Study On The Construction Of Kidney Bean High-efficiency Regeneration System And Related Mechanism

Plant regeneration technology is the basis of genetic transformation,and also promotes the development of agricultural production,plant protection and germplasm resources innovation.In this study,different genotypes(Phaseolus vulgaris L.)were used as experimental materials to build a kidney bean regeneration system through direct and indirect organogenesis—providing a technical solution for the genetic transformation of kidney beans and other studies,using real-time fluorescent quantitative PCR to generate different tissues for indirectorgans.Measurement and analysis of gene expression patterns related to middle regeneration—provide a theoretical reference for further research on the mechanism of kidney bean indirect organogenesis Below are key research findings:1.Direct organogenesis:(1)A18-1 cluster bud regeneration rate(72.25%),average number of buds(4.28),A18-1 is an ideal material for direct organogenesis.(2)The induction rate and average number of buds in the hypocotyl are the highest,which are 88.89% and 8.59 respectively,which is the ideal type of explant for direct organ regeneration.(3)Add 6-BA(7mg·L-1)+NAA(0.2mg·L-1)during the induction of cluster buds to get the highest cluster bud induction rate and average number of buds,respectively:74.21% and 2.91,As the best bud induction medium for direct organogenesis of cotyledonary nodes;(4)The best rooting medium is 1/2MS+IBA(0.5mg·L-1).2.Indirect organogenesis:(1)A18-1 callus induction rate(84.72%)and callus fresh weight value(0.520±0.003g)are the highest,which is the ideal material for indirect organogenesis.(2)Callus fresh weight value of the entire cotyledon node The highest is 0.673±0.117 g,and the callus induction rate is 90.67%,which is the ideal explant type for indirect organ regeneration.(3)The highest callus induction rate of TDZ(0.5mg·L-1)is 91.51%,is the best callus induction medium for indirect organogenesis cotyledonary nodes.(4)In consideration of comprehensive bud induction rate(93.33%)and average bud number(4.27±0.15),indirect organogenesis kidney bean The optimal concentration of 6-BA in the induction culture of variety P16165 cotyledonary node bud is 1.0mg·L-1.(5)The observation of tissue section proves the nature of indirect organogenesis.3.Analysis of gene expression related to regeneration in vitro:(1)Both ClV3 and WUS have the highest expression levels in the subcultured callus,and their expression can promote the formation of cell dedifferentiation callus.(2)During the regeneration of invitro seedlings,the expression level of TFL1 showed a gradual increase,and the expression level in buds was significantly higher than that of callus and subsequent callus,indicating that TFL1 played a vital role in the differentiation stage of buds in vitro.(3)The expression of PIN1 in different tissue parts is different,and the expression level in callus is the highest,indicating that PIN1 plays a vital role in the process of callus induction.(4)The expression of AHK2,AHK3,ARR1 and ARR10 in bud tissue is significantly higher than othe rtissues,indicating that they play an important role in the bud regeneration stage in vitro.