In Vitro Culture Of Embryos And Genetic Diversity Analysis Of The Derived Plants Using RAPD In Peach

Certain frequency of abnormal seeds exists in peach.In this research,such abnormal seeds were classified by shape,and the in vitro culture system was established.Then seedlings were analyzed by RAPD marker to test whether genetic diversity exists in the accessions.The results are summarized below.1.The surface sterilization of explantsThe best sterilization method was immersion in 75%alcohol for 30s,then transfer into 0.1%HgCl₂ for 10min followed by wash in sterile water for 5 times.The contamination rate was 15.52%in this protocol.2.The germination of seed embryosThe best inoculation media was MS+6-BA2.0mg/L+2,4-D0.25mg/L in which the percentage of germination reached 96.67%.Seedlings on this media had many lateral buds which benefited the later subculture multiplication and the average number of buds could reaches 2.54 per seedling.3.Subculture multiplicationMS+6-BA1.5mg/L+IBA0.3mg/L was the most suitable proliferation media in which the average multiplication coefficient could reach 3.73.Meanwhile,the quality of bud seedlings on this media is better than others,it could ensure lowest rate of vitrification and browning.4.Seedling strengthening and rooting(1)The optimal media for strengthening was MS,in which its Ca²⁺ was doubled, supplemented with 6-BA0.75mg/L+IBA0.3mg/L+PP₃₃₃0.2 mg/L.The average height of bud seedling was 2.98cm,which well meeted the requirements of rooting.(2) 1/2MS added 0.1%AC in combination with soak in 100mg/L IBA for 30min and 16℃dark treatment for 7 days was the best method of inducing roots.Within 1 week,the rooting rate and the average number of root could reach 80%and 6.50,respectively,and the average length of roots was 7.13cm.5.Seedling hardening and transplantingThough reducing humidity gradually and three-step acclimatization procedure,the survival rate of plantlets was over 70%.6.Extract of DNA and RAPD reaction systemImproved CTAB method was suitable for DNA extraction.The best reaction system was established.The total volume was 25μl,with 2μmol/L primer,25mmol/L Mg²⁺, 10mmol/LdNTP,2.5U Taq DNA polymerase,25mmol/L buffer and 40ng templet DNA.(3) The suitable amplification procedure was 93℃for 2min,36℃for 1min,72℃for 2min.93℃for 1min,36℃for 1min,72℃for 2min,for 40 cycles,Followed by 93℃for 1min,36℃for 1min,72℃for 10min.The amplified products were saved at 4℃.7.RAPD analysis12 random primers were selected from 62 random primers,which had more clearer polymorphic bands.2621 clear bands were got in total,and specific bands were 248, accounting for 9.44%.The RAPD result showed that specific bands existed between normal and abnormal seedlings,but didn't appear among normal seedlings.Therefore,the diversity was not from the tissue culture but the seed embryos themselves.Thus it can be concluded that the genetic diversity indeed exist in abnormal seed seedlings.