (Retulaceae), Barringtonia (Lecythidaceae), Brownlowia (Tiliaceae), Bombax (Bombacaceae), Crudia (Caesalpinaceae), and Liquidambar (Hamamelidaceae) and several other genera (Raven and Axelrod 1974).

Classification and phylogeny of ancient and modern Angiosperms. Ancient Angiosperms (primitive) that is still survive until now generally has been present at the beginning of the Cretaceous (110-90 mya), where Africa and South America is only 800 km and is still connected by volcanic islands, so the movement of species is still allowed, while Modern Angiosperms is generally present before the unification of Africa and Eurasia that occurred in the early Paleocene (about 63 mya). Ancient Angiosperms has evolved to adapt to various environmental conditions, until now the diversity, abundance, and has been spread over other plant groups.

Figure 6. Evolution scenario Angiosperms and relatives; (a) is a close relative anthophyta Angiosperms other, (b) Angiosperms derived from Caytonia; (c) Angiosperms derived from Benettitales. T = Triassic, J = Jurassic, K = Cretaceous; Ct = Caytonia, Ag = angiospernae, Bn = Benettitales, Gn = Gnetales (Doyle and Donoghue 1988).

Â

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Table 2. Reproductive character of Angiosperm (Friedman and Williams 2004) Early Angiospermae (20th century) Pollen grains 2-cell Composition of the female gametophyte when ripe 7-cell, 8-nuclei Position of the cell cycle at fertilization of gametes Unknown Ploidy endosperm 3n The pattern of endosperm development Free nuclei or cellular

Early Angiospermae (21st century) 2-cell 4-cell, 4-nuclei G1 2n Cellular

The phylogenetic character of basal Angiosperms (ancient) and non-basal (modern) strongly influences the composition of its classification. The reproductive character of ancient Angiosperms can be traced by comparing among the Angiosperms members that present at this time, and compiling them in a proper phylogenetic among basal Angiosperms, so their origins history can be traced to about 130 mya, the year which Angiosperms began to emerge to replace gymnospemae and ferns domination (Table 2) (Friedman and Williams 2004).

common ancestor (Figure 7). This discovery answered the question of Darwin in 1903 on the mystery that the origin of flowering plants is confusing, so it is called "abominable mystery." The exact determination of phylogenetic line as the true picture of evolution will guide the preparation of appropriate systematic (Friedman and Williams 2004). The result of this evolution shows that the plant always tries to adapt to changing environmental conditions, including major changes due to global warming or glaciation, to create new types that are more dominant and much different than the parent (Walther et al. 2002; Thomas 2005).

Character

Modern Angiospermae (kebanyakan) 2-cell 7-cell, 8-nuclei Unknown 3n Free nuclei

Arabidopsis thaliana (eudicot) 3-cell 3-cell 7-cell, 8-nuclei 7-cell, 8-nuclei G1 G2 3n 3n Free nuclei Free nuclei Zea mays (monocot)

CONCLUSION

Figure 7. Angiosperms phylogeny based on recent molecular analysis. Amborella, Nymphaeales, and Karis Austrobaileyales have a more ancient lineage than monokot origin, eumagnoliid, and eudikot (Friedman and Williams 2004).

Throughout the 20th century, taxonomic experts agree that the character of ancient Angiosperms reproduction is represented by magnoliid, where all other Angiosperms is considered to be originated from it. But in 1999, a number of phylogenetic analyses showed that the Amborella, Nymphaeales and Austrobaileyales develop a more basal part of another Angiosperms; and appeared before the ancestors, i.e. monokot, eudikot, and eumagnoliid, so it had an older lineage, and may still represent the biological character of the most ancient Angiosperms. All other Angiosperms, but those three, shares the properties of a

Lessons from the past show that global warming and glaciation is a repeated natural cycle. The trigger factor is not always the same, but global warming is always accompanied by elevated levels of CO2 in the atmosphere.

The existence and the destruction of various plants, animals and other organisms continue to occur from time to time.

Every era has its own life form, as a mirror showing that global environmental conditions are always changed.

Biodiversity is not always the same between one period of global warming with the other period of global warming, or from one glaciation to the next glaciation, although new breeds always show traces of evolution of his ancestors.

Therefore, the impacts of climate change which is caused by global warming needs to be anthropogenically wary of.

Even if global warming is still going on and is followed by mass extinction, it is believed that various forms of life will still be present on the earth, but such life forms is almost certainly different from the one at this moment. Living organisms which can survive are likely to evolve into new taxa that are different from existing taxa. Humans who were present at that time probably is not a man who is present at this time, because Homo sapiens may have been extinct for not being able to adapt or otherwise has evolved into a new man who may no longer shows characteristics of human wisdom.

ACKNOWLEDGEMENTS The author thanks to Prof. Dr. Mien A. Rifai from LIPI Jakarta, which has provided inspiration for the preparation

SETYAWAN – Global warming effect on the plant evolution and diversity

of this paper. Thanks are also extended to Dr. Himmah Rustiami from Research Center for Biology, LIPI Cibinong Bogor and Dr. Charly Danny Heatubun from the State University of Papua, Manokwari who have read and give correction on the early manuscript.

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Names of sub-species until phylum should be written in italic, except for italic sentence. Scientific name (genera, species, author), and cultivar or strain should be mentioned completely at the first time mentioning it, especially for taxonomic manuscripts. Name of genera can be shortened after first mentioning, except generating confusion. Name of author can be eliminated after first mentioning. For example, Rhizopus oryzae L. UICC 524, hereinafter can be written as R. oryzae UICC 524. Using trivial name should be avoided, otherwise generating confusion. Mentioning of scientific name completely can be repeated at Materials and Methods. Biochemical and chemical nomenclature should follow the order of IUPAC-IUB, while its translation to Indonesian-English refers to Glossarium Istilah AsingIndonesia (2006). For DNA sequence, it is better used Courier New font.

