Highly Efficient Doubled-Haploid Production in Wheat (Triticum aestivum L.) via Induced Microspore Embryogenesis

haploid plant per floret (Devaux, 1988). Androgenesis induction in microspores may be affected by various An efficient doubled-haploid production technology that induces factors which cause low induction efficiency and by gehomozygosity can greatly reduce the time and cost of cultivar developnotype dependence (Dunwell, 1985). Most advances toment. Low efficiency of doubled-haploid production previously has limited exploitation of this method for crop improvement. This study ward improving anther–microspore culture methods aimed to develop a more efficient and effective isolated microspore have been focused primarily on the concept of using culture system for generating doubled-haploid wheat (Triticum aesti“stress” treatments to induce androgenesis from the vum L.) plants. We report here the development and testing of a new preprogrammed gametophytic to the sporophytic pathchemical formulation for its efficiency to induce microspore emway (Touraev et al., 1996, 1997; Hu and Kasha, 1999; bryogenesis, and the development of a system for double haploid Zhou and Konzak, 1997; Zheng and Konzak, 1999; Siproduction, in which the induction of embryogenesis in microspores monson et al., 1997; Reynolds, 1997). Those culture was followed by isolating embryogenic microspores, and culturing systems have been effective only for a narrow range them under optimized growth conditions to produce high embryoid of responsive genotypes, and other genotypes remain yields. Up to 50% of the total treated microspores in the whole recalcitrant. Thus, more effective methods are needed spike were converted from their preprogrammed gametophytic to a sporophytic pathway by a chemical inducer formulation consisting of for inducing androgenesis in large populations of micro0.1 g L 1 of 2-hydroxynicotinic acid, 10 6 mol L 1 2,4-dichlorophenoxyspores for a wide range of genotypes. acetic acid, and 10 6 mol L 1 6-benzylaminopurine. The isolated emA relationship between microspore embryogenesis bryogenic microspores were cocultivated with live wheat ovaries in and chemical treatment was observed in our experia liquid NPB 99 media with an osmolality of about 300 mOsmol kg 1 ments and by others (Konzak et al., 2000; Bennett and H2O, resulting in the regeneration of 50 to 5500 green plants per Hughes, 1972; Rowell and Miller, 1971; Picard et al., single spike of eight wheat genotypes. The high efficiency and simplic1987). Although Picard et al. (1987) described improveity make the system practical for biological research and for acceleratments in androgenesis with wheat (T. aestivum) anther ing cultivar development in wheat breeding programs. cultures, their treatments were less effective than those in use for anther culture at that time (Zhou and Konzak, 1989). We envisioned that according to the signal system A the process by which pseudoembryos concept of Ryan and Balls (1962) and Constabel et al. (embryoids) able to germinate into plants are pro(1995) some chemical formulations could effectively induced from microspores (pollen embryogenesis), is of duce a large proportion of microspores to become emsignificant interest for developmental and genetic rebryogenic, if the correct formulations were developed. search as well as for plant breeding and biotechnology, We recognized, however, that after embryogenesis was since the process is a means for producing genetically induced, the induced microspores require an optimal true-breeding, doubled-haploid (DH) plants. By prophysiological environment to develop further into emducing DH progeny, the number of possible gene combibryoids able to germinate and develop into green plants. nations for inherited traits is more manageable (Konzak The objectives of this work were to develop a method et al., 1987). An efficient DH technology can greatly for efficiently initiating microspore embryogenesis by a reduce the time and the cost of cultivar development chemical inducer formulation, and for producing large (Hu and Yang, 1986; Hu, 1997). quantities of microspore-derived green plants from a Low efficiency in DH production previously has limwide spectrum of genotypes under optimal culture conited exploitation of this potentially powerful method ditions. for crop improvement. Several methods of haploid production have been investigated and reported in the litMATERIALS AND METHODS erature, including microspore and/or anther culture (androgenesis), ovule culture (gynogenesis), Hordeum Growing Wheat Plants bulbosum L. or maize (Zea mays L.) pollination methThe spring wheat genotypes ‘Chris’, ‘Pavon 76’, WED 202ods (alien species chromosome elimination), and an 16-2,‘Yecora Rojo’, ‘Calorwa’, ‘Waldron’, ‘Wawawai’, and alien cytoplasm system (Dunwell, 1985; Kasha, 1989). winter wheats ‘Capo’ and ‘Svilena’, were used. These genoMicrospore and