Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen.

Reversible male sterility and doubled haploid plant production are two valuable technologies in F(1)-hybrid breeding. F(1)-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F(1)-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter.

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.