| Cotton fiber is an important raw material of textile industry. Usinggenetic engineering methods to transform some genes related to fiberdevelopment is an important method to improve the quality of cottonfiber. GbMYB2 gene cDNA was an open reading frame by 747bp long. Itwas cloned from Gossypium barbadense L. Cotton. Previous study in ourlaboratory confirmed that over-expression of GbMYB2 in transgenicArabidopsis caused abnormal phenotypes including more dense leaftrichomes and longer roots. In order to do further study of GbMYB2 genein cotton, we optimize the Agrobacterium-mediated transformationsystem of Gossypium hirsutum L. while simultaneously transformGbMYB2 into cotton.Conventionally, Agrobacterium-mediated transformation usedhypocotyls as explants. Its conversion process include callus,embryogenic callus and embryogenesis, which is a typical representativeof indirect embryogenesis. This protocol was the preferred method fortransgenic cotton and have made a great contribution to cottontransformation history. Unfortunately, this transformation protocol was severely restricted by genotype-dependent response, a prolonged cultureperiod, low embryogenesis rate, high frequency of abnormal embryodevelopment as well heavy work load. Therefore, we optimize not onlythe standard transformation protocol, but also the protocol usingembryonic callus and shoot apex as expants. Results of the study are asfollows:1. Optimized the standard transformation system: First, facing theproblem of low conversion efficiency, the kind of explants,Agrobacterium concentration, co-culture time, Agrobacterium strains, Csource and coagulants were studied and compared. The results show that:50 mg/L Kan can screen kan-resistant callus effectively; Hypocotyls isbetter than cotyledon as explants; both LBA4404 and EHA105 can be theengineering bacteria; Agrobacterium concentration is OD600=0.5;co-culture time is 48h; glucose is the best C source. Then, facing theprolonged culture period and heavy work load, subculture time and thecharacteristics of non-embryogenic callus were analyzed. Results showthat: the best subculture time on MSB5 is twice; granular,gray and greencallus grow fast and have high embryogenesis capacity; differentiate thenon-embryogenic callus timely can shorten the transformation period. Inthe last place, facing the high incidence of abnormal embryos, theconversion process was optimized. Results show that: subculturing theembryos onto the differential medium without filter paper can reduce incidence of abnormal embryos.2. Optimized the protocol using embryonic callus and as expants: Inthe first place, facing the problem of high contaminate rate and lowconversion efficiency, antibiotic concentration, kind of explants,Agrobacterium strain and concentration were explored. Results show that:50 mg/L Kan can screen kan-resistant embryonic callus effectively; bothhypocotyls and cotyledon can be used as explants; LBA4404 is betterthan EHA105; using high concentration of Cef to wash theAgrobacterium can reduce the rate of pollution. Secondly, facing theproblem of low differentiation subculture time was studied. It wasshowed that subculture can make the embryonic callus lost itsembryogenesis capacity.3. Optimized the protocol using embryonic callus and as expants:First of all, facing the problem of low root-inducing rate, plant growthregulators and root-inducing time was analysed. Results show that: 1.5mg/L NAA can not only promote root-inducing rate but also accelerate itsgrowing speed; reserve of hypocotyls is in favor of root-inducing;root-inducing time is very important to root-inducing rate.In the nextplace, facing the problem of low conversion efficiency, antibioticconcentration, type of explants and acupuncture was studied. Resultsshow that: 100 mg/L Kan can screen kan-resistant plants effectively;acupuncture shoot apex can increase Kan-risitant plant occuring rate; shoot apex from 5 day sterile seedlings excel etiolated ones. In addition,inducing shoot apex and root simultaneously was first adopted. |
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