Plant male germ line transformation

Abstract

A new method to produce transgenic plants - male germ line transformation (MAGELITR) is reported. Unicellular tobacco microspores were isolated from excised anthers, and DNA carrying two marker genes was transferred biolistically. The bombarded microspores were matured in vitro for 6 days, and the mature pollen was used for in vivo pollination. Seeds were recovered and putative transformants were selected on the basis of their antibiotic resistance. Five kanamycin-resistant plants were chosen for further analysis, four contained the first construct, one the second. Parallel experiments with bicellular immature pollen did not produce any transgenic plants. A detailed DNA blot and expression analysis confirmed the transgenic nature of the five plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations. MAGELITR is a fast, regeneration-independent method, not prone to chimerism and somaclonal variation which should be genotype-independent and may be applicable in a wide range of species once in vitro maturation of pollen is established.