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 To 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.
Highlights
Transformation of many plant species has become routine, permitting the introduction of characteristics that would not be possible by conventional breeding
We investigated the possibility of transforming and obtaining transgenic cowpea (Vigna unguiculata L Walp) plants using the particle bombardment process
In this paper we describe a protocol for stable transformation of cowpea via particle bombardment, using the selectable marker bar
Summary
Transformation of many plant species has become routine, permitting the introduction of characteristics that would not be possible by conventional breeding. Most legume transformation studies have used Agrobacterium tumefaciens to generate for example transgenic soya bean (Hinchee et al, 1994), chickpeas (Fontana et al, 1993), peas (Puouti-Kaerlas et al, 1990; Schroeder et al, 1993; Grant et al, 1995) and cowpea (Muthukumar et al., 1996; Kononowicz et al, 1993, 1997). Mature embryos (Penza et al, 1991; Akella and Lurquin 1993), leaf discs (Garcia et al, 1986, 1987) and cotyledons (Muthukumar et al, 1996; Kononowicz, et al, 1997) have been used. Neither Garcia and co-workers (1986, 1987) nor Penza and co-workers (1991) were able to recover plants from transformed tissues
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