TRANSFER GEN   -1,3-GLUCANASE DARI JAMUR Trichoderma asperillum PADA KALUS ABAKA UNTUK KETAHANAN TERHADAP PENYAKIT LAYU FUSARIUM
Authors: PURWATI RULLY DYAH , SULISTYOWATI LILIEK 
Published: June 2020

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