BOGOR
2007
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Dengan ini saya menyatakan bahwa disertasi saya yang berjudul
”INDUKSI VARIASI SOMAKLONAL DAN SELEKSI IN VITRO MENGGUNAKAN
PEG UNTUK IDENTIFIKASI VARIAN KACANG TANAH YANG TOLERAN CEKAMAN KEKERINGAN” adalah hasil penelitian saya yang merupakan bagian dari serangkaian penelitian HIBAH TIM PASCASARJANA angkatan I, tahun ke- 1, 2 dan 3 (2003-2005) yang berjudul ”Rekayasa Genetika dan Seleksi in vitro
untuk Mendapatkan Plasma Nutfah Kacang Tanah dengan Novel Characters –
Toleran terhadap Stres Kekeringan dan Resisten Penyakit Busuk Batang Sclerotium” yang diketuai oleh Prof.Dr.Ir. Sudarsono, M.Sc. dan didanai oleh Departemen Pendidikan Nasional. Disertasi ini belum pernah diajukan untuk memperoleh gelar dalam program sejenis di perguruan tinggi manapun.
Sumber informasi yang berasal atau dikutip dari karya yang diterbitkan maupun yang tidak diterbitkan dari penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di bagian akhir disertasi ini.
Bogor, Juli 2007
Enni Suwarsi Rahayu NRM A.361020041
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ENNI SUWARSI RAHAYU. Induction of Somaclonal Variation and In Vitro
Selection Using PEG for Identification of Drought Tolerant Peanut Variants.
Under direction of SUDARSONO, HAJRIAL ASWIDINNOOR, SATRIYAS ILYAS and EDI GUHARDJA.
Drought stress tolerance of peanut cultivar having certain mechanism without decreasing the yield needed to be developed. Somaclonal variation has been successfully used to obtain variant lines with improved drought stress tolerance. In this case, induction of somaclonal variation is followed by in vitro selection on selective medium containing polyethylene-glycol (PEG). The objective of this research were 1) to develop suitable in vitro selection method to obtain peanut somatic embryo that can tolerate stress due to addition of PEG in the selective medium, 2) to determine somaclonal variant indication of peanut, 3) to obtain plant population having somaclonal variation regenerated from in vitro selected somatic embryo, 4) to obtain somaclonal variant plants that are drought stress tolerance, and 5) to identify physiological mechanism involved in peanut drought stress tolerance.
In order to develop suitable in vitro selection method, several experiments were conducted to evaluate the effectiveness of polyethylene glycol (PEG)-6000
as in vitro selective agent; to determine the effective concentration of PEG to
inhibit growth and development of seedling, epycotyl and somatic embryo; to evaluate tolerance of the peanut cultivars against PEG stress; and to determine changes in total proline content due to PEG stress. Results of the experiment indicated that addition of PEG 6000 into MS-0 medium inhibited growth and development of peanut seedling, epycotyl, and somatic embryo, but increased the tissue damage score and total proline content of epicotyl. Addition of PEG 6000 might be used to simulate drought stress under in vitro condition. PEG at 15% concentration was effective for inhibiting growth and development of peanut tissue. Based on these results, an in vitro selection method was developed to screen peanut somatic embryo that was drought stress tolerant, by maintaining somatic embryo for three months with three times sub-culturing in selective media MS with addition of 16 µM picloram and 15% PEG 6000.
A number of PEG induced stress insensitive somatic embryos were identified after culturing 500 clumps of embryogenic tissue of peanut cv. Kelinci with three consecutive passages on medium containing 15% PEG. Germination of selected somatic embryos and regeneration of plantlets resulted in 24 peanut R0 lines, nine lines of them produced normal R0:1 seed. In addition, a number of somatic embryos were identified after culturing clumps of embryogenic tissue with three consecutive passages on medium without PEG. Germination of cultured somatic embryos and regeneration plantlets resulted in 38 peanut R0 lines, 20 lines of them produced normal R0:1 seeds. The R1 somaclonal population of both was obtained by planting R0:1 seeds in glass-house, and then R2 somaclonal population was obtained by planting R1:2 seeds produced by R1 somaclones. These R0, R1 and R2 population were evaluated for both qualitative and quantitative characters.
The results showed that phenotypic variation on both qualitative and quantitative characters were observed among R0, R1 and R2 generation of somaclonal lines. Variant phenotype on qualitative characters observed included,
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from in vitro selected somatic embryo. Variant phenotype of quantitative
characters included plants with significantly higher plant dry weight, plant height, root dry weight and pod weight. There were four lines with significantly higher root dry weight, and three lines with significantly higher pod weight.
The R1 and R2 populations were also evaluated to identify the drought stress tolerant lines. Drought stress was induced by pouring 15% PEG solution and by reducing watering. To induce stress by pouring 15% PEG, variant peanut seedlings were grown individually in plastic pot (600 ml) containing a mixture of burst rice-hull and manure (1:1, v/v). The seedlings were poured with PEG solution (15%) every two days since four leaves stage seven weeks after planting. Inducing drought stress by reducing watering was conducted by growing plants in polybag (50 cm) containing a mixture of top soil, sand and manure (2:1:1, v/v). These plants were divided into two groups. One group was subjected to stress condition periodically during vegetative and generative periods (12 – 80 days after planting) by watering them only after their 75% leaves have wilted; the other group was grown optimally by watering every two days.
