AgNO3对橡胶树花药愈伤组织形态及体胚发生的影响

Effects of AgNO3 on morphology and somatic embryogenesis of anther callus of Hevea brisiliensis

关键词:  橡胶树, 花药愈伤组织, AgNO₃, 细胞形态, 体胚发生

Key words:  Hevea brisiliensis, anther callus, AgNO₃, cell morphology, somatic embryogenesis

摘要:

橡胶树的花药愈伤组织在长期继代过程中,胚性易下降甚至丧失; 而AgNO₃作为乙烯活性抑制剂,被广泛应用于植物组织培养中。该研究以继代培养4 a以上的热研7-33-97花药愈伤组织为材料,在继代培养基中添加2.5 mg·L^-1 AgNO₃预培养35 d后,观察预培养前后愈伤组织表形及其细胞形态的变化,并设计不同浓度AgNO₃及不同处理时间对其进行体胚诱导,90 d后分别统计胚状体总数和正常胚数。结果表明:浅黄色质地柔软的愈伤组织在含AgNO₃的培养基上预培养后能转变成鲜黄色易碎愈伤组织,在倒置显微镜下前者大多表现为不规则多边形,细胞内含物较稀薄; 而后者则呈圆形或椭圆形,细胞内含物丰富,属于典型的胚性细胞。在体胚诱导的第1个月添加5 mg·L^-1 AgNO₃能显著促进体胚的发生,AgNO₃浓度升至10 mg·L^-1时体胚发生受到抑制,且畸形胚的形成率显著增加; 在含5 mg·L^-1AgNO₃的培养基中培养2个月以上,体胚发育明显受阻,大部分形成畸形胚。该研究结果在一定程度上恢复了橡胶树长期继代花药愈伤组织的胚性能力,并提高了其体胚发生频率,为橡胶树花药胚性愈伤组织长期继代培养过程中胚性的保持提供了参考。

Abstract:

It is a common problem that the embryogenic competence of rubber tree anther callus might be gradually declined or even completely lost during long-term subculture. As a potent inhibitor of ethylene action, AgNO₃ is widely used in plant tissue culture. In this study, more than four years old anther callus was used as a material for the research of the appearance and cell morphology changes by pre-culturing on subculture medium supplemented with 2.5 mg·L^-1 AgNO₃. In order to promote the frequency of somatic embryogenesis, different concentrations of AgNO₃ were added to somatic embryo induction medium, and different treatment time with AgNO₃ was tested. Total number of embryoid and number of normal somatic embryos were respectively counted after 90 d. The results showed that the pale yellow soft callus could turn into bright yellow fragile callus after pre-culture on medium containing 2.5 mg·L^-1 AgNO₃. Under an inverted microscope, the former was mostly irregular polygon with relatively thin cytoplasm, and the latter was seen to be sphere or ellipsoid, rich in cytoplasm, which was considered to be typical embryogenic cell. Adding 5 mg·L^-1 AgNO₃ to somatic embryo induction medium during the first month, significantly enhanced somatic embryo production, while 10 mg·L^-1 AgNO₃ inhibited somatic embryogenesis and more abnormal embryos emerged. Development of somatic embryo was obviously hindered when callus was cultured on the induction medium containing 5 mg·L^-1 AgNO₃ for more than 2 m, and most of them were deformity. In conclusion, this study recovered the embryogenic characteristics of long-term subculture anther callus to some extent, and increased the frequency of somatic embryogenesis, which provides references for maintaining embryogenic capability during long term subculture of anther callus in Hevea brisiliensis.