摘要
植物体细胞转变为胚性细胞进而发育成为成熟体胚的过程是一个复杂的生理生化过程,该过程既被外界环境因素的影响,如蔗糖、ABA、氮源等,也受到植物体内许多基因的调控。本实验以铁皮石斛原球茎为外植体诱导出的愈伤组织在黑暗中继代培养2次后得到的胚性愈伤组织为实验材料,研究不同浓度蔗糖和ABA对铁皮石斛胚性愈伤组织生长发育及生理生化特性的影响。
以继代2次的胚性愈伤组织为材料,固液两种条件下不同分化培养基上培养3、4、5、6个星期后,对获得的体胚进行形态观察发现,中等浓度的蔗糖(5%和6%)和ABA的组合最利于胚性愈伤组织的发育,且随着ABA浓度的增加,体胚一致性趋于整齐,长势较好,次生褐化程度低。对体胚进行统计发现,在6%蔗糖和0.76μmol/LABA的组合中体细胞胚胎的产量最高,该培养基上单位外植体平均生成32个体胚,产胚率高达59%。
在不同处理对铁皮石斛胚性愈伤组织生长的影响的基础上,研究了固体条件下不同分化培养基培养3、4、5、6个星期后总蛋白图谱,发现培养5个星期后蛋白表达量到达高峰,该时期蛋白条带多而密；0.76μMABA和蔗糖的组合处理后组织中蛋白含量比0.57μMABA和0.95μMABA与蔗糖的组合处理要高,且出现了更多的小分子蛋白条带,尤其是一条分子量大约为44KDa的蛋白条带在这几个处理中强表达。
研究固体培养条件下不同分化处理5个星期后的铁皮石斛体细胞胚的同工酶酶谱,我们发现POD酶和EST酶随着外界培养环境中激素和蔗糖的变化而发生了明显的变化。相对于对照组而言,经过分化培养基处理后得到的体细胞胚POD酶存在或多或少的酶带缺失现象,这表明处理组中铁皮石斛体细胞胚呼吸作用减弱；与之相反,处理组中EST酶谱相对对照组有新的酶带出现,这说明有新的基因表达,贮藏物质增加。
通过分析体细胞胚胎发育调控LEC基因的性质,发现4种LEC蛋白中,有完全相似的理化性质,而对其功能结构域的生物信息学分析发现这几种基因在植物胚胎发育过程中起着重要的调控作用,其中LECl和LIL基因主要在胚胎发育早期发挥作用,LEC2和FUSCA3基因在胚胎发育成熟阶段能调控储藏蛋白的合成和积累,并使种子获得抗干燥胁迫的能力。
综上所述,我们认为6%蔗糖和0.76μMABA组合最有利于铁皮石斛体细胞胚胎的产生,同时蛋白等贮藏物质积累最多,在0.76μMABA和中高浓度蔗糖的胁迫下,铁皮石斛体细胞胚胎在球形胚后期进入休眠期。
Somatic embryogenesis which is complicated has been an important pathway for studying plant embryogenesis. It is affected by some outer factors such as sucrose, ABA and nitrogen source, and regulated by genes. In this study, the embryonic calli was used as the original materials, which were induced by protocorm-like bodies (PLBs) of Dendrobium candidum Wall ex Lindl. The effect of sucrose and abscisic acid (ABA) were tested on the regulation of somatic embryos of Dendrobium candidum Wall ex Lindl.
The embryogenic calli are cultured under the conditions of solid-liquid differentiation medium for 3,4,5,6 weeks, the results indicated that the combination of ABA and sucrose (5% and 6%) were most efficient for the development of embryogenic callus. And with the increase of ABA concentration, embryos development tends to be consistency, growing well, and with the low level of secondary browning. On the other hand, the combination of 6% sucrose and 0.76μmol/L ABA yields the highest level of somatic embryo, the medium of explants unit generated an average of 32 individual embryos, embryo production rate as high as 59%.
On the basis of the results obtained above, the total protein pattern was tested with the embryonic callus growth on the solid differentiation medium. We found that after 5 weeks culture the protein expression reaches its peak, and the protein bands are more; The protein content of the treatment of 0.76,μM ABA and sucrose combination is higher than the treatment of 0.57μM ABA and 0.95μM ABA, and there are more small molecule protein bands. Particularly a molecular weight of about 44KDa protein band is strong expression under these treatments.
We find that POD and EST enzyme activity outside the training environment with the hormone and changes in sucrose significant changes have taken place. Compared with the control group, the differentiation medium was obtained after somatic embryos POD enzyme deficiency exists more or less with the phenomenon of enzyme, indicating that treatment group Dendrobium somatic embryos decreased respiration; In contrast, treatment EST zymogram group relative control group emerged with a new enzyme, indicating that new gene expression, increase the storage material.
Analysis of somatic embryogenesis by regulating the nature of LEC genes and found four kinds of LEC protein, there are totally similar physical and chemical properties, and its functional domain of Bioinformatic analysis found that several genes in plant embryo development plays an an important regulatory role, which LEC1 and L1L genes play a major role in early embryonic development, LEC2 and FUSCA3 genes in embryonic development stages of maturity can control storage protein synthesis and accumulation, and make seeds resistant to desiccation stress capacity.
In summary, we believa that 6% sucrose and 0.76μM ABA Dendrobium most beneficial combination of somatic cell production, and storage protein accumulation largest,0.76μM ABA and high concentration of sucrose stress, somatic embryos Dendrobium. In the late globular embryo into the dormant period.
引文
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