In Vitro Conservation Of Germplasm By Tube Plantlets And The Related Physiological And Anatomical Studies In Citrus In Fujian Province

The tube plantlets of 19 accessions of germplasm from embryo culture were used as the materials for in vitro conservation in citrus L. in Fujian Province. Through a series of comparisons of the conservation of restricting growth for prolonging the subculture cycle, the key factors of restricting growth and the best dosage were selected in the representative cultivar Ponkan from Yongchun County. 19 accessions of citrus germplasm were conserved under the optimized conditions. Moreover, the changing law of physiological indices of tube plants and the changes of leaf ultra-structure were analysed during conservation. The major results were as follows:1. Establishment of the conservation materials of tube plantlets from the mature nucellar embryos or zygotic embryos in 19 accessions of main citrus cultivars (lines) in Fujian Province19 accessions of main citrus cultivars (lines) in Fujian Province were collected for in vitro conservation. The results indicated the basal medium MS was suitable for the culture of all mature embryos of citrus species. On one hand, the survival rate of Citrus sinensis was up to 90% after in vitro conservation for 12 months; on the other hand, the survival rates of the other citrus, such as Sugan in Youxi and the hybrid between manderine and pummelo in Jianou, were less than 30% after in vitro conservation for 12 months. On the subculture, the plantlets could root 1-2 weeks earlier by adding IAA 0.2mg/L in rooting medium, and the rooting rate was also improved generally.2. Selection of the key factors for restricting growth in the germplasm conservation in vitro of citrus tube plantletsThe plantlets from the nucellar embryos of Ponkan in Yongchun were used as materilas, three restricting growth factors (mannitol,PP333,CCC) were selected to study the growth and conservation of tube plantlets. The results indicated that all of them suppressed the height and the weight of the plantlets more or less, and the effect of PP333 was the best. The media adding PP333 0.5mg/L or PP333 1.0mg/L both prolonged the subculture time, and the survival rates after conservation for 12 months were 83.3% and 80%, respectively, both of which were higher than the check obviously. The best concentration of CCC was 15 mg/L, and the mannitol was 1 -5g/L.All of the 19 accessions of Fujian citrus germplasm were conserved under the optimized conditions (adding PP333 0.5mg/L to the MS medium from) from March in 2006, and the tube plantlets all grew well.3. The physiology changes in the restricting growth conservation of the citrus tube plantletsThe plantlets from the nucellar embryos of Ponkan in Yongchun were used as materials. Adding different concentrations of PP333 or CCC in the media, the activities of SOD began to rise and the accumulation of MDA descended as the concentration increased. When the concentrations of PP333 were more than 1mg/L and CCC more than 20 mg/L, the activities of SOD turned to descend as the concentration increased while the accumulation of MDA turned to rise, which showed that the concentration of PP333 should be less than 1 mg/L and CCC less than 20 mg/L, or the ability of cleaning the oxygen free radicals would descend. Adding different concentrations of mannitol in the media, the activities of SOD were all lower than those of the check while the accumulation of MDA was higher than that of the check, which indicated that adding mannitol in the medium could prevent the plantlets from absorbing the nutrient and water, and the plantlets would accelerate the membrane lipid peroxidation because of 'hunger'.Adding the best concentrations of PP333,CCC and mannitol in the media and performing the dynamic analysis of accumulation of MDA and the activities of SOD showed that the conservation time could be extended by adding PP333 and CCC in the media, and the activities of SOD were thoroughly higher than those of the check, but the accumulation of MDA was thoroughly lower than that of the check. Therefore, PP333 and CCC could weaken the cell membrane peroxidation, delay the caducity of the plantlets in subculture and extend the conservation time. Adding mannitol in the media, the activities of SOD descended as the extension of the conservation time which were thoroughly lower than those of the check and the accumulation of MDA was higher than that check. It was obvious that mannitol was propitious for suppressing the growth of the plantlets in vitro, and extending the subculture time had no accordance with clearing the oxygen free radicals. The above results indicated that, the suppressing effects of the three restricting growth factors were similar, while PP333 and CCC were plant growth inhibitors and mannitol was permeate regulator, so the physiological mechanism of the two functions was different. 4. The changes of the ultrastructure of mesophyll cells during restrictinggrowth conservation of citrus tube plantletsThe observation of the transmission electron microscope(TEM) showed that, as the conservation time of the plantlets in vitro extended, the characteristics of ultrastructure of mesophyll cells under stress appeared, i.e., the number of starch grains and osmiophilic droplets in the chloroplast increased, some chloroplasts were out of shape, and the host space expended slightly and the base grains were fuzzy. In one single cell, sensitivity of mitochondria and cell nucleus to the external environment was not so good as that of the chloroplast. According to the infer, as the conservation time of the plantlets in vitro extended, the caducity of the mesophyll cells appeared more obvious. After the membrane system was destroyed, the permeanace of the cell increased, which would accelerate cell caducity, and then resulted in the deaths of the plantlets in vitro.