甜瓜自交系‘羊角蜜’植株再生及遗传转化体系的建立

Establishment of Tissue Culture and Genetic TransformationSystems for Cucumis melo L.Inbred Line‘YJM’

组培再生体系是进行植物遗传转化、开展功能基因组学研究的前提条件,然而目前甜瓜的组培再生及遗传转化体系仍不成熟,限制了该领域基因的功能研究和育种应用。本研究以薄皮甜瓜(Cucumis melo L.)自交系‘羊角蜜(YJM)’为试材,系统比较和分析了外植体类型、基础培养基、激素浓度、抗生素和农杆菌菌株等因素对其组培再生和遗传转化的影响。结果表明播种后2 d子叶节的不定芽分化率显著高于5 d后子叶节和下胚轴;相较B5、N6、WHITE和SH四种诱导培养基,外植体在MS或LS上具有更高的愈伤诱导率及不定芽分化率;当培养基中6-BA浓度为0.5 mg L^(-1)时,不定芽诱导率最高、生长状态最好;据此提出‘YJM’播种后2 d子叶节的最佳诱导培养基配方为(MS/LS基础培养基+0.5 mg∙L^(-1)6-BA+1 mg∙L^(-1)ABA+30 g∙L^(-1)蔗糖+9 g∙L^(-1)Agar)。随后,我们比较了三种生根培养基(MS+9 g∙L^(-1)Agar、MS+0.5 mg∙L^(-1)6-BA+1 mg∙L^(-1)ABA+9 g∙L^(-1)Agar和MS+0.8 mg∙L^(-1)IAA+9 g∙L^(-1)Agar)对‘YJM’不定芽生根的影响,发现MS+0.8 mg∙L^(-1)IAA+9 g∙L^(-1)Agar培养基上不定芽生根率显著高于另外两种。进一步分析了外植体对潮霉素、双丙氨膦和卡那霉素三种抗生素的响应,发现潮霉素和双丙氨膦对子叶节的筛选效果显著优于卡那霉素;相较农杆菌菌株GV3101,EHA105转化‘YJM’外植体效果更佳。

Tissue culture is considered a prerequisite for plant genetic transformation and functional genomic research,whilethe current tissue culture regeneration and genetic transformation system of melon is still immature,which limits thefunctional research and breeding application of genes in this field.We systematically compared and analyzed the effects of explants type,basal medium,hormone concentration,antibiotics and agrobacterium strain on tissue culture regeneration andgenetic transformation of Cucumis melo L.inbred line YJM.Adventitious bud differentiation rate of cotyledon segment was significantly higher at 2D after sowing than that of the cotyledons-and hypocotyls-5DAS.In comparison to the explants onB5-,N6-,WHITE-and SH-based ones,the explants had higher callus induction rate and adventitious bud differentiation rateon MS or LS.When the concentration of 6-BA was 0.5 mg L^(-1),the adventitious bud induction rate was the highest andthegrowth state was the best.Therefore,the optimum medium formula for cotyledon node induction 2 days after sowing'YJM'was(MS/LS base medium+0.5 mg∙L^(-1)6-BA+1 mg∙L^(-1)ABA+30 g∙L^(-1)sucrose+9 g∙L^(-1)Agar).Rooting rate was subsequently investigated for the regenerated shoots that were cultured on three different root-initiation media(MS+9 g∙L^(-1)Agar,MS+0.5 mg∙L^(-1)6-BA+1 mg∙L^(-1)ABA+9 g∙L^(-1)Agar,and MS+0.8 mg∙L^(-1)IAA+9 g∙L^(-1)Agar),and the best performance was revealed for those on the medium of MS+0.8 mg∙L^(-1)IAA+9 g∙L^(-1)Agar.We further analyzed the responseof the explants to hygromycin,dialanine and kanamycins,and found hygromycin and dialanine were more effective thankanamycins in screening cotyledon nodes,the transformation efficiency of EHA105 was apparently superior to that of GV3101.