TRANSFER GEN -1,3-GLUCANASE DARI JAMUR Trichoderma asperillum PADA KALUS ABAKA UNTUK KETAHANAN TERHADAP PENYAKIT LAYU FUSARIUM
No Thumbnail Available
Date
2012-03-06
Journal Title
Journal ISSN
Volume Title
Publisher
Pusat Penelitian dan Pengembangan Perkebunan
Abstract
Description
ABSTRAKKendala utama dalam budidaya tanaman abaka (Musa textilis Nee.)adalah penyakit layu Fusarium yang disebabkan oleh Fusarium oxysporumf.sp cubense (Foc). Upaya perbaikan sifat ketahanan tanaman abakamelalui persilangan sulit dilakukan karena keragaman genetiknya sempitakibat pola perbanyakan secara vegetatif yang terus-menerus.Transformasi gen ketahanan β-1,3-Glucanase merupakan salah satualternatif untuk memperbaiki sifat ketahanan tanaman dengan bantuanvektor Agrobacterium tumefaciens. Gen -1,3-Glucanase diisolasi darijamur endofit Trichoderma asperillum yang diketahui antagonis terhadapFusarium oxisporum. Penelitian bertujuan untuk mengintroduksi gen β-1,3 Glucanase pada tanaman abaka, sebagai tahap awal untuk memperolehtanaman abaka tahan terhadap penyakit layu Fusarium. Penelitiandilaksanakan di Laboratorium Bioteknologi Fakultas Pertanian, Univer-sitas Brawijaya dan Laboratorium Kultur Jaringan Balai PenelitianTanaman 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 planletabaka transforman, dan konfirmasi planlet abaka transforman yangmengandung gen Gus dan -1,3-Glucanase. Transfer gen dilakukanmelalui vektor A. tumefaciens strain LBA4404 yang mengandung plasmidpB2GW7 berisi gen-gen -1,3-Glucanase, Gus ( -glucuronidase) sebagaigen pelapor dan Bar (Basta resistance) sebagai gen penyeleksi. Kalusabaka klon UB-13 embriogenik berukuran 3 x 3 x 3 mm 3 direndam dalamsuspensi A. tumefaciens, kemudian ditanam pada media kokultivasi selama2 hari. Setelah kokultivasi, kalus dipindahkan ke media MS cair+Timentin100 ppm selama 2 minggu. Selanjutnya kalus dipindahkan ke mediainduksi kalus (MK) yaitu MS + BAP 5 mg/l + Thidiazuron 0,4 mg/l +vitamin C 100 mg/l + Basta 50 ppm + Timentin 100 ppm. Regenerasitunas dilakukan dengan memindahkan kalus transforman ke media induksitunas (MT): MS+BAP 0,5 mg/l + vitamin C 100 mg/l dengan penambahandan tanpa Timentin 100 ppm. Tunas transforman dengan tinggi 2-3 cmdipindahkan ke dalam media induksi akar (MA) : MS + arang aktif 2 g/ldengan penambahan dan tanpa Timentin 50 ppm. Keberadaan gen Gusdideteksi dengan reaksi histokimia, dan konfirmasi keberadaan gen -1,3-Glucanase dilakukan dengan Polymerase Chain Reaction (PCR). Daripenelitian berhasil diperoleh 2% kalus transforman yang lolos seleksiBasta. Hasil konfirmasi keberadaan gen Gus pada planlet transformanmenunjukkan 9 dari 20 (45%) planlet yang diuji, positif mengandung genGus. Konfirmasi keberadaan gen -1,3-Glucanase dengan PCRmenunjukkan hanya 2 dari 20 planlet transforman, positif mengandung -1,3-Glucanase. Pengujian ketahanan dari plantlet transgenik tersebut perludilakukan terhadap Fusarium oxisporum f.sp cubense (Foc).Kata kunci: Musa textilis Nee., transformasi gen, -1,3-Glucanase,Agrobacterium tumefaciens, penyakit, jamur patogen,FusariumABSTRACTThe main constraint of abaca (Musa textilis Nee.) cultivation isinfection of wilt disease caused by Fusarium oxysporum f.sp cubense(Foc). The effort to improve abaca resistance through hybridization is stilldifficult due to narrow genetic variability resulted from continuousvegetative multiplication. Transformation of -1,3-Glucanase resistancegene is an alternative way to improve character of genetic resistance withhelp of Agrobacterium oxisporum. The research aimed at introducing -1,3-Glucanase gene to abaca plants prior to obtaining the plants resistanceagainst Fusarium wilt diseases. The research was conducted inBiotechnology Laboratory, Faculty of Agriculture Brawijaya Universityand Tissue Culture Laboratory of Indonesian Tobacco and Fibre CropsResearch Institute, from June 2007 to May 2009. This experimentconsisted of three steps, namely: -1,3-Glucanase gene transfer onto abacaembriogenic 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. tumefaciensvector strain LBA4404 with pB2GW7 containing genes of -1,3-Glucanase and Gus ( -glucuronidase) as reporter, and Bar (Bastaresistance) as selector marker. Embriogenic calli of abaca clone UB-13were soaked in A. tumefaciens suspension and then cultured in co-cultivation medium for two days. After co-cultivation, calli weretransferred 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 ppmBasta + 100 ppm Timentine. Shoots regeneration was conducted bytransferring transgene calli to shoot induction medium : MS + 0.5 mg/lBAP + 100 mg vitamin C/l with and without addition of 100 ppmTimentine. Transgene shoots with 2-3 cm height were sub-cultured to rootinduction medium : MS + 2 g active charcoal/l with and without additionof 50 ppm Timentine. Detection of Gus gene was conducted usinghistochemical reaction, while confirmation of -1,3-Glucanase gene wasperformed by PCR. This project resulted in 2% transgene calli passingBasta selection. Nine out of 20 plantlets (45%) confirmed the existance ofGus gene. PCR results showed that only 2 out of 20 transformed plantlets positively contained -1,3-Glucanase gene. The plantlets resistanceagainst 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