湿地松体细胞胚胎发生胚性愈伤组织诱导条件优化

Optimization of Embryogenic Callus Induction Conditions for Somatic Embryogenesis of Pinus elliottii

【目的】提高湿地松(Pinus elliottii E.)体细胞胚胎发生过程中胚性愈伤组织的诱导能力,优化胚性愈伤组织的诱导培养条件,建立稳定高效的湿地松胚性愈伤组织诱导培养技术体系。【方法】以湿地松未成熟合子胚作为外植体,探究基因型、球果采集日期、植物生长调节剂和碳源等因子对湿地松胚性愈伤组织诱导的影响;观察和划分湿地松合子胚的发育阶段,并测定不同发育阶段的合子胚的生长素(IAA)、细胞分裂素(ZR)、脱落酸(ABA)和赤霉素(GA3)含量,探究合子胚发育过程中内源激素含量的动态变化。【结果】内因和外因等因素对胚性愈伤组织诱导均有显著或极显著影响,通常7月中上旬是采集湿地松球果的最佳时期,选取处于2~4阶段即多胚头、裂生多胚和球形胚发育阶段的合子胚作为诱导材料,胚性愈伤组织诱导率较高;以16号基因型的湿地松球果的未成熟合子胚为材料,能够稳定保持较高的胚性愈伤组织诱导率。在DCR基本培养基中添加2.2 mg/L激动素(KT)+2.2 mg/L 2,4-二氯苯氧乙酸(2,4-D)+2.2 mg/L 6-苯甲基腺嘌呤(6-BA)为湿地松胚性愈伤组织诱导的最佳植物生长调节剂浓度组合;如以1.5 g/L松三糖代替蔗糖作为碳源,16号基因型球果的胚性愈伤组织诱导率可达40%。此外,通过观察合子胚发育过程中内源激素含量的动态变化,推测合子胚内源脱落酸(ABA)对胚性愈伤组织的诱导有重要调控作用。【结论】本研究得到了较为稳定高效的湿地松胚性愈伤组织诱导培养技术体系,为进一步建立完整高效的湿地松体细胞胚胎发生和植株再生体系提供依据。

[Objective]To improve the ability of embryogenic callus induction during somatic embryogenesis of Pinus elliottii,optimize the conditions for embryogenic callus induction,and establish a stable and efficient technical system of embryogenic callus induction of P.elliottii[.Method]Immature zygotic embryo of P.elliottii was used as explant to investigate the effects of genotype,cone-collecting date,plant growth regulator and carbon source on embryogenic callus induction of P.elliottii.The developmental stages of zygotic embryos of P.elliottii were observed and classified,the contents of auxin(IAA),cytokinin(ZR),abscisic acid(ABA)and gibberellin(GA3)in zygotic embryos at different development stages were determined,and the dynamic changes in endogenous hormone content during the development of zygotic embryo were investigated.[Result]Both internal and external factors had significant or extremely significant effects on embryogenic callus induction.The best time to collect P.elliottii cones was usually mid-early July,when zygotic embryos were in stage 2-4,including polyembryonic head,schizoid polyembryo and sphericalembryo,the embryogenic callus induction rate was higher.Immature zygotic embryos of P.elliottii cones of genotype 16 were used as the material,which could maintain a high embryogenic callus induction rate.The optimum concentration combination of plant growth regulators induced by embryonic callus of P.elliottii was 2.2 mg/L kinetin(KT)+2.2 mg/L 2,4-Dichlorophen oxyacetic acid(2,4-D)+2.2 mg/L 6-Benzylaminopurine(6-BA)in DCR basic medium.If 1.5 g/L pine triose was used as carbon source instead of sucrose,the embryogenic callus induction rate of genotype 16 cones could reach 40%.In addition,by observing the dynamic changes in endogenous hormone content during the development of zygotic embryo,it is speculated that endogenous abscisic acid(ABA)in zygotic embryo plays an important role in regulating embryogenic callus induction.[Conclusion]In this study,a relatively stable and efficient technical system of embryogenic callus induction and culture was obtained,which laid a foundation for the further establishment of a complete and efficient system of somatic embryogenesis and plant regeneration of P.elliottii.