RESUMEN

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Asunto(s)

Toxinas Bacterianas , Gossypium/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/genética , Endotoxinas/genética , Vectores Genéticos , Gossypium/embriología , Gossypium/fisiología , Proteínas Hemolisinas , Control Biológico de Vectores , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Plantas Modificadas Genéticamente , Regeneración , Rhizobium/genética , Transformación Genética

RESUMEN

A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton ( Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20-24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.

Asunto(s)

Adenina/análogos & derivados , Gossypium/fisiología , Semillas/fisiología , Ácido 2,4-Diclorofenoxiacético/farmacología , Adenina/farmacología , Cotiledón/fisiología , Medios de Cultivo , Técnicas de Cultivo/métodos , Germinación/efectos de los fármacos , Giberelinas/farmacología , Gossypium/efectos de los fármacos , Gossypium/embriología , Hipocótilo/fisiología , Cinetina , Maltosa/farmacología , Regeneración/efectos de los fármacos , Semillas/efectos de los fármacos , Semillas/embriología

RESUMEN

Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 µM N6-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 µM indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid and 8.90 µM BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 µM thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field.