| Cowpea(V.unguiculata L.Walp.)is an important legume crop in the world with strong tolerance to salt and drought stresses.While transgenic technology has been widely applied to various types of crops,its use in cowpea is much limited due to low bud regeneration efficiency and poor transgene integration efficiency.In this thesis,we explored several key factors influencing Agrobacterium tumefaciens-based genetic transformation efficiency in cowpea,including seed sterilization method,genotype of explant and selective gene.In addition,we established a simple but efficient system for the induction of hairy roots by Agrobacterium rhizogenes in cowpea.The main results are as follows:1.Three methods of seed disinfection were compared,including chlorine treatment,sodium hypochlorite treatment and mercuric chloride treatment.It showed that 0.1%mercuric chloride treatment performed the best,as it resulted in the highest seed germination rate after 48 hours of treatment with no pollution of seeds.2.A total of 51 cowpea genotypes were screened for bud regeneration frequency under Agrobacterium mediated transformation.In the first round of screening,we identified 10 genotypes with a bud differentiation rate greater than 80%.Theses genotypes were subjected to a second round of screening each in three replicates.The reproduction coefficient of the 10 genotypes ranged from 1.17 to 1.93.It was exceptionally high in"II7E0815" and" White August Cowpea ",which were 1.70 and 1.93,respectively.The induction rate of adventitious buds of the 10 genotypes ranged from 22.51%to 86.40%."White August Cowpea" showed the highest induction rate of adventitious buds,which was 86.40%.As "white cowpea August" showed a satisfied reproductive coefficient as well as adventitious bud induction rate in Agrobacterium mediated transformation,it is suggested to be used as a suitable explant genotype for future transgenic research.3.The impacts of the selective marker genes nptII and bar on callus induction rate and gene transformation rate were compared.It showed that the callus induction rate in the glufosinate and bar screening system was significantly greater(by 12.98%)than that of the kanamycin and nptII screening system.PCR amplification of the transgene sequence showed an 100%and 98.61%positive rate using callus tissues selected by kanamycin and glufosinate,respectivey,whereas no significant differences were noted statistically.After GUS staining,it was observed that the callus obtained by kanamycin and nptII screening system were all chimeras,while the callus obtained by the glufosinate and bar screening system were partial chimeras.Further studies found that the proportion of callus transformants was 77.2%after screening with glufosinate and bar screening system.However,the ratio of callus transformation obtained by kanamycin and nptII screening system was less than 50%,and the difference between them was significant.Therefore,in terms of balancing callus induction rate and the transformation rate,the glufosinate and bar screening system is considered more suitable for cowpea.4.To develop a new and simple method for hairy root induction in cowpea,the traditional tube-based and the novel seed germination bag-based transformation protocols were compared.Using the cowpea genotype "JX6",it showed that the average hairy root number induced using the seed germination bag-based method was 10.12,which was a bit lower than that of the tube-based method.However,the rate of transgenic hairy root was much higher in the former than in the later method(33.12%in comparison to 17.63%).We then further compared the efficiency of transgenic hairy root induction among 14 cowpea genotypes using the seed germination bag-based method."II7E0826" was found to have the best performance by showing the number of hairy of 16 and the transgenic hairy root rate of 55.67%.This genotype was hence suggested to be a good choice for Agrobacterium rhizogenes mediated induction of hairy roots.This study has laid a foundation for creating transgenic cowpea plants,which is prerequisite for many downstream tasks such as functional genomic studies and transgenics-based cowpea breeding. |
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