| 1.sanguinea and I.ensata is a very cold resistant perennial herb in Iris of Iridaceae.It has a unique flower type and high ornamental value.It has a broad application prospect in the northern region.In order to carry out genetic engineering breeding in the future,our research group has carried out the construction of regeneration system of these two species in the early stage,but the efficient regeneration system in genetic engineering breeding is more conducive to improving the composition of transgenic materials.In this study,we try to use different explant materials to construct the regeneration system with high callus induction rate and differentiation rate,and try to establish its genetic transformation system.The research results can lay a foundation for the future molecular breeding work.The main research results are as follows:1.Establish an efficient tissue culture system with the root segment of I..ensata as the explant:select the mature seeds of I ensata,disinfect the seeds and peel off the seed coat.Then put the treated seeds into solid medium MS+3.5 g/L agar for 20 days,and obtain sterile seedlings.The seedlings with 3-4 leaves were put into solid medium MS+active carbon 1.0 g/L+4.0 g/L agar and cultured for 20-25 days.Then select the part with young fibrous root in the middle of adventitious root as explant,and callus was produced at the fibrous root incision.The best medium for callus induction was MS+6-BA 1.0 mg/L+ 2,4-D 2.0 mg/L+NAA 0.2 mg/L+7.0 g/L agar,the induction rate was 100.00%;The best medium is MS+6-BA 0.2 mg/L+2,4-D 0.1 mg/L+NAA 0.2 mg/L+8.0 g/L agar with a proliferation coefficient of 9.83.If endophyte occurs during callus proliferation,callus was cultured after it was added to the bacteriostatic medium,and it is MS+6-BA 0.2 mg/L+2,4-D 0.1 mg/L+NAA 0.2 mg/L+penicillin G sodium 100 mg/L,the bacteriostatic rate is 86.67%.After the transparent and thick endophytic bacteria disappeared,the callus was transferred back to the proliferation medium.The best differentiation medium was MS+6-BA 2.0 mg/L.+NAA 0.1 mg/L+ 8.0 g/L agar,the differentiation rate is 73.33%.The best rooting medium is MS+NAA 0.5 mg/L+activated carbon 1.0 g/L+ 6.0 g/L agar,the highest rooting coefficient is 9.37 and the rooting rate is 100.00%.After 3 days of seedling cultivation,the seedlings are cultivated in a substrate of peat soil:vermiculite=1:1.During seedling cultivation and growth after transplanting,water needs to be sprayed around the seedlings and in the air,and spraying frequency is 1 day per time.The result is that the survival rate of the seedlings is 100.00%after 30 days.2.Establishing an efficient tissue culture system with different organs of I.sanguinea as explants:taking I.sanguinea hypocotyls as explants,the best medium for callus induction is MS+6-BA 1.0 mg/L+2,4-D 1.5 mg/L+7.0 g/L agar,the highest induction rate is 95.38%.The suitable proliferation medium was MS+6-BA 0.5 mg/L+2,4-D 0,3 mg/L+8.0 g/L agar,and the proliferation coefficient is 5.79.The adventitious bud differentiation medium was MS+6-BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.2 mg/L+8.0 g/L agar.After 50 days of culture,the highest callus differentiation rate was 71.67%.The rooting medium of adventitious buds is the same as above,the rooting coefficient is 8.53,and the rooting rate is 1 00.00%Taking the parts with young fibrous roots in the middle of adventitious roots of I.sanguinea as explants,callus induction medium MS+6-BA1.0 mg/L+ 2,4-D 1.0 mg/L+ NAA 0.4 mg/L,the induction rate is 91.1 1%after 30 days of culture.The medium MS+6-BA 0.5 mg/L+2,4-D 0.5 mg/L+penicillin G sodium 1 00 mg/L is a suitable medium for bacteriostasis during callus proliferation,the bacteriostatic rate 93.94%.The differentiation medium is MS+6-BA 2.0 mg/L+ NAA 0.1 mg/L,and the highest differentiation rate was 71.67%.The rooting medium of adventitious buds is the same as above,the rooting coefficient is 8.53,and the rooting rate is 100%.The seedlings were transplanted in the substrate after 3 days,and the survival rate was 100.00%after 20 days.The seedling cultivation method,cultivation substrate and management method after transplantation were the same as I.ensata,the survival rate was 100.00%after 20 days.The methods of seedling refining,culture medium and management after transplanting were the same as I.ensata's.3.Preliminary study on the genetic transformation system of I.sanguinea:Taking the parts with young fibrous roots in the middle of adventitious roots of I.sanguinea as the transformation acceptor,the pre culture medium is MS+6-BA 1.0 mg/L+2,4-D 1.0 mg/L+NAA 0.4 mg/L,and the pre culture for 2 days is suitable.After the transformation acceptor is infected by bacterial solution for 2.0 minutes,then add to the co-culture medium which is MS+6-BA 1.0 mg/L+ 2,4-D 1.0 mg/L+NAA 0.4 mg/L,with 100 umol/L as added.The medium of recovery induction was MS+6-BA 1.0 mg/L+2,4-D 1.0 mg/L+NAA 0.4 mg/L,with 100 mg/L Card added,for 30 days,after identification with Gus staining solution,the callus was all blue,with a conversion rate of 51.11%.The callus was differentiated and screened,with MS+6-BA 2.0 mg/L+NAA 0.1 mg/L,with Kana 30 mg/L and Card 300 mg/L as added,and the differentiation rate of adventitious buds was 3.33%after 40 days of differentiation.After adventitious buds were stained and identified,blue spots appeared,and they were determined to be positive adventitious buds.However,adventitious buds died later,and no resistant plants were producedThe callus induced from hypocotyls of I.sanguinea was used as the transformation receptor.After 2.0 min of infection with the bacterial solution,the cells were co-cultured at 28? for 2 days,and the culture medium was MS+6-BA 0.5 mg/L+2,4-D 0.3 mg/L,with 100 umol/L as added.After co-culture,the transformed receptor is connected to differentiation screening medium MS+6-BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.2 mg/L,with Kana 30 mg/L and Card 300 mg/L as added.Gus staining was carried out,after 20 days of culture.The positive tissue was stained blue,but adventitious roots and abnormal differentiation appeared at 35 days of culture.After staining,GUS gene was not found in this tissue.4.Preliminary study on the genetic transformation system of I.ensata:taking the parts with young fibrous roots in the middle of adventitious roots of I.ensata as the transformation receptor,the pre culture medium is MS+6-BA 1.0 mg/L+2,4-D 2.0 mg/L+NAA 0.2 mg/L.pre culture for 2 days is suitable.After the transformation acceptor is infected by bacterial solution for 1.5 minutes,then add to the co-culture medium which is MS+ 6-BA 1.0 mg/L+2,4-D 2.0 mg/L+NAA 0.2 mg/L,with 100 umol/L as attached.The medium of recovery induction was MS+6-BA 1.0 mg/L+ 2,4-D 2.0 mg/L+NAA 0.2 mg/L,with 100 mg/L Card added,for 30 days,after identification with Gus staining solution,the callus was all blue,with a conversion rate of 31.11%.The callus was differentiated and screened,with MS+6-BA 2.0 mg/L+ NAA 0.1 mg/L,with Kana 30 mg/L and Card 300 mg/L as added.After 20 days of differentiation,green spots appeared on the surface of callus,and there was a trend of differentiation,but after 40 days of culture,the callus did not further differentiate,but gradually brown and died. |
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