| Cowpea is an important crop worldwide, and in parts of the developing world it provides a vital source of protein for human diets. Plant biotechnological techniques offer useful approaches that can aid plant breeders in crop improvement. The primary aim of this project was to develop a tissue culture system for cowpea regeneration with applicability to U.S. genotypes.; The in vitro response of shoot tip and leaf disk explants to various levels of Murashige and Skoog (MS) macro and micro nutrients, vitamins, and iron were evaluated. In another study, shoot tip cultures were initiated on MS medium supplemented with ascorbic acid, thymine, casein hydrolysate, 6-benzylaminopurine (BAP), or activated charcoal. The effect of the media constituents were genotype dependent and proved useful in the development of a regeneration system.; A micropropagation system was developed utilizing shoot tips isolated from in vitro grown seedlings. Shoot tip cultures were established on MS medium supplemented with BAP and 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin. Individual shoots were excised from the shoot clusters and rooted on MS medium containing naphthaleneacetic acid (NAA). Plantlets were transferred to soil, acclimatized in a growth chamber, and grown to maturity in a greenhouse where they exhibited healthy growth and set seed. The addition of gibberellic acid (GA3) to the multiplication medium enhanced shoot proliferation and was found useful for micropropagation.; A plant regeneration system was developed utilizing cowpea cotyledon explants. Cotyledons were initiated on 1/3 NIS medium supplemented with 15 mg/L BAP and then transferred to medium containing 1 mg/L BAP for shoot induction. Shoots were transferred to hormone-free NIS medium for rooting. Plantlets were transferred to soil, acclimatized and grown to maturity in the greenhouse. Seeds collected from the regenerants germinated producing healthy plants.; In another study, the regeneration medium was supplemented with the ethylene inhibitors, silver nitrate, 2,5-norbomadiene, and cobalt chloride (CoCl 2). Silver nitrate and 2,5-norbomadiene improved the frequency of shoot regeneration from cotyledon explants. The addition of coconut water to the regeneration medium also resulted in increased shoot formation from cotyledon explants.; The tissue culture systems developed were used in the genetic transformation of cowpea. Agrobacterium tumefaciens EHA 105 harboring the plasmid pBI121, containing kanamycin resistance and β-glucoronidase (GUS), were used to transform cowpea. Two different transformation approaches were evaluated. In the first approach, cotyledon explants were co-cultured with A. tumefaciens and then allowed to produce shoots utilizing the regeneration system. Putative transformants were obtained. In the second approach, compact mature plants were produced using the micropropagation system. The entire foliar portion of the plants were vacuum infiltrated with A. tumefaciens and the resulting seeds were screened for transformants. GUS expression was observed in the cotyledons of seeds from the vacuum-treated plants.; To our knowledge this is the first report of micropropagation and regeneration from U.S. cowpea genotypes, and furthermore, the first report of GUS-positive plants from U.S. genotypes. The results obtained from this project will be vital to scientists as they strive to improve U.S. cowpea genotypes by introducing novel genes into the germplasm. |