The results of the experiments indicated 1) stress induced by PEG 15% solution at vegetative period reduced shoot growth, but did not affect negatively on root growth, 2) effect of drought stress at vegetative and generative periods on root and shoot growth were different between one population to another, 3) plant regenerated from in vitro cultured and in vitro selected somatic embryo have higher tolerance level to stress induced by PEG than the standard plant, 4) nine lines of progeny of plants regenerated from in vitro cultured and in vitro selected embryo somatic had drought stress tolerance character, and two of them had higher pod number than standard plant, both in optimal and stress condition, 5) the reduction of stomata density and the increase of leaf total proline content play sufficient role, while root/shoot ratio and primary root length did not play a significant role in plant tolerance to drought stress.
In conclusions, the induction somaclonal variation followed or didn’t follow by in vitro selection using PEG 15% were effective to obtain somaclonal variant that tolerate to drought stress without intensive root growth mechanism. Evaluation of drought stress tolerance resulted in four lines (K0-2, K0-11.3, K0- 30.1 and K15-4) that tolerate to drought stress and had higher pod weight than the standard plant.
Keywords : somaclonal variation, in vitro selection, PEG 6000, drought tolerance, tolerance mechanism without intensive root growth, proline,
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ENNI SUWARSI RAHAYU. Induksi Variasi Somaklonal dan Seleksi In Vitro
Menggunakan PEG untuk Identifikasi Varian Kacang Tanah yang Toleran
Cekaman Kekeringan. Dibimbing oleh SUDARSONO, HAJRIAL
ASWIDINNOOR, SATRIYAS ILYAS dan EDI GUHARDJA.
Kultivar kacang tanah yang toleran terhadap cekaman kekeringan dengan mekanisme yang tidak menurunkan hasil panen masih perlu dikembangkan. Variasi somaklonal telah berhasil dimanfaatkan untuk menginduksi galur varian yang meningkat toleransinya terhadap kekeringan. Dalam penelitian ini induksi variasi somaklonal diikuti oleh seleksi in vitro dalam media selektif yang mengandung PEG. Penelitian bertujuan untuk 1) mengetahui metode seleksi in
vitro yang efektif dalam rangka memperoleh ES kacang tanah yang toleran
terhadap potensial air rendah, 2) mengetahui indikasi varian somaklonal kacang tanah, 3) memperoleh populasi tanaman varian somaklonal hasil seleksi in vitro, 4) memperoleh populasi tanaman varian somaklonal yang toleran terhadap cekaman kekeringan, dan 5) mengetahui mekanisme toleransi tanaman kacang tanah terhadap cekaman kekeringan secara fisiologis.
Untuk mengembangkan metode seleksi in vitro yang efektif dilakukan
percobaan yang bertujuan menguji efektivitas PEG 6000 sebagai bahan penyeleksi dalam media in-vitro dengan mengevaluasi respon kecambah, tunas dan embrio somatik kacang tanah terhadap kondisi cekaman oleh PEG, menentukan konsentrasi PEG yang efektif menghambat pertumbuhan dan perkembangan jaringan eksplan, menentukan konsentrasi PEG sub-letal, dan mengukur perubahan kandungan prolina total jaringan akibat cekaman PEG. Dari hasil percobaan disimpulkan bahwa penambahan larutan PEG dalam media in-vitro memberikan kondisi cekaman yang ditandai dengan terhambatnya perkembangan eksplan dan peningkatan kandungan prolina dalam jaringan seperti respon terhadap cekaman kekeringan. Konsentrasi PEG 15% efektif menghambat pertumbuhan dan perkembangan jaringan eksplan dan merupakan konsentrasi sub letal yang dapat menapis jaringan dengan sifat yang toleran dari jaringan lain dengan sifat peka terhadap cekaman akibat PEG.
Berdasarkan hal ini dikembangkan metode seleksi in vitro untuk menapis embrio somatik kacang tanah yang toleran terhadap cekaman kekeringan, yaitu dengan memelihara embrio somatik varian selama tiga bulan dalam media selektif MS dengan fitohormon pikloram 16µM ditambah PEG 15%.
Sejumlah embrio somatik yang insensitif terhadap cekaman akibat PEG
telah diperoleh dengan mengkulturkan 500 clump kalus embriogenik kacang
tanah cv. Kelinci dalam medium yang mengandung PEG 15% selama tiga kali berturut-turut. Perkecambahan embrio somatik hasil seleksi dilanjutkan regenerasi plantlet menghasilkan 24 galur tanaman R0, sembilan di antaranya menghasilkan benih R0-1 normal. Selain itu, sejumlah embrio somatik juga telah diperoleh dengan mengkulturkan kalus embriogenik dalam medium yang tidak mengandung PEG. Perkecambahan embrio somatik yang diperoleh tanpa seleksi ini menghasilkan 38 galur tanaman R0, 20 galur di antaranya dapat menghasilkan benih R0-1 normal. Populasi tanaman R1 diperoleh dengan menanam benih R0-1 di rumah kaca, dan selanjutnya populasi tanaman R2 diperoleh dengan menanam benih R1-2 di rumah kaca pula. Populasi R0, R1 dan R2 dievalusi untuk mengetahui variasi somaklonal pada karakter kualitatif dan kuantitatif.