摘要
本论文对金钗石斛和铁皮石斛的组织培养和ABA对铁皮石斛类原球茎脱水耐性的诱导及其机理研究进行了研究,主要结果如下:
一.铁皮石斛和金钗石斛的组织培养
1.利用铁皮石斛成熟的种子为外植体,在无激素培养基上获得了无菌苗,在含有TDZ和NAA的1/2MS培养基上,诱导了铁皮石斛类原球茎,而6-BA的使用会导致材料进一步的分化。铁皮石斛类原球茎的增殖,以1/2MS+0.1mg/LNAA+3%蔗糖为佳,尽管在不含NAA的培养基上也有高的增殖率,但类原球茎的质量却较差。
2.金钗石斛的外植体以幼茎基部效果最好,腋芽易萌发,但材料数量少;以老茎为外植体时,接种前严格的清洗和预处理,能够有效地提高消毒效率,降低材料的污染。通过正交试验,对老茎腋芽萌发的研究表明,NAA和BA对腋芽的萌发都有一定促进作用,以0.1mg/L NAA和1mg/L BA混用效果最好,而GA抑制腋芽萌发。
二.ABA对铁皮石斛类原球茎脱水耐性的诱导及机理研究
1.50 μ mol/L以下浓度的ABA预培养,都能够在一定程度上提高铁皮石斛类原球茎对脱水的耐性,其中以5 μ mol/L ABA的效果最佳,预培养时间以24h为好,经2h快速脱水后,成活率较对照提高10倍左右。
2.保护性溶质含量的测定结果表明,在24h内,ABA预培养并不能明显引起蔗糖、脯氨酸等物质的积累,但可溶性多糖的含量有一定程度的增加。因此,认为脱水耐性的增强,可能与可溶性多糖含量的增加有关。
3.显微结构观察发现,ABA预处理过程中,淀粉粒逐渐降解。证实ABA诱导的脱水耐性,可能与石斛多糖的积累相关。
4 超显微结构的观察发现,ABA预培养不会引起叶绿体、线粒体及液泡的
明显变化,但脱水后,经ABA预培养的抗性类原球茎,其膜系统的完
好性明显强于未经ABA预培养的敏感性类原球茎。虽然在结构上,抗
性类原球茎细胞中线粒体、叶绿体及液泡都出现一定程度的皱缩,但内
外膜均保持较好,而在敏感材料的细胞中,线粒体、叶绿体膜的降解则
较为严重。此外,预处理引起了细胞大范围的轻度质壁分离,对照中则
未见。脱水后,抗性材料中质壁分离现象严重,但细胞结构基本保持完
好,细胞膜完整,而对照细胞己经严重损伤,结构破坏,细胞膜己无祛
清晰辨认。因而,我们认为这种由ABA引起的轻度的质壁分离,很可
能与细胞生理状态的调整及对脱水的耐性相关。
5.通过DD-RTPCR技术,在ABA预培养6h和 12h,共筛选出 116个差异
条带,通过 Reverse Northern筛选及对部分差异片段的 Northern检验,
从中得到了三个阳性片段,一个由ABA诱导表达,一个增加表达还有
一个表达降低。
6 在进行同源性分析时,发现以上阳性片段都无可以进行比较的同源基
因,因此我们难于推断其可能的功能,但由于其与ABA对脱水耐性的
诱导相关,进一步的研究很可能会获得非常有意义的结果。
In this thesis,tissue culture of Dendrobium nobile L .and Dendrobium candidum Wall, ex Lindl. as well as the desiccation tolerance of protocorm like body(PLB) of D. candidum induced by ABA and its mechanism were studied.The results are as follows: 1.TISSUE CULTURE OF D. NOBILE L. AND D.CANDIDUM Wall.ex Lindl.
We have got germ-free seedlings of D. candidum at MS medium without any phytohormone by mature seed. It was better for PLB induction on medium of 1/2MS with TDZ and NAA, 6-BA would result in differentiation at the same time. The repropagation rate was almost the same while PLB was transfered to 1/2MS + 0.1mg/L NAA + 3%sucrose and 1/2MS + 3%sucrose.But the PLB on medium without NAA showed worse in quality.
The best result was obtained while young stems were used as explant . Although it was easy to induce the growth of axillary buds, the small quantity might restrict in practice use. There were many disadvantages when old stems were used as explant, because of the difficulty in sterilizing and to inducing the growth of axillary buds. But the quantity advantage was showed up by pretreatment and improved sterilizing method. The growth rate of Axillary buds was rose while a medium choised from a cross test, contained 0.1mg/L NAA and 1mg/L 6-BA, was used.
2.STUDIES OF DESICCATION TOLERANCE OF D. CANDIDUM PLB INDUCED BY ABA AND ITS MECHANISM
Desiccation tolerance of D candidum PLB was induced by ABA preculture at a concentration below 50μmol/L. The best result was obtained while ABA concentration is 5μmol/L and the duration of preculture was 24h. PLB viability rate was 10 times higher than the control after 2h desiccation.
Detection results of cytoplasmic osmoticums showed that ABA preculture could not induce the accumulation of sucrose and proline etc. But the content of
polysaccharide rose conspicuously. So we think that the accumulation of polysaccharide may play an important role in desiccation tolerance induced by ABA preculture.
Starch grain degradation were obtained in microstructure research during ABA preculture . It may relate to the accumulation of substances which take role in desiccation tolerance. As detection results of cytoplasmic osmoticums showed that only polysaccharide was accumulated. It suggested that the accumulation of polysaccharide may take role in desiccation tolerance induced by ABA.
The mitochondrion, chloroplast and vacuolus membrane of ABA precultured PLB kept a better status than control after desiccation. Although slight plasmolysis was occurred, they have almost no changes in shape after ABA preculture. The cellstructure retained better after desiccation but plasmolysis became more seriously. In spite of the plasmolysis was not obtained in control PLB, plasma membrane was seriously destructed after desiccation. This results showed that the plasmolysis during ABA preculture may be related to the desiccation tolerance induced by ABA.
116 differential cDNA fragments were choiced from DD gels. After examining by Northern blot of some differential fragments, 3 cDNA fragments were proved to be positive. One of which was inhibited by ABA, another was promoted and the third was induced by ABA preculture.
Homologous analysis showed that none of this three cDNA fragment has
homologous gene. So it is difficult for us to suggestion its possible function. As it may relate to desiccation tolerance induced by ABA , further studies would give us more result significance.
引文
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