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Seed Transformation System using Hygromycin-B Selection for Malaysian Chili Varieties via Agrobacterium tumefaciens

Ismanizan Ismail, and Zamri Zainal, and Hisham Zainal Ariffin, (2005) Seed Transformation System using Hygromycin-B Selection for Malaysian Chili Varieties via Agrobacterium tumefaciens. Malaysian Journal of Biochemistry and Molecular Biology, 12 (1). pp. 1-7. ISSN ISSN 1511-2616

Full text not available from this repository.

Official URL: http://ejum.fsktm.um.edu.my/ArticleInformation.aspx?ArticleID=604

Affiliations

Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology

Abstract

An improved transformation system for transgenic chilli in three Malaysian varieties was developed by combining strategies to enhance Agrobacterium tumefaciens-mediated T-DNA delivery by seeds infection with the development of a rapid, efficient selection protocol based on hygromycin-B. Seeds of chili cultivars were precultured and infected with Agrobacterium tumefaciens strain LBA 4404 carrying the pCAMBIA 1301 binary vector. This plasmid contains ß-glucuronidase (GUS) as a reporter gene and hygromycin phosphotransferase (hpt) gene which confer resistance to hygromycin-B. Direct transformation approach was used and callus phase was omitted. The optimal hygromycin concentration for selection was shown to be at 15 mgl-1 based on its effect on germination, plantlet formation and necrosis. Seeds were cultured on Murashige and Skoog (MS) medium containing hygromycin-B for selection. Transformants were confirmed by GUS histochemical analysis and polymerase chain reaction (PCR). GUS activity was exhibited in the individual plantlet as indicated by blue color. In PCR analysis using specific primers for gus and hpt genes, DNA fragments of 789 and 591 bp in length were amplified respectively from the total DNA of young leaves of mature transgenic plants. Seeds of To plants were then grown in greenhouse, left to mature and seeds collected to produce T1 regenerants. Molecular analysis were carried out in the T1 generation to study the integration and expression stability of transformed genes. Polymerase chain reaction showed that both gus and hpt genes were present in T1 generation and expression confirmed by GUS histochemical analysis.

Item Type:Journal
Additional Information:The authors are grateful to Dr. Richard Jefferson, Plant Biotechnology Centre, Australia for providing the pCAMBIA 1301. This work was supported by a Universiti Kebangsaan Malaysia grant (320316005) awarded to Ismanizan Ismail and in part by IRPA grant 09-02-02-0054 from the Ministry of Science, Technology and Environment, Malaysia awarded to Zamri Zainal
Keywords:Seed transformation, Agrobacterium tumefaciens, C. annuum, direct shoot regeneration
Subjects:Q Science, Computer Science
R Medicine, Dentistry, Pharmacy, Nursing
ID Code:2253

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