| Hybrid Cymbidium is a new variety with big bright-color flowers and a long-lasting flowering,which inherit from Cym.hybridum,and pleasant fragrance and graceful gesture,which come from Chinese Cymbidium.It has very high ornamental value,economic value and broad market prospect.In recent years,there were some reports on genetic transformation of Cymbidium,however,few reports were concerned on genetic transformation of hybrid Cymbidium.In this study,the rhizomes of Cymbidium‘Xiaofeng'and protocorm-like bodies?PLBs?of the Cymbidium‘Junhong'were employed to study factiors influencing on genetic transformation efficiency of rhizomes and protocorm-like bodies?PLBs?in order to establish efficient genetic transformation system of hybrid Cymbidium.The main research results are as follows:1 Construction of genetic transformation receptor system of Cymbidium‘Junhong'Cutting had not significant impact on the induction of PLBs using‘Junhong'stem tip;Medium had significant effect on proliferation,bud?seedling?differentiation of protocorm-like bodies,the best medium for proliferation and bud?seedling?differentiation were MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+AC 0.3 g/L and MS+6-BA 1.5 mg/L+NAA 0.1 mg/L+AC 0.1 g/L,on which the proliferation rate and bud?seedling?differentiation rate were 3.3%and 83.3%,respectively.Medium had significant effect on seedling height,but not significant effect on the average number of leaf and root.When seedlings with 4 cm height were cultured on 1/2MS+6-BA 0.1 mg/L+NAA 0.5 mg/L+AC 0.5 g/L for 60 d,the average seedling height was 7.1 cm,the average number of leaf and root was 3.7 and 3.8 per plantlet,respectively.Seedlings with 7 cm height were transplanted in bark and peat soil mixed substrate?2:1?for 120 d,the transplantation survival rate of plantlets was 96.1%.The browning mortality rate of‘Junhong'PLBs was increased with the increase of the concentration of hygromycin,and the smaller the PLBs diameter was,the more sensitive the reaction was.When PLBs with 0.3 cm and 0.6 cm diameter cultured on medium with 90 mg/L and 110 mg/L hygromycin,respectively,the browning mortality rates were 51.1%and 50%in the first generation,and both were 100%the second generation,respectively.When PLBs with 0.3 cm diameter cultured on medium with 50 mg/L hygromycin,the browning mortality rate of the first and second generation were 49.9%and 72.2%,respectively.Therefore,50 mg/L hygromycin was subjected for the screen of transformant.Cefotaxim had obvious inhibitory effect on growth of Agrobacterium.When the concentration of cefotaxim was 200 mg/L,the growth of Agrobacterium tumefaciens was completely suppressed.So 200 mg/L cefotaxim was employed to inhibit the growth of Agrobacterium tumefaciens strain EHA101 in culture of PLB after infection.2 Genetic transformation of‘Junhong'Bacterium concentration and co-culture time had significant effect on the genetic transformation efficiency of‘Junhong'PLBs.When OD600 was 0.2,both the survival rate and GUS transient expression rate were the highest,which were 66.3%and53.3%,respectively,and when co-culture time was 3 d,both the survival rate and GUS transient expression rate were the highest,which were 60.2%and 30.5%,respectively.3 The genetic transformation of‘Xiaofeng'Bacterium concentration and infection time had obvious effect on the genetic transformation efficiency of‘Xiaofeng'rhizome.When OD600 was 0.8 and infection time was 40 min,the survival rate of‘Xiaofeng'rhizome was the highest?14.9%?.When 0.3 cm long rhizomes without stem tip were immersed in bacterium concentration(OD600=0.8)for 40 min and then co-cultured for 10 d,42 transgenic plantlets were obtained from 162 regenerated plantlets,the genetic transformation rate was 25.9%.Through the above research,Agrobacterium-mediated genetic transformation systems of‘Xiaofeng'and‘Junhong'were established,and 42 transgenic plantlets of‘Xiaofeng'were obtained.These laid a foundation of further research on AtDREB1A gene function and creation of Hybrid Cymbidium varieties using transgenic breeding method. |
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