Methods for corn transformation

#The present invention relates to the field of plant biotechnology. More specifically, it concerns methods for incorporating genetic components into a plant via genetic engineering techniques. In particular, provided herein are systems for genetically transforming corn.

#During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products for food, feed and industrial uses.

#Agrobacterium-mediated transformation is one method for introducing a desired genetic element into a plant and is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry a desired piece of DNA into many plant species. The major events marking the process of T-DNA mediated pathogenesis are induction of virulence genes, processing and transfer of T-DNA.

#FIG. 1 is a plasmid map of pMON30113.

#FIG. 2 is a plasmid map of pMON42071.

#FIG. 3 is a plasmid map of pMON65324.

#The present invention is particularly directed to the transformation of any cereal plant, particularly corn lines. The present invention provides for a rapid and efficient process for obtaining transformed corn plants by reducing the time the transformable explants spend in the selection phase, which in this system is the callus induction phase, of a typical transformation process and by increasing the temperature during the selection phase. It has been discovered that placing the transformed corn explant on a tissue culture media containing a selective agent for a total period of 2-4 weeks is sufficient to select for transformed plants and to obtain fertile transgenic plants therefrom. Moreover, the transformed explants may be placed in a single vessel containing the selection media for the entirety of the selection period with efficacious results, thus eliminating the need for repeated subculturing and transfer of the explants to fresh media. In this method, it is often desirable to maintain selection pressure during the regeneration phase as well.

#It has further been discovered that the transformation efficiency may be increased about twofold by maintaining the temperature during the selection phase, or at least a portion of the selection phase, at a temperature greater than 27° C., a typical corn tissue culture temperature. Preferably, the selection temperature is maintained between about 28.5° C. and about 35° C., more preferably the temperature is maintained between about 30° C. and about 34° C., and most preferably the temperature is maintained between about 30° C. and about 32° C. This elevated temperature is maintained for a period of not more than three weeks and more preferably not more than two weeks during the selection phase.

#It has also been discovered that limiting the amount of submersion during Agrobacterium inoculation of the transformable explants increases the transformation frequency. The explants can be isolated directly into inoculation medium and then removed from the medium within 20 minutes. Explants could also be isolated into co-culture medium and then spotted with 1 μL of Agrobacterium solution or contacted with filter paper saturated with Agrobacterium solution for 5 to 60 minutes. It is believed that the anaerobiosis of being in solution is detrimental to the explants. Thus limiting or eliminating the anaerobiosis increases the transformation efficiency. It has been observed that immersion in the inoculation medium for more than 60 minutes has a detrimental effect on the explant. Thus eliminating the anaerobiosis by altering the manner or the time in which the Agrobacterium contacts the explant increases the transformation efficiency.