RINGKASAN Pada kedelai cekaman kekenngan pada fase genetatif dilaporkan menyebabkan penurunan produksi biji hingga 50 %. Penggunaan varietas kedelai tahan terhadap kekeringan merupakan salah satu alternatif untuk mengatasi masalah tersebut. Perakitan tanainan kedelai tahan kering telah dilaporkan oleh beberapa peneliti. Berbagai metode telah digunakan antara lain dengan perlakukan kekeringan di lapang dan dilanjutkan dengan seieksi ataupun seleksi in vitro dengan memberikan simulasi kekeringan menggunakan polietilena glikol (PEG) seperti yang dilakukan oleh Widoretno et nl. (2002 a'b). Seleksi tersebut inenghasilkan galur-galur talian kering. Berdasarkan uji fisiologis dalam skala laboratorium terhadap galur-galur tersebut, telah terbukti bahwa ketahanan terhadap kekeringan yang ditunjukkan oleh galur-galur tersebut berkaitan dengan peningkatan kandungan prolin tanaman. Simulasi kekeringan dengan PEG di glass house terhadap galur-galur yang secant fisiologis tahan kering, membuktikan bahwa galur-galur tersebut memang benar tahan kering dengan tingkat ketalianan medium. Secara genetis, telah diketahni bahwa ketahanan terhadap cekaman kekeringan dikontrol oleh banyak gen (Pastori & Foyer, 2002). Diantara berbagai gen tersebut gen yang telah diidentifikasi antara lain gen DREBl yang berperanan dalain pembentukan drought responsive element binding protein, GmDREB2 yang berperanan dalam pembentukan drought responsive element binding transcription factor (Chen et al, 2004; 2005), gen Mn-sod yang berperanan dalam pembentukan manganese-superoxide dismutase (Porcel et al., 2002), gen PSCS yang mengkode pembentukan delta 1 -pyrroline-5-carboxylate Synthetase yang berperanan dalam akumulasi prolin (Porcel et al., 2004), gen PIP I dan PIP2 yang bertanggungjawab terhadap pembentukan aquaporin, suatu protein yang terkait dengan ketersediaan air tanaman (Porcel et al., 2006) dan gen leaH yang meugkode pembentukan dehydrin selama cekaman kekeringan (Porcel et al., 2005), gen HVAi terakumulasi pada saat desikasi biji (Hong et al. 1988). Xu et al. (19%), gen 77*57 mengkode trehalose-6-phosphate synthetase dan terlibat dalam biosintesis trehalose (Hohnstrom et al., 19% dan Romero et al., 1997), gen SacB (Holmstrom et al. 1994). Gen SOD (superoxide dismutase) (Sengupta et. al., 1993), gen betA mengkode enzim choline dehydrogenase dan betB yang mengkode betaine aldehyde dehydrogenase yang berperanan dalain biosintesis glycine betaine. Akumulasi glycine betaine menyebabkan ketahanari terhadap kekeringan (Holmstrom et al. 1994). Dengan demikian perlu dibuktikan bahwa ketahanan terhadap kekeringan dari galur-galur yang telah dihasilkan dari penelitian Widoretno et al. 2002 ^b tersebut memang disebabkan karena faktor genetis. Mengingat bahwa banyak gen yang berperanan dalam mengontrol ketahanan terhadap kekeringan, maka dalam penelitian ini dibatasi pada gen yang ekpresinya telah dikaji secara fisiologis oleh Widoretno et al. 2002 ** yaitu gen pengliasil prolin PSCS, gen DREB] yang berperanan dalain pembentukan drought responsive element binding protein. Pendekatan awal (tahun I) dilakukan dengan tekiiik random amplified polymorphic DNA (RAPD) untuk memastikan adanya perbedaan sekuen antara galur talian kering dan tidak talian kering serta mendapatkan marker RAPD yang berkaitan dengan sifat tahan kering. Selanjutnya dilakukan identifikasi gen talian kering tersebut dengan tekuik PCR-sekuensing menggunakan primer gen tahan kering tanaman lain (tahun II). Flaal sekuensing digunakaii sebagai dasar pembentukan gene-base PCR marker (probe) gen tahan kering (tahun III). BaJian tanaman yang digunakan adalah kedelai varieias tahan kering Dicrig, varietas tidak tahan kering Burangrang, galur tidak tahan kering liasil seleksi Balitkabi MSC8606 dan MLG 2999, galur medium tahan keiing B3731, dan hasil perlakuan PEG 3S4 dan 2S3 galur tidak tahan kering hasil perlakuan PEG 8B6. Tahapan yang dilakukan adalah isolasi DNA genom dari daun muda kedelai dengan nietode CTAB (Doyle &Doyle, 1987) dilanjutkan dengan Polymerase Chain Reaction (PCR) dengan menggunakan pasangan primer DREB 1 F= 5'-ATGTTTCCCCTGAGCTCTCA-3' dan DREB 1 R= 5'TCCAACACCTGCTCTTCTlC-3',serta P5CS F=5'-ATGGCAA GGCTTGTGCTGCT-3' dan P5CS R= 5'-TTCCAATCCGTTGTCCTTGG-3\ Hasit PCR selanjutnya disekuensing dan dianalisis homologinya
dengan gen yang sama yang telali di laporkan dalam gene Bank dan dianalisis alignment antar varietas dan galur. Hasil penelitian menunjukkan bahwa gen tahan kering DREBI dan P5CS dapat dideteksi keberadaannya dengan teknik PCR pada ke deiapan varietas,galur dan somaklon yang diteliti. PCR untuk gen DREBI memperlihatkan pita liasil ampliflkasi sekitar 500 bp, sedangkan untuk gen P5CS memperlihatkan pita amplifikasi sekitar 1400 bp. Gen DREBI tersebut homolog dengan gen DREBI pada berbagai knltivar kedelai yang telah di data dalam gene Bank. Perbedaan si fat ketahanan dari varietas Dieng yang bersifat toleran dengan 7 varian tanaman kedelai yang bersifat peka dan medium kekeringan disebabkan oleh adanya beberapa point mutation yang menyebabkan frameshifi mutation. Adanya poJyraorfisme pada satu nukleotida mi ( Single Nucleotide Polymorphism, SNP) dapat digunakan sebagai ,dasar pembentukan probe.
