A Preliminary Study On The Regeneration And Genetic Transformation System Of Paeonia Lactiflora Pall

Chinese herbaceous peony is a traditional famous flower in our country,it’s called’flower prime minister in ancient time.It is a common decorative plants.Chinese herbaceous peony can be appreciated as a landscape,some species also have medicinal value.Because of the complex dormancy characteristics,It has a long breeding cycle and a low breeding coefficient.The traditional breeding method is hard to quickly improve breed,but molecular breeding provides a feasible method for improve Chinese herbaceous peony character.The establishment of the regeneration system lay a theoretical foundation for breeding and variety improvement.And the research of genetic transformation technology provide reference basis for molecular breeding.And there is few research on Chinese herbaceous peony regeneration and genetic transformation technology at home and abroad,so we should as quickly as possible to establish a perfect regeneration and genetic transformation system.This experiment puts Chinese herbaceous peony as material,gets aseptic seedling through embryo culture.This experiment puts embryo,no vaccine cotyledon,euphylla,hypocotyl as test materials,in order to research the inducement,proliferation and differentiation of callus.At the same time,this experiment puts embryogenic callus as acceptors to research the genetic transformation,and filtrate the transform condition.The primary results of this research are as followings:1.The method of seed disinfection:soaked 30 seconds with the 75%alcohol,soaked 5 minutes with HgCl₂.The best preliminary culture medium to break the embryo dormancy:MS+GA₃ 0.5 mg·L^-1,the best medium for seedlings growth:MS + GA₃ 0.5 mg·L^-1 + KT 1.0 mg·L^-1 and GA₃ 0.5 mg·L^-1+ 6-BA 1.0 mg·L^-1.2.Embryo and cotyledon are the best test materials for the induction of callus,the best method to induce is through the culture medium:1/2MS + TDZ 0.5 mg L^-1 + 2,4-D 0.2 mg·L^-1.And through this culture medium,embryo could induce embryonic callus.The best proliferation medium of callus:1/2MS + TDZ 0.2 mg·L^-1 + 2,4-D 0.5 mg·L^-1 + NAA 0.5 mg·L^-1.The medium of induce adventitious bud differentiation:1/2MS + TDZ 0.2 mg·L^-1 +NAA 0.5 mg·L^-1.Add 0.1 mg·L^-1 IAA to basic medium can induce the differentiation of adventitious root.3.The Kan with 5 mg·L^-1 concentration is the resistance selection pressure of embryonic callus.The Cef with 200 mg·L^-1 concentration is fungicide concentration,in this case we can effectively contol the excessive breeding of agrobacterium tumefaciens,and it has a less influence on plant.The best conditions of Agrobacterium mediated embryonic callus for concentration of bacterium is OD600=0.6,invasion time is 25 min,co-culture is 3 d,delay culture is 2 d.4.The test is on the Kan 5 mg·L^-1 pressure condition,after 2 times training,receiving 13 pieces of embryonic callus.By testing of PCR,the results shows that the target gene has integrated to the embryonic callus cells.