Establishment Of Genetic Transformation System Of Walnut (Juglans Regia L.) And RolB Gene Introduction

Walnut (Juglans regia L.) is a difficult-to-root woody plant. There is large difference amongdiffernet species, which restrict factory production of seedlings and standard garden growing. Rooting ispressing problem urgently need to be sloved as well as significant breeding aim in walnut industry. Theprimary objective of this research was to develop protocols for the Agrobacterium tumefaciens mediatedtransformation of an elite walnut genotype. Because of Juglans regia cv. Lvbo juvenile nature, in vitroembryos have a high potential for regeneration used as experimental materials. Their stems with budsand leaves were taken as explants so that an experiment could be carried on the callus formation,adventitious buds induction and differentiation, the growth and development, and the rooting. In thetissue culture, different treatment methods and the combination of various type and mass concentrationhormones were used for each stage. The growth and development of media was investigated, therebythe optical combination of plant growth regulators (PGRs) was acquired through the survey. On thisbasis, a regeneration system of walnut was established. Through adding Kanamycin and Cefotaximeinto medium of adventitious buds formation, the influence of these on the growth of explants wereobserved in order to confirm the minimum lethal mass concentration. Then the synthesis andmodification of the target gene fragment were completed. After restriction enzyme digestion ofexpression vector PENR(K)-LNY, the target gene fragment connecting with expression vector wastransferred to Escherichia Coli. The plant expression vector was built successfully. Genetictransformation conditions of Agrobacterium mediated were explored, including the comparativeanalysis of various influencing factors, the choice of appropriate preincubation time, the suitableinfection time, co-cultivation time and antibiotic concentration. Genetic transformation system forwalnut was established. Finally, transgenic plants on the filter were detected by fluorescent proteindetection and PCR detection in order to check integrated situation of rolB gene.The main research result and innovation point as follows:1. The stage of in vitro germination of walnut embryos: after the disinfecting treatment with0.1%mercuric chloride adding0.1%Tween-80for five minutes, the highest germination rate is68.49%.After30days the best result achieved was84.56%using the DKW medium. During thegermination process it can be seen that the maximum value of germination achieved was after10-15days, with no increase after that.2. The optimal differentiation medium of adventitious buds was DKW+6-BA0.5mg/L+IBA0.05mg/L+sucrose30g/L+agar6.5g/L, and the regeneration rate is2.96.3. The stage of rooting of walnut seeding: a two-phase in vitro rooting protocol was used. For rootinduction phase, the best dark treatment time was between6and8days. The sucrose content andnitrogen concentration of the medium were40g/L and1416mg/L in root development phase, andthe rooting rate was53.26%and45.14%respectively.4. The stage of callus culture of leaves: there was a significant influence on leaves callus formation for dark incubation time. After2weeks of dark treatment, callus rate of leaves could be up to100%.The increase of dark incubation time (three weeks) would inhibit the formation of leaves callus.Low concentration of TDZ (2mg/L to4mg/L) promoted the formation of leaves callus, but the highconcentration of TDZ (8mg/L to10mg/L) inhibited the formation and browning of leaves callusand made the structure of callus more porous. Besides, there was a positive role in advancing theformation of callus for2,4-D, IBA and NAA, but the performance was different. Lowconcentration of2,4-D (1.0mg/L) promoted the formation of leaves callus, but high2,4-D(2.0mg/L)inhibited the formation of callus with its concentration enrichment. Low concentration ofIBA (0.01mg/L to0.5mg/L) was conducive to leaves callus formation, and the high (1.0mg/L) wasbad for leaves callus formation. The concentration of NAA in the range of the test (0.1mg/L-0.5mg/L) had been promoting the formation of callus.5. Plant expression vector construction of rolB: cDNA sequence of rolB from Agrobacterium Riplasmid was found from GenBank in the test. After ORF optimization, the target gene fragmentwas cloned into pSPORT1vector, which was sequenced for verification. The cDNA fragment ofrolB was successfully ligased to the downstream of CaMV35S promoter in plant expression vectorPEZR(K)-LNY containing YFP marker gene by gene recombination technology.6. The vector was transformed into walnut by Agrobacterium mediated method. After10and15dayspreincubation time, the transformation rate was up to3%or1.33%. When the concentration of Kanwas0mg/L, the induction rate of adventitious buds was90%. There was only a very small numberof adventitious, when the concentration of Kan was100mg/L, and the stem segment with leavesgradually went yellow. The200mg/L or400mg/L mass concentration of Kan made the explantsdied. Therefore, The100mg/L mass concentration of Kan was used for screening transgenic plantsserving as nomal level.7. Identification of transgenic plants: By100mg/L Kan and500mg/L Cef screening, the living bodyleaves were studied by laser scanning confocal microscope (LSCM), and the YFP could be seen.After further PCR amplification testing, the result showed3plants were positive, which stated rolBgene had been integrated into walnut genome.