Prosiding Seminar Tahun 2001_03

PROSIDING TAHUN 2001
Seminar Hasil Penelitian Rintisan dan Bioteknologi Tanaman

Bogor,
30-31 Januari 2001
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Transformasi, Studi Molekular, dan Bioasai Tanaman Ubi Jalar
untuk Ketahanan terhadap Hama atau Penyakit


(
Alberta Dinar Ambarwati, Dwinita W. Utami, Diani Damayanti, Atmitri Sisharmini,
Tri Joko Santoso, M. Herman, Minantyorini)

ABSTRAK

Perakitan tanaman ubijalar transgenik tahan hama boleng atau penyakit virus dilakukan dengan menyisipkan gen proteinase inhibitor (pin)II atau gen coat protein (CP) dari Sweet Potato Feathery Mottle Virus (SPFMV) ke dalam genom tanaman. Penguasaan teknik transformasi, molekuler, dan bioasai akan sangat mendukung keberhasilan dalam perakitan tanaman transgenik. Pada tahun 2000 dilakukan penelitian transformasi dengan gen pinII dan gen CP-SPFMV melalui penembakan partikel maupun Agrobacterium tumefaciens, serta mempelajari teknik dasar molekuler dan bioasai. Eksplan daun dan petiol ubijalar cv. Jewel digunakan sebagai jaringan target. Ekstraksi DNA tanaman dilakukan dengan beberapa modifikasi CTAB (Varadarajan & Prakash, 1991; Porebski et al., 1997) dan bioasai dilakukan pada umbi ubijalar putativ transgenik dan non transgenik (kontrol). Transformasi melalui penembakan partikel dengan ko-transformasi pTwa (pinII, bar) dan pRQ6 (gus, hpt) belum menghasilkan transforman yang tahan pada seleksi higromisin 25 mg/l. Sedangkan melalui Agrobacterium tumefaciens dihasilkan 18 tanaman putativ transgenik yang ditransformasi dengan gen pinII dan 6 tanaman putativ transgenik dengan gen CP-SPFMV. Hasil kuantitas konsentrasi DNA berkisar 0,7-1,8 µg/µl. Optimasi PCR diperoleh pada kondisi 95oC selama 30 detik, 50oC selama 60 detik, dan 72oC selama 45 detik. Pengujian bioasai pada 32 umbi berdasarkan jumlah lubang gerekan menunjukkan rata-rata skor kerusakan umbi 4,4-4,5 dengan persentase mortalitas Cylas pada tanaman putativ transgenik lebih tinggi (30,8%) dibandingkan tanaman non transgenik (15%).

Kata kunci : transformasi, molekuler, bioasai, pinII, CP-SPFMV, ubijalar

ABSTRACT

Transformation, molecular study, and bioassay on sweet potato for resistance to pest or disease. The production of transgenic sweet potato resistant to pest or virus disease was done by inserting proteinase inhibitor (pin)II gene or coat protein (CP) of Sweet Potato Feathery Mottle Virus (SPFMV) gene into sweet potato genome through transformation technique. Knowledge of transformation, molecular, and bioassay is therefore important in contributing the success to produce transgenic plant. Experiment was conducted in 2000, including some activities : transformation with pinII and CP-SPFMV gene via particle bombardment and Agrobacterium tumefaciens, study on basic molecular, and bioassay. Explants from petiole and leaf pieces of sweet potato cv. Jewel were used as target tissues. DNA extraction used the modification of CTAB method (Varadarajan & Prakash, 1991; Porebski et al., 1997) and bioassay was conducted on storage roots of putative transgenic plants and non transgenic plants (control). Transformation via particle bombardment with co-transformation of pTwa (pinII, bar) and pRQ6 (gus, hpt) have not produced the transformant yet surviving in hygromycin selection (25 mg/l). Transformation via Agrobacterium tumefaciens produced 18 and 16 putative transgenic plants transformed with pinII and CP-SPFMV gene, respectively. The quantity of the DNA concentration was about 0.7-1.8 µg/µl. Optimation of PCR was obtained at 95oC for 30 sec, 50oC for 60 sec, and 72oC for 45 sec. Bioassay on 32 storage roots based on the number of punctures, showed the score damaged roots was 4.4-4.5 and the percentage of Cylas mortality on putative transgenic plants (30.8%) was higher than on untransformed plants (15%).

Key words : transformation, molecular, bioassay, pinII, CP-SPFMV, sweet potato

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