Transgene expression in cowpea (Vigna unguiculata (L.) Walp.) through Agrobacterium transformation of pollen in flower buds

Abstract

Pollen transformation shows potential as a fast and easy means of obtaining transformed plants carrying desirable transgenes. Agrobacterium tumefaciens has been suggested as the best natural plant genetic engineering system. Laboratory and screenhouse studies were undertaken to investigate the possibility of obtaining transformed progeny in cowpea by Agrobacterium-mediated transformation of pollen. Flower buds of selected cowpea accessions were inoculated the evening before opening with a late log phase culture of A. tumefaciens strain pGV 2260 carrying the transgene vector ptjk 142. The vector is a disarmed Ti plasmid carrying a chimeric fusion of the UidA and Bar genes for β-glucuronidase expression and bialaphos resistance respectively. Seedlings resulting from inoculated flowers were screened for expression of the genes by β-glucuronidase (GUS) assay and Basta spraying. GUS positive plants were analyzed by polymerase chain reaction and Southern hybridization. Up to 90% of inoculated flowers and developing pods aborted. Pod set from inoculated flower buds ranged between 8.92 and 10.51% with no significant difference in pod set among accessions. Nine seedlings showed positive GUS expression. None was tolerant to Basta. Seven seedlings showing positive GUS expression also showed positive signals under PCR and Southern analysis giving indicative evidence of transgene presence. Overall transformation frequency was 0.36%.

Key words: Agrobacterium tumefaciens, b-glucuronidase, bialaphos resistance, cowpea, pollen transformation.