Sains Malaysiana 38(6)(2009): 921–928

Pengaktifan Gen Pelapor di dalam Tumbuhan Transgenik Tomato Melalui Kaedah Agro-Infiltrasi Vektor Penandaan Tanpa Promoter

(Activation of Reporter Gene in Transgenic Tomato by Agro-Infiltration of the Promorterless Tagging Vector)

Safarina Ahmad

Fakulti Sains Sumber dan Teknologi, Universiti Malaysia Sarawak

Kota Samarahan 93400, Kuching, Sarawak, Malaysia

Ismanizan Ismail* & Zamri Zainal

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor D.E., Malaysia

NurulFarhana Zakaria

Makmal Genetik, Institut Penyelidikan Perhutanan Malaysia

52109 Kepong, Kuala Lumpur, Malaysia

Diserahkan: 19 Januari 2009 / Diterima: 24 April 2009

ABSTRAK

Kajian ke atas kaedah inokulasi eksplan menggunakan vektor penandaan tanpa promoter, pCAMDIN ke atas tomato varieti tempatan MT1, bagi tujuan mutagenesis penyelitan T-DNA menggunakan kaedah infiltrasi agro telah dijalankan. Bahagian daun muda disuntik dengan larutan yang mengandungiAgrobacterium tumefaciens, 100 mg/l Str, 50 mg/l Kan, 10mM MES dan 100 μM Acetosyringone. Infiltrasi dilakukan ke atas sampel daun dengan menggunakan medium LB, bahagian permukaan daun dilukakan dan infiltrasi agro dilakukan ke atas sampel daun pokok tomato yang berada di dalam rumah kaca. Tisu yang diinfiltrasi tidak mengalami kecederaan dan kekal hijau selepas 5 hari. Infiltrasi yang mudah dilakukan dan pengekspresan transien GUS yang dapat dicerap berlaku pada sampel yang dianalisa adalah antara kebaikan kaedah ini. Seterusnya, fragmen gen gus telah berjaya diamplifikasi. Kejayaan integrasi gen gus ke dalam tomato juga dapat dilihat melalui penghibridan Southern. Berdasarkan keputusan ini, dapat dicadangkan bahawa penyelitan transgen daripada vektor rekombinan pCAMDIN telah berlaku dalam genom tomato.

Kata kunci: A. tumefaciens; agro-infiltrasi; tomato transgenik; transformasi

ABSTRACT

A study on explant inoculation method using promoterless tagging vector, pCAMDIN, on local tomato (MT1 variety) for the purpose of T-DNA insertional mutagenesis by agroinfiltration have been carried out. The injection of young leaves with a solution containing Agrobacterium tumefaciens, 100 mg/l Str, 50 mg/l Kan, 10mM MES and 100μM Acetosyringone. The infiltration was carried out with the use of LB medium by gently wounding the surface of the leaves and every procedures were carried out in the glass house. Infiltrated tissues were not damaged and remained green even after 5 days. Advantages of the technique included ease of infiltration and the observed transient expression on the sample. gus gene fragments were successfully amplified. Successful integration of gus gene into the tomato can also be observed in the results of Southern hybridization. Thus, it can be suggested that transgene insertion from recombinant vector pCAMDIN has occurred in tomato genome.

Keywords: A. tumefaciens; agro-infiltration; transformation; transgenic tomato

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*Pengarang untuk surat-menyurat, email: maniz@ukm.my