Immature Embryo Rescue And Genetic Transformation Of ’Manaohong’ Cherry

’Manaohong’cherry（Prunus pseudoceresus’Manaohong’）is a new cultivar with longer shelf life and matures early in Guizhou Province.However,there is a serious problem of embryo abortion in this cultivar,which greatly restricts the acquisition of hybrid seedlings in cross breeding.How to improve the germination rate of seeds in cross breeding has become one of the urgent problems to improve the efficiency of cherry cross breeding.Therefore,the establishment of the’Manaohong’cherry embryo rescue system is of great significance for its cross breeding.Generally,stone fruit trees,e.g.cherry,peach etc have been recognized as the difficult regeneration in vitro,thereby,the genetic tranformation are considerably lagging behind other fruit trees.Currently,the development of young embryo,as well as the the embryo abortion stage of’Manaohong’cherry were investigated via anatomical observation and germination experiments.Also,the best embryo rescue stage was determined,and the effects of plant growth regulators（PGR）in medium on embryo germination,shoot multiplication,as well as root induction were optimized.Furthermore,the callus was induced using immature embryos of’Manaohong’cherry,and the genetic transformation system was established via Agrobacterium-mediated methods.The main contents and results in present study are as follows:1.The results showed that,during the development the abortion rate of cherry embryos increased in a pattern that slowly from day 15 to day 25 while sharply from day 25 to day 30,and no significant change was observed from day 30 to day 35.Therefore,the large-scale abortion of immature embryos started at the day 30 after full-blossom.By morphological observation,the normal embryos were found full,smooth and round,while the abortive ones were shriveled.Germination test was carried out on the embryos at different developmental stages after the full-blossom,from day 15 to day 25,the normal embryo development index is less than 0.30,and the embryo germination rate is 0.Thus,it is difficult to rescue the embryo at this period.On the day 25,the germination rate was the highest,reaching to 34%,which was significantly higher than the germination rate in other developmental stages.Therefore,the best embryo rescue time was the day 25 after the full-blossom.2.In the embryo rescue system,the immature embryos at the day 25 after the full-blossom were transferred into the medium containing different proportioning of growth regulators for embryo germination,and the growth regulator ratio was determined by L9（3⁴）orthogonal experiment.The best medium for embryo germination was MS+0.5 mg/L TDZ+0.5 mg/L 6-BA+1.0 mg/L IBA,which gave70%of germination rate.After germination and growth of young embryos,cherry seedlings were obtained.The seedlings were transferred into medium with MS as the basic medium and differential concentrations of growth regulators were added to screen out suitable medium for multiple bud induction and proliferation.After 30 days of cultivation,the best medium for shoot multiplication was MS+0.5 mg/L TDZ+0.1mg/L 6-BA+1.5 mg/L IBA+0.5 mg/L GA₃,by which the growth coefficient was as high as 5.45.Buds from 2 to 4 cm in length were transferred into the rooting medium for 30 days.Acclimated seedlings were then selected for indoor cultivation and transplanted into the sterilized nutrient soil and the survival rate reached up to90%.3.The optimal conditions for the callus induction of’Manaohong’cherry were determined by experiments with different plant growth regulators:MS+1.5 mg/L TDZ+1.5 mg/L IBA+7 g/L Agar+30 g/L Sucrose.In the late-proliferation culture,it is most beneficial to the growth and proliferation of the callus after the dark culture for25 days and then transferred to the light.4.Using callus as a transforming receptor,the sensitivity of callus to Kan and Tim was determined,The screening strength of Kan was 80 mg/L,and the inhibitory concentration of Tim was 150 mg/L.Genetic transformation system of cherry callus mediated by Agrobacterium tumefaciens was established:Agrobacterium tumefaciens infection for 20 min,bacterial liquid OD₆₀₀00 value of 0.8,and the concentration of AS in the co-culture medium was 20 mg/L,the callus was in the screening medium containing 80 mg/L Kan,150 mg/L Tim,After screening culture,the transformed cherry callus with the most Kan resistance was obtained.After GUS chemical staining and PCR detection,the highest conversion rate was 1.33%.