The Establishment Of Biolistics Transformation Systems Of Apical Meristem Of Gossypium Barbadense L.

Cotton is one of the important economic crops in the world. Traditional breedingmethods on cotton faces many problems today, such as poverty on germplasm resources,difficulties in distant hybridization. It is concerned about making use of genetic engineeringtechnology to upgrade varieties of cotton. The genetic transformation technique mediated bygene gun is developing rapidly in recent years. It has many advantages, such as no limitedhost and wider target materials, highly control degree, faster, etc. However, there are stillmany problems that the somatic embryogenesis from sea island cotton is difficulty anddepends on genotype. The efficiency is low and the cycle is long, et al. Therefore, theestablishment of efficient genetic transformation system mediated by gene gun on cotton is ofgreat significance to the transgenic breeding of cotton.In this paper, Biolistics transformation systems were established to study the factors oftransformation with apical meristems of Xinhai No.13, a breed of Gossypium barbadense L.cultured in Xinjiang and with embryonic calli of Xinluzao No.33. An effective way wasexplored of the callus differentiation of Gossypium barbadense L. and the jod lays a certainfoundation for the breeding of Gossypium barbadense L. in Xinjiang. The main results are asfollows:1. Biolistics transformation system was established with apical meristem of Xinhai No.13:Apical meristems as target material were from aseptic seedlings which were cultivated for4din dark and for1d in light.The microcarriers were covered up with a diameter of1.0μm andwith a DNA concentration of1.5μg/μL.The target materials were bombarded with the heliumpressure of1100psi, and with the bombardment distance of6cm, and with the vacuumpressure of28inch Hg after apical meristems were cutured for12h in the osmotic medium ofMSB_GS. After16h in osmotic medium of MSB_GS, the target materials recovered for24h in therecovery medium of MSB_HF. Then moved to kanamycin screening medium of MSB_SX-1containing concentration of120mg/L for35days. In the induction rooting medium of MSB_SG-1adding4g/L activated carbon, the seedlings were induced to take roots. After PCR andRT-PCR detection,6transgenic regenerated plants were obtained in the end. The gene EPSPSwere expressed in the transgenic regenerated plants.2. Embryonic calli of Xinluzao No.33were transformed by Biolistics after cultured for4h and for16h after transformation in the osmotic medium of MSBGS. The target materialsrecovered for24h in the medium of MSBHF. Then moved to kanamycin screening mediumcontaining kanamycin concentration of60mg/L to screen. Finally7PCR positive seedingswere obtained after taking root. It preliminarily proved the evidence that EPSPS genes wereintegrated into the genome of the regenerated seeding of Xinluzao No.33.3. Five varieties of Gossypium barbadense L. all can be induced callus in six differentcallus culture medium called Y0-Y5. The induction of callus is strongly influenced bygenotype of cotton. But there are obvious differences in a same induction medium of thecallus growth for different varieties.The radicle, hypocotyl and cotyledon of seedings ofXinhai No.13all were induced loose calli in the medium of Y5（0.1mg/L KT,0.1mg/L2,4-D and0.1mg/L NAA）. The growth speed is different that hypocotyl’ is faster than cotyledon’,and that cotyledon’ is faster than radicle’. The effects of the concentration and the proportionof hormone on callus’ differentiation of Xinhai No.13is great. And the loose calli can beproliferated and may be good for differentiation in an callus induction medium called Y1-1（0.05mg/L KT and0.1mg/L2,4-D）.