Symbols of standard chemical and abbreviation of chemistry name can be applied for common and clear used, for example, completely written butilic hydroxytoluene to be BHT hereinafter. Metric measurement use IS denomination, usage other system should follow the value of equivalent with the denomination of IS first mentioning. Abbreviation set of, like g, mg, mL, etc. do not follow by dot. Minus index (m-2, L-1, h-1) suggested to be used, except in things like “per-plant” or “per-plot”. Equation of mathematics can be written separately. Number one to ten are expressed with words, except if it relates to measurement, while values above them written in number, except in early sentence. Fraction should be expressed in decimal.

In text, it should be used “%” rather than “gratuity”.

Title of article should be written in compact, clear, and informative sentence preferably not more than 20 words (generally 135 characters including spaces). Name of author(s) should be completely written. Running title is about five words, refelcting the idea of the manuscript. Name and institution address should be also completely written with street name and number (location), zip code, telephone number, facsimile number, and e-mail address. Manuscript written by a group, author for correspondence along with address is required. First page of the manuscript is used for writing above information.

Abstract should not be more than 250 words, written in English, on page two of the manuscript. Keywords is about five words, covering scientific and local name (if any), research theme, and special methods which used. Introduction is about 400-600 words, covering background and aims of the research. Materials and Methods should emphasize on the procedures and data analysis. Results and Discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into sub titles. Thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences. Conclusion should preferably be given at the end of the discussion. Acknowledgments list and funding sources are expressed in a brief. Dedications are rarely allowed.

Figures and Tables of maximum of three pages should be clearly

presented. Title of a picture is written down below the picture, while title of a table is written in the above the table. Colored picture and photo can be accepted if information in manuscript can lose without those images. Photos and pictures are preferably presented in a digital file. JPEG format should be sent in the final (accepted) article. Author could consign any picture or photo for front cover, although it does not print in the manuscript. There is no appendix, all data or data analysis are incorporated into Results and Discussions. For broad data, it can be displayed in website as Supplement.

Citation in manuscript is written in “name and year” system; and is arranged from oldest to newest and from A to Z. The sentence sourced from many authors, should be structured based on the year of recently. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the family (last) name of the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided, and suggested to refer an original reference.

References. APA style in double space is used in the journal reference.

Only published or in-press papers and books may be cited in the reference list. Unpublished abstracts of papers presented at meetings or references to "data not shown" are not permitted. References should be cited in alphabetic order. All authors should be named in the citation (unless there are more than five). If there are more than five, list the first author's name followed by et al.

Include the full title for each cited article. Authors must translate foreign language titles into English, with a notation of the original language (except for Spanish, France, and Germany). For Indonesian manuscript, translation of Indonesian title to English is not necessary. For correct abbreviations of journal titles, refer to Chemical Abstracts Service Source Index (CASSI).

Provide inclusive volume, number, and page ranges for journal articles, but not for book or book chapters.

Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam.

Chapter in book: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. WileyBlackwell, New York.

Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007.

Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island.

Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesia] Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [Dissertation].

Brawijaya University, Malang. [Indonesia] Information from internet: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem.

Mol Syst Biol 4: 187. www.molecularsystemsbiology.com Progress of manuscript. Notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. Manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. Author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. Offprint or reprint is only available with special request.

NOTE: Author(s) agree to transfer copy right of published paper to NUSANTARA BIOSCIENCE, the ISEA Journal of Biological Science.

Authors shall no longer be allowed to publish manuscript completely without publisher permission. Authors or others allowed multiplying article in this journal as long as not for commercial purposes. For the new invention, authors suggested to manage its patent before publishing in this journal.

NOTIFICATION: All communications are strongly recommended to be undertaken through email.

| Nus Biosci | vol. 1 | no. 2 | pp. 53‐103 | July 2009 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

Blood cholesterol levels of hypercholesterolemic rat (Rattus norvegicus) after VCO treatment MARTI HARINI, OKID PARAMA ASTIRIN

53‐58

Effect of manure and NPK to increase soil bacterial population of Azotobacter and Azospirillus in chili (Capsicum annum) cultivation MUJIYATI, SUPRIYADI

59‐64

Nitrogen content, nitrate reductase activity, and biomass of kimpul (Xanthosoma sagittifolium) on shade and nitrogen fertilizer variation ISNAINI CHOIRUL LATIFA, ENDANG ANGGARWULAN

65‐71

Characterization of white grub (Melolonthidae; Coleoptera) in salak plantation based on morphology and protein banding pattern KRISNANDARI TITIK MARYATI, SUGIYARTO

72‐77

Variation of morphology, karyotype and protein band pattern of adenium (Adenium obesum) varieties DWI HASTUTI, SURANTO, PRABANG SETYONO

78‐83

Variability analysis of Sukun durian plant (Durio zibethinus) based on RAPD marker ISMI PUJI RUWAIDA, SUPRIYADI, PARJANTO

84‐91

Diversity of Sonneratia alba in coastal area of Central Java based on isozymic patterns of esterase and peroxidase AHMAD DWI SETYAWAN

92‐103

Published three times in one year PRINTED IN INDONESIA

ISSN 2087‐3948 (print)

ISSN 2087‐3956 (electronic)