CAJ | 학술논문

为了建立玉米高频再生及高效遗传转化体系,对影响玉米胚性愈伤组织诱导的11个因素及影响胚性愈伤分化的9个因素用正交实验方法进行研究.结果显示,基因型对胚性愈伤诱导有极显著影响.6-BA.培养基、AgNO3、2,4-D、ABA对胚性愈伤诱导的影响达到显著水平.多重比较分析显示ABA 2 mg/L每间隔1代添加对胚性愈伤诱导率有显著影响.在影响分化的因素中,基因型和6-BA浓度表现出极强的主效应,NAA、培养基、KT、2,4-D对分化产生显著影响.Southern blotting分析表明,25 mg/L潮霉素选择压下抗性愈伤率作为转化体系优化指标是可靠的.在影响转化效率的因素中,acetosyringone(AS)使用浓度因基因型不同而表现出敏感度差异,共培养温度24～25℃、农杆菌浓度和浸泡时间0.7 OD×15 min,以及pH值5.5～6.2是最高转化率的优选组合.在整合后的玉米遗传转化体系中,黄早4和综31自交系以抗性愈伤率为指标的GUS基因稳定转化率分别达到48.6%和46.2%.
위료건립옥미고빈재생급고효유전전화체계,대영향옥미배성유상조직유도적11개인소급영향배성유상분화적9개인소용정교실험방법진행연구.결과현시,기인형대배성유상유도유겁현저영향.6-BA.배양기、AgNO3、2,4-D、ABA대배성유상유도적영향체도현저수평.다중비교분석현시ABA 2 mg/L매간격1대첨가대배성유상유도솔유현저영향.재영향분화적인소중,기인형화6-BA농도표현출겁강적주효응,NAA、배양기、KT、2,4-D대분화산생현저영향.Southern blotting분석표명,25 mg/L조매소선택압하항성유상솔작위전화체계우화지표시가고적.재영향전화효솔적인소중,acetosyringone(AS)사용농도인기인형불동이표현출민감도차이,공배양온도24～25℃、농간균농도화침포시간0.7 OD×15 min,이급pH치5.5～6.2시최고전화솔적우선조합.재정합후적옥미유전전화체계중,황조4화종31자교계이항성유상솔위지표적GUS기인은정전화솔분별체도48.6%화46.2%.
In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25℃ for co-culture temperature, 0.7 OD×5 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively.