Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/1/2002
Publication Date: 12/2/2002
Citation: SHOU, H., PALMER, R.G., WANG, K. IRREPRODUCIBILITY OF THE POLLEN-TUBE PATHWAY TRANSFORMATION PROCEDURE. PLANT MOLECULAR BIOLOGY REPORTER. 2002. V. 20. P. 325-334. Interpretive Summary: Plant breeders and geneticists use many techniques to introduce traits into plants. Their objective is to improve the quality or quantity of plants. Traditional methods have been very successful and have included traits from very distant relatives. Tissue culture can also be used to introduce traits, that can be very diverse, but the subsequent regeneration is difficult in many plants, e.g. soybean. Our objective was to use a method that avoids tissue culture, but could still introduce traits from diverse plants, or even from animals or microorganisms. This method, called pollen-tube pathway uses the male (pollen) as it grows (pollen tube) down the female tissue to fertilize the egg. The introduction of traits (now a DNA application) via the pollen tube in soybean, has been successful in China, but not successful outside China. Working with the Chinese soybean line and U.S. soybean lines, we obtained a total of 4793 progeny seed from 5590 individually treated soybean flowers. Biochemical tests indicated that none of the seed carried the pollen-tube introduced DNA. Our results, over a three-year period, with seven soybean lines, and four different DNA constructions, indicated that the pollen-tube pathway technique (transformation) is either not effective in soybean, or that the efficiency is too low to be detected in our experimental conditions. This information will be useful to plant breeders, plant geneticists, and plant tissue culture researchers in public and private industry.
Technical Abstract: There is growing interest in the development of tissue culture independent genetic transformation methods for plants. The pollen-tube pathway transformation technique is one of the alternative methods. Because of its poor repeatability and the insufficient molecular evidence for confirming transformation, the technique is controversial. The objective of this research was to evaluate the robustness of the pollen-tube pathway technique in soybean [Glycine max (L.) Merr.]. Solutions of purified DNA constructs carrying a selectable marker gene bar and a reporter gene gus or a gene of interest (npk1) were applied to severed styles of flowers to six to eight hours after self-pollination. The experiment was repeated for three summers in the field in which four different DNA constructs and seven soybean genotypes were tested. A total of 4793 progeny seeds were harvested from 5590 individually treated soybean flowers. All seeds were germinated. Plantlets were sprayed with the herbicide Liberty¿. While no glufosinate-resistant plantlets were identified, 2% of progenies showed partial resistance to the herbicide. Histochemical GUS assay and Southern blot analysis indicated that the partially resistant progeny plant were escapes from the herbicide screening. Our results indicate that the pollen-tube pathway transformation technique is either not effective in soybean, or that the efficiency is too low to be detected in experimental conditions reported here.