The Establishment Of Transformation System Of Cotton (Gossypium Hirsutum L.) Via Particle Bombardment

In this paper, many transgenic cotton plants which were introduced an insect-resistant gene via partical bombardment of apical meristems were obtained successfully. Simultaneously, a large amount of kanamycin-resistant somatic embryos of cotton in which a GUS gene was integrated via partical bombardment of embryogenic callus were also obtained. A new transformation system that combined Agrobacterium-mediated and particle bombardment transformation was attempted in cotton.Shoot-tip culled from 3-day-old seedlings was opmtimal to the holistic transformation system. Thereafter, a more efficient regeneration system was established by comprehensive contrastes and the parameters combinations were as follows: Helium pressure at 1 lOOpsi, the vaccum pressure at 28 inches Hg, target distance at 9cm, 1 time bombardment. Subsequently, the meristems tissue bombarded were cultured in the selective media containing kanamycin, and then the concentration of kanamycin was steped to a higher amount in the series of subculture media. In the end, many regenerated resistant plants to kanamycin were obtained, with a transformation rate reaching 4.23%, and the cycles of transformation were approximate 80 days. The surviving rates of transplanting of the transgenic plants exceeded 90% by grafting, thereafter; the plants grafted were confirmed by PCR assay.Embryogenic callus particle bombardment system was improved through GUS expression, and get a more efficient system: firstly, osmotic treated to embryogenic callus in solid medium with 0.3mol/L mannitol for 4hr before and 16hr after bombardments. Secondly, bombard the calli treated with the parameters showed as follow: vaccum pressure at 28 inches Hg, target distance at 9cm, 2 times bombardments, target DNA of 0.5-1.0 g per bombardment. After these, the calli was transferred to culture in selective medium containing kanamycin 150mg/L after recovery culture of 10-15days, and two months later, the somatic embryos resistant to kanamycin were obtained while a stable expression of GUS was detected in the embryogenic callus, which showed the GUS gene had been introduced the genome of cotton.The apical merstems of cotton were very difficult to germinate after treatment of combination of Agrobocterium-med\a.ted transformation and particle bombardment in my exploration. So the new method need further research.This research added new content and germplasm to modern cotton molecular breeding. Meanwhile, it was significant to the construction of a technical system of transformtion on a large-scale and insect-resistant gene engineering in cotton.