【作者】 高翔；

【导师】 王丽；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2010， 博士

【摘要】 对香雪兰3个品种进行了种间杂交的初步研究,共尝试了3个组合,选取14个杂交子代使用AFLP分子标记技术进行杂种鉴定,根据条带统计结果、聚类分析及主成份分析发现,子代全部是3个香雪兰品种的杂交后代,而且大部分都属于偏母本类型的杂种,子代AFLP扩增结果出现了亲本都没有的DNA条带,说明种间杂交产生了遗传变异。MSAP(Methylation Sensitive Amplified Polymorphism)技术检测了香雪兰亲本和杂交后代的甲基化水平,在荷白与荷粉杂交体系中用了16对引物,共产生了711条带,甲基化水平在14.09%-15.77%之间,荷粉与荷黄的杂交体系中,21对引物共扩增出619条带,甲基化水平为11.38%-12.75%,两者都是以内侧胞嘧啶全甲基化为主。进一步将MSAP数据分为甲基化敏感多态性(Methylation-Sensitive Polymorphism,MSP)和甲基化不敏感多态性(Methylation-Insensitive Polymorphism,MISP),与AFLP一样,对MSAP数据做了聚类和主成份分析,验证了杂种的真实性。尽管亲本向杂种的DNA甲基化模式的遗传传递主要遵循孟德尔的遗传方式,香雪兰种间杂交仍然可以导致杂种一代发生不同程度的DNA甲基化变异,在荷白与荷粉的杂交后代中,变异率为0.84%,荷粉与荷黄的杂交后代的变异率更低,仅为0.16%,杂种后代与亲本相比,主要表现为甲基化程度的降低。通过Mantel’s检测,分析了AFLP、MSP和MISP之间的相关性,发现MSP与MISP之间相关性极显著,在荷白和荷粉杂交体系中,AFLP与MSP和MISP之间具有相关性,但是不显著,在荷粉与荷黄的杂交体系中,则没有相关性。以幼花序和花序轴做为外植体,根据所用激素不同,香雪兰既可以通过直接体细胞胚胎发生途径由周缘细胞直接再生,还可以经过胚性愈伤组织间接再生。在直接体细胞发生途径中,最佳的激素配比是IAA:6-BA=2:1mg/L,幼花序的再生率为84%,花序轴的再生率为100%。在间接体细胞发生途径中,激素比例为6-BA:2,4-D为5:1mg/L时,愈伤组织再生的效率最高,其中幼花序为92.4%,花序轴为100%。当胚性愈伤组织转移到含有2mg/L IAA和3mg/L 6-BA的MS培养基上后,可以分化成完整的植株。AFLP和MSAP两种分子标记技术被用来检测再生植株的遗传和表观遗传稳定性,在直接体细胞再生途径中,20对引物在亲本和11个再生植株中共扩增出916条清晰条带,其中8条表现为多态性,比率为0.87%,在间接体细胞再生途径中,18对引物共扩增出1075条带,其中3条为多态性条带,比率为0.27%,与亲本相比,再生植株变异模式有两种,即再生植株新带的增加和亲本条带的消失。MSAP分析表明,组织培养可以导致CG和CNG两种模式的甲基化水平的变异,变异率在直接和间接途径中分别为1.1%和1.3%。以幼花序为外植体,在N6培养基上可以诱导黄原间接体细胞胚胎发生途径,SDS-PAGE技术检测这一形态发生过程中的可溶性蛋白变化,发现6种多肽与体细胞胚发育有关,分子量为45,53,55kD的多肽存在于胚性愈伤组织、球形胚、和胚芽鞘时期,83kD的多肽则存在于后两个时期,即胚芽鞘期和真叶期胚中,37kD的多肽存在于胚性愈伤组织和球形胚期胚状体中,35kD的多肽存在所有的时期当中。利用静态顶空的方法收集了6个香雪兰栽培品种及其中两个品种9个正反交F1代的花香成分,经过GC-MS分析后,共检测到43种花香成分,每个品种的花香成分在3-25个之间,主要成分都仅仅为2-4种,包括异戊二烯类物质和脂肪族衍生物,同时还有少量的含氮和含硫化合物。在所有的成分当中,沉香醇的含量最高,在亲本和子代的花香研究中发现,亲本主要花香成分可以稳定的遗传给后代,此外,研究还发现花瓣和雌蕊是香雪兰花香的主要释放部位。基于S?rensen’s相似指数,进行了非度量多维测度分析,结果表明,被测品种可以分为两类,同时本论文还讨论了香雪兰花香在授粉中的作用,主要表现在吸引蝴蝶、娥类和蜜蜂等昆虫方面的作用。更多还原