SUMMARY Drought stress at generative phase has been reported responsible to the decrease of seed production up to 50 % in soybean. The utilization of drought tolerance variety is one alternative to solve the problem. Breeding on drought tolerance variety has been reported, Many method has been employed including drought treatment in field continued by selection or in vitro simulation of drought using certain salt such as polyetilene glycol (PEG) as has been conducted by Widoretno et al. (2002 a>b). Research by Widoretno et al. (2002 fl'b) has produced drought resistent line. Physiological testing to those lines in laboratoriurn showed that the tolerance was associated with the increase of prolin content of the plant. PEG simulation in the glass house, has proved the tolerance habit of the plant with medium level of tolerance. Genetic research has reported that drought tolerance was converted by many genes ( Pastori & Foyer, 2002). Some of them has been identified including DREBl which has a role on the formation of drought responsive element binding protein, GmDREB2 which confer the formation of drought responsive element binding transcription factor (Chen et al, 2004; 2005), Mn-sod ■•■ which control the formation of manganese-superoxide dismutase (Porcel et al., 2002). P5CS which code the formation of delta l-pyrroline-5-carboxylate Synthetase which has a role on prolin acumulation (Porcel el al., 2004), PI PI dan PIP2 which responsible on the formation of aquaporin, a protein related to water availability on plant (Porcel et al., 2006) Iea8 which has a role on the formation of debydrin during drought stress (Porcel et al, 2005), HVA1 which accumulated when seed was desiccated (Hong et al. 1988). Xu el al. (1996), TPS1 which code trehalose-6-phosphate synthetase and involved in the biosynthesis of trehalose (Holmstrom et al., 1996 dan Romero et al., 1997), and SacB (Holmstrom et al. 1994). Genes SOD (superoxide dismutase) (Sengupta et. al., 1993), he/A code the formation of choline dehydrogenase and hetB which code betaine aldehyde dehydrogenase which has a role on the biosintesis of glycine betaine. Acumulation of glycine betaine convert the drought tolerance (Holmstrom et al. 1994). So, it is needed to prove that drought tolerance showed by lines produced by Widoretno et al. 2002 a'b was genetically controlled. Since there are many genes involved in the conversion of drought tolerance, in this experiment was restricted to two genes i.e gene that its expression has been studied physiologically by Widoretno et al. 2002 a'b that is prolin producing gene P5CS, and other gene, DREBl which has a role in the formation of drought responsive element binding protein. In the first year of this research program, sequence differences between drought tolerance and drought susceptible variety/lines was identified using random amplified polymorphic DNA (RAPD). RAPD marker will be developed from the polymorphic bands resulted. In the second year, identification of drought tolerance gene P5CS and DREBl will be done using PCR-sequencing technique using primers derived from drought tolerance gene which has been identified by other researcher. In the third year, sequencing result will be used as the base of development of gene-base f'CR marker (probe) for drought tolerance. Plant material used was drought tolerance variety Dieng, drought susceptible variety Burangrang, medium tolerance line 2S3, drought susceptible line, 8B6, MSC8606 and MLG 2999. DNA was isolated from the young leaf of those plants using CTAB method (Doyle &Doyle, 1987) continued with Polymerase Chain Reaction (PCR) using primer pair DREB 1 F= 5'-ATGTTTCCCCTGAGCTCTCA-3' and DREB 1 R= 5'TCCAACACCTGCTCTTCTTC-3',and P5CS F-5'-ATGGCAA GGCTTGTGCTGCT-3' and P5CS R= 5'TTCCAATCCGTTGTCCTTGG-3'. PCR. result was continued with sequencing and analyzed their homolog with the same that reported in gene Bank and analyzed alignment among variety and genotype was done. The result showed that drought tolerance gene DREBl m&P5CS can detected by PCR technique in eight variety, genotype and somaclone of research materials. PCR for DREBl gene showed the ribbon from amplification about 500 bp, P5CS gene showed the ribbon from amplification about 1400 bp. DREBl gene homolog with DREBl gene in varoius soybean cultivar that collected a) gene Bank. The difference of tolerance of variety Dieng which have tolerance with 7 variant of soybean which have susceptible and medium drought caused by several point mutation which cause frameshift mutation. Polymorphism in this one nucleotide ( Single Nucleotide Polymorphism, SNP) can used as basic probe tbrming.
