Sains Malaysiana 35(1): 61-66 (2006)

The Development of a Suitable Cabbage Tissue Culture

System for Gene Transformation Studies

(Penyediaan Kultur Tisu Kobis yang Sesuai untuk Kajian Transformasi Gen)

Latifah Amin

Pusat Pengajian Umum

Universiti Kebangsaan Malaysia

43600 UKM Bangi Selangor D.E. Malaysia

Mohd Azib Salleh

Fakulti Sains dan Teknologi Sumber

Universiti Malaysia Sarawak

94300 Kota Samarahan, Sarawak Malaysia

Abdul Karim Abdul Ghani

Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia

43600  UKM Bangi, Selangor D.E. Malaysia

Mohd Nazir Basiran

Institut Penyelidikan Teknologi Nuklear Malaysia

Bangi, 43000 Kajang, Selangor D.E. Malaysia

ABSTRACT

Experiments were carried out to establish optimum culture condition and to identify the most responsive hypocotyls segments to regenerate shoots in the presence of slight callus for the local cabbage cultivar, Brassica oleracea L. var capitata subvar. kkcross. Results of the study showed that only the upper segment of the hypocotyls (1 cm and 2 cm from both  shoot apex) excised from in vitro seedlings, regenerate shoots while the segment nearest to the apex (1 cm) regenerate both shoots and roots. Optimum regeneration was obtained when the seedlings' age were 3 weeks old as compared to younger or older explants. Types of culture vessels did not exhibit any significant effect on the hypocotyls culture. Multiple shoots with the presence of slight healthy callus (0.5 - 2.0mm diameter) were successfully regenerated at a frequency of 42 and 50% respectively from hypocotyls sections cultured on a basal Murashige and Skoog (MS) medium supplemented with 3-lndole acetic acid (1AA) [1 µg/L (w/v)], kinetin [2 µg/L(w/v)] and casein hydrolysate [500 µg/L(w/ v)] (designated as Ms1 medium) and B5 medium supplemented with adenine sulphate [40 mg/L(w/v)], glutamine (6mM) and 2, 4,5-trichlorophenoxyacetic acid [2,4,5- T (0.1 mg/L( w/v)] (designated as cs23 medium). Clonal propagation from axillary buds was induced on a basal MS medium supplemented with low concentration of kinetin [0.05-0.2mg/L(w/v)]. Plantlets with complete roots were successfully transferred into a mixture of soil and vermiculate (1: 1) and grown into normal mature cabbage plants in the glasshouse.

Keywords: Tissue culture, gene transformation

ABSTRAK

Keadaan pengkulturan optimum dan keratan hipokotil yang paling responsif untuk meregenerasikan pucuk dan akar dalam kehadiran sedikit kalus bagi kultivar kobis tempatan, Brassica oleracea L. var capitata subvar, kk cross telah dikenalpasti. Hasil kajian menunjukkan hanya keratan daripada bahagian atas hipokotil (1 cm dan 2 cm daripada hujung pucuk) anak cambah in vitro meregenerasikan pucuk sementara segmen paling hampir kepada hujung pucuk (1 cm) meregenerasikan pucuk dan akar. Gerakbalas regeneratif optimum diperolehi apabila eksplan diambil daripada anak cambah in vitro yang berumur 3 minggu berbanding eksplan yang lebih muda atau tua. Bekas pengkulturan tidak memberikan kesan yang nyata pada kultur hipokotil. Suatu medium asas Murashige dan Skoog (MS) yang diperkaya dengan kombinasi hormon 1M [l µg/L(b/i)], kinetin [2 µg/L(b/i)] dan kasein hidrolisat [500 µg/L(b/i)] (dikenali sebagai medium Ms1) dan medium B5 yang diperkaya dengan aden in sulfat [40 mg/L(b/i)], glutamin (6.8 mM) dan 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) [0.1 mg/L(b/i)] (dikenali sebagai medium cs23) telah dapat memberikan kadar regenerasi pucuk berganda masing-masing sebanyak 42 dan 50% di samping kehadiran sedikit kalus yang sihat (diameter 0.5-2.0 mm). Perambatan klon dari tunas aksilari dilakukan atas suatu medium asas MS dengan penambahan kinetin pada kepekatan rendah (0.05 - 0.2 mg/L). Plantlet dengan akar yang lengkap telah berjaya dipindahkan ke campuran tanah dan vermikulit (1: 1) dan telah dapat menghasilkan pokok kobis matang yang normal di dalam rumah kaca.

Kata kunci: Kultur tisu, transformasi gen

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