African Journal of Agribusiness Research

African Journal of Tropical Agriculture Vol. 1 (2), pp. 014-020, October, 2013. © International Scholars Journals

Full Length Research Paper

Stable gene transformation in cowpea (Vigna unguiculata L. walp.) using particle gun method

J. Ikea^1,2*, I. Ingelbrecht², A. Uwaifo¹ and G. Thottappilly^2,λ

¹Biochemistry Department, University of Ibadan, Ibadan, Nigeria

²International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria

*Corresponding author, Present address: CSIRO Plant Industry, Black Mountain Laboratories, G.P.O Box 1600, Canberra ACT 2601, Australia. Email: j[email protected]

Accepted 18 July 2013

Abstract

We investigated the possibility of transforming and obtaining transgenic cowpea (Vigna unguiculata L Walp) plants using the particle bombardment process. Meristematic explants that could give rise to whole fertile plants were used in transformation experiments with reporter and selectable marker genes driven by a 35S CaMV promoter. Conditions for optimal delivery of DNA to explants were established based on transient gus expression assays two days after bombardment. The size of microcarriers, microflight distance and helium pressure significantly affected transient expression of reporter genes. A total of 1692 explants were bombarded with DNA-coated particles and placed on 3 mg/l bialaphos selective medium. Only 12 regenerated shoots produced seeds eventually, and all were Gus negative even though 7 gave positive PCR signals with the bar primer. Eight out of 1400 seeds from T_o plants were GUS positive. DNA from eight of the GUS positive seedlings were amplified with both the gus and bar primers in PCR analysis but only two gave a positive Southern signal. Only two of the 3557 T₂ seedlings obtained were GUS positive. However, 3 seedlings survived Basta spray. The two GUS positive and 3 Basta surviving seedlings gave positive Southern hybridisation signals. Twelve T₃ seedlings from these were GUS positive and also gave positive Southern hybridisation signals. The positive reaction of T₁, T₂ and T₃ seedlings under Southern analysis confirms the stable integration of introduced genes and the transfer of such genes to progenies. However, the level of expression of introduced genes in cowpea cells is very low and this accounted for the high mortality rate of progenies under Basta spray.

Key words: Transformation, particle bombardment, gus assay, transient expression, reporter gene, basta, bar gene.