Establishment Of Tissue Culture System And Preliminary Study On Genetic Transformation System Of Iris Laevigata

I.laevigata is a very precious cold resistant perennial flower in Northeast China.In order to realize the development and utilization of wild resources and carry out the molecular breeding research in the future,it is necessary to establish a regeneration system.In this paper,the aseptic seedlings germinated from the seeds of I.laevi gata were used as experimental materials to study the effects of different concentrations of hormone combinations on their proliferation and rooting,and were successfully transplanted.In addition,the callus regeneration system is established by using the root segment,hypocotyl and leaf sheath of I.laevigata as explants.At the same time,the transformation process of GUS gene by Agrobacterium mediated method is studied based on the hypocotyl callus regeneration system of I.laevigata and the hypocotyl callus as explant.The main results are as follows:1.The germination rate of the seed of I.laevigata collected from Daxinganling is obviously different with different treatment methods.From October to December of the same year,60 days of stratification sand storage(breaking dormancy by using the change of temperature in the natural environment of Harbin area),the germination rate increased from 1.11%to 38.89%.After the seeds were immersed in 2%NaClO solution for 30 minutes,the germination rate of the seeds increased from 1.11%to 70.00%.The germination rate of the seeds stored in stratified sand(60 days,natural environment temperature changes)is increased to 93.33%after being soaked in NaCIO solution(2%concentration,30 minutes).2.Direct regeneration system of adventitious buds of I.laevigata is established with the seedlings(2-3 leaves)germinated from the seeds of I.laevigata as explants.The optimal disinfection method of I.laevigata seeds is immerse the seeds in the detergent water,shake for 1min and immerse for 10min,wash under running water for 30min,and then immerse the seeds in tap water(room temperature)for 24h,75%alcohol disinfection for 10s,then washed with sterile water for three times,then soaked in 2%NaCIO solution for 25min,and then washed with sterile water for five times.When inoculated for 5 days,the culture dish is replaced once,and the pollution rate is 0,the germination rate is 90%after 20 days,and the seedling grew well in the later stage.The optimum medium for induction and proliferation of adventitious buds of I.laevigata is MS+IBA 0.5mg/L+6-BA 1.5mg/L+NAA 1.0mg/L,the induction rate is 73.34%,and the proliferation coefficient is 4.10.The optimum medium for rooting is MS+NAA 0.2mg/L,the rooting rate is 93.33%,and the rooting coefficient is 7.17.The best way to transplant I.laevigata tissue culture plantlet is to open the lid of the tissue culture bottle at room temperature,pour sterile water(cover the surface of the culture medium)into the plantlet for 3 days,then wash the culture medium of the root with tap water,trim the rotten root and the withered yellow leaf,after 20 days of water culture,trim the withered yellow leaf again,and finally transplant it to the substrate of black soil:vermiculite=3:1,and water every 2-3 days.The survival rate of transplanting can reach 90.00%.3.The callus regeneration system is established by taking the root segment(Cut off the thin adventitious root on the root segment to make a wound)of I.laevigata as the explant.The most suitable medium for callus induction is MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.4mg/L,the induction rate is 73.33%.The optimal medium for callus proliferation is MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.2mg/L,and the proliferation rate is 73.33%.Penicillin G sodium 100mg/L is used to inhibit the endophytic bacteria produced in the process of callus proliferation,the inhibition rate is 46.28%.The most suitable medium for differentiation is MS+6-BA 2.0mg/L+NAA 0.4mg/L+KT 1.0mg/L,the differentiation rate is 49.52%.Rooting and transplanting refer to Chapter ?.4.The callus regeneration system is established by using the hypocotyl of I.laevigata as explant.The most suitable agar content for callus induction of I.laevigata hypocotyls is 7g/L.The optimum medium for callus induction of I.laevigata is MS+6-BA 0.5mg/L+2,4-D 1.0mg/L+NAA 0.4mg/L,Induction rate is 75%.The medium of callus proliferation is MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.2mg/L.The medium for callus differentiation is MS+IBA 0.5mg/L+6-BA 1.5mg/L+NAA 1.0mg/L,the differentiation rate is 39.72%.Rooting and transplanting refer to Chapter ?.5.The optimum medium for callus induction from leaf sheath of I.laevigata is MS+6-BA 0.5mg/L+2,4-D 1.5mg/L+NAA 0.4mg/L,the induction rate is 76.67%.The medium of callus proliferation is MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.2mg/L.Callus differentiation is induced by MS+6-BA 2.0mg/L+NAA 0.4mg/L+KT 1.0mg/L,the differentiation rate is 23.33%.Rooting and transplanting refer to Chapter ?.6.Medium MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.4mg/L induced callus from the new root tip of I.laevigata,the induction rate is 80%.In the process of callus induction from the hypocotyl of I.laevigata.Medium MS+6-BA 0.5mg/L+2,4-D 0.5mg/L+NAA 0.4mg/L can induce callus of young leaves of I.laevigata.7.Based on the regeneration system of I.laevigata hypocotyl callus and the callus as receptor material,the influence of different factors on transformation efficiency is explored,and the following conclusions are preliminarily drawn.The optimal kana screening concentration is 38.21 mg/L(half lethal concentration).The optimum concentration of carb is 400mg/L,and the growth rate is 0.The optimal infection time of GUS gene mediated by Agrobacterium is 3 minutes,the resistant callus rate is 41.52%.The optimal co-culture time is 2 days,the resistant callus rate is 50.00%.