Inheritance and expression of genes for kanamycin and chloramphenicol resistance in transgenic cotton plants

To fully evaluate and characterize the stability of traits transferred via Agrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic cotton (Gossypium hirsutum L.) plants (no. 1397, no. 1427, and no. 1438) for inheritance and expression of their foreign genes. Following regeneration from tissue cultures transformed using A. tumefaciens, primary regenerated plants were tested for expression of T-DNA marker genes encoding the proteins neomycin phosphotransferase II (NPT-II) and chloramphenical acetyltransferase (CAT). To test for transmission of marker traits flowers were either self-pollinated or crossed to the cotton cultivar Coker 312. Selfed progeny and backcrosses were germinated in vitro and selected on medium containing 50 mg L-1 kanamycin. At the three-leaf stage of development, enzymatic activity of NPT-II and CAT was measured on seedlings capable of germination and survival in the presence of kanamycin. Progeny derived from selfed and backcrossed pollinations of plants no. 1397 and no. 1427 both segregated 3:1 and 1:1, repsectively, as expected for a single gene trait. No CAT activity was detected in the plants or their progeny. Southern blot analysis of regenerated platns, selfed progeny and backcrosses of no. 1397 and no. 1427 confirmed the inheritance of only the NPT-II coding sequence portion of the T-DNA. In plant no. 1438 and its backcross progeny, enzyme expression of both NPT-II and CAT was detected. Segregation analysis of no. 1438 backcross progeny showed a 1:1 pattern of inheritance. Southern blot analysis of no. 1438 and its progeny showed that the entire T-DNA coding region was present. Despite the presence and function of the CAT gene in backcross progeny of no. 1438 these plants did not display chloramphenicol resistance. Germination of seeds obtained from no. 1397, no. 1427, and no. 1438 on kanamycin-containing (50 mg L-1) medium efficiently selected for transgenic seedlings expressing kanamycin resistance. Our data support the conclusion that the single gene insertion of foreign traits in cotton is inherited and expressed in a Mendelian fashion.