Optimization Of In Vitro Regeneration System Using Cotyledon Node From Different Genotypes Of Cucumber

Cucumber（Cucumis sativus L.）has a long history of cultivation.It is a horticultural crop of the Cucurbitaceae Melon genus and one of the important vegetable crops.In vitro regeneration of cucumber is affected by many factors such as genotype,explant type,hormones and so on.Domestic and foreign researchers have conducted in-depth discussions on it for a long time,but genotype has always been a key factor restricting the efficiency of cucumber in vitro regeneration.Breaking through the genotype barrier is essential to establish an effective in vitro regeneration system.At the same time,an efficient and stable in vitro regeneration system is the basis for cucumber genetic transformation.Therefore,further research on various factors affecting cucumber in vitro regeneration and optimization of the cucumber regeneration system have important theoretical and practical significance.Based on previous studies,this study used different genotypes of cucumber cotyledon node explants as materials to study the regenerative ability of different genotypes of cucumbers,and determined the changes in endogenous hormone content of different genotypes of cucumbers after in vitro culture.In order to explore the key hormones in the process of cucumber adventitious bud induction and differentiation,as well as the influence of exogenous hormones on the regeneration ability of cucumbers,a complete cucumber regeneration system in vitro was established,laying a certain foundation for the genetic transformation of cucumbers.The results of the study are as follows:1.Comparative study on the in vitro regeneration ability of different genotypes of cucumberOn the basis of the cucumber cotyledon node regeneration system established in our laboratory,10 genotypes of cucumber cotyledon node explants were used as materials,and the differentiation ability and endogenous hormone content of different genotype cucumber materials were analyzed.Correlation,reveal the key hormone factors that affect the in vitro regeneration of different genotypes of cucumber,and further optimize the cucumber regeneration system.The results showed that in the MS medium without exogenous hormone addition,the differentiation rate of the 10 materials was significantly different,and the endogenous CTK level was significantly positively correlated with the differentiation rate,and the correlation coefficients were 0.4544（cultivation 0 d）and 0.6110（cultivation 14 d）.Real-time fluorescent quantitative PCR also showed that the expression of the key cytokinin metabolism gene CYP735A has the greatest difference.After exogenous addition of 1.5 mg·L^-16-BA to MS medium,the differentiation rate of different genotypes increased from 5.95%-20.45%to 42.39%-80.43%.Observation of adventitious bud morphology revealed that two different types of adventitious buds were formed during the regeneration of explants:type I normal buds and type II abnormal buds.The ratio of different genotype materials to differentiate into type I and type II adventitious buds is significantly different.The genotypes with a high ratio of type I/type II adventitious buds have a significantly higher rate of regenerated seedlings than those with a low ratio.The results indicate that cytokinin is a key hormone that affects the differentiation vigor of different cucumber genotypes,and the formation of different types of adventitious buds is an important reason for the acquisition rate of cucumber explants regenerated seedlings.2.Changes of Endogenous Hormones in Cucumbers of Different Genotypes in Vitro CultureIn this study,the ELISA hormone determination method was used to determine the explants of cucumbers of different genotypes.They were cultured on MS basic medium and MS⁺induction medium(MS+1.5 mg·L^-16-BA)for 0 d and 14 d.The content of endogenous IAA,CTK,ABA,GA and JA hormones in cotyledon nodes.The results showed that compared with the endogenous hormone content at the initial culture（0 d）,the cotyledon node was cultured on MS minimal medium for 14 d,and the endogenous hormone content showed a different trend,which can be roughly divided into rising,there are three kinds of decline and basically unchanged.Compared with the endogenous hormone content on MS medium in the same period,the change trend of endogenous hormone after adding exogenous cytokinin was similar.It shows that exogenous cytokinin not only affects the content of endogenous CTK,but also affects the synthesis and metabolism of other endogenous hormones.3.Optimization of Exogenous Hormone ratio for Improving the Regeneration Ability of Cucumber in VitroAccording to the changing law of endogenous hormones in cotyledon node explants,the effect of adding different types and proportions of exogenous hormones on the differentiation of cucumber cotyledon node explants was studied.The results showed that the ratio of type I/type II explants produced in different materials had the highest correlation with the ratio of IAA/ABA（R²=0.4214）,and the ratio of type I/type II adventitious shoots was significantly positively correlated with the rate of regenerated shoots（R²=0.5623）.For genotypes C5 and C6 with a low ratio of type I/type II explants（0.52 and 0.46）,a low ratio of adventitious shoots,and a low rate of regenerated seedlings,the induction was carried out by adding different ratios of exogenous NAA and ABA,and C5 and C6 were found When the NAA/ABA ratio was 0.01:0.1,the ratio of type I/type II explants（1.65 and 1.15）,the ratio of adventitious buds（1.81 and 1.17）and the rate of regenerated seedlings（48.57%and53.33%）were significantly improved.According to the existing research foundation in the laboratory,the differentiation medium was optimized by orthogonal experiment using C1 as the material.The results found that:NAA/ABA ratio has a significant effect on the differentiation rate of cucumber cotyledon nodes,and the combination with better results on the differentiation rate the optimal concentration of cytokinin is 0.5 mg·L^-1,the ratio of NAA/ABA is 0:1.0,the ratio of NH₄⁺/NO₃^-is 1:2,and the sampling period of cotyledon nodes is 3 d of germination.This result indicates that the hormone ratio of auxin to abscisic acid has an important effect on the regeneration of cucumber explants.