There are only a few cells in the plant kingdom that are as exaggerated in their size or composition as cotton fibers. It is precisely their highly elongated structure and exceptional chemical make-up that establishes cotton fiber as an ideal model for studies of plant cell elongation and cell wall

Cotton Fiber Growth in Planta and in Vitro. Models for Plant Cell Elongation and Cell Wall Biogenesis

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.

Insect resistant cotton plants.

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

Genetically engineered plants of commercial cotton varieties were obtained by Agrobacterium–mediated transformation. Inoculated tissues selected on kanamycin–containing medium gave rise to transformed calli that were resistant to the antibiotic and expressed the neomycin phosphotransferase enzyme. Amplified tissues were plated onto hormone–free medium to promote embryogenesis. Somatic embryos germinated and whole plants also expressed the marker enzyme.

Genetically Transformed Cotton ( Gossypium Hirsutum L.) Plants

Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a ‘Double Gene/Single Selection (DGSS)’ plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than ‘Single Gene/Single Selection (SGSS)’ vector (aphA-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops ($ 120 billion US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.

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Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

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.).

Calli of cotton (Gossypium hirsutum L) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media High inorganic salts (MS), low salt (2/3 MS), excess KNO3, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA3) and kinetin were tested for their effects on somatic embryo maturation Long-term embryo proliferation and maturation were best on medium containing MS plus 19g/l KNO3 Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 01 mg/l indoleacetic acid (IAA) Plants were recovered from 106% of the embryos When ≥5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 01 mg/l GA3 Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period

Somatic embryogenesis in cotton ( Gossypium ). II. Requirements for embryo development and plant regeneration

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.

Joe R. Goodin

Papers

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

Somatic embryogenesis in cotton ( Gossypium ). II. Requirements for embryo development and plant regeneration

Somatic embryogenesis in cotton (Gossypium) I. Effects of source of explant and hormone regime

Swelling of Etiolated Oat Protoplasts Induced by Phytochrome, cAMP and Gibberellic Acid: A Kinetic Study

Swelling of etiolated oat protoplasts induced by cAMP and red light