DAFTAR PUSTAKA Ariffin. 2002. Cekaman Air dan Kehidupan Tanainan. Fakultas Pertanian. Universitas Brawijaya Chen,M., Wang,Q.Y., Cheng,X.G., Xu,Z.S., Li,L.C, Ye,X.G., Xia,L.Q. dan Ma,Y.Z. 2005. GmDREB2, a soybean DRE-binding transcription factor, conferred drought and highsalt tolerance in transgenic plants, www.ncbi.nih.gov, Diaksesl5Maret2007 Chen,Y., Chen,P. dan de los Reyes,B-G. 2004. Analysis of the expression of DREB1 gene orthologs in cultivated and wild species of soybean, www.ncbi.nih.pov. Gardner, F.P., R.B Pearce and R.L Mitchel. 1991. FisiologiTanaman Budidaya. Ed. bahasa Indonesia. Universitas Indonesia Press. Hanum, Chairani , W. Q. Mugnisjah , S. Yaliya , D. Sopandy , K. Idris , A. Sahar. 2007. Pertumbuhan Akar Kedelai pada Cekaman Aluminium, kekeringan dan Cekaman Ganda Aluminium dan Kekeringan. Agritwp, 26(1): 13 — 18 Holmstrom, K. O., Mantyia, E., Welin, B., Mandal, A., Palva, E. T., Tunnela, O. E. and Londesborough, J., Nature, 1996,379, 683-684. Maestri, M., F.M. Da Matta, AJ. Regazzi & R.S Barros. 1995. Accumulation of proline and quartenary ammonium compounds in mature leaves of water stressed coffee plants {Coffea arabica and C. canephora). J. Hart. Sci., 70(2), 229-233 Mitra, J. 2001. Genetics and Genetics Improvment of Drought Resistance in Crop Plants. Current Science. 80(6):758-763. Pastori, G.M. & C.H. Foyer. 2002. Common Components, Networks, and Pathways of Cross-Tolerance to stress. The Central Role of' Redox" and Abscisic Acid-Mediated. Plant Physiol. 129:460468. Porcel,R., Aroca,R, Azcon,R. and Ruiz-Lozano,J.M. 2006. PIP aqiiaporin gene expression in arbuscular mycorrhizal Glycine max and Lactuca sativa plants in relation to drought stress tolerance. Plant Mol. Biol. 60 (3), 389-404 Porcel.R., Azcon,R and Ruiz-LozanoJ.M. 2005. Evaluation of the role of genes encoding for dehydrin proteins (LEA D-11) during drought stress in arbuscular mycorrhizal Glycine max and Lactuca sativa plants. J. Exp. Bot. 56(417), 1933-1942 Porcel,R., Azcon,R. dan Ruiz-Lozano,J.M. 2004. Evaluation of the role of genes encoding for delta lpyrroline-5-carboxylate synthetase (P5CS) during drought stress in arbuscular myconiiizal Glycins max and Lactuca sativa plants. www.iicbi.THh.gov. Diakses 15 Maret 2007 Porcel,R., Jose Miguel,B- dan Ruiz-Lozano,J. 2002. Evaluation of antioxidant activities in mycorrhizal soybean plants under drought stress and its relationship to the process of nodule senescence, www.ncbi.nih.gov. Diakses 15 Maret 2007 Purwanto, E. 2003. Photosynthesis activity of soybean {Glycine max L.) under drought stress. Agrosains 5 (1 ):13-18 Romero, C, Belles, J. M., Vaya, J. L., Serrrano. R. and CuJianez-Macia, F. A., 1997, : 293-297. Sengupta, A., Heinen, J. L, Holaday, A. S., Barke, J. J. and Allen, R. D., 1993, Proc. Xu, D.P., Duan, X.L., Wang, B.Y., Hong, B.M.. Ho, T.H.D. and Wu, R. 19% Expression of a laic embryogcnesis abundant protein gene, IIVAI, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol. 110,249-257.
