【作者】 天晓；

【导师】 侯占铭； 李凤民； 熊友才；

【作者基本信息】 内蒙古师范大学 ， 植物学， 2008， 硕士

【摘要】 植物体细胞胚发生的机理研究和多倍体诱导的技术优化一直是植物分子及细胞生物学中的两个热点问题。已有的研究表明,不同价位金属离子分别对宁夏枸杞（Lycium barbarum L.）体细胞胚发生的调节效应和作用机制差异显著,但在同一条件下的比较试验严重不足,且以体细胞胚为基底的同源四倍体诱导非常少见。本研究以宁夏枸杞代表品种—宁红1号为试验材料,以不同价位的金属离子Ag⁺、Ca²⁺和Eu³⁺的可溶性化合物按照不同浓度梯度作为外源影响因子,试验研究了三种不同外植体根基部、茎和叶切块体细胞胚发生的效率和相对生长速率;同时试验比较了体细胞胚、愈伤组织和激活种子在不同浓度梯度的秋水仙素和DMSO混合液诱导下枸杞同源四倍体发生效率,并对育成的同源四倍体进行了细胞学和形态学分析。以上两个方面为今后枸杞体细胞胚诱导的高效体系的建立和植物同源多倍体技术优化提供理论基础,为将来名贵中药材高效同质多倍体的培育、减少或避免嵌合体干扰的产生、提高诱变效率提供实践途径。主要试验结果如下:1.枸杞无菌苗长至3-4片真叶后,将根基部、茎和叶切成小块,在MS+2,4-D0.2mg/L的诱导培养基上进行愈伤组织诱导,发现真叶被诱导得到的胚性愈伤组织长势最好、相对生长速率最高,转入无激素MS培养基时得到的体细胞胚数最多。叶愈伤组织比起根基部和茎被诱导得到的愈伤组织质地紧密,相对生长速率达到0.04g/（d·外植体）、而根基部和茎的都是0.02g/（d·外植体）;转入无激素MS培养基15天时,每块胚性愈伤组织所诱导的体细胞胚数分别是根39.1个、茎42.8个、叶44.9个。2.根基部、茎和叶为外植体诱导得到的愈伤组织在含2,4-D 0.2mg/L的MS培养基上4次继代培养后,转入含不同浓度梯度的Ag⁺、Ca²⁺和Eu³⁺三种外源离子的MS培养基进行分化培养,发现三种离子在低浓度情况下均可促进愈伤组织的体细胞胚发生,而高浓度抑制体细胞胚的形成,且体细胞胚数在三种外植体中具有相同的变化趋势。当Ag+浓度为1mg/L时,每块叶愈伤组织形成的体细胞胚数为对照组的2倍多,Ca2+和Eu3+在其较适宜浓度下均无达到这个水平。3.枸杞多倍体诱导:愈伤组织:枸杞无菌苗叶片在MS+0.1mg/L6BA+0.5mg/LNAA激素条件下诱导出来的无绿色芽点、质地紧密的愈伤组织为试验材料,在0.2%秋水仙素和2%DMSO混合液处理48h时诱导率高达66.7%,且嵌合体少、同质性高,是较好的枸杞多倍体诱导的方法。激活种子:将枸杞种子经常规消毒后浸于无菌水中,待种子露白时用0.2%秋水仙素和2%DMSO混合液浸泡24h时诱导率最高达8%,但嵌合体现象严重,多倍体的特征不稳定,表现出不同程度的回复突变。体细胞胚:枸杞无菌苗叶片在含0.2mg/L2,4-D的MS培养基上诱导出来的愈伤组织在相同培养基上继代培养4次后,转入不含激素的MS培养基上分化10天所得到的球形胚为试验材料,在20mg/L秋水仙素和1%DMSO混合液处理24h时诱变率6%、无嵌合体现象。本文首次报道,在体细胞胚的离体条件下,通过秋水仙素处理诱导获得了枸杞同源四倍体。4.染色体计数鉴定表明,正常二倍体植株染色体数为24,所得到的四倍体植株染色体增加一倍,为48条染色体。5.从形态学和细胞学方面对四倍体植株和二倍体植株进行了比较。结果显示,形态上四倍体比二倍体植株根系较少,但粗壮。四倍体植株与二倍体植株在气孔大小、频度具极显著差异。在40倍镜下,四倍体气孔长度31.9μm,二倍体植株气孔长度24.3μm;四倍体宽度29.0μm,二倍体宽度23.9μm。而在10倍镜视野内,气孔频度四倍体的21.6个,二倍体的是27个。四倍体气孔叶绿体数分布范围在24—50个,其中叶绿体数为38的占17%左右;二倍体气孔包围细胞叶绿体数分布范围在12—28个,其中叶绿体数为20的占25%。四倍体和二倍体气孔叶绿体数之比约等于2。更多还原

