| Anthurium andraeanum is a perennial evergreen plant of the genus Araceae.Its elegant shape,unique flower pattern,bright colors,long flowering period,and can be used to potted and cut flowers,It is the second largest tropical flower products after tropical orchids.However,in the large-scale production process of anthura,bacterial leaf spot disease occurs frequently.Once occurred,it is difficult to control with drugs,which is one of the factors hindering the development of anthura industry.It is a huge threat in the development of anthura industry.Therefore,It is means great deal to the development of anthura industry to carry out research on disease-resistant cultivar cultivation.In the early stage of the laboratory,an intermediate expression vector containing the antimicrobial peptide gene AaAMP was constructed and transferred into Agrobacterium,On this basis,in vitro culture regeneration system of anthura,Agrobacterium-mediated genetic transformation system and antimicrobial peptide gene AaAMP were studied,and the following results were obtained.1.In order to further optimize the in vitro culture regeneration system of anthura.Firstly,the aseptic seedlings of A.andraeanum 'Alabama' were used as test materials,The effects of explants from different parts of aseptic seedlings on callus induction were discussed.The results showed that the stem segments with leaves were the best callus-inducing explants,The rate of callus was 100%after 60 days of inoculation and the average size of the callus was 14.3 mm.Secondly,the stem segments with leaves of different varieties of anthura were used as explants to explore the differences among the varieties induced by callus.The results showed that the rate of callus of the six varieties were more than 70%after 60 days of inoculation,Among them,'Tali Red','Trenza','Pink Champion' and 'Alabama',the rate of callus reached 100%,It was concluded that callus induction using stem segments with leaves as explants of anthura aseptic seedlings was suitable for most anthura varieties.Thirdly,the effects of different kinds and concentrations of Cytokinins(6-BA,ZT,TDZ)on callus redifferentiation of A.andraeanum 'Alabama'were studied.The results showed that the callus differentiation effect was better when the concentration of ZT was 0.5 mg/L.The adventitious bud differentiation rate reached 93.3%when subcultured for 60 days,and the adventitious bud morphology was normal and strong.2.In order to detect the infective activity of the test Agrobacterium and the expression characteristics of the antimicrobial peptide gene AaAMP.First,Agrobacterium-mediated transient transformation technique was used to transform tobacco and anthura callus.The results showed that after the histochemical stain of Gus,there were obvious blue plaques in both tissues,indicating that the Agrobacterium strains had strong infective vigor,and the foreign genes could be expressed normally in plants and showed it's biological function.Next,the prokaryotic expression strain Escherichia coli BL21 containing the antimicrobial peptide gene AaAMP was subjected to IPTG induction culture.The results showed that the target gene could be expressed smoothly.When cultured for 2 h,4 h,6 h and 8 h,there was a band between 6.5 and 9.5 KD corresponding to the size of the target protein.After QE identification,antimicrobial peptide proteins were detected in the target protein bands.The in vitro bacteriostatic test was carried out on the broken prokaryotic expression strain,and the results showed that the broken strain had certain bacteriostatic effect.3.In order to detect the expression and biological function of the target gene in plants,the wild type Arabidopsis thaliana(columbia-0)was used as the material to transform target gene using the inflorescence infection method.T3 generation Arabidopsis thaliana was obtained after multi-generation screening.After Gus histochemical staining and PCR detection,the target gene AaAMP has been transferred to Arabidopsis thaliana.Detection by RT-PCR showed that the target gene could be normally transcribed in transgenic Arabidopsis thalianam in vitro bacteriostatic test of transgenic Arabidopsis thaliana plant cells was carried out after cell fragmentation.The results showed that the crude liquid of transgenic Arabidopsis thaliana cells had no bacteriostatic function.4.In order to further optimize the genetic transformation system of anthura,The effects of explant types of anthura aseptie seedlings on genetie transformation efficiency were discussed.The results showed that among the four receptor materials of aseptic seedling leaves,petiole,stem segments and stem segments with leaves,the transformation effect of stem segments was better,and the rate of callus was 60%after 60 days bacteriostatic culture.The survival rate of callus was 26.67%.Secondly,after co-culture for different time(2-6 weeks)antimicrobial cultureand then callus transferred to the screening medium.the results showed that the effect of four weeks of bacteriostasis culture was better,and the survival rate of callus could still reached 17.36%after 60 days of screening culture.Thirdly,the recipient callus was infected by Agrobacterium tumefaciens in different ways.The results showed that the transformation effect of callus scratch treatment was better.The relative growth of callus was the largest at 30 days of culture,and the survival rate was 52.56%at 60 days of screening culture.Fourthly,different concentrations of ascorbic acid(15-45 mg/L)were added to the medium at different stages of transformation test.The results showed that 30 mg/L ascorbic acid had a better transformation effect.The survival rate of callus was 35.6%after 60 days of screening culture,which was significantly higher than that of other regions.The increase in conversion may be due to the browning degree of receptor material was reduced by ascorbic acid.5.Based on the optimized genetic transformation system,50 resistant callus and 17 resistant aseptic seedlings were obtained.The results of PCR showed that 18 resistant callus and 3 resistant aseptic seedlings amplified the target bands.In the experiment,600 anthura receptor materials were transformed,and the total conversion rate was 3.5%. |
PDF Full Text Request