阔叶猕猴桃抗生素敏感性及遗传转化的研究

以阔叶猕猴桃(Actinidia latifolia)叶片为外植体,研究了各种抗生素对形态分化和器官形成的影响,并以叶盘为受体,通过根癌农杆菌介导法获得了抗性愈伤组织和抗性芽,组织化学染色表明gus基因已在植物组织中表达。研究结果表明,对不定芽分化来说,头孢霉素(Cef)效果明显好于羧苄青霉素(Carb)。Cef浓度为300mg.L-1时效果最好,再生频率达100%,平均再生芽数达最大为9.76个芽/外植体。生根过程中高浓度的Cef(≥200mg.L-1)明显抑制生根,而Carb对生根没有显著影响。在卡那霉素(Kan)和Carb组合处理中,Carb对生根没有显著影响,但Kan明显抑制生根。随着Kan浓度的升高,生根率和平均根数迅速减少,当浓度升高到50mg.L-1时,生根完全被抑制。因此,农杆菌介导的阔叶猕猴桃遗传转化中愈伤组织和不定芽诱导过程宜选用300mg.L-1Cef来抑制农杆菌生长,Kan筛选的临界浓度为20mg.L-1。在抗性芽生根过程中Kan浓度应该控制在50mg.L-1以内,并改用Carb作为杀菌剂,生根效果会更好。该研究确定了农杆菌介导的阔叶猕猴桃叶片遗传转化中使用抗生素的种类和用量,为通过基因工程对阔叶猕猴桃进行遗传改良奠定了基础。

STUDIES ON ANTIBIOTICS SENSITIVITY AND GENETIC TRANSFORMATION OF Actinidia latifolia

Effects of antibiotics on in vitro regeneration and organogenesis from leaf explants of Actinidia latifolia were studied.Genetic transformation calli of A.latifolia were generated after co-cultivation of leaf segments with Agrobacterium tumefaciens strain.Successful transformation was confirmed by histochemical analysis of gus activity in kanamycin-resistant calli and leaves.Results showed that cefotaxime is superior to carbenicillin for shoot differentiation.When cefotaxime concentration was 300mg·L~(-1),shoot differentiation rate and mean number of shoots per explant both reached the highest levels,100% and 9.76 shoots/explant.However,a negative effect on in vitro rooting of regenerated shoots by higher cefotaxime concentrations(≥200mg·L~(-1)) was also seen.Carbenicillin had no marked influence on rooting.In the treatments of different combinations or alone of kanamycin and carbenicillin on rooting,carbenicillin had no marked influence on rooting but kanamycin inhibited markedly rooting.With the increase of kanamycin concentration,the rooting rate decreased sharply.Rooting was completely inhibited on the media containing 50mg·L~(-1) kanamycin.It was considered that for A.latifolia 300mg·L~(-1) cefotaxime was suitabie for elimination of Agrobacterium tumefaciens during transgenic shoot induction;20mg·L~(-1) kanamycin was useful in the selection of transgenic versus false-positive shoots;during rooting phase kanamycin concentration should be controlled under 50mg·L~(-1),and carbenicillin substitute for cefotaxime to elimination of Agrobacterium facilitated rooting.This study had determined the type and concentration of antibiotics in Agrobacterium-mediated transformation of A.latifolia,and laid a good foundation for germplasm improvement through genetic engineering.