Transgenic soybean plants derived from the half-seed explants have been produced and confirmed in the R0 and R1 generations through molecular analysis, and ISU is seeking partners interested in commercializing this technology.
Commonly used methods for soybean transformation such as the cotyledonary node (“coty node”) or embryogenic callus (“callus”) method have various disadvantages. For example, the coty node method requires precise wounding of seedlings to introduce genetic material and in vitro germination that can result in low transformation efficiencies, poor reproducibility and non-germline transformation. The callus method—in which embryonic tissue is bombarded with DNA-coated carrier particles—has the drawbacks of requiring a prolonged tissue culture period, and can also result in the complex insertion of genes into the plant genome and may result in the regeneration of sterile plants. To overcome these disadvantages, ISU researchers have developed a method for efficient soybean transformation and regeneration using half-seed explants. In this method, half-seed explants (separated cotyledonous tissues derived from mature soybeans) are infected with Agrobacterium tumefaciens carrying a transgene of interest and regenerated in vitro using tissue culture medium. Transgenic plants can be obtained within 9-10 months, and any suitable genotype of soybean can be used with this method. Because this approach does not require technical precision or prolonged tissue culture, it is more efficient and robust than other soybean transformation methods.
“Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation”, M. Paz, J.C. Martinez, A. Kalvig, T. Fonger, and K. Wang, 2006, Plant Cell Reports 25: 206-213.
Paz et al., “Agrobacterium-mediated transformation of soybean and recovery of transgenic soybean plants,” including affidavit.
This article, labeled as updated on March 22, 2010, was available to the public in 2010, and was uncoverable by searching, at http://agron-www.agron.iastate.edu. Included is an affidavit of Damien Bolin attesting to the fact that the March 22nd version of Paz et al. was uploaded to http://agron-www.agron.iastate.edu on April 17, 2010, and was available to the public on that date. The protocol discloses, among other things, trimming of the embryonic axis found at the nodal end of the cotyledons to approximately 3 mm. This reference cites, among others, Paz et al., “Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation,” Plant Cell Reports 25: 206-213 (2006), and Paz and Wang, “Soybean transformation and regeneration using half-seed explants,” U.S. Patent No. 7,473,822, which issued January 6, 2009. Keywords: Kan Wang, Margie Margarita M. Paz, Dow Agrosciences, Dayakar Pareddy, Siva Chennareddy, Tatyana Minnicks, Olga Karpova, David Griffin, Jayakumar Pon Samuel, Kelley Smith, Rodrigo Sarria-Millan, Tejinder Mall