Plant Tissue Cult. 13(1) : 1-12, 2003 (June)

Agrobacterium-mediated Transformation of Lentil (Lens culinaris Medik.)

R. H. Sarker, Ashapurno Biswas, Barkat Murtaja Mustafa, Shirin Mahbub and M. I. Hoque

Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Key words: Lentil, Transformation, GUS expression, Kanamycin resistance, PCR analysis

Abastract

Agrobacterium-mediated transformation system was developed for two microsperma varieties of lentil (Lens culinaris Medik.) namely, Barimasur-2 and Barimasur-4. Transformation ability of different explants like cotyledonary node, decapitated embryo, immature embryo and epicotyl were tested with Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Highest percentage of GUS positive regions were found in epicotyl explants followed by decapitated embryo as detected by transient assays. Decapitated embryo was found to be more effective in formation of multiple shoots on MS medium supplemented with 0.5mg/l BAP, 0.5mg/l Kn, 0.1 mg/l GA3 and 5.5 mg/l tyrosine following Agrobacterium infection and selection. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l. Stable expression of the GUS gene was observed in various parts of the transformed shoots. Genomic DNA was isolated from these shoots and stable integration of GUS and nptII genes was confirmed by polymerase chain reaction (PCR) analysis.

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