Plant Biotechnology Journal (2007) 5, pp. 483–494 doi: 10.1111/j.1467-7652.2007.00256.x © 2007 The Authors 483 Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Oxford, UK PBI Plant Biotechnology Journal 1467-7644 © 2007 Blackwell Publishing Ltd ? 2007 ? Original Article Reversible male sterility and doubled haploids Alexandra Ribarits et al. Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen Alexandra Ribarits 1 , A. N. K. Mamun 1, †, Shipeng Li 2 , Tatiana Resch 1 , Martijn Fiers 2 , Erwin Heberle-Bors 1 , Chun-Ming Liu 2 and Alisher Touraev 1, * 1 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Plant Molecular Biology, Dr Bohrgasse 9/4, 1030 Vienna, Austria 2 Plant Research International, PO Box 16, 6700 AA, Wageningen, the Netherlands Summary 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. Received 12 December 2006; revised 27 February 2007; accepted 1 March 2007. *Correspondence (fax +43 14277 9546; e-mail alisher.touraev@univie.ac.at) †Present address: Plant Biotechnology Division, IFRB, AERE Bangladesh Atomic Energy Commission, GPO Box No. 3787, Dhaka-1000, Bangladesh Keywords: doubled haploid plants, glutamine synthetase, male sterility, microspore, tapetum, tobacco. Introduction Today, many important seed (corn, rape), vegetable (tomato, pepper) and foliage (tobacco, forage grasses) crops are available as F 1 -hybrid cultivars. They have great commercial value as a result of heterosis (Birchler et al., 2003) and the protection of breeder’s rights. Two inbred lines are crossed to produce F 1 -hybrid seeds, and a reliable system of pollination control is mandatory to enforce cross-pollination (Perez-Prat and van Lookeren Campagne, 2002). However, the lack of a universal technology for reversible male sterility is a major barrier to the exploitation of the advantages of F 1 -hybrid seed production in a wider range of plant species (Perez-Prat and van Lookeren Campagne, 2002). Male sterility avoids the labour of manual emasculation, and serves as a molecular strategy for transgene containment by preventing pollen release to the environment (Daniell, 2002; Perez-Prat and van Lookeren Campagne, 2002). In practice, tight sterility and efficient fertility restoration to maintain the male-sterile lines are musts for the successful incorporation of the sterility trait into F 1 - hybrid breeding (Budar and Pelletier, 2001; Perez-Prat and van Lookeren Campagne, 2002). The most commonly used male sterility system is cytoplasmic male sterility (CMS) (Kaul, 1988). However, it is not available in any given crop, and requires tedious breeding procedures. Near-isogenic male-fertile plants carrying the appropriate restorer genes need to be available for the nuclear restoration of fertility, and, unless using naturally occurring CMS, potential parental inbred lines must be transformed into male-sterile lines, which is usually achieved through a series of backcrosses (Hanson and Bentolila, 2004). Genetically engineered reversible CMS has recently been presented as a promising alternative