摘要
为了丰富栽培小麦转化受体基因型,优化农杆菌转化小麦幼胚的技术体系,本研究评价了21份四川优良小麦品种(系)的幼胚再生能力,并以筛选到的最佳受体为材料,利用正交设计研究了预培养时间、菌液浓度、侵染时间、抑菌剂浓度、共培养方式和农杆菌菌株等易互作的6个因素对幼胚抗性愈伤再生率的影响,并探讨了筛选剂卡那霉素(Kanamycin)的适宜浓度。结果表明,21份材料中‘5157’、‘川农16’、‘R364’、‘川麦42’‘、内麦8号’的绿苗率超过了30%,可作为优良转基因受体加以利用。以‘5157’为受体材料建立农杆菌转化体系,正交试验表明菌液浓度和农杆菌菌株对幼胚抗性愈伤再生率的影响达到显著水平,而其他因素影响不显著。经优化后的遗传转化体系为:农杆菌菌株用C58C1,幼胚不经预培养,在菌液OD值为0.4的农杆菌中侵染40 min,共培养介质选用培养基,抑菌剂为200 mg/L羧卞青霉素(Carbenicillin),适宜的卡那霉素筛选浓度为50 mg/L。本研究为四川小麦遗传转化提供了5个候选基因型,为建立和优化小麦幼胚转化技术体系提供了理论依据,同时初步建立了农杆菌转化小麦幼胚的技术体系。
In order to enrich the genotype of wheat transformation receptor and optimize the technical system of wheat immature embryo transformation by Agrobacterium tumefaciens,the regeneration ability of immature embryos of 21 Sichuan wheat varieties(lines)was evaluated,and the optimal receptors were selected as materials.The pre-culture time,concentration of bacterial solution,infection time and bacteriostasis were studied by orthogonal design.The effects of six factors,such as concentration,co-culture mode and Agrobacterium strains,on the regeneration rate of immature embryo-resistant callus were studied.The suitable concentration of kanamycin was also discussed.Results indicated that the green plant regeneration rate of varieties(lines)'5157','Chuannong 16','R364','Chuanmai 42',and'Neimai 8'were more than 30%,which could be used as superior genotypes in immature embryos culture and transformation.The transformation system of Agrobacterium tumefaciens was established with'5157'as acceptor material.The orthogonal test showed that the concentration of bacterial solution and Agrobacterium tumefaciens strain had significant effect on the regeneration rate of immature embryo resistant callus,but other factors had no significant effect.The optimized genetic transformation system was as follows:Agrobacterium strain C58C1,immature embryo without pre-culture,Agrobacterium strain with OD value of 0.4 was infected for 40 minutes,co-culture medium was used,inhibitor was 200 mg/L carbenicillin,and the suitable concentration of kanamycin was 50 mg/L.This study provided five candidate genotypes for Sichuan wheat genetic transformation.Also,it provided theoretical references for establishment and optimization of the transformation system of wheat immature embryo,and preliminarily established the technical system of Agrobacterium mediated transformation of wheat immature embryos.
作者
蒋云
张洁
宣朴
余桂容
张军
陈谦
王颖
尹春蓉
郭元林
Jiang Yun;Zhang Jie;Xuan Pu;Yu Guirong;Zhang Jun;Chen Qian;Wang Ying;Yin Chunrong;Guo Yuanlin(Biotechnology and Nuclear Technology Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu,610061;Institute of Agro-products Processing Science and Technology,Sichuan Academy of Agricultural Sciences,Chengdu,610061;Triticeae Research Institute,Sichuan Agricultural University,Wenjiang,611130;Sichuan Academy of Botanical Engineering,Neijiang,641200)
出处
《分子植物育种》
CAS
CSCD
北大核心
2019年第4期1203-1212,共10页
Molecular Plant Breeding
基金
四川省财政创新能力提升工程项目(2016ZYPZ-004)
"十三五"四川省公益性育种研究专项项目(2016NYZ-0030)
国家重点研发计划"主要农作物诱变育种"项目(2016YFD0102101)共同资助
关键词
小麦
农杆菌
遗传转化
正交设计
幼胚培养
Wheat
Agrobacterium
Genetic transformation
Orthogonal test design
Immature embryos culture