【Abstract】 The experiment of interspecific hybridization had been conducted with 3 cultivars, a total of three combinations were tried, and the molecule identification of 14 offspring from the cross-combinations by AFLP showed that most offspring were real and mother-closed hybrids. In offsprings, most of the AFLP bands were inherited from had different from their parents but some bands were novel bands, which meaned that the offsprings not only inherited the characteristics of parents, but also had new variation.MSAP technique was used to detect the DNA methylation level of freesia parents and hybrids, in white Freesia hybridaⅡ×pink Freesia hybrida hybrid system, 16 primer pairs generated 711 sites, the DNA methylation level was between 14.09%-15.77%, in pink Freesia hybrida×yellow Freesia hybridaⅡhybrid system, 21 primer pairs generated 619 sites, and the DNA methylation level was between 11.38%-12.75%, CG methylation sites was the principal methylation pattern. Furthermore, the MSAP data were dissected into methylation-sensitive (MSP) and methylation -insensitive polymorphisms (MISP), cluster analysis and principal component analysis were also been done,and the results was similar to AFLP.Although in the majority of CCGG sites, DNA methylation showed Mendelian inheritance from parental lines to hybrids, alterations in methylation pattern were detected in some hybrids studied at certain frequencies, the variation ratio was 0.84% and 0.16% in white Freesia hybridaⅡ×pink Freesia hybrida hybrid system and in pink Freesia hybrida×yellow Freesia hybridaⅡrespectively. Compared with the mid-parent, DNA methylation level of most hybrid offspring was a little lower.We analyzed the various correlations of the AFLP and MSAP data including methylate sensitive polymorphism (MSP) and methylate insensitive polymorphism (MISP), the correlation of MSP and MISP was significant in both hybrid system, certain association between MSP and AFLP or MISP and AFLP was detected in white Freesia hybridaⅡ×pink Freesia hybrida hybrid system, but none of the correlations of them were detected in another hybrid system.For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2mg/L indole acetic acid (IAA) and 1mg/L 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 5 mg/L 6-BA and 1mg/L 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 2mg/L IAA and 3mg/L 6-BA, they could develop into plantlets with roots.In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybridaUsing the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used for the analysis of soluble proteins produced during the somatic embryogenesis of this plant. There are six polypeptides, which might play some roles in the process of somatic embryo development. Three polypeptides (45, 53 and 55 kD) were detected in the stages of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf. One polypeptide (83 kD) was specific for the stages of embryoid with coleoptiles and embryoid with leaf. One polypeptide (37 kD) was detected in the first two stages, namely, embryogenic callus and globular embryoid. One polypeptide (35 kD) was regularly synthesized in each stage, from embryogenic callus to embryoid with leaf.Floral fragrance compounds of one freesia specie, four cultivars and ten hybrids of two cultivars were studied using static headspace extraction for sample preparation followed by GC-MS analysis. In total, 43 compounds were identified and the number of compounds per species ranged between 3 and 25. Main compounds dominated by only 2–4 compounds in most species were isoprenoids and fatty acid derivatives accompanied by sulphur-containing compounds and nitrogen-containing compounds. Among the compounds, linalool (26.6-85.4%) was the major component and was detected from all the flowers. In the parents and the studied progeny, the laws of segregation of the scent traits in this cross were unveiled, stable inheritance of the main components was observed. Furthermore, In vitro analysis showed that the pistil and petal were the main producers of the floral fragrance compoundsBased on the measurement of S?rensen’s index of similarity (Is) nonmetric multidimensional scaling (MDS) was used to detect meaningful underlying dimensions and to visualize similarities between the investigated species. The MDS analysis showed two groups of species. The results are discussed in relation to pollination, especially by butterflies, moths, and bees.更多还原