Establishment Of Receptor System For Genetic Transformation Of Fraxinus Mandshurica Rupr. And Study On Genetic Transformation

Fraxinus Mandshurica Rupr.Rupr.is a tall deciduous tree of the genus of Fraxinus,and it is one of the important precious broad-leaved tree species in the forest area of Northeast China.It is one of the main afforestation tree species in Heilongjiang Province,and it is also an important industrial timber tree species and ecological forest construction tree species,with high ecological and economic value.In order to improve the micropropagation efficiency of Fraxinus Mandshurica Rupr.Rupr.,the laboratory has obtained the genes ESR1,CUC1 and the photoperiod gene LWD1 related to the differentiation of Fraxinus Mandshurica Rupr.adventitious buds.Based on the previous research of this study,the establishment of different genetic transformation receptor systems of Fraxinus Mandshurica Rupr.and the genetic transformation mediated by Agrobacterium were carried out,in order to further improve the genetic transformation system of different explants in Fraxinus Mandshurica Rupr.,explore the influence of double gene overexpression on the genetic transformation efficiency of Fraxinus Mandshurica Rupr.,and provide technical support and theoretical basis for the genetic improvement of Fraxinus Mandshurica Rupr..The following results were obtained through research:1.The establishment of Fraxinus Mandshurica Rupr.spherical embryo induction system:2 mg/L 2.4-D or 2 mg/L 2.4-D and 3 g/L potassium citrate induced suspension cells,both of which could produce dense spherical cell masses,which induced a larger number of spherical cell masses and an induction rate of 67.33%.Potassium citrate alone 3 g/L induces the proliferation of spherical embryos best,and the number of new cell clusters can reach about85/g.2.The genetic transformation condition of Fraxinus Mandshurica Rupr.leaves and petioles as receptors was optimized:the best methods for the genetic transformation of leaves were as follows:Agrobacterium concentration OD ₆₀₀=0.6,ultrasound 90 s+immersion for 5min+vacuum for 5 min,culture for 3 days,kanamycin and cephalosporin combination concentrations of 30 mg/L and 200 mg/L were selected for screening.The combination concentrations of hygromycin and cephalosporin were selected as 20 mg/L and 350 mg/L.The best methods for the genetic transformation of petioles were as follows:OD ₆₀₀=0.6concentration of Agrobacterium,ultrasound 120 s+immersion for 1 min+vacuum for 7 min,culture for 3 d in total,kanamycin concentration of 20 mg/L,and the rest was the same as that of leaves.3.Expression of Fraxinus Mandshurica Rupr.FmESR1,FmLWD1 and FmCUC1 genes and construction of interference vectors:The interference vectors of FmLWD1 and FmCUC1:they are named RNAi-FmLWD1 and RNAi-FmCUC1.;The expression vectors of the dual gene in FmESR1,FmLWD1 and FmCUC1 genes:they are named FmESR1-LWD1,FmLWD1-CUC1and FmESR1-CUC1.4.Genetic transformation of different plant receptor tissues of Fraxinus Mandshurica Rupr.:In the genetic transformation of suspension cells and spherical embryos,the transformation effect of spherical embryos was better,among which FmLWD1-CUC1,double gene overexpression made the growth amount up to 0.96g.Among the genetic transformation of leaves,the callus induction rate of FmESR1-LWD1 double gene overexpression was 48.89%.In the genetic transformation of petioles,FmESR1 single gene overexpression had high induction rates on callus,which were 63.33%,respectively.In the genetic transformation of the hypocotyl,overexpression of FmESR1 gene contributed to the formation of adventitious buds,and the statistical results of 17 days showed that it was twice that of non-transgenic shoots.Among the genetic transformation of stem segments with bud points,the germination rate of FmCUC1 monogene overexpression was the highest,95%,and the lowest germination rate of FmCUC1 silent bud points was 38.15%.5.Quantitative analysis of genetic transformation of different receptors of Fraxinus Mandshurica Rupr.:In the resistance callus of leaves and petioles,the highest relative expression was 1.62 of the FmLWD1-CUC1 double gene overexpression;the highest relative expression in the hypocotyl was 2.87 of the FmESR1-LWD1 double gene overexpression;and the highest relative expression was 3.06 of the LWD1 gene in the overexpression of the FmESR1-LWD1 double gene in the bud point stem segment.