丹参花药组织培养研究

Study on Anther Tissue Culture of Salvia miltiorrhiza

为了建立丹参花药培养再生体系,进行了丹参花药组织培养技术研究。以丹参花蕾为试验材料,研究了小孢子发育时期的确定方法,明确了小孢子处于单核靠边期的花蕾长度,结果显示,丹参花蕾长度为5.0~7.9 mm时小孢子处于单核靠边期。并利用小孢子处于单核靠边期的花药做外植体,采用正交试验设计,分别比较了不同激素种类及配比对愈伤组织诱导和不定芽诱导的影响。结果显示,6-BA浓度为1.50 mg/L、2,4-D浓度为0.25 mg/L时,愈伤组织诱导率最高,达到83.33%,且愈伤组织长势较快,体积均匀,呈明显的颗粒状;6-BA浓度为1.25 mg/L、NAA浓度为0.05 mg/L、IBA浓度为0.25 mg/L时,不定芽诱导率最高且明显>其他处理,达到70.00%,且愈伤组织大部分呈鲜绿色,颗粒状明显,丛生苗较多,芽生长速度较快。丹参花蕾长度为5.0~7.9 mm时,小孢子处于单核靠边期,适合进行花药培养;适宜愈伤组织诱导的最佳培养基为MS+6-BA 1.50 mg/L+2,4-D 0.25 mg/L+糖30 g/L+琼脂6.5 g/L;适宜不定芽诱导的最佳培养基为MS+6-BA 1.25 mg/L+NAA 0.05 mg/L+IBA 0.25 mg/L+糖30 g/L+琼脂6.5 g/L。

In order to establish the anther culture regeneration system of Salvia miltiorrhiza,the anther culture technology of S.miltiorrhiza was studied.Using S.miltiorrhiza flower bud as the experimental material,the method for determining the developmental stage of microspores was studied,and the length of flower buds with microspores in the mononuclear edge stage was determined.The results showed that when the length of flower bud was 5.0-7.9 mm,microspores were in the mononuclear edge stage.And using anthers with microspores in the mononuclear edge stage as explants,an orthogonal experimental design was used to compare the effects of different hormone types and ratios on callus induction and adventitious shoot induction.The results showed that when the concentration of 6-BA was 1.50 mg/L and the concentration of 2,4-D was 0.25 mg/L,the induction rate of callus tissue was the highest,reaching 83.33%,and the callus tissue grew rapidly,with uniform volume and obvious granular shape.When the concentration of 6-BA was 1.25 mg/L,NAA was 0.05 mg/L,and IBA was 0.25 mg/L,the induction rate of adventitious buds was the highest and significantly higher than other treatments,reaching 70.00%.The callus tissue was mostly fresh green,with obvious granules,more clustered seedlings,and faster bud growth rate.When the length of flower buds was 5.0-7.9 mm,the microspores were in the mononuclear edge stage and suitable for anther culture.The optimal medium for inducing callus tissue is MS+6-BA 1.50 mg/L+2,4-D 0.25 mg/L+sugar 30 g/L+agar 6.5 g/L.The optimal medium for inducing adventitious buds is MS+6-BA 1.25 mg/L+NAA 0.05 mg/L+IBA 0.25 mg/L+sugar 30 g/L+agar 6.5 g/L.