【作者】 沈晓燕；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2009， 硕士

【摘要】 本研究以中国水仙（Narcissus tazetta var. chinensis）鳞茎盘切块、子房切片为外植体,在建立高效离体培养体系的基础上,对中国水仙离体形态发生,特别是愈伤组织分化过程中的生理变化以及遗传变异进行了研究。主要研究结果如下:1.中国水仙高效离体培养体系的建立以中国水仙鳞茎盘切块为外植体,对其离体培养条件进行优化研究。结果表明,中国水仙鳞茎在3%多菌灵中浸泡6h是无菌体系建立的关键措施。天然添加物中,5%香蕉泥最有利于小鳞茎直接诱导;200 mg·L^-1为用于小鳞茎直接诱导的最适甜菜碱浓度。愈伤组织诱导的适宜培养基为MS+2.0 mg·L^-1 2,4-D+1.0 mg·L^-16-BA及MS+1.0 mg·L^-1 2,4-D+0.5 mg·L^-1 KT;愈伤组织分化的适宜培养基为MS+3.0 mg·L^-16-BA+2.0 mg·L^-1KT +0.1 mg·L^-1 NAA和MS+3.0mg·L^-16-BA +5.0 mg·L^-1KT。以子房切片为外植体,研究了中国水仙再生系统建立的有效途径。结果表明,发育时间较短的子房切片在MS+15.0mg·L^-16-BA +1.0mg·L^-1NAA培养基上经初代培养后可直接诱导出小鳞茎;诱导形成三种不同类型的愈伤组织（Ⅰ白色,表面瘤状突起;Ⅱ呈淡黄色,表面颗粒状不明显;Ⅲ呈淡黄色,表面颗粒状明显）,分别在培养基MS+0.5 mg·L^-1 2,4-D+1.0 mg·L^-1 KT、MS+1.0 mg·L^-1 2,4-D+0.5 mg·L^-1 6-BA及培养基MS+2.0 mg·L^-1 2,4-D+0.5 mg·L^-1 6-BA上诱导率达最高;愈伤组织类型Ⅰ、Ⅱ在分化培养基MS+5.0 mg·L^-16-BA+5.0 mg·L^-1 KT +0.1 mg·L^-1 NAA上分化效果较佳,而分化培养基MS+3.0mg·L^-16-BA+5.0 mg·L^-1 KT较适合愈伤组织类型Ⅱ、Ⅲ的分化。本研究打破常规的培养方法,首次将试管小鳞茎的膨大培养先于其继代增殖培养而进行,为小鳞茎的继代增殖培养提供健壮的鳞茎球。其中单因素试验结果表明,当添加PP333的浓度为3.0 mg·L^-1时试管小鳞茎膨大效果最好;多因素试验结果表明,各因素对试管小鳞茎膨大培养的影响排序为PP333> 2,4-D> NAA>2,4-D×NAA,筛选出的最佳膨大培养基为MS+1.0 mg·L^-1 2,4-D +2.0 mg·L^-1 NAA+3.0 mg·L^-1 PP333。双因素试验结果发现,在培养基中添加6-BA 5.0 mg·L^-1、白砂糖30 g·L^-1时最有利于小鳞茎的继代增殖培养;采用1/2MS添加活性炭0.5 g·L^-1的组合最适合于试管小鳞茎壮苗生根培养;以不经任何处理的田园土:泥炭土=2:1为基质最适合小鳞茎的移栽成活,成活率达到91.67%。2.中国水仙子房切片愈伤组织的细胞组织学观察石蜡切片的观察结果表明,发育时间较长的子房切片诱导出来的愈伤组织细胞排列松散,细胞非常大,细胞形状不固定,而发育时间较短的子房切片诱导出来的三种类型愈伤组织细胞在细胞排列上均比前者要紧密些,细胞形状也变得更加规则:细胞紧密度排序为类型Ⅲ>类型Ⅱ>类型Ⅰ;细胞的大小排序为类型Ⅰ>类型Ⅱ>类型Ⅲ;细胞的均一度排序为类型Ⅲ>类型Ⅰ>类型Ⅱ。3.中国水仙愈伤组织分化过程中的若干生理变化对中国水仙愈伤组织分化过程中POD、SOD和CAT活性及可溶性蛋白、葡萄糖、果糖、蔗糖和淀粉含量变化的研究发现,愈伤组织类型和来源不同,分化过程中各生理指标的变化存在一定差别:发育时间较短的子房切片诱导形成的愈伤组织类型Ⅰ随着分化时间的延长,淀粉含量变化先降低后升高,其余各生理指标变化均与此相反;愈伤组织类型Ⅱ在分化过程中,SOD活性和淀粉含量变化曲线都呈“W”型,而可溶性蛋白含量变化趋势为降低—升高—降低,其余各生理指标变化呈单峰型变化;愈伤组织类型Ⅲ在分化过程中POD活性变化呈单峰型,CAT活性和淀粉含量变化都呈双峰型,葡萄糖和果糖含量均先降低后升高,其余各生理指标变化都呈“W”型变化;发育时间较长的子房切片诱导形成的愈伤组织分化过程中, POD活性总体呈下降趋势,SOD活性变化呈双峰型,葡萄糖含量变化呈“W”型变化,可溶性蛋白含量和淀粉含量呈降低—升高—降低的变化趋势,其余各生理指标变化均呈单峰型;鳞茎盘切块来源的愈伤组织在分化过程中果糖和淀粉含量变化趋势均为降低—升高—降低,CAT活性和葡萄糖含量变化呈“V”型,其余各生理指标变化均呈单峰型变化。4.中国水仙离体形态发生的遗传稳定性检测染色体数目鉴定结果表明,鳞茎盘切块、子房切片不同离体器官发生途径再生小鳞茎壮苗生根培养后的根尖细胞染色体数目有不同程度的变异发生。直接器官发生途径来源的非三倍体所占比例范围为3.92%⁴.95%;间接器官发生途径来源的非三倍体所占比例范围为10.38%¹8.10%。ISSR分子标记检测结果表明,鳞茎盘切块、子房切片直接器官发生途径中各培养阶段培养物与供体植株具有相对一致的DNA图谱;间接器官发生途径诱导形成的愈伤组织在引物扩增后出现一些变异谱带,其余各培养阶段培养物几乎没有变异谱带出现,在培养终阶段变异率范围仅为0.00¹.69%。可见,中国水仙离体形态发生中间接器官发生途径的遗传稳定性不及直接器官发生途径。本研究还发现,中国水仙鳞茎盘切块愈伤组织分化形成的玻璃化小鳞茎和正常小鳞茎的DNA图谱不完全一致;DNA水平上检测由子房切片诱导形成的三种类型愈伤组织的遗传稳定程度发现,类型Ⅱ>类型Ⅲ>类型Ⅰ;由子房切片愈伤组织类型Ⅰ来源的玻璃化小鳞茎、产生花苞的小鳞茎都产生有别于供体植株的DNA扩增谱带,而正常小鳞茎则保持了相对较高的遗传稳定性。更多还原

【Abstract】 The slivers of bulbs and slices of ovaries were used as explants to carry out studies on the highly-efficient culture in vitro system, physiological changes during callus differentiation especially, as well as genetic variation during in vitro morphogenesis in Narcissus tazetta var. chinensis. The main results were described as follows:1. Establishment of the highly-efficient culture in vitro system of Narcissus tazetta var. chinensisThe slivers of bulbs were used as explants to carry out studies on optimizing conditions of in vitro culture in Narcissus tazetta var. chinensis. The results showed that the bulbs of Narcissus tazetta var. chinensis were soaked in 3% carbendazim for 6 hours, which was the key measure to establish the aseptic system. As the best natural additive, 5% banana slurry was favorable for inducing bulblets directly. 