【作者】 阳永学；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 园林植物与观赏园艺， 2007， 博士

【摘要】 地锦（Parthenocissus tricuspidata Planch．）是城市垂直绿化中重要的攀缘木本植物之一，因其生长迅速、攀缘力强、夏叶浓绿、秋叶紫红、适应性广，在世界上应用极为普遍，主要用于楼房墙体，道路坡面等的垂直绿化。目前，国内外最常栽培该属植物还有五叶地锦（Parthenocissus quinquefolia Planch．），它与地锦相比抗性强、生长快，但吸盘不发达，附着力较差。为了创造地锦属新的种质资源，丰富垂直绿化的植物材料，需对地锦特有性状如耐盐碱和抗干旱性能、生长速度等进行遗传改良，但是由于五叶地锦和地锦的种间不亲和使得通过传统有性杂交进行地锦遗传改良未能成功，现代植物生物技术的应用为地锦的遗传改良提供了新方法。围绕利用生物技术提高地锦耐盐碱性能这一目标，本研究在前人研究的基础上，首先以地锦叶柄为外植体，建立了有效的地锦叶柄体胚发生途径的植株再生体系，并获得了胚性愈伤组织。其次，长期保存了地锦的胚性愈伤组织，并从胚性愈伤组织诱导大量体胚发生和植株再生，提高了地锦体胚再生频率；在此基础上，以胚性愈伤为转化受体，通过衡量抗性愈伤组织率的高低，对根癌农杆菌介导GUS报告基因转化地锦遗传转化的影响因素进行了研究，建立了地锦胚性愈伤组织遗传转化体系，为耐盐目的基因转化奠定了一定的基础。另外，以胚性愈伤组织为外植体，利用组织培养过程中发生的体细胞变异来筛选耐盐的愈伤组织和体胚，并在含NaCl的培养基上获得地锦耐盐再生植株。本研究的主要研究结果如下：1．以地锦无菌苗40d龄的叶柄为外植体，建立了地锦叶柄体胚发生途径的植株再生体系，并成功地获得了胚性愈伤组织。研究结果表明：将叶柄接种在B5+0.5-2.0mg／L 2，4-D+500mg／L CH+0.1g／L AC的培养基，不同的基因型均可以诱导胚性组织，然后将胚性组织接种在B5+1.0-2.0mg／L BA+0.1-0.3mg／L NAA+500mg／L CH的培养基上可诱导体胚产生。90％的正常体胚在不加激素的MS培养基上均可一次性长成根茎叶完整的植株，畸形胚在MS+1.0mg／L BA+0.01mg／L NAA+500mg／L CH的培养基中可先诱导芽后在1／2MS+0.5mg／L IBA上诱导根的形成，不同基因型形成体胚和再生植株的能力存在显著性差异。通过组织切片技术，观察了地锦体胚发生过程中的不同形态的胚性培养物和体胚，证实了地锦植株再生过程是经过体胚发生途径进行再生。将根系发育完好的再生植株移栽后，植株生长和外观形态表现正常。2．探讨了胚性愈伤组织长期保存的方法，并从长期继代的胚性愈伤组织中诱导了大量的体胚，提高了体胚再生频率。此外，利用ISSR标记分析了再生植株的变异情况。结果表明，在MS+0.1mg／L 2，4-D+0.1mg／L NAA+500mg／L CH培养基上，在弱光照培养条件下，胚性愈伤组织能快速增殖并保持较好的胚胎发生能力。培养基中添加适量的BA与生长素（NAA或2，4-D）可促使长期继代的胚性愈伤组织形成体胚，形成体胚的数目最高可达61／g FW（鲜重Fresh Weight），ISSR分析结果表明再生过程中存在体细胞变异。3．通过对农杆菌介导地锦胚性愈伤组织遗传转化的诸多影响因素如农杆菌菌株、共培养时间、农杆菌侵染液浓度、侵染时间和AS的浓度等进行比较研究，建立了根癌农杆菌介导地锦胚性愈伤组织遗传转化体系。研究结果表明：菌株EHA105与菌株C58相比具有较强的侵染力，胚性愈伤组织侵染最适宜的农杆菌菌液浓度为OD₆₀₀=0.7，侵染时间为40min，4d共培养时间和在共培养培养基中添加100μM AS有利于提高转化效率，利用这些参数能使抗性愈伤组织率达到42.6％左右。GUS组织化学染色、PCR分析和Southern杂交证实外源GUS报告基因已整合到转化植物基因组中。4．利用组织培养技术筛选耐盐愈伤组织并获得了耐盐植株。首先探讨了不同NaCl浓度对地锦胚性愈伤组织增殖的影响，发现NaCl对胚性愈伤组织的增殖、存活率和愈伤的状态都有极大的影响，确定了盐半致死浓度和致死浓度分别为10g／L和20g／L，随后通过筛选耐盐愈伤得到了耐盐的体胚，并诱导其再生植株，所得植株与对照相比，表现出一定的抗盐性状，初步判断为耐盐植株。5．对体胚发生过程发生的畸形胚和次生胚现象、体胚再生体系遗传稳定性的影响因素、胚性愈伤组织遗传转化的影响因素及假转化体的问题和细胞工程技术提高地锦耐盐性能的可行性进行了讨论，并对应用基因工程提高地锦耐盐性的应用前景和下一步工作重点进行了分析。更多还原

【Abstract】 Parthenocissus tricuspidata (Boston ivy) is one of the most important climbing woody vines used for urban vertical greening. Owing to its many desirable traits such as fast growth, good holdfasts, large and dark green foliages that turn red-purple in autumn and wide adaptability, it is widely cultivated in many countries and usually used to cover walls, trellises, fences or for erosion control on slopes. Currently, there is another species widely cultivated in the world named P. quinquefolia in Parthenocissus. Comparing to P. tricuspidata, this species has stronger resistance and grows faster, but its cupule is undeveloped, so the adsorbability is weaker. In order to create new germplasm in Parthenocissus and enrich the plant materials used for vertical greening, genetic improvement of the species is necessary for the traits of fast growth, drought and salt tolerance in particular. However, conventional cross breeding experiments between P. tricuspidata and P. quinquefolia proved to be unsuccessful because of interspecific incompatibility. Application of plant biotechnology may offer great potential for genetic improvement of P. tricuspidata.For the objective of improving the salt tolerance of P. tricuspidata, firstly, an efficient system of plant regeneration from petiole explants of P. tricuspidata via somatic embryogenesis was successfully developed, and friable embryogenic callus was obtained. Secondly, friable embryogenic callus can be maintained for a long time and showed strong capability of embryogenesis, and was also able to produce a large number of somatic embroys. Thus, the frequency of plant regenerated from somatic embryos was improved. On the basis of these results, we comprehensively studied and optimized parameters that affecting the efficiency of Agrobacterium-mediated transformation of embryogenic callus of P. tricuspidata by assessing the frequency of resistant callus, and established an efficient