高羊茅高效组织培养再生体系的建立

Establishment of Efficient Tissue Culture Regeneration System for Festuca aruudiuacea

植物分子育种与基因功能鉴定是建立在高效的组织培养再生体系基础上的。本研究以高羊茅(Festuca arundinacea Schreb)为材料,探讨了不同外植体状态、基本培养基类型、外源激素组合与固化剂种类与浓度对愈伤组织诱导的影响,并进行了愈伤组织分化长芽与生根研究。结果表明:愈伤组织诱导过程中,离体成熟胚比完整种子作外植体愈伤组织出现早、质量优、诱导率高;基本培养基的选择上以N6诱导率最高,MS最低;激素组合研究中高羊茅三个品种(SAFari, FirewarⅡ, Barleduc)在无6-BA培养基中,生长激素选择上都是2,4-D优于IAA,2~2.5 mg/L 2,4-D在三个品种中诱导率都在50%以上,而在添加0.5 mg/L 6-BA时诱导率都偏低;固化剂选择上则以Phytagel与Agarose二都最优,Phytagel固化剂最佳诱导浓度为2~4 g/L。分化长芽最佳培养基为MS+0.1 mg/L 2,4-D+0.1 mg/L 6-BA+0.5 mg/L NAA+20 mg/L AgNO3+2%蔗糖+0.6%琼脂,生根壮苗最佳培养基为1/2MS+0.5 mg/L NAA+3%蔗糖+0.3%Phytagel。本研究为高羊茅的高效转基因体系建立与CRISPR基因功能研究提供准备。

Plant molecular breeding and gene function identification are based on efficient tissue culture regeneration system. This study used Festuca arundinacea Schreb as material to explore different explant status, basic medium type and exogenous hormone. The effects of hormone combination and gell agent types and concentrations on callus induction, and callus differentiation studies were carried out. The results showed that in the process of callus induction, the mature embryos showed earlier appearance, higher quality and higher induction rate than the intact seeds as explant callus. The selection of basic medium was the highest in N6 and the lowest in MS. In the combined study, three varieties of tall fescue(SAFari, FirewarⅡ, Barleduc) in the absence of 6-BA medium, the growth hormone selection is 2,4-D better than IAA, 2~2.5 mg/L 2,4-D in the induction rate of all three varieties was above 50%, while the induction rate was lower when 0.5 mg/L 6-BA was added. Both Phytagel and Agarose were best gell agents. The best concentration of Phytagel is 2~4 g/L. The shoot regeneration medium is MS+0.1 mg/L 2,4-D+0.1 mg/L 6-BA+0.5 mg/L NAA+20 mg/L AgNO3+2% sucrose+0.6% agar, rooting medium is1/2 MS+0.5 mg/L NAA+3% sucrose+0.3% Phytagel. This study will lay the foundation for the establishment of a highly efficient transgenic system and gene function by CRISPR for tall fescue.