Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker�310�FR)

Abstract

In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were beta-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR�2)

“…Coker 310. To date, only limited reports of high-frequency regeneration in cotton somatic embryos are available due to a genotype-dependent response (Rangan and Zvala 1984;Mitten 1985;Shoemaker et al 1986;Trolinder and Goodin 1987;Finer 1988;Cousins et al 1991;Kolganova et al 1992;Firoozabady and DeBoer 1993;Rangan and Rajasekaran 1996;Sakhanokho et al 2000;Rajasekaran et al 2000;Zhang et al 2001;Chaudhary et al 2003;Kumria et al 2003;Mishra et al 2003). Several problems have been highlighted during the regeneration of cotton from somatic embryos, including the excretion of secondary metabolites from the explants into the medium, browning of callus after a short period of culture, a low frequency of embryo maturation and abnormal somatic embryo germination.…”

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

“…In this procedure, the transformation efficiencies are generally low due to the low frequency of embryogenesis and the difficulty in germination of transformed embryos (Firoozabady et al 1987;Umbeck et al 1987Umbeck et al , 1989. Also, lack of proper root development among the germinated embryos lowers the transformation frequency still further (Chaudhary et al 2003).…”

The Uses of Somatic Embryogenesis for Genetic Transformation

“…Agrobacterium tumefaciens-mediated genetic transformation of cotton (Gossypium hirsutum) was achieved using the line Coker 310FR, which readily regenerate plants through somatic embryogenesis (Chaudhary et al 2003). Hypocotyls or cotyledonary leaves from 7-day-old seedlings were used as the explant sources.…”