A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens

There is a great potential for genetic manipulation of crop and medicinal plants to enhance productivity through increasing pest and microbial disease resistance and environmental stress tolerance, and also for studying gene function and regulation of physiological and developmental processes$^1$. Transgenic plants that transmit the introduced trait to progeny generations are produced using various DNA delivery methods such as particle acceleration$^2$, electroporation$^3$ and polyethylene glycol permeabilization of protoplasts$^4$. However, most commonly used method for obtaining transgenic plants is by the Agrobacterium tumefaciens-mediated transformation$^{5,6}$. Agrobacterium can transfer DNA to a remarkably broad group of organisms – numerous dicot and monocot angiosperm species$^5$, gymnosperms$^6$ and fungi, including yeast$^7$, ascomycetes$^8$ and basidiomycetes$^9$. Recently, Agrobacterium was reported to transfer DNA to human cells$^{10}$.