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

RULLY DYAH PURWATI, LILIEK SULISTYOWATI

Abstract


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


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DOI: http://dx.doi.org/10.21082/littri.v18n1.2012.24%20-%2030

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