TRANSFER GEN -1,3-GLUCANASE DARI JAMUR Trichoderma asperillum PADA KALUS ABAKA UNTUK KETAHANAN TERHADAP PENYAKIT LAYU FUSARIUM

ABSTRAK Kendala utama dalam budidaya tanaman abaka (Musa textilis Nee.) adalah penyakit layu Fusarium yang disebabkan oleh Fusarium oxysporum f.sp cubense (Foc). Upaya perbaikan sifat ketahanan tanaman abaka melalui persilangan sulit dilakukan karena keragaman genetiknya sempit akibat  pola  perbanyakan  secara  vegetatif  yang  terus-menerus. Transformasi gen ketahanan β-1,3-Glucanase merupakan salah satu alternatif untuk memperbaiki sifat ketahanan tanaman dengan bantuan vektor Agrobacterium tumefaciens. Gen   -1,3-Glucanase diisolasi dari jamur endofit Trichoderma asperillum yang diketahui antagonis terhadap Fusarium oxisporum. Penelitian bertujuan untuk mengintroduksi gen β- 1,3 Glucanase pada tanaman abaka, sebagai tahap awal untuk memperoleh tanaman abaka tahan terhadap penyakit layu Fusarium. Penelitian dilaksanakan di Laboratorium Bioteknologi Fakultas Pertanian, Univer- sitas Brawijaya dan Laboratorium Kultur Jaringan Balai Penelitian Tanaman Tembakau dan Serat, mulai Juni 2007 sampai dengan Mei 2009. Penelitian terdiri atas tiga tahap sebagai berikut: transfer gen   -1,3- Glucanase pada kalus abaka embriogenik, regenerasi tunas dan planlet abaka transforman, dan konfirmasi planlet abaka transforman yang mengandung gen Gus dan   -1,3-Glucanase. Transfer gen dilakukan melalui vektor A. tumefaciens strain LBA4404 yang mengandung plasmid pB2GW7 berisi gen-gen   -1,3-Glucanase, Gus (  -glucuronidase) sebagai gen pelapor dan Bar (Basta resistance) sebagai gen penyeleksi. Kalus abaka klon UB-13 embriogenik berukuran 3 x 3 x 3 mm 3 direndam dalam suspensi A. tumefaciens, kemudian ditanam pada media kokultivasi selama 2 hari. Setelah kokultivasi, kalus dipindahkan ke media MS cair+Timentin 100 ppm selama 2 minggu. Selanjutnya kalus dipindahkan ke media induksi kalus (MK) yaitu MS + BAP 5 mg/l + Thidiazuron 0,4 mg/l + vitamin C 100 mg/l + Basta 50 ppm + Timentin 100 ppm. Regenerasi tunas dilakukan dengan memindahkan kalus transforman ke media induksi tunas (MT): MS+BAP 0,5 mg/l + vitamin C 100 mg/l dengan penambahan dan tanpa Timentin 100 ppm. Tunas transforman dengan tinggi 2-3 cm dipindahkan ke dalam media induksi akar (MA) : MS + arang aktif 2 g/l dengan penambahan dan tanpa Timentin 50 ppm. Keberadaan gen Gus dideteksi dengan reaksi histokimia, dan konfirmasi keberadaan gen   -1,3- Glucanase dilakukan dengan Polymerase Chain Reaction (PCR). Dari penelitian berhasil diperoleh 2% kalus transforman yang lolos seleksi Basta. Hasil konfirmasi keberadaan gen Gus pada planlet transforman menunjukkan 9 dari 20 (45%) planlet yang diuji, positif mengandung gen Gus. Konfirmasi keberadaan gen   -1,3-Glucanase dengan PCR menunjukkan hanya 2 dari 20 planlet transforman, positif mengandung   - 1,3-Glucanase. Pengujian ketahanan dari plantlet transgenik tersebut perlu dilakukan terhadap Fusarium oxisporum f.sp cubense (Foc). Kata kunci: Musa textilis Nee., transformasi gen,   -1,3-Glucanase, Agrobacterium tumefaciens, penyakit, jamur patogen, Fusarium ABSTRACT The main constraint of abaca (Musa textilis Nee.) cultivation is infection of wilt disease caused by Fusarium oxysporum f.sp cubense (Foc). The effort to improve abaca resistance through hybridization is still difficult due to narrow genetic variability resulted from continuous vegetative multiplication. Transformation of   -1,3-Glucanase resistance gene is an alternative way to improve character of genetic resistance with help of Agrobacterium oxisporum. The research aimed at introducing   - 1,3-Glucanase gene to abaca plants prior to obtaining the plants resistance against Fusarium wilt diseases. The research was conducted in Biotechnology Laboratory, Faculty of Agriculture Brawijaya University and Tissue Culture Laboratory of Indonesian Tobacco and Fibre Crops Research Institute, from June 2007 to May 2009. This experiment consisted of three steps, namely:   -1,3-Glucanase gene transfer onto abaca embriogenic calli, regeneration of transgene abaca shoots and plantlets, and confirmation of transgene abaca plantlets containing Gus and   -1,3- Glucanase genes. Gene transfer was performed using A. tumefaciens vector strain LBA4404 with pB2GW7 containing genes of   -1,3- Glucanase and Gus (  -glucuronidase) as reporter, and Bar (Basta resistance) as selector marker. Embriogenic calli of abaca clone UB-13 were soaked in A. tumefaciens suspension and then cultured in co- cultivation medium for two days. After co-cultivation, calli were transferred to liquid of MS medium + 100 ppm Timentine for two weeks. Furthermore, the calli were sub-cultured into callus induction medium : MS + 5 mg BAP/l + 0.4 mg Thidiazuron/l + 100 mg vitamin C/l + 50 ppm Basta + 100 ppm Timentine. Shoots regeneration was conducted by transferring transgene calli to shoot induction medium : MS + 0.5 mg/l BAP + 100 mg vitamin C/l with and without addition of 100 ppm Timentine. Transgene shoots with 2-3 cm height were sub-cultured to root induction medium : MS + 2 g active charcoal/l with and without addition of 50 ppm Timentine. Detection of Gus gene was conducted using histochemical reaction, while confirmation of   -1,3-Glucanase gene was performed by PCR. This project resulted in 2% transgene calli passing Basta selection. Nine out of 20 plantlets (45%) confirmed the existance of Gus gene. PCR results showed that only 2 out of 20 transformed plantlets positively contained   -1,3-Glucanase gene. The plantlets resistance against Fusarium oxisporum f.sp cubense (Foc) needs to be evaluated. Key words: Musa textilis Nee, gene transformation,   -1,3-Glucanase, Agrobacterium tumefaciens, plant disease, fungal disease, Fusarium