小麦小孢子培养影响因素研究

Study on Factors Affecting Microspore Culture in Wheat

为优化小麦小孢子培养技术,建立实用化的小麦小孢子培养技术体系,以冬小麦品种新麦35和西农059及春小麦品种Bobwhite为供试材料,研究了预处理方式和不同分离液对小麦小孢子活力及形成胚性小孢子的影响,并对不同培养密度下小孢子形成愈伤组织的诱导率进行分析,比较了两种分化培养基对胚状体和愈伤组织分化率的影响。结果表明,与高温加甘露醇预处理相比,对麦穗进行低温预处理12 d的胚性小孢子数量多,小孢子活力最高;采用NPB-99培养基作为小孢子分离液不影响梯度离心,得到的小孢子活力较高,活力小孢子比率达31.96%,远高于单一甘露醇分离液和NPB-99无机盐分离液的3.62%和4.59%;小孢子培养密度为0.5×10^(4)个·mL^(-1)时诱导产生的愈伤组织较多,诱导率为60.22%;在GEM培养基上愈伤组织分化率相对较高,分化率为6.80%。因此,低温预处理12 d的麦穗以NPB-99培养基作为小孢子分离液操作简单、效果好,小孢子培养密度对胚状体/愈伤组织诱导率具有显著性影响,基因型对愈伤组织诱导率和分化率具有极显著影响,培养基对分化率有显著影响。

In order to optimize the wheat microspore culture technology and establish the practical wheat microspore culture technology system,two winter wheat varieties Xinmai 35 and Xinong 059,and a spring wheat variety Bobwhite were used as test materials to study the effects of different pretreatment methods and pollen cell separation solution on the formation of embryogenic cells and pollen viability.The effect of pollen cell culture density and differentiation medium on the induction rate and regeneration rate of callus was analyzed.The results showed that compared with the pretreatment of high temperature and mannitol,the number of embryonic pollen was higher and the microspore viability was the highest when wheat spikes were pretreated at low temperature for 12 days.When NPB-99 medium was used as cell separation solution,the microspore viability ratio was 31.96%,which was much higher than that of the other two kinds of separation solution(3.62%and 4.59%).When the microspore culture density was 0.5×10^(4)·mL^(-1),more embryoids or callus were induced,and the induction rate reached 60.22%.The differentiation rate of shoots was relatively high on GEM medium,and the regeneration rate was 6.80%.Pretreatment at low temperature for 12 days,using NBP-99 medium as microspore separation solution was a simple and effective method.Statistical analysis showed that genotype had a significant effect on induction efficiency of callus,while microspore culture density had a significant effect.Genotype had a significant effect on regeneration efficiency of callus,while differentiation medium had significant effect on the differentiation rate of callus.