基于高遗传转化率及低幼苗玻璃化率的黑果枸杞叶片组合培养体系  

A combined culture system for leaf explants of Lycium ruthenicum with high genetic transformation rates and low seedling vitrification rates

作　　者：闫婷[1,2] 乌日恒 鲁敏 杨荣 王美珍[1] 刘雪锋 YAN Ting;WU Riheng;LU Min;YANG Rong;WANG Meizhen;LIU Xuefeng(Inner Mongolia Academy of Forestry Sciences,Hohhot 010010,China;Black Wolfberry EngineeringResearch Center,Inner Mongolia Autonomous Region,Hohhot 010010,China;Key Laboratory of SandyEcosystem and Ecological Engineering,Inner Mongolia Autonomous Region,Hohhot 010010,China)

机构地区：[1]内蒙古自治区林业科学研究院,呼和浩特010010 [2]内蒙古自治区黑果枸杞工程[技术]研究中心,呼和浩特010010 [3]内蒙古自治区沙地[沙漠]生态系统与生态工程重点实验室,呼和浩特010010

出　　处：《西北植物学报》2024年第1期53-62,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：内蒙古自治区自然科学基金项目(2022QN03020);内蒙古林业与草原局科研能力提升项目(104004001)。

摘　　要：【目的】建立高效稳定的黑果枸杞遗传转化体系,有效降低其再生幼苗玻璃化率,促进其基因功能研究和提高遗传改良效率。【方法】以黑果枸杞叶片作为外植体,利用农杆菌(LBA4404、EHA105)介导的遗传转化法,通过调整基础培养基类型并添加相应浓度的植物激素,筛选出最适愈伤组织诱导培养基、分化和选择培养基、生根诱导培养基,将黑果枸杞遗传转化率提高到65%以上,同时降低幼苗玻璃化率至10%以下。【结果】(1)黑果枸杞叶片高效组合培养体系中最佳农杆菌侵染浓度(OD_(600))为0.6,侵染时间为25 min,在此条件下侵染叶片抗性愈伤诱导率达78.2%~96%;(2)黑果枸杞遗传转化中最适分化和选择培养基为:MS+肌醇50 mg/L+烟酸0.25 mg/L+维生素B60.25 mg/L+铁盐母液1 mL/L+甘氨酸1.0 mg/L+维生素B10.05 mg/L+6-BA 0.25 mg/L+蔗糖30 g/L+琼脂6 g/L+卡那霉素30 mg/L+特美汀300 mg/L(pH=6.0);最适生根诱导培养基为:WPM+IBA 0.25 mg/L+蔗糖30 g/L+琼脂6 g/L+卡那霉素30 mg/L+特美汀300 mg/L(pH=6.0);(3)在最适分化和选择培养基中,农杆菌LBA4404-pBI121侵染叶片外植体产生的幼苗玻璃化率约为65%,而农杆菌EHA105-pBI121侵染处理则低于10%;(4)采用木本植物低盐WPM培养基可使黑果枸杞再生幼苗生根效率达到81.2%左右。(5)将阳性愈伤数量与总接种叶片数量比值作为遗传转化效率的评价指标,在最适遗传转化体系中,农杆菌LBA4404-pBI121和EHA105-pBI121侵染后遗传转化率分别为51%和65.2%。【结论】黑果枸杞叶片高效组合培养体系能够显著提高其遗传转化率,降低玻璃化幼苗的发生率。[Objective]We aim to establish an efficient and stable genetic transformation system of Lycium ruthenicum and reduce the vitrification rate of the regenerated seedlings in order to promote gene function study and genetic improvement.[Methods]L.ruthenicum leaves were used as explants and Agrobacterium(LBA4404,EHA105)was used to transform L.ruthenicum.By adjusting the types of basic medium and adding plant hormones,we screened the optimal callus-induction medium,differentiation and selection medium,and rooting-induction medium.The transformation rate of L.ruthenicum was increased to over 65%,while the seedling vitrification rate was decreased to below 10%.This combined culture system laid a foundation for the molecular breeding of L.ruthenicum.[Results](1)The optimal infection concentration of Agrobacterium(OD600)was 0.6 and the infection time was 25 min for the combined culture system of L.ruthenicum.Under this condition,the callus-induction rate was 78.2%-96%;(2)The optimal differentiation and selection medium contained:MS+inositol 50 mg/L+nicotinic acid 0.25 mg/L+vitamin B60.25 mg/L+Fe salt storage solution 1 mL/L+glycine 1.0 mg/L+vitamin B10.05 mg/L+6-BA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L+Kanamycin 30 mg/L+Timentin 300 mg/L,pH 6.0.The optimal rooting medium contained:WPM+IBA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L+Kanamycin 30 mg/L+Timentin 300 mg/L,pH 6.0.(3)On the optimal differentiation and selection medium,the seedling vitrification rate infected by Agrobacterium LBA4404-pBI121 was about 65%,while that infected by Agrobacterium EHA105-pBI121 was below 10%.(4)The rooting efficiency of the regenerated seedlings reached 81.2%using a low-salt WPM medium of woody plants.(5)The ratio of the positive callus to the total number of inoculated leaves was used to evaluate the transformation efficiency.Using the optimal transformation system,the transformation rates of Agrobacterium LBA4404-pBI121 and EHA105-pBI121 were 51% and 65.2%,respectively.[Conclusion]The combined culture system of L.ruthenicum leaves can improve t

关 键 词：黑果枸杞 农杆菌 组合培养体系 转化效率 幼苗玻璃化 

分 类 号：Q945.5[生物学—植物学] S567.19[农业科学—中草药栽培]