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

Abstract

SummaryReversible 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.

Proteome Analysis of the Wild and YX-1 Male Sterile Mutant Anthers of Wolfberry (Lycium barbarum L.)

“…The importance of GS1 in pollen reproduction has been shown in rice [59] and maize [60]. In tobacco, GS1 was inhibited by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters, and pollen aborted close to the first pollen mitosis in the transgenic plants, resulting in male sterility [61]. YX-1 anthers also showed lower GS activity relative to WT (Fig.…”

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants

“…Subsequently, expression of a ribonuclease inhibitor gene (barstar) from B. amyloliquifaciens in tapetal cells eliminated the ribonuclease activity and restored fertility in the male sterile lines (Mariani et al 1992). Using similar rationale and strategy, male sterile plants have been produced by expression of degradative enzymes (Worrall et al 1992;Kriete et al 1996;Cho et al 2001;Ruiz and Daniell 2005), by inhibition of particular enzymes by antisense strategy (van der Meer et al 1992;Groot et al 2004;Kapoor and Takatsuji 2006;Nawaz-ul-Rehman et al 2007;Ribarits et al 2007;Sandhu et al 2007), by expression of hormone encoding genes (Schmulling et al 1988), by expressing unedited form of mitochondrial genes in nucleus and targeting the product into mitochondria using a mitochondrial transit peptide Hernould et al 1998), by expressing the male sterility-associated mitochondrial DNA from well-characterized CMS systems (He et al 1996;Kim et al 2007;Yamamoto et al 2008), and also by expressing transcription factors and anther-speciWc receptors (Takada et al 2005;Ishimaru et al 2006;Mitsuda et al 2006;Li et al 2007). In many of these cases the restoration also has been achieved by selectively blocking the expression of the gene causing male sterility (Mariani et al 1992;Schmulling et al 1993;Kriete et al 1996;Zabaleta et al 1996;Bisht et al 2004;Busi et al 2006;Li et al 2007).…”

Plant Cell Culture – Present and Future

“…A variety of transcriptomic and metabolomic methods have now been applied [15,[69][70][71][72]. These investigations have been complemented by a range of other molecular investigations [68,73,74]. Such approaches include the use of gene expression profiling in Brassica napus, a technique that has revealed the expression of several embryogenesis-related genes like the BABY BOOM ERF/AP2 transcription factor [29], LEC1 and LEC2 [25][26][27][28] as early as 48-72 h after the initiation of microspore culture [70].…”

“…These findings represent a major breakthrough and will facilitate the study of plant embryogenesis in an isolated system. Other recent improvements in methodology [68,74] include the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide range of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment [75]. The regeneration frequency and percentage of green plants using this protocol are significantly greater than is found with the culture of shed microspores.…”