A method is disclosed to achieve genetic transformation of cotton plants and lines. Immature cotton tissues are genetically transformed in vitro, by Agrobacterium-mediated genetic transformation. The resultant cotton tissues are subjected to a selection agent or agents to screen for transformants. The transformed cultures are then induced to commence somatic embryogenesis. One possible regime for regenerating such somatic embryos into whole cotton plants is disclosed.

Genetic engineering of cotton plants and lines

A method of genetically engineering a fiber-producing plant is disclosed. This method first comprises the step of constructing a plant expression vector that comprises a protein coding sequence and a DNA sequence capable of promoting gene expression in fiber cells, wherein the DNA sequence is homologous to a sequence selected from the group consisting of 12 different genomic sequences. The method next involves introducing the expression vector into a fiber-producing plant wherein the protein coding sequence is expressed in the fiber cells of the fiber producing plant. A method of obtaining a DNA sequence capable of promoting gene expression in fiber cells is also disclosed.

Genetically engineering cotton plants for altered fiber

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants.

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.).

Biotechnology Applications of Plant Callus Cultures

Seventeen cultivars of cotton (Gossypium hirsutum L.) were evaluated for callus initiation and maintenance using 3 initiation media and 3 maintenance media. After a series of transfers of a 3% glucose media, calli were placed on a 3% sucrose medium. After several weeks calli were observed for the presence of embryo-like structures. Cultivars Coker 201 and Coker 315 were identified as embryogenic. Embryogenic callus has since been routinely obtained within 6 weeks by initiating callus on glucose media for 3-4 weeks followed by transfer to sucrose media. Histological examination has shown that embryos are derived from isodiametric, densely cytoplasmic cells and follow predictable patterns of development. Upon maturity, transfer to auxin-free media with reduced sucrose levels results in embryo germination. Regenerated plants can be transferred to greenhouse within 90 days of callus initiation.

https://link.springer.com/content/pdf/10.1007/BF00269112.pdf

L. J. Couche

Papers

Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.).