A simple and rapid Agrobacterium -mediated transformation protocol for cotton ( Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

Abstract

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.

Agrobacterium tumefaciens Mediated Genetic Transformation in Coker-312 (Gossypium hirsutum L.) Using Hypocotyls Explants

“…Although optimal co-cultivation duration was critical for transformation in some crops, in case of cotton it was found uncertain ranging from 36 to 72 hours (Sunilkumar and Rathore, 2001;Leelavathi et al, 2004). Jin et al, (2005) suggested that co-cultivation at 19 0 C for 48 hours was effective for the efficient transformation which was confirmed earlier by Dillen et al, (1997) and Sunilkumar and Rathore (2001).…”

“…Co-cultivation duration was found uncertain in cotton ranging from 36 to 72 hours, mainly fascinated by the genotype variability (Sunilkumar and Rathore, 2001;Leelavathi et al, 2004;Jin et al, 2005). In present report, hypocotyls were co-cultivated in MS media (0.1 mg/l 2, 4-D + 0.5 mg/l kinetin) (Fig.…”

Development of Transgenic Rice (Oryza sativa L.) Plant Using Cadmium Tolerance Gene (YCFI) through Agrobacterium Mediated Transformation for Phytoremediation

“…Res., 9 (4): 139-154, 2015 (a) Tra nsformed C (negative control) T11 T10 T9 T8 T7 T6 T5 T4 T3 T2 T1 1 lines (particularly T1, T2, T3 and T4) showed similar sizes of bands. Some researchers (Babic et al, 1998;Leelavathi et al, 2004) also reported alike findings of same size bands of transgenes in different transgenic plants in Southern blot analysis, even transgenic plants were obtained from different transformation events of Agrobacterium-mediated genetic transformation of B. carinata and Gossypium hirsutum.…”

Genetic engineering of cotton with a novel cry2AX1 gene to impart insect resistance against Helicoverpa armigera

“…Friable embryogenic calli were cultured with Agrobacterium strain LBA4404 (p2300-tp2AX1) (Leelavathi et al 2004). After cocultivation, the calli were subcultured on MS medium supplemented with 1.9 gL -1 KNO 3 , 25 mgL -1 kanamycin, and 250 mgL -1 cefotaxime until initiation and maturation of somatic embryos.…”