菊花‘金不凋’再生及遗传转化体系的构建

Construction of Regeneration and Transformation System of Chrysanthemum ’Jinbudiao’

‘金不凋’属于典型的日中性菊花,可作为日中性菊花开花分子机理的理想研究材料,目前尚未建立‘金不凋’的再生及遗传转化体系。本研究以‘金不凋’叶片为外植体,分别对影响再生及转化体系的主要因素进行试验。结果表明‘:金不凋’叶盘愈伤组织的不定芽分化最佳培养基为MS+6-BA 1.5 mg/L+NAA 0.5 mg/L,不定芽分化率为90.0%,平均再生不定芽数达5.5556个;相关性分析表明褐化率与愈伤诱导率以及平均再生愈伤组织数之间存在显著的负相关关系;试管苗最佳生根培养基为MS,生根率为100%。在‘金不凋’再生体系建立的基础上,初步建立了其遗传转化体系:‘金不凋’叶盘经过24 h预培养,在菌液浓度OD600=0.6的农杆菌中侵染10 min,共培养2 d,延迟培养5 d,叶片不定芽分化潮霉素临界浓度为2 mg/L,生根筛选潮霉素临界浓度为5 mg/L时,转化效率最高。通过潮霉素抗性筛选和PCR鉴定,获得3株PCR阳性菊花苗,转基因阳性苗频率为2.5%,本研究为‘金不凋’的基因工程育种提供了技术基础。

’Jinbudiao’is a typical day-neutral chrysanthemum,which can be used as an ideal research material for the molecular mechanism of day-neutral chrysanthemum flowering.However,the regeneration and genetic transformation system of’Jinbudiao’has not been established.In this study,the leaves of’Jinbudiao’were used as explants,and the main factors influencing regeneration and transformation systems were tested.The results showed that the optimal medium for adventitious bud differentiation of’Jinbudiao’leaf callus was MS+6-BA 1.5 mg/L+NAA 0.5 mg/L,the adventitious bud differentiation rate was 90.0%,and the average number of regenerative adventitious buds was 5.5556.The correlation analysis showed that there were significant negative correlation between the browning rate and the callus induction rate and the average number of regenerative callus.The best rooting medium of seedlings was MS,rooting rate was 100%.The genetic transformation system was preliminary established:the’Jinbudiao’leaf disk was pre-cultured for 24 h,and infected for 10 min in Agrobacterium with bacterial concentration OD600=0.6,co-culture for 2 days,and delayed screen time for 5 days,the critical concentration of hygromycin in adventitious bud differentiation and rooting was 2 mg/L,5 mg/L respectively.3 transgenic chrysanthemum lines were obtained by hygromycin resistance screening and PCR analysis,and the transgenic frequency of transgenic plants was 2.5%,which laid the foundation for the genetic engineering breeding of’Jinbudiao’.