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United States Department of Agriculture

Agricultural Research Service

Title: Stable Transformation of Embryogenic Cell Suspension Cultures of Cotton (Gossypium Hirsutum) by Particle Bombardment

Authors
item RAJASEKARAN, KANNIAH
item Hudspeth, R - BOSWELL COTTONSEED CO.
item CARY, JEFFREY
item Anderson, D - BOSWELL COTTONSEED CO.
item CLEVELAND, THOMAS

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 10, 1999
Publication Date: N/A

Interpretive Summary: Infection of cotton bolls by the fungus, Aspergillus flavus, results in reduced cottonseed value, because the fungus invades the seed in the bolls and produces aflatoxin, a poison and potent carcinogen. The presence of aflatoxin in cottonseed endangers the health of livestock consuming cottonseed used in animal feeds and the health of humans consuming products (e.g., milk) derived from the affected livestock. The objective of our research is to genetically engineer commercial varieties of cotton with antifungal genes in order to inhibit infection of cotton bolls by aflatoxin producing fungi. Genetic engineering or cotton involves time consuming, laborious procedures involving growing cotton cells and tissues in nutrient containing media to regenerate fully developed cotton plants. One of the ways to overcome this problem is to use cotton cells that are continually producing cotton embryos from which cotton plants can be regenerated immediately following genetic engineering. DNA containing the gene of interest is introduced using the gene gun method, which involves coating microscopic gold particles with the DNA and firing them into the embryogenic cell suspension cultures with a blast of helium. High frequency gene incorporation into the embryos has been demonstrated by using a visual marker gene which gives blue color upon staining. The research will benefit the cotton industry through providing the technology to introduce valuable traits into cotton, such as resistance to aflatoxin contamination or for improved agronomic characters, such as fiber quality.

Technical Abstract: Stable transformation with high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures of cotton (Gossypium hirsutum L.). Transient and stable expression of B-glucuronidase (GUS) gene was monitored in cell suspension cultures. Stable expression was observed in about 5% of the transiently expressing cells. The increased efficiency is due to (1) the use of rapidly dividing cell suspension cultures; (2) multiple bombardment of cells during the logarithmic phase of growth; and (3) selection for transformed cells in gradually increasing concentrations of the antibiotic G418. Southern analysis of 10 randomly selected plants indicated transgene copy number of one to four.

Last Modified: 7/28/2014
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