| Chinese jujube(Ziziphus jujuba Mill.),originated in China,belongs to the Rhamnaceae genus.It is one of the characteristic and dominant fruit tree in China,occupying the largest cultivated area comparable to other dried fruit trees.In recent years,genetic engineering technology has been supplied to fruit tree breeding more widely,especially transgenic technology exhibiting powerful tendency to be the high-priority choice on fruit tree breeding.However,as the complexity of genetic background in Chinese jujube and hinders in jujube tissue regeneration,the progress of its gene transformation is still limited.Although there are few reports on jujube gene transformation,the system is incomplete and immature,as problems like poor efficiency and reproducibility strictly constraining the technology in jujube trees.Based on the current knowledge of jujube gene transformation,this experiment was implemented with tissue culture seedlings of different genotypes.Parameters,incorporating placement status of co-cultural leaf,hormones in the medium,were valued by testing resistant callus rate to optimizing the system of jujube gene transformation.The results are as follows:1.Influence of leaf placement status(paraxial surface and abaxial surface)on the transformation rate of resistant callus during co-cultivation.The resistant callus on the abaxial surface contacted the medium grew better with more fresh color during co-cultivation;etiolation emerged around the resistant callus on the abaxial surface contacting to medium,and ultimately died after a long time.In addition,adaxial surface contact co-cultivation of leaves also performed higher resistant callus transformation rate.Therefore,in the follow-up investigation of other factors should be treated with the adaxial surface contacting medium.2.Effect of antibiotics on the transformation efficiency.The results of preliminary experiments show that the antibacterial effect is better when the concentration of cephalosporin(Cef)is 350 mg/L.Statement of resistant callus also supported the condition of 350 mg/L Cef was more suitable compring to 250 mg/L carbenicillin(Carb).3.Effect of hormone types on the transformation rate of resistant callus.The test results show that the resistant callus induced by the hormone NAA displayed better condition with bright green color and large callus;yet callus by the hormone IBA exhibited yellowish-brown edge and low vitality.In addition,the resistant callus transformation rate was higher when addling NAA than IBA.Adding NAA at the concentration of 0.5 mg/L to cultural medium had a better effect on jujube resistant callus induction.4.The jujube genetic transformation system was established and optimized:the leaf adaxial surface contacted the medium during co-cultivation,the growth regulator combination in the medium was 1.0 mg/L TDZ+0.5 mg/L NAA,and the antibiotic used 350 for the selection of resistant buds.mg/L Cephalosporin(Cef).5.Screening of target gene.Our research team previously found that ZjCNGC2 plays an vital role in the response of jujube to low temperature stress and ZjAC2 as the key gene regulating cAMP synthesis in jujube.Thusly,ZjCNGC and ZjAC2 were selected for transformation by the construction of plant overexpression vectors pCAMBIA 1302-ZjCNGC2 and PBI121-ZjAC2 and Agrobacterium tumefaciens(GV3101)mediated gene transformation.6.Screening of different genotypes of jujube for transformation.Based on the provious research mentioned before,the reformed process,consisting of adaxial surface contact medium,350 mg/L cephalosporin(Cef),0.5 mg/L NAA resistance medium,was used to find out suitable jujube genotypes for gene transformation.The results demonstrate that:of all 11 jujube genotypes infected by the bacterial liquid GV3101 containing ZjCNGC2,5 genotypes grew resistant callus,a total of 8 pieces of callus;of all 27 jujubes infected by GV3101 embracing ZjAC2,there were 11 genotypes with resistant calli,a total of 60 pieces of callus.The callus was identiied by amplifying GFP gene,peculiarly existing in positive callus,with PCR-agarose electrophoresis at first and the LUROR-3415 handheld dual-band fluorescent protein excitation lamp was used for further identification which screened 6 ZjAC2 positive callus.Finally,three jujube genotypes that are relatively easy to transform by ZjAC2 were screened out,namely 5-⑨-12-28,DP1 and DP2. |
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