Studies On Germplasm Conservation In Vitro Of Plantlets And The Changes Of Endogenous Hormones In The The Process Of Conservation In Citrus Plants

In this experiment,42 accessions of citrus germplasm were collected for in vitro conservation.The main research contents were as follows:citrus mature embryos were used as the explants for establishing the in vitro regeneration system;the tube plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as the conservation materials for obtaining the effective parameters of restricting growth conservation and the genetic stability of conserved plantlets were detected by ISSR technique;42 accessions of citrus germplasm were conserved under the optimized conditions;some physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD and CAT),as well as free proline contents were measured,and the changes of endogenous hormones contents were also measured by HPLC for discussing the in vitro preservation mechanism,which would provide a scientific basis for the better in vitro preservation of citrus germplasm.The main results were as follows:1.Establishment of the in vitro regeneration system of citrus plants.The plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as materials to study the best conditions of the proliferation,rooting and transplanting.The results showed that the best proliferation medium was the MS medium supplemented with 1.0 mg/L 6-BA and 0.01 mg/L NAA,and the proliferation coefficient was 9.70;the plantlets cultured on the MS medium supplemented with 1.0 mg/L IBA rooted best;and the best transplanting medium was the mixture of sawdust and husk(1:1).2.Study on germplasm conservation in vitro of plantlets in citrus plants.The stem segments which from the nucellar embryos of Citrus sinensis cv.Xuegan growing for 3 months,were used as materials,and the effective parameters of restricting growth conservation was optimized by adjusting different concentrations of sucrose,different concentrations of agar,different concentrations of sucrose and mannitol,and PP₃₃₃.The results showed that the survival rate after conservation for 12 months was 85.6%on the MS medium supplemented with 50～80 g·L^-1of sucrose;the survival rate after conservation for 12 months was 86.8%on the MS medium supplemented with 0.5mg·L^-1PP₃₃₃. Both of the preservation effects were the same.Considering from the effects of conservation and preservation costs,the MS medium supplemented with 0.5mg·L^-1PP₃₃₃was the best for conservation in vitro of plantlets in citrus plants.After preservation for 18 months,citrus plantlets rooted and transplanted well,and the plants grew robustly.3.Establishment of the examining system of genetic stability of the conserved plantlets by ISSR-PCR.The tube plantlets of citrus conserved before and after preservation for 18 months were used as materials to analyze the stability of the genetic variation by ISSR-PCR.The results showed that the patterns of ISSR were completely identical,which suggested that there be no variation in the DNA level.,the plantlets from the preservation with the genetic stability,and which also showed the feasibility of the restricting conservation.4.Establishment of the conservation materials of tube plantlets from the mature embryos in 42 accessions of main citrus plants.42 accessions of citrus germplasm were collected for in vitro preservation.The results showed that the MS medium supplemented with 0.5mg·L^-1PP₃₃₃applied for the mature embryos in vitro preservation of most citrus cultivars,and the survival rate of Citrus sinensis cv.Xuegan after conservation for 15 months was as high as 89.1%.The transplanting seedlings of the preservation in vitro grew normally and robustly.5.Measurement of the physiology indexes during the restricting growth conservation of the citrus tube plantlets.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L^-1PP₃₃₃and in the medium without PP₃₃₃, were used to measure those physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD,CAT),as well as free praline contents.The results showed that in the preservation process,the soluble protein content,activity of POD and activity of CAT increased after the first downward trend,and the free proline content of the upward trend.However,the plantlets in the medium supplemented with 0.5mg·L^-1PP₃₃₃,the soluble protein content,activity of POD and activity of CAT showed as a parabola-shaped curve,and the fall time of the plantlets in the medium adding PP₃₃₃postponed by 1 to 2 months.And the trend of free proline content in the medium adding PP333 was upward,while the growth rate was lower than that of the control.6 Measurement of endogenous hormones by HPLC during restricting growth conservation of tube plantlets in citrus plants.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L^-1PP₃₃₃and in the medium without PP₃₃₃,were used to determinate the changes of endogenous hormones of GA₃,IAA,ABA and ZT by HPLC method.The results showed:(1)At the preservation of the early stage,the contents of GA₃,IAA and ZT were rising,and the plantlets in vitro grew rapidly.AT the preservation of the late stage,the contents of GA₃,IAA and ZT were slowing down,and the pIantlets in vitro grew fast;and the ABA showed an upward trend.(2)At the preservation of the early stage,the contents of GA₃,IAA and ZT of the plantlets in the medium adding PP333 were lower than that of control,and at the preservation of the late stage,which started to rise,and higher than that of the control.The content of ABA in the preservation of the first six months was higher than that of the control,then lower than that of the control.(3)The ratio of IAA/ABA in the preservation of the first four months was up,and that the preservation of the first four months,the maximum,and then the ratio began to drop.At the preservation of the early stage,the ratio of IAA/ABA of the plantlets in the medium adding PP₃₃₃ was lower than that of control,and then declined slowly,and higher than the control.(4)At the whole preservation stage,the ratio of IAA/ZT was the upward trend.The ratio of pre-preservation of the plantlets in the medium adding PP333 changed gently,late growth rapidly;and the ratio of the control,with a growth rate slightly faster,then more gently.