Transformation Of Populus Tomentosa Carr. With RolB-pttGA20ox Double Genes And Genetic Stability Of Transgenes

Chinese white poplar(Populus tomentosa Carr.)is a native tree species of section Leuce which is widely employed for forest production,forestation and ecological environment construction in the north of China.However,the difficulty of rooting in hardwood cuttings has seriously hindered the practical and industrial process of P.tomentosa.The major objective of this study was to breed new P.tomentosa varieties with improved rooting ability and apical dominance through Agrobacterium tumefaciens-mediate transformation of rolB-pttGA20ox double genes.Meanwhile,the inheritance and expression pattern about rolB-pttGA20ox double genes were also studied by transgenic tobacco.The major results of this study were introduced as follows:1)Optimization of A.tumefaciens-mediated genetic transformation system.Kanamycin(Kan)-resistance test shows that the critical kanamycin sensitive concentrations for inducing shoots and roots of P.tomentosa(PT-16 and PT-19)were 25 and 20 mg/L respectively.The effective genetic transformation process was as follows:pre-culture of leaf discs for 2 d→culture of leaf discs on the regeneration medium containing 200μmol/L AS(pH 5.0)for 4 h→immersed in the agrobacterium solution(OD₆₀₀=0.3-0.4)for 25 min→culturing leaf discs on the co-culture medium containing 200μmol/L AS(pH 5.0,without COCl₂·6H₂O)for 5 d→initial application of Kan selection culture for 3 d.The Kan-resistance shoot regeneration rate reached to 16%with the optimized transformation system.The infection ability of nopaline strain C58 with P.tomentosa was better than that of octopine strain LBA4404.2)Molecular detection oftransgenic poplar.Twenty-nine transgenic plantlets with Kan resistance were obtained through identification of rooting and inducing shoots ability on rooting medium and regeneration medium containing 30 mg/L Kan.The 65.52%positive rate was indicated by PCR analysis.Southern blotting shows that the target genes were integrated into the poplar genome and the integration mode was single-copy.Tissue-specific expression indicates that expression levels of rolB gene were root＞stem＞leaf,and pttGA20ox gene was expressed similarly in different parts of transgenic plantlets.3)Demonstration of the transgene expression in transgenic poplar.The transgenic plantlets exhibited a high sensitivity to auxin and possessed remarkably higher rooting ability compared to control at low auxin levels,rolB gene could be regulated by a short-period induction of exogenous auxin,whereas pttGA20ox gene was not regulated by exogenous auxin.The content of endogenous hormones(GA₃,IAA and ZR)in the transgenic plantlets were significantly different from control.4)Characterization of the function of rolB-pttGA20ox double genes in transgenic poplar.Phenotypic measurement shows that the amount and the length of roots were remarkably higher than those of the control.The average taproot length of transgenic plantlets was 43.3 cm,which was 12.7 cm longer than control.The average height of transgenic plantlets was 2.18 times as large as that of the control.5)Validation of the stable expression of rolB-pttGA20ox double genes in transgenic tobacco.The physiological detection shows that the target genes could steadily express in transgenic tobacco.The environment variations,such as transplantation,did not induce the silencing of transformed genes.6)Demonstration of the genetic stability of rolB-pttGA20ox double genes in transgenic tobacco.PCR screening of T₁ plantlets shows the stable inheritance of genes in the transgenic plantlets.The segregation ratio between transgenic versus non-transgenic plants was observed to be 3:1,which conformed to the Mendelian Law of single gene segregation.This also suggests that there was one insertion locus of transgene in the genome of transgenic tobacco.Physiological detection indicates that rolB-pttGA20ox double genes were expressed in T₁ transgenic tobacco.