200 mg·L^-1 was the optimal concentration of betaine for bulblet induction. Both the MS medium supplemented with 2.0 mg·L^-1 2,4-D and 1.0 mg·L^-16-BA and the MS medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 KT were suitable for callus induction. Both the MS medium supplemented with 3.0 mg·L^-16-BA, 2.0 mg·L^-1 KT and 0.1 mg·L^-1 NAA and the MS medium supplemented with 3.0 mg·L^-1 6-BA and 5.0 mg·L^-1 KT were facilitated to callus differentiation.The studies on the establishment of the efficient regeneration system of Narcissus tazetta var. chinensis from slices of ovaries were carried out. The results showed that in the initial culture, slices of ovaries that had shorter development time could induce bulblets directly on the MS medium supplemented with 15.0 mg·L^-1 6-BA and 1.0 mg·L^-1 NAA. Three different types of calli were induced （TypeⅠcallus was white and its surface had strumae; TypeⅡcallus was light yellow and its surface had no obvious granular; TypeⅢcallus was also light yellow and its surface had obvious granular.）. The induction rates of the three different types of calli reached highest respectively on the MS medium supplemented with 0.5 mg·L^-1 2,4-D and 1.0 mg·L^-1 KT, the MS medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA and the MS medium supplemented with 2.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Both TypeⅠcallus and TypeⅡcallus cultured on the MS medium supplemented with 5.0 mg·L^-16-BA, 5.0 mg·L^-1 KT and 0.1 mg·L^-1 NAA had better differentiation effects. The MS medium supplemented with 3.0 mg·L^-1 6-BA and 5.0 mg·L^-1 KT was facilitated to differentiation of TypeⅡcallus and TypeⅢcallus.In this study, the method of subculture multiplication of bulblets in vitro following with bulblet expansion culture was used firstly, which broke the routine cultural method and specially could provide strong bulbs in order to have better results after subculture multiplication of bulblets in vitro. The single-factor test results showed that when the concentration of PP333 was 3.0 mg·L^-1 , bulblet expansion culture had the best effects; the multi-factor test results showed that the effect of different factors on bulblet expansion culture was as the order——PP333> 2,4-D> NAA>2,4-D× NAA. The best medium which was selected for bulblet expansion culture was MS medium with 1.0 mg·L^-12,4-D and 2.0 mg·L^-1 NAA and 3.0 mg·L^-1 PP333. The double-factor test results showed that the medium with 3.0 mg·L^-1 6-BA and 30 g·L^-1 white granulated sugar was in favor of subculture multiplication of bulblet in vitro; the 1/2 MS medium supplemented with 0.5 g·L^-1 Ac was most suitable for shoot growth and rooting of bulblets in vitro; the survival rate of bulblets in vitro was up to 91.67% after being transferred into the mixture of rural soil and peat soil （2:1） , which was without any treatment but most facilitated to transplantation.2. The histiocytic observation of callus induced by slices of ovaries of Narcissus tazetta var. chinensis by paraffin sectionThe results indicated that the microstructural characteristics of the cells of callus induced by slices of ovaries that had longer development time were loose, large and irregular. In contrast, the cells of the three types of calli induced by slices of ovaries that had shorter development time were closer and more regular: The tightness of cells was as the order——TypeⅢ> TypeⅡ> Type; the size of cells was as the order——TypeⅠ> TypeⅡ> TypeⅢ; the homogeneous degree of cells was as the order——TypeⅢ> TypeⅠ> TypeⅡ.3. Some physiological changes during differentiation of callus from Narcissus tazetta var. chinensisBy means of studies on the changes of POD, SOD and CAT activities and the contents of soluble proteins, glucose, fructose, sucrose and starch during differentiation of callus from Narcissus tazetta var. chinensis, the results showed that when