菜心EMS诱变条件优化与突变体库构建

Optimization of EMS mutagenesis condition and construction of mutant library in flowering Chinese cabbage

【目的】明确甲基磺酸乙酯(EMS)诱变处理菜心种子的最佳浓度和处理时间,为EMS诱变技术在菜心种质资源创新利用方面及丰富菜心种质资源选育提供支持。【方法】设4种不同浓度的EMS溶液(0.2%,0.4%,0.6%和0.8%)和3种不同处理时间(8、12和16 h)共12个组合,诱变处理2个菜心自交系(C40和小80天)的种子,并对诱变后代出苗率和结籽株比例进行分析,筛选EMS诱变菜心种子的最佳处理条件。【结果】随着EMS浓度的增加,菜心种子成苗率和结籽株比例逐渐降低。诱变处理8 h,EMS浓度为0.6%时,C40和小80天菜心的成苗率分别65%和70%,结籽株比例分别为13%和31%;EMS浓度升高至0.8%时,C40菜心的成苗率降至50%,2个菜心材料的结籽株比例均低于20%。随着EMS处理时间的延长,菜心种子成苗率和结籽株比例逐渐降低。EMS处理浓度为0.4%时,诱变12 h,2个菜心材料(C40和小80天)的成苗率均为75%,结籽株比例分别为30%和45%;诱变16 h,C40和小80天菜心的成苗率分别降至60%和55%,结籽株比例均低于20%。EMS诱变处理不仅抑制成苗率,还严重影响结实情况。根据成苗率和M_1群体植株的结籽株比例,并保证最大诱变效率和突变体群体,确定菜心种子EMS诱变处理的最佳条件为0.4%EMS诱变处理12 h。不同基因型菜心材料对EMS的耐受性不同,生长势强的菜心材料EMS处理的时间或浓度可适当增大或降低。采用最佳诱变条件处理3000粒小80天菜心种子,在M_1群体中出现黄化、白化、皱缩、矮化及嵌合等变异性状,M_1群体包含500个突变株系。随机选取种子量大的M_1群体中176个家系,构建包含2110个单株的M_2群体,在M_2群体中发现94个突变单株,总的突变频率为4.3%。其中,叶片叶色或形状突变的单株61株,株型变异的单株共31株,花色突变的单株2株。【结论】利用EMS诱变菜心种子有明显效果,构建的菜心EMS突变体库表型变异丰富,尤其是出现株型紧凑,叶色浓绿的有益突变,可用于菜心功能基因组学研究和育种。

【Objective】This research aimed to clarify the optimal concentration and time of ethylmethylsulfone(EMS)mutation treatment of flowering Chinese cabbage,it provided support for the innovation and utilization of EMS mutagenic technology in flowering Chinese cabbage germplasm resources and enriching the selection of flowering Chinese cabbage germplasm resources.【Method】Two high-generation inbred lines C40 and Xiao 80 Days were mutated by EMS solution of different concentrations(0.2%,0.4%,0.6%and 0.8%)under different treatment times(8,12 and 16 h).The adult plant rate and rate of seed-setting plant after mutation were investigated to determine the best mutagenesis conditions.【Result】Under the same EMS treatment time,the adult plant rate and seed-setting plant rate of flowering Chinese cabbage decreased gradually with the increase of EMS concentration.When the EMS concentration was 0.6%,the adult plant rate of C40 and Xiao 80 Days were 65%and 70%,respectively;and the seed-setting plant rate of two materials were 13%and 31%respectively.When EMS concentration was 0.8%,the adult plant rate of C40 decreased to 50%,and the seed-setting plant rate of both materials was less than 20%.Under the same concentration of EMS,the longer the mutagenesis time was,the worse the adult plant rate and seed-setting plant rate.When EMS concentration was 0.4%,mutagenesis time was 12 h,the adult plant rates of C40 and Xiao 80 Days were both 75%,and the the seed-setting plant rate of two materials were 30%and 45%respectively.After 16 h,the adult plant rates were reduced to 60%and 55%,and the seed-setting plant rates were reduced,both below 20%.EMS treatment not only inhibited the adult plant rate,but also seriously affected the seed-setting plant rate.According to the adult plant rate and the seed-setting plant rate of M1 population,and to ensure the maximum mutation efficiency and mutant population,the optimal EMS mutatgenic condition was determined as 0.4%EMS mutation treatment for 12 h.The tolerance of different genotypes of flowering Chinese cabbage to EMS was different,and the treatment time or concentration of EMS could be appropriately increased or decreased on plants grew well.3000 seeds of Xiao 80 Days were treated with the best mutagenic conditions,and 500 M1 mutant lines were obtained.In M1 population,there were some variation characters such as yellowing,albinism,shrinkage,dwarfing and chimerism.176 M1 families with large seed quantity were randomly selected to construct M2 population with 2110 individuals.94 mutant individuals were found in M2 population,and the total mutation frequency was 4.3%.There were 61 plants with leaf color or shape mutation,31 plants with plant type mutation and 2 plants with flower color mutation.【Conclusion】EMS mutagenesis of flowering Chinese cabbage seed has obvious effect.The EMS mutant library is rich in phenotypic variation,especially the beneficial mutation of compact plant type and dark green leaf color,which can be used in the functional genomics research and breeding of flowering Chinese cabbage.