transformation system, which will be valuable for genetic engineering of salt tolerance of P. tricuspidata. Furthermore, for selecting salt-tolerant plant in vitro via somacolonal variation during tissue culture, embryogenic callus of P. tricuspidata was transferred to the medium containing different concentration of NaCl. Salt tolerant plant has been regenerated from the salt tolerant somatic embryos. The major results of this study were introduced separately as following:1 An efficient system of plant regeneration from 40-old-day petiole explants of P. tricuspidata via somatic embryogenesis was successfully developed and friable embryogenic callus was obtained. Embryogenic tissue was induced on B5 basal medium supplemented with 0.5-2.0 mg/L 2, 4-D, 500 mg /L CH and 0.1 g/L AC. Somatic embryos were induced on B5 medium containing various concentrations of BA (1.0, 1.5, 2.0mg/L) and NAA (0.1, 0.3mg/L) plus 500mg/LCH. Ninety percent of normal somatic embryos converted into plantlets directly on MS medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 1.0mg/L BA, 0.01mg/L NAA and 500mg/L CH and rooted on half strength MS medium containing 0.5mg/L IBA. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse, and all plants showed normal morphological characteristics.2 The ways of embryogenic callus maintainence and induction of somatic embryos were studied. The ISSR marker was used to analyze the genetic stability of regeneration system. The results showed embryogenic callus can proliferate rapidly and show strong capability of embryogenesis on the MS medium supplemented with 0.1 mg/L 2, 4-D, 0.1 mg/L NAA and 500mg/L CH under the dim light. The proper addition of BA and 2, 4-D or NAA can promote the formation of somatic embryos, and the highest number was 61/g FW. ISSR analysis showed somaclonal variation in the process of plant regeneration.3 An efficient transformation protocol was developed by studing parameters that affecting the efficiency of Agrobacterium-mediated transformation of embryogenic callus of P. tricuspidata such as co-culture time and bacterial density and so on. The results showed the strains EHA105 has a superior virulent than the strains C58, the optimal bacterial density is OD6oo=0.7, and the ideal infection time is 40 min. In addition, 4 day of co-culture and 100μM AS in the co-culture medium can promote the T-DNA transfer. By infection according to this optimized T-DNA transformation and selection on the selection medium, the highest frequency of resistant callus was 42.6%. GUS histochemical staining and polymerise chain reaction and southern blot analysis confirmed the integration of the T-DNA into the plant genome.4 We obtained NaCl tolerant plants via in vitro selection during the process of P. tricuspidata tissue culture. Embryogenic callus were transferred to medium supplemented with different NaCl concentrations for callus selection, the results showed the growth, frequency of survival and morphology of callus were affected significantly by diffferent NaCl concentrations, and the half lethal concentration of NaCl was about 10g/L, the lethal concentration of NaCl was about 20 g/L. Salt tolerant somatic embryos were obtained during subculture of NaCl tolerant callus on the callus proliferation containg NaCl. Plants regenrated from these tolerant somatic embryos, and showed salt tolerant morphology on the medium supplimented 6 g/L NaCl compared to the control. Thus, these plants can be judged preliminarily as the NaCl tolerant plant.5 Some phenomena that occured during the process of regeneration, for example abnormal embryos formation and secondary somatic embryogenesis, and factors affecting the regeneration system stability and the efficiency of Agrobacterium-mediated genetic transformation and so on were disscussed. The prospect of plant gene engineering on the improvement of salt tolerant of P. tricuspidata and the further work of this study were also disscussed.更多还原