Establish And Optimize Regeneration System Of Cowpea(Vigna Unguiculata L. Walp.) Cotyledonary Nodes And Immature Embryos

Cowpea (Vigna unguiculata L. walp.), an annual herb, is widely grown in tropical and template regions of the world. Insect pest is the main restrictive factor of cowpea cultivation and storage. Researchers tried to put insecticidal genes of wild varieties into cultivars of cowpea by transgenic technology, so it is very important to establish a steady and repeatable and efficient in vitro regeneration system.This study adopted Xiang-feng Ⅷ, Cheng-jiang Ⅶ, Gua-mianwang and Cui-lv cowpea as experimental material, and used cotyledonary nodes and immature embryos as explants to establish and optimize in vitro regeneration system. The main results are as follows:1. Optimize regeneration system of cotyledonary nodes1) There was significant difference of four genotypes(Xiang-feng Ⅷ, Cheng-jiang Ⅶ, Gua-mianwang and Cui-lv cowpea) of cotyledonary nodes at the all stages of culture in vitro. As a whole, Xiang-feng Ⅶ was bestter than others, and the worst was Gua-mianwang.2) For5different cutting modes of cotyledonary nodes(cotyledonary nodes without cotyledon, cotyledonary nodes with one cotyledon, cotyledonary nodes with two cotyledons, dissected cotyledonary nodes without cotyledon, dissected cotyledonary nodes with one cotyledon), the number of differentiated adventitious bud of dissected cotyledonary nodes with one cotyledon was higher than others explants.3) For3different basal culture medium(MS,B5and MSB5),the seed germination rate and uniformity were highest on B5culture medium, and MS culture medium was best for differentiated adventitious bud and rooting.4) After1week culture, cut off axillary buds could got more adventitious buds than cue off it at early inoculation.5) Low concentration (0.005g/ml) of seed extract could stimulate the seed germination of cowpea in the range of0-0.2g/ml, but seed extract above0.005g/ml could inhibited seed germination. Any concentration of seed extract could restrain differentiated adventitious bud. 6) The activated carbon is good for rooting of tissue culture seeding in the range of0-2.0g/L activated carbon. When l.Og/L activated carbon was added into medium, rooting was better than other treatments, and high concentration of activated carbon (2.0g/L) could restrain rooting.7) Low concentration of salts is good for rooting of tissue culture seeding. Rooting was best and on1/2MSB5than MSB5and1/4MSB5.2. Plantlet regeneration directly from immature embryo explants1) Comparing different time processing between different immature embryo in75%ethanol and0.1%mercuric chloride, the results showed that the optimum sterilizing method of explants is that the immature embryos with pod are surface-sterilized by immersion in75%alcohol for60s, follow by immersion and agitation in0.1%HgC12for15min, washed by sterile water for3-5times at least.2) Comparing culture result of10～25days immature embryos, the result showed that with the increasing age of immature embryos, seed coats were isolated easily and rate of regenerated plantlet is higher and higher. Culture medium, appended with2.0mg/L6-BA, could improve the rate of regenerated plantlet and make the plants more vigorous and strong.3) Comparing culture result of1-5times content of trace elements, the result showed that with the increasing content of MS tract elements, the rate of regenerated plantlet increased gradually and then decreased, and the highest rate was58%on culture medium adding3times content of trace elements.