IN VITRO REGENERATION AND OVEREXPRESSION OF PEA DNA HELICASE 45 (PDH45) GENE THROUGH AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION IN OILSEED BRASSICA SPP.

Abstract

An efficient genotype independent method for in vitro regeneration and
genetic transformation of Brassica spp. viz. Brassica juncea and Brassica napus have
been developed for the local varieties using cotyledonary leaf with petioles and
hypocotyls as explants. Explants obtained from 6-day old aseptically grown
seedlings were inoculated and co-cultivated with Agrobacterium tumefaciens strain
EHA105 harbouring a binary vector with PDH45 gene under the regulatory
control of 35s promoter and terminator sequences, permitting transformed
shoots to be selected on hygromycin containing medium. Well rooted
transformed plants were transferred to pots containing soil and after
acclimatization the plants were maintained under controlled environmental
condition. A total of 9 transformed plants were obtained from BARI Sarisha-8
variety, presumably indicating this protocol is more amenable to genotype
independent genetic transformation. Integration and expression of the
introduced transgene (PDH45 and hptII) were analysed by polymerase chain
reaction (PCR). Factors influencing transformation efficiency include explant age,
optical density of Agrobacterium culture for infection, duration of infection and
co-cultivation with Agrobacterium were also assessed. Genetic transformation
method developed in this study certainly could be utilized for introducing
abiotic stress resistance in the local cultivars of oilseed Brassica.