Establishment Of A Transient Grape Callus Transformation System And Functional Analysis Of Candidate Genes For Cold Tolerance

Grapes(Vitis L.)are economically significant fruit crops worldwide,offering considerable economic,social,and ecological benefits.With the completion of the grape genome sequencing in 2007,functional genomics has emerged as a focal point in grape research.However,efficient and straightforward transformation techniques are crucial in advancing grape gene function studies.Transient genetic transformation serves as an essential tool for functional research and a vital step in gene function analysis.Due to the highly heterozygous grape genome,extended periods of vegetative growth,difficulties in regeneration,and substantial challenges in genetic transformation,progress in functional grape gene research has been constrained.Typically,grape gene function studies are conducted through heterologous expression in model plants such as Arabidopsis and tobacco.Consequently,establishing an efficient and rapid method for gene identification is indispensable.In this study,various grapevine tissues,including ’Cabernet Sauvignon’ small flower buds,leaves,petioles,shoot tips,and ’Chardonnay’leaves,petioles,and shoot tips,were used to examine the effects of different culture media on the induction and proliferation of callus tissues in different cultivars.The optimal conditions for callus induction and proliferation were identified,and an Agrobacterium-mediated grape transient transformation system was established through screening various transient transformation parameters,such as infection methods,Agrobacterium suspension concentrations,infection times,acetosyringone(AS)concentrations,and co-cultivation durations.Additionally,the feasibility of this transient transformation system was validated by transiently expressing the grape cold-tolerant gene VaCIPK18 and evaluating its expression levels and fresh weight changes under low temperatures.This research provides valuable insights and foundations for studying the functions of key grape genes.The main research findings are as follows:1.The optimal culture media for grape callus induction and proliferation were optimized.The best induction medium was B5-Ⅱ,with callus induction rates of 100%for ’Cabernet Sauvignon’ small flower buds,leaves,petioles,shoot tips,and ’Chardonnay’ leaves,petioles,and shoot tips in B5 medium(B5-Ⅱ)containing 0.05 mg·L-1 NAA,0.5 mg·L-1 2,4-D,and 2.0 mg·L-1 KT.The optimal proliferation medium was B5-I,where callus tissues from ’Chardonnay ’ and’Cabernet Sauvignon’ leaves exhibited the most favorable proliferation effects in B5 medium(B5-I)containing 0.5 mg·L-1 NAA and 2.0 mg·L1 6-BA,with fresh weight approximately doubling every seven days.2.An Agrobacterium-mediated transient transformation system for ’Chardonnay’ callus tissues was established.The plant expression vector pCAMBIA2301,containing the GUS reporter gene,was transferred into callus tissues using Agrobacterium tumefaciens strain EHA105,and transformation conditions were screened via GUS histochemical staining and GUS enzyme activity analysis.Optimal infection conditions were determined to be sonication followed by vacuum infiltration.Further orthogonal design experiments were conducted to study the main factors influencing the transient transformation system,including bacterial suspension concentration(OD600=0.6,0.8,1.0,1.2),AS concentration(0,100,200,300 μM),infection time(3,9,15,21 min),and co-cultivation time(1,2,3,4 d).The results indicated that the optimal parameters were an Agrobacterium strain EHA105 with OD600=0.8,subjected to 4 min of sonication followed by 5 min of vacuum infiltration and 1 day of cocultivation.Under these conditions,the GUS enzyme activity reached 352.96 ± 33.95 mol 4MU/mg/min.Moreover,it was found that callus tissues were maximally inhibited at a concentration of 75 mg·L-1 kanamycin.3.The feasibility of the Agrobacterium-mediated transient transformation system for ’Chardonnay’callus tissues was validated using VaCIPK18.Under 4℃ and-4℃ treatments,the relative gene expression of VaCIPK18 in transformed ’Chardonnay’ callus tissues was significantly higher than in untransformed callus tissues.After 10 days of treatment at 4℃ and-4℃,the net weight of the transformed callus tissues was significantly higher than that of the untransformed tissues.These results suggest that the transiently transformed ’Chardonnay’ callus tissues expressing VaCIPK18 exhibited enhanced stress resistance,demonstrating the applicability of the Agrobacterium-mediated transient transformation system for studying grape stress resistance gene functions.