| Restricting conservation and cryopreservation are two main methods of plant germplasm in vitro preservation. The target of restricting conservation was to restrict the growth of in vitro shoots and reduce the frequency of subculture basing on the maintenance of genetic stability. Cryopreservation was a method for long-term preservation of plant materials at ultra-low temperature in liquid nitrogen, which stagnated the metabolism of plants. In this research, the effective parameters of restricting growth conservation or cryopreservation and stability of regenerated plants were examined, and suitable in vitro preservation methods were established for lilium cultivar 'Siberia' and Lilium lancifolium Thunb. The main results were as follows:1. Effects of the medium with different chemical elements or different concentration of osmotic reagents (such as mannital or sucrose) or inhibitors (such as ABA or CCC) on the growth of in vitro shoots of lily were studied. An appreciate protocol of lily in vitro preservation by restricting growth method was established as follows: adventitious buds derived from the plantlets of bulbs, stems or stamens were subcultured in the medium of MS+BA2.0mg/L+NAA0.1mg/L then preserved in the restricting growth medium. It showed that MS or half strength MS medium with 50~90g/L sucrose restricted in vitro growth of the shoots efficiently and prolonged the interval between subcultures over 11 months. The in vitro shoots had been conserved in the medium with 1.0~3.0mg/L ABA for more than 9 months. And a high survival rate of over 80% was achieved. Otherwise, 10~50g/L mannitol or 10~40mg/L CCC could not restrict the growth of in vitro shoots effectively.2. The vitrification method was examined to cryopreserve apical meristems of lily. Dominant parameters such as sucrose concentration of preculture medium, duration of preculture and duration of exposure to PVS2 were examined respectively. And orthogonal design was utilized to analyzed the effects of the dominant parameters on survival rate. An appreciate vitrification cryopreservation protocol was developed as follows: Apical meristems excised from in vitro plantlets of lily, which were subcultured for 45~60d and cold-hardened at 4℃ for over one week, were precultured in liquid MS medium supplemented with 0.1~0.3mol/L sucrose for l~2d. And then loaded with the mixture consisted of MS medium, 2mol/L glycerol and 0.4mol/L sucrose for 20 min. The apical meristems were exposed to the cryoprotectant (PVS2) at 0℃ for 60~120min to dehydrate sufficiently before plunged into liquid nitrogen directly. At least 24h later, after rapid thawing in a water bath at 40℃, the apical meristems were rinsed in liquid MS medium supplemented with 1.2mol/L sucrose for 10 min. And then placed on theregeneration medium and subcultured for two weeks in dark prior to exposure to the light. The survival rate was over 50%.3. Apical meristems of lily were cryopreserved by encapsulation-dehydration. And orthogonal test was carried out to analyze the effects of the main parameters on the survival rate. As a result, this experiment achieved a low survival rate. So an appreciate technique of encapsulation-dehydration cryopreservation needed to be further studied.4. Compared the regenerated plants with the control, no variation was displayed on the level of morphology, protein or isozyme, which suggested the feasibility of restricting conservation and cryopreservation to conserve lily germplasm.
|
PDF Full Text Request