types and sources of callus were different, the changes of every physiological index during differentiation existed some differences. As the differentiation time past, the starch contents of the TypeⅠcallus induced from slices of ovaries that had shorter development time trend toward firstly decrease then increase, on the contrary, the rest of the physiological indexes trend toward firstly increase then decrease. During the TypeⅡcallus differentiation, both SOD activity and starch contents presented the changing curve of“W”shape, whereas, the soluble protein contents decreased at first and increased subsequently then decreased, and all the other physiological indexes changed as a single peak curve. During the TypeⅢcallus differentiation, the POD activity changed as a single peak curve, whereas, the CAT activity and starch contents both showed a double-peak curve, both glucose and fructose contents trend toward firstly decrease then increase, and the rest of the physiological indexes presented the changing curve of“W”shape. During differentiation of callus induced from slices of ovaries that had longer development time, the POD activity was totally descending trend, but the SOD activity showed a double-peak curve, the glucose contents presented the changing curve of“W”shape, the contents of soluble proteins and starch both decreased at first and increased subsequently then decreased, whereas, the rest of physiological indexes changed as a single peak curve. During differentiation of callus induced from slivers of bulbs, the contents of fructose and starch both decreased at first and increased subsequently then decreased, whereas, both CAT activity and glucose contents presented the changing curve of“V”shape, and the rest of physiological indexes changed as a single peak curve.4. The detection of genetic stability during in vitro morphogenesis of Narcissus tazetta var. chinensisThe root tip cells of bulblets, which were induced from slivers of bulbs and slices of ovaries by different in vitro morphogenesis pathways and had been completed shoot growth and rooting culture, were used to investigate the chromosome number. The results showed that there were some variations at a certian degree. The non-triploids from direct organogenesis and indirect organogenesis ranged from 3.92% to 4.95% and from 10.38% to 18.10% respectively. Using ISSR markers, with regard to donor plants, the genetic integrity of cultures that derived from slivers of bulbs and slices of ovaries by way of direct organogenesis at different culture stages was validated. In the way of indirect organogenesis, there were some abnormal bands in calli; the cultures at the rest of culture stages had rare abnormal bands; at the final stage, the variant rates of cultures only ranged from 0.00 to 1.69%. In a word, the genetic stability of indirect organogenesis was lower than that of direct organogenesis during in vitro morphogenesis of Narcissus tazetta var. chinensis. At the same time, this study showed that DNA pattern of ISSR was not entirely consistent between vitrified bulblets and normal bulblets which both derived from callus of slivers of bulbs. On the DNA level, the degree of genetic stability of the three types of calli induced from slices of ovaries was followed by TypeⅡ> TypeⅢ> TypeⅠ; the DNA pattern was not entirely consistent among vitrified bulblets, bulblets with buds and normal bulblets which all derived from TypeⅠcallus; both vitrified bulblets and bulblets with buds had some variations in the DNA pattern as compared with their donor plants and the normal bulblets were diploid mostly.更多还原