In Vitro Conservation And Cloning Of SODs Of Persimmons(Diospyros Spp.) Germplasm Resources In Fujian Province

Diospyros spp. are known as famous fruits. In this experiment,30persimmon germplasmresources in Fujian province were collected as the materials for embryo culture and in vitroconservation. Moreover,28partial or complete sequences of SODs were obtained in Diospyrosoleifera. The main results were summarized as follows:1. Immature embryo culture and obtaining of in vitro plantlets in persimmons.The best medium for immature embryo culture was: MS+sucrose30g.L^-1+agar9g.L^-1+AC0.5g.L^-1; the medium promoting the growth of height was: MS+sucrose30g.L^-1+agar5g.L^-1+AC1g.L^-1; the medium for getting more roots was: MS（1/2N）+sucrose45g.L^-1+agar9g.L^-1+AC0.5g.L^-1; the suitable medium for the length of root was: MS+sucrose15g.L^-1+agar9g.L^-1+AC0.5g.L^-1; the appropriate medium to get more leaves was: MS+sucrose30g.L^-1+agar7g.L^-1+AC0.5g.L^-1. The phenomenon of browing could be prevented on the medium with0.5g.L^-1AC.14.4%‘abnormal’ plantlets occurred in the culture of immature embryo.2. Mature embryo culture and obtaining of in vitro plantlets in persimmons.The best medium for mature embryo culture was: MS（1/2N）+sucrose45g.L^-1+agar9g.L^-1+AC0.5g.L^-1; the medium promoting the growth of height was:1/2MS+sucrose45g.L^-1+agar5g.L^-1+AC0.5g.L^-1; the medium for getting more roots was: MS（1/2N）+sucrose45g.L^-1+agar9g.L^-1+AC0.5g.L^-1; the suitable medium for the length of root was: MS（1/2N）+sucrose15g.L^-1+agar5g.L^-1+AC0.5g.L^-1; the appropriate medium to get more leaves was: MS（1/2N）+sucrose45g.L^-1+agar9g.L^-1+AC1g.L^-1. Compared with immature embryo, the lower mineralelements reqiured in the culture of mature embryo. No vitrification occurred in mature embryoculture. The etiolation rate of mature embryo culture was lower than that of immature embryoculture.3. Subculture and rooting culture of persimmon in vitro plantlets.For Diospyros oleifera, the suitable medium for proliferation was: MS（1/2N）+sucrose30g.L^-1+agar7g.L^-1+ZT2mg.L^-1+TDZ2mg.L^-1+AC0.5g.L^-1; appropriate medium for rootingwas: MS（1/2N）+sucrose30g.L^-1+agar7g.L^-1+TDZ1mg.L^-1+AC0.5g.L^-1. The obviousfunction of ZT was promoting proliferation, and the effects of activated carbon was not only preventing browning but also rooting.4. Callus induction and plantlet regeneration in persimmon.The best medium for callus induction from plantlet leaves was: MS（1/2N）+sucrose30g.L^-1+agar7g.L^-1+2,4-D0.2mg.L^-1+TDZ0.2mg.L^-1+ZT1mg.L^-1. A high rate of differentiationwas obtained when the callus was thansfered on the medium of MS（1/2N） with TDZ. In addtion,the formation time of callus from the wild persimmon was earlier than that of thecultivars（Diospyros oleifera）, and the differentiation ability of the wild persimmon was higherthan that of Diospyros oleifera. Different degrees of browning occurred in the process of callusinduction and differentiation.5. In vitro conservation of persimmon germplasm resources in Fujian province33accessions of persimmon germplasm resources in Fujian province were collected. Thesubculture cycle of in vitro conservation could be more than5months under the alternative culturebetween MS（1/2N） and MS（1/2N）+ZT1.0mg.L^-1. Etiolation was not found in the culture of thewild persimmon while the etiolation rates could be10-20%in Diospyros oleifera. Shoot-tip andleaf necrosis occurred in both the wild and the cultivar persimmons to some extent, which directlyled the reduction of conservation time and survival rates.6Cloning of SODs in Diospyros oleifera6.1Fe-SOD gene cloning in Diospyros oleiferaIn vitro leaves of Diospyros oleifera were used as the materials for the cloning of SODs. Acomplete sequence of885bp named Do-FSD（JQ797750）, encoding209amino acids, wasobtained. According to bioinformatics analysis, unstable hydrophilic protein Do-FSD without thesignal peptide probably located in the cytoplasm, and its phosphorylation occurred mainly onserine, followed by followed by thyosine and threonine residues, regulating the metabolism ofperoxide process on persimmon in vitro tculture hrough influencing the specificity, activity andmolecular conformation of protease. One conserved sequence（JN997420）and5transcripts namedDo-FSD1to Do-FSD5（accession numbers were JQ797745, JQ797746, JQ797747, JQ797748,JQ797749）were obtained.6.2Obtaining of Mn-SOD transcripts in Diospyros oleiferaThe cDNA of leaves from Diospyros oleifera in vitro was as the template for cloningMn-SOD genes,11MSD transcripts among which, Do-MSD1to Do-MSD10（JQ797735toJQ797744）, with the polymorphism of3’UTR, and all the sequences might be the different members of MSD in Diospyros oleifera. An unknown function protein DUF3767was obtainedafter sequence assembly between Do-MSD1and conservative sequence （JQ245697）.6.3Obtaining of Cu/Zn-SOD transcripts in Diospyros oleifera2sequences, with different bases and amino acids but the same length of conservativedsequences, named Do-CSD1（JQ002570） and Do-CSD2（JQ797751）, were obtained by using thesame primers. Designing primers in the same and differences between Do-CSD1and Do-CSD2,8sequences, with different length and the number of nucleotides and amino acids, were obtained,named as Do-CSD3to Do-CSD10, their accession numbers in Genbank were JQ797752toJQ797759. All these transcripts might be caused by alternative splicing.