枸杞体细胞胚发生中Ca^(2+)和ATPase的超微结构定位研究

ULTRACYTOCHEMICAL LOCALIZATION OF CALCIUM AND ATPase ACTIVITY ON THE 2,4-D INDUCED SOMATIC EMBRYOGENESIS OF LYCIUM BARBARUM L

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca^(2+)含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca^(2+)和ATPase活性的时空分布动态。结果表明:2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca^(2+)沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca^(2+)在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca^(2+)呈弥散性分布。在此过程中,ATPase活性时空分布与Ca^(2+)的定位变化具有高度一致性,仅仅稍滞后于Ca^(2+)出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca^(2+)和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca^(2+)含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca^(2+)和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca^(2+)和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。

In order to research the function mechanism of the 2,4-D during the development of plant somatic embryogenesis, we studied its function mechanism and relationship with the space-time distributing of Ca2+ content and ATPase activity on somatic embryogenesis of Lycium barbarum L. The possible effects on 2,4 - dichlorophenoxyacetic acid (2,4-D) induced somatic embryogenesis and changes of Ca2+ and ATPase active at different development period of somatic embryogenesis. The result showed: The 2,4-D was a key hormone for induced embryonic state of Lycium barbarum L. The embryonic callus and non-embryonic callus was separately obtained in the medium that contains the auxin 2,4-D and lack 2,4-D. In the present study, we have observed the Ca2+ was more abundant in the further intercellular matrix and on the cell wall at the multi-cellular stage, and Ca2+ was concentrated in the plasma membrane and vacuoles membrane during embryonic cell differentiate and division, to the globular embryo, more Ca2+ was seen in the nucleus. Afterward, it was also observed to be distributed in the thicken cell wall and intercellular matrix. At the same process, the variations of ATPase activity and Ca2+ were highly similar, ATPase activity was mainly located on the plasma membrane in early embryogenic cells. With further development, it was also observed to be distributed inendoplasm, nucleus and vacuoles, with the thickening of embryogenic cell wall, ATPase activity was found in the thickened region and the intercellular space. However, the variations of ATPase activity and Ca2+ have not clearly observed variety dynamics at the nonembryogenic callus, and with further vacuolation of nonembryogenic cell, Ca2+ content and ATPase activity gradually drop. It was indicated there was a closely relationship between the dynamics of Ca2+ and ATPase activity in somatic embryogenesis by 2,4-D induced. And the space-time distribution of Ca2+ and ATPase activity play a key role on signal transmission and the regulation of relevant gene expression.