Study On Factors Influencing Regeneration System And NP-1 Gene Transformation In Colored Cotton

Naturally colored cotton, is considered as a "green product" and "ecological treasure". Because of its unique advantage and characteristic, colored cotton becomes one of the fields of research at present. The use of transgenic technology to raise the disease-resistance of the plant, has offered a new way for breeding of plant. We discussed some factors influencing the callus induction and growth in a regeneration system. Preliminary studies on gene transformation of colored cotton via Agrobacterium tumefaciens carrying NP-1 was done, in order to enhance the disease-resistance.Explants derived from hypocotyls and cotyledons segments of 5-6 day seedlings , and hypocotyls were superior to cotyledons in the induction of callus. Different hormones and concentrations play an important role in the callus induction and growth. 2,4-D was an important hormone of induced callus. State of the callus growth was better on basic medium MSB medium containing 0.1mol/L2,4-D and 0.1mol/L KT. The effects of various sugar sources were different on the formation and growth of cotton callus. The results indicated that glucose was advantageous to the induction and proliferation of cotton callus, sucrose came second. Maltose and lactose were not advantageous to the induction and growth of callus. The addition of mannitol restrained the induction of callus. The medium MSB containing twice of KNO3 and NH4NO3 reduced by half could induce a number of embryogenic callus.At the same time, the callus occurrence and cell configuration of the callus were observed via paraffin slice method and scanning electron microscope technology. Two kinds of callus formed in cultures. One was light yellow or grey and loose, while the other was hard and white or dark green. The former grew rapidly and eventually developed into embryogenic callus. On the contrary, the latter grew slowly. And the former was derived from the cortex tissue.Furthermore, we discussed Agrobacterium-mediated transformation andsome factors affecting the transformation frequency. For example, the optimum concentration of selective antibiotic kanamycin, infection time, coculture time of explants and Agrobaclerium, and effect of AS. It were appropriate for Agrobacterium infecting explants 10min and coculturing 48h, then transferred into the medium supplemented with 500mg/L Cefotaxime and 50mg/L kanamycin to select resistant callus.