In vitro regeneration and transient gene expression in mango cv. 'Vellaikolumban'.
Samanta S., Ravindra M. B., Dinesh M. R., Anand L., Mythili J. B.
Author Affiliation: Department of Biotechnology, University of Agricultural Sciences, GKVK, Bangalore - 560 065, India.
Journal of Horticultural Science and Biotechnology 82 : 275-282
Abstract : A protocol to regenerate shoots through somatic embryogenesis was developed in the polyembryonic mango cv. 'Vellaikolumban', and the feasibility of gene transfer using the ?-glucuronidase (GUS) reporter gene was demonstrated. Young, 25-30 d-old fruits were found to be ideal for culture initiation. MS and B5 media were equally effective for initiation of nucellar cultures. A higher percentage of somatic embryo induction was obtained from nucellar cultures using B5 or MS medium supplemented with 20% (v/v) coconut water compared to MS medium without coconut water. Maturation of somatic embryos, 1.0-1.5 cm in length, was optimised in M3 medium composed of B5 salts, 400 mg l-1 L-glutamine and 1 mg l-1 abscisic acid. Faciation and necrosis of embryogenic cultures could be effectively controlled through the use of 0.1 mg l-1 salicylic acid. For germination of mature somatic embryos, with well-developed roots and shoots, a semi-solid medium was found to be superior to a bilayer medium. Various stages of nucellar culture were transformed with Agrobacterium tumefaciens strain LBA 4404 containing the binary vector, pCambia 2301, harbouring the npt II gene as a selectable marker and GUS as a reporter gene. During kanamycin sensitivity tests, it was found that 200 mg l-1 kanamycin completely inhibited regeneration of fresh new callus in nonco-cultivated embryogenic callus derived from nucellus. Among the various stages of nucellar culture tested, embryogenic callus was found to be amenable to Agrobacterium-mediated gene transfer. Maximum transformation was obtained using 150 µl bacterial culture with 3 d of co-cultivation. The expression of the reporter transgene was confirmed by GUS assay.