白刺花胚性愈伤组织诱导及体细胞胚发生

Embryonic Callus Induction and Somatic Embryogenesis of Sophora davidii

【目的】探讨不同植物生长调节剂对白刺花胚性愈伤组织诱导的作用,以及培养基中氮源和无机盐浓度对白刺花体细胞胚发生和植株再生的影响,以期建立白刺花体细胞胚发生、发育及调控技术体系,为白刺花种苗快速繁殖体系建立及遗传转化研究提供参考。【方法】以白刺花叶片为外植体,研究生长调节剂2,4-D(1.0、2.0、3.0、4.0 mg ·L -1 )、NAA(0、0.5、0.8、1.0 mg ·L -1 )、6-BA(0.2、0.5、1.0、2.0 mg ·L -1 )和TDZ(0、0.2、0.5、1.0 mg ·L -1 )组合对胚性愈伤组织诱导,及NAA(0、0.2、0.5 mg ·L -1 )、6-BA(0、0.5、1.0 mg ·L -1 )和TDZ(0、0.2、0.5 mg ·L -1 )组合对体细胞胚发生的调控作用,筛选最优生长调节剂组合;并研究培养基中KNO 3和NH 4NO 3比例对体细胞胚发生的作用,及MS培养基中无机盐浓度(1/5MS 、1/4MS、1/3MS、1/2MS)对体细胞胚萌发的影响,筛选最佳的体细胞胚发育及成熟萌发条件。【结果】白刺花叶片外植体胚性愈伤组织诱导适宜培养基为MS + 2,4-D 3.0 mg ·L -1 + NAA 0.5 mg ·L -1 + 6-BA 0.2 mg ·L -1 + TDZ 1.0 mg ·L -1 +蔗糖40 g ·L -1 +琼脂7.0 g ·L -1 ,诱导率为42.0%。采用MS基本培养基时,最佳的体细胞胚发生培养基为MS + NAA 0.5 mg ·L -1 + 6-BA 1.0 mg ·L -1 + TDZ 0.5 mg ·L -1 +蔗糖40 g ·L -1 +谷氨酰胺100 mg ·L -1 +琼脂7.0 g ·L -1 ,体细胞胚发生率为78.46%,总胚数为对照的3.6倍;MS培养基中,KNO 3浓度提高1倍、NH 4NO 3降至1/2时,体细胞胚发生率可提高至91.33%,总胚数为采用MS基本培养基时的1.4倍;1/3MS培养基有利于体细胞胚的萌发,萌发率为82.75%,幼苗长势良好,单株平均鲜质量为76 mg,幼苗驯化移栽1个月后成活率达90%以上。【结论】白刺花叶片接种于添加2,4-D、NAA、6-BA和TDZ不同组合的诱导培养基上,可脱分化形成愈伤组织或胚性愈伤组织,2,4-D浓度对愈伤组织形态和质地有较大影响。TDZ有利于体细胞胚的形成,适宜浓度的生长素与细胞分裂素组合及硝态氮和铵态氮的比例对体细胞胚的形成和发育具有调控作用,降低MS无机盐浓度可提高体细胞胚萌发率,本试验体系的再生植株移栽成活率达90%以上。

【Objective】 The study is to explore the effect of different plant growth regulators on embryonic callus induction and the effect of nitrogen source and mineral concentration in MS medium on somatic embryogenesis and plant regeneration of Sophora davidii , and to establish a technical system of somatic embryogenesis,development and regulation, which would provide a basis for the establishment of rapid propagation system and genetic transformation of S. davidii.【Method】 Leaves from S. davidii were used as explant to study the effects of growth regulators 2,4-D(1.0,2.0,3.0,4.0 mg ·L -1 ), NAA( 0,0.5,0.8,1.0 mg ·L -1 ), 6-BA(0.2,0.5,1.0,2.0 mg ·L -1 ) and TDZ(0,0.2,0.5,1.0 mg ·L -1 ) on embryonic callus induction, and NAA(0,0.2,0.5 mg ·L -1 ), 6-BA(0,0.5,1.0 mg ·L -1 )and TDZ(0,0.2,0.5 mg ·L -1 )on somatic embryogenesis, which aimed to screen optimum medium. Ratio of KNO 3and NH 4NO 3, mineral concentration(1/5MS, 1/4MS, 1/3MS, 1/2MS)in MS medium were tested to obtain a good culture condition for somatic embryogenesis and germination.【Result】 The optimum medium for embryonic callus induction was MS + 2,4-D 3.0 mg ·L -1 + NAA 0.5 mg ·L -1 + 6-BA 0.2 mg ·L -1 + TDZ 1.0 mg ·L -1 + 40 g ·L -1 sucrose + 7.0 g ·L -1 agar,and the rate of embryonic callus induction reached to 42.0%. MS medium supplied with 0.5 mg ·L -1 NAA, 1.0 mg ·L -1 6-BA, 0.5 mg ·L -1 TDZ, 40 g ·L -1 sucrose, 100 mg ·L -1 glutamine and 7.0 g ·L -1 agar was suitable for somatic embryo formation, somatic embryogenesis rate was 78.46%, the number of somatic embryos was 3.6-fold compared with control. Improved MS supplied with 2-fold KNO 3 and half NH 4NO 3 was beneficial to somatic embryogenesis, somatic embryogenesis rate was 91.33%, and the total number of embryos was 1.4-fold compared with the optimal basic medium. 1/3MS was beneficial to somatic embryo germination, and somatic embryo germination rate was 82.75%. Regenerated plantlets were developed well in 1/3MS medium, and mean fresh weight was 76 mg. Plantlets were successfully acclimatized with a survival rate of 90% and grew vigorously.【Conclusion】 Different growth regulators,such as 2,4-D, NAA, 6-BA and TDZ combinations were helpful for inducting callus or embryonic callus, 2,4-D had a great influence on morphology and texture of callus. TDZ was helpful for the formation of somatic embryos. Proper auxin combination with cytokinin and proportion of nitrate and ammonium nitrogen can regulate the formation and development of somatic embryos. The rate of somatic embryo germination can be increased by reducing MS inorganic salt concentration. The survival rate of transplanted plantlets reached 90% in this experimental system.