| 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. |
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