| In this study,sterile seedlings were obtained through the germination of U.rhynchophylla seeds.Using its leaves,petioles and stems as test materials,the process and influencing factors of callus induction and differentiation and regeneration of U.rhynchophylla were explored,and suitable callus induction was screened and determined.And the differentiation culture conditions,the tissue culture regeneration system of U.rhynchophylla was established.Based on the regeneration system,the leaf disc method was used to screen to determine the effective screening pressure of Kanamycin in Agrobacterium-mediated genetic transformation of U.rhynchophylla,and to determine the optimal inhibitory concentration;by exploring the pre-culture time and Agrobacterium The influence of infection concentration,infection time,co-cultivation time and AS concentration in the co-culture medium on the instantaneous transformation efficiency of U.rhynchophylla,determined the optimal genetic transformation conditions,and initially established an U.rhynchophylla genetic transformation system.The main research results are as follows:1.Establishment of tissue culture regeneration system for U.rhynchophyllaUse 0.1%Hg Cl,75%ethanol and 10%Na Cl O to set different levels of disinfection time,disinfect and sterilize U.rhynchophylla seeds and germinate to obtain sterile seedlings.Using sterile seedling leaves,petioles and stems as explants to induce callus,firstly determine the best basic medium,sucrose concentration and light conditions for callus induction through a single factor test,and then design based on the results of the single factor test Three-factor three-level orthogonal test to determine the best hormone combination for callus induction;transfer the induced callus to the differentiation medium,and select the best hormone combination for adventitious bud formation by adding different concentrations of exogenous hormones;Different basic medium,sucrose concentration and exogenous hormone ratio were explored,and the best rooting induction medium was screened.The results showed that the best combination for disinfection and sterilization of U.rhynchophylla seeds was 75%ethanol 15 s+10%Na Cl O 8 min.The best explants for inducing U.rhynchophylla callus are stem segments,and the best callus induction medium and culture conditions are WPM+3 mg·L-1 2,4-D+2mg·L-1 6-BA+0.5 mg·L-1 NAA+30 g·L-1 sucrose,shading culture,the highest callus induction rate is 97.96%.The best hormone combination for adventitious bud induction was 0.5 mg·L-1NAA+3.5 mg·L-1 KT,and the budding rate was 87.60%.The best basic medium for rooting is WPM,and a sucrose concentration of 20 g·L-1 can effectively reduce the degree of vitrification of U.rhynchophylla seedlings.The best hormone combination is 3 mg·L-1 IAA+1 mg·L-1NAA,The highest rooting rate is 98.33%.2.Establishment of U.rhynchophylla Genetic Transformation SystemUsing U.rhynchophylla leaves,petioles and stems as transformation receptors,single-factor experiments were conducted to explore the effects of different concentrations of kanamycin,Cefalotin and Timentin on the callus induction rate of U.rhynchophylla.The results showed that the sensitivity of stems to Kan was weaker than that of leaves and petioles;the effective kanamycin selection pressure for genetic transformation of leaves was 50 mg·L-1,and the effective kanamycin selection of petioles for genetic transformation The pressure is 70mg·L-1,and the effective kanamycin selection pressure when the stem is genetically transformed is 90 mg·L-1;The damage of Cefalotin is less than that of Cefalotin.Therefore,when sterilizing in the late stage of U.rhynchophylla genetic transformation,Tim is preferred to sterilize explants,and the concentration of Tim is 90 mg·L-1 as the inhibitory concentration.By exploring the effects of different pre-cultivation time,Agrobacterium dip concentration,dip time,co-cultivation time and AS concentration in the co-culture medium on the genetic transformation of U.rhynchophylla leaves,petioles and stems,the GUS transient expression rate is used to determine the best genetics Transformation conditions,the results showed that the best explants for genetic transformation of U.rhynchophylla are leaves,which have the highest transient expression rate of GUS,followed by petioles,and stems have the worst transformation effect.The best combination of genetic transformation factors for U.rhynchophylla leaves is:pre-culture for 2 d,OD600=0.8 soaked for 30 min,add 120μmol·L-1AS to the co-culture medium and co-culture for 2 d;the best genetic transformation factors for U.rhynchophylla petiole The combination is:pre-culture for 2 days,OD600=0.8 soak for 40min,add 120μmol·L-1 AS during co-cultivation,and co-culture for 2 days;the best combination of genetic transformation factors for U.rhynchophylla stems is:pre-culture for 2 days,OD600=0.8 Soak for 40 min,add 150μmol·L-1 AS to the co-culture medium,and co-culture for 2 d.Then use the leaves of sterile U.rhynchophylla seedlings as explants,use the best combination of factors to carry out genetic transformation operations,obtain resistant seedlings through resistance screening,extract the resistant seedling RNA,reverse transcription to synthesize c DNA and then carry out PCR amplification The results show that the GUS gene has been successfully integrated into the genome of U.rhynchophylla and successfully expressed,and the positive rate is about 20%.This experiment successfully constructed the genetic transformation system of U.rhynchophylla,which is of great significance to the research of transgenic U.rhynchophylla. |
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