【Abstract】 In recent years, the study of development of plant somatic embryo and technique of polyploid induction have become two important subjects in cytobiology and molecular biology. Many researches indicated that different valent metal ions had significantly different effect on adjusting somatic embryogenesis and had not same way of regulating for Lycium barbarum L.. However, the meaningful data was not sufficient to support it under same experimental condition. For polyploid induction, using somatic embryo to induce autotetraploid was also rarely reported.In this study, Lycium barbarum L.（ninghong 1 hao） in Ningxia was used as materials for studying induction efficiency and relative growth rate of somatic embryo with three different explants （ root, stem and leaf） and different concentration of Ag⁺、Ca²⁺ and Eu³⁺ as exogenous factor.To induce the autotetraploid, somatic embryo, callus and seed were treated with different concentration of colchicines and DMSO mixture and was induced successfully into autotetraploidy plant from all of them. The inducing efficiency was compared for the three source of explant and some cytological and morphological analysis were also done to confirm the tetraploidy.The objective of this study was to establish efficient somatic embryo induction system, optimize the technic of inducing plant autopolyploid and provide a method of cultivating homogeneity autotetraploid without chimera. The results are as follows:1.When axenic Lycium barbarum L. had 3-4 leaves, the pieces of root , stem and leaf were induced on MS+2,4-D0.2mg/L. The result showed that the callus from leaf had highest of growth and the largest numbers of somatic embryo on MS medium without hormones. Leaf callus was more tight than that of stem and root. The relative growth rate of leaf callus reached 0.04g/（d·explant）, while stem callus was （0.02g/（d·explant）） and root callus was （0.02g/（d·explant））; After cultured for 15 days on MS medium without hormones, the numbers of somatic embryo of a piece of callus was: 39.1 （root）,42.8 （stem）, 44.9 （leaf）.2.After 4 generation subculture on MS+2,4-D0.2mg/L, the callus induced from root, stem and leaf were transfered to MS medium with different concentration Ag⁺、Ca²⁺ and Eu³⁺ for comparing the metal ion effect on embryogenesis. The result showed that low level of Ag⁺、Ca²⁺ and Eu³⁺ raised the number of the embryo, but high level reduced it. The change tendency was same for three different explants.When the concentration of Ag⁺ was 1mg/L, it raised the number of somatic embryo as 2 times as that of control group. The promoting effect of Ca²⁺ and Eu³⁺ was not as good as Ag⁺ when in their best level.3.polyploid induction From Callus, which was induced from leaves cultured on MS+ 6-BA0.1mg/L+ NAA0.1mg/L, the tight and colourless primordium cut from this callus was treated with0.2%colchicines and2%DMSO mixture for 48h and the 66.7% polyploid induction rate was get after cultured on the original medium, more than this, little chimera to be find.From germinated seed, which was sterilized and germinated in sterile water, the rooting seed was treated with 0.2% colchicine and 2%DMSO mixture for 24h and 8% polyploid induction rate was get after cultured on the MS medium, but the polyploid was unstable because of chimerism and reversal mutation.From somatic embryo, the globular embryoid induced from leaf by above mentioned method was treated with 20mg/L colchicine and 1%DMSO mixture medium for 24h, 6% polyploid induction rates was get without chimerism. This is the first report for inducing autotetraploid of Lycium barbarum L. from somatic embryo.4.Chromosome number counting assay showed that the chromosome number of normal diploid is 24 and that of the polyploid is 48. This is confirmed the tetraploidy of the polyploid get in this experiment.5.The morphological and cytological study indicated that the tetraploid grown slowly, the green color of its leaf was deep, its stem was stronger, its root was fewer but stronger, compared to diploid.Comparing stomata of tetraploid and diploid also gave significant difference in number and size of pore. The data is as follows:Under light microscope with 40×, for length of pore, tetraploid （31.9um）. diploid.（ 24.3um）, for width, tetraploid （29.0um）. diploid.（23.9um）.Under light microscope with 10×, for frequency （ number of pore in one field）:tetraploid （21.6）. diploid.（27）. For amount of chloroplasts in one stomata , tetraploid （ 24 50） , 17% stomata was 38; diploid （12 48）, 25% stomata was 20. So, the amount of chloroplast in defence cell of tetraploid is as about 2 times as that of diploid.更多还原