Malaysian Applied Biology Journal

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05_IsmanizanI

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Malays. Appl. Biol. (June 2004) 33(1): 27-35

RECOVERY OF TRANSGENIC CHILI USING COTYLEDONARY NODES VIA AGROBACTERIUM TUMEFACIENS-MEDIATED

TRANSFORMATION

ISMANIZAN, I*, ZAMRI, Z. and SITI- NORLIDAH, T.

School of Biosciences and Biotechnology, Faculty of Science and Technology,

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

Email : This e-mail address is being protected from spambots. You need JavaScript enabled to view it

ABSTRACT

Agrobacterium-mediated transformation in chilli (Capsicum annuum) has been limited mainly due to difficulties in regeneration of explant material. By using cotyledonary nodes, an improved protocol was developed. Agrobacterium lumefaciens LBA 4404 strain harboring the binary vector pCAMBIA 1301, carrying the uid A gene encoding GUS ((3-glucuronidase) enzyme and hygromycin phosphotransferase (HPT) gene as a selectable marker, was used to inoculate cotyledonary nodes in wounded site. After 21 days of incubation in solid Murashige and Skoog medium without growth regulators (MSO), 80% of the explants generated shoots. Selection of the transformants was carried out at the rooting phase where 10 mg T1 hygromycin-B was added to the rooting medium. About 40% of the shoots survived and produced numerous roots in this medium. Transient p-glucuronidase (GUS) activity was used to monitor the transformation event. Approximately 8% of the putative transformed lines analysed were GUS positive with histochemical assay. The presence of introduced gus and hpt genes in transgenic plants was confirmed by polymerase chain reaction analysis.

ABSTRAK

Kaedah transformasi cili berperantarakan Agrobacterium didapati amat terhad kerana kesukaran dalam proses regenerasi. Dalam kajian ini, satu protokol telah di bina dengan menggunakan nodul kotiledon. Bakteria Agrobacterium tumefaciens strain LBA 4404 yang membawa vektor binari pCAMBIA 1301 serta mengandungi gen uid A yang mengkodkan enzim GUS dan gen higromisin fosfotransferase (HPT) sebagai penanda pemilihan telah digunakan untuk menginokulasi nodul totiledon pada kawasan terluka. Selepas eraman selama 21 hari dalam media Murashigee and Skoog tanpa pengawalaturan rertumbuhan (MSO), 80% eksplan berjaya menjana pucuk. Pemilihan transforman di lakukan pada peringkat pengakaran di mana 10 mg/ml telah di tambah ke dalam medium pengakaran. Hampir 40% pucuk terus hidup dan menghasilkan ikar yang banyak dalam medium ini. Aktiviti transien P-glucuronidase (GUS) di gunakan untuk memantau proses iransformasi. Lebih kurang 8% titisan transforman putatif yang di analisis memberikan keputusan positif dalam asai histokimia GUS. Kehadiran gen gus dan hpt dalam tumbuhan transgenik cili di sahkan melalui analisis tindakbalas :┬╗limerase.

Key words: Capsicum annuum, b-Glucuronidase, in vitro regeneration, organogenesis, T-DNA

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