TRANSFORMABILITY OF COWPEA (VIGNA UNGUICULATA L.WALP) BY PARTICLE BOMBARDMENT

Abstract

This study was conducted to investigate 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. Reporter and selectable marker genes driven by a 35S CaMV promoter were used in transformation experiments. Conditions for optimal delivery of DNA to plants were established based on transient gus expression assays two days after bombardment. Introduced genes were detected in T₀ plants and progenies by gus assays, polymerase chain reaction (PCR) and southern analyses. Two to three weeks culture of explants in the dark on high concentrations (5µM) of 6-benzylaminopurine (BAP) followed by culture in the light on low BAP resulted in the formation of shoots. Up to six shoots were formed adjacent to one another in a de novo fashion within a month from one primary explant. Subsequent Kanamycin or bialaphos selection pressure resulted in regeneration of transgenic T₀ chimeras. Kanamycin (50mg/l) was able to cause significant chloiosis in non-transformed shoots. Minor differences were found based on the type of particle (gold/tungsten)) used. However. the size of microcarriers, microflight distance and helium pressure significantly affected transient expression of reporter genes. Embryos (125 in all) each attached to one cotyledon were cocultured with Agrobacterium containing kanamycin gene after bombardment with non DNA-coatcd particles. These were put straight to soil and 13 survivors gave 1500 T1 seedlings. Seedlings which survived 100mg/1 kanamycin for 25 days without bleaching were rooted on 50mg/1 kanamycin. Only one of seven surviving seedlings gave a positive southern signal. However, DNA samples from six seedlings were amplified in PCR analysis using Bt gene specific primers. The subsequent PCR/Southern analysis with a Bt-gene probe hybridized all amplified bands although the signals were very faint. A total of 1692 explants were also bombarded with DNA-coated particles and placed on 3mg/1 bialaphos selection. Only 12 of these explants produced seeds eventually. All 12 T₀ plants were gus negative even though 7 gave positive PCR signals with bar primer. Subsequent PCR/Southern analysis with a bar gene probe detected five of the seven tested. Eight out of 1400 seeds harvested from T₀ 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. Three thousand, five hundred and fifty seven T₂ seedlings were obtained but only 2 were gus positive. However, 3 seedlings survived basta (glufosinate ammonium) spray. The two gus positive and 3 basta surviving seedlings gave positive Southern hybridization signals. Twelve T3 seedlings from these were gus positive and also gave Southern hybridization signals. Ninety-nine plants obtained from embryos bombarded and grown up without any form of selection gave 4,587 seedlings. Only 28 survived two basta sprays, but 13 produced seeds as the rest died due to fungal attack. Six of these gave positive PCR signals with gus primer and 3 with bar primer. Two out of 1,991 seedlings from these survived basta spray and gave positive southern signals. Twelve T3 seedlings from these also gave positive southern 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.