%0 Journal Article
%@ ISSN 1511-2616
%A Ismanizan Ismail,  
%A Zamri Zainal,  
%A Hisham Zainal Ariffin,  
%D 2005
%F myais:2253
%I Malaysian Society for Biochemistry and Molecular Biology
%J Malaysian Journal of Biochemistry and Molecular Biology
%K Seed transformation, Agrobacterium tumefaciens, C. annuum, direct shoot regeneration
%N 1
%P 1-7
%T Seed Transformation System using Hygromycin-B Selection for Malaysian Chili Varieties via Agrobacterium tumefaciens
%U http://myais.fsktm.um.edu.my/2253/
%V 12
%X 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 ?-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.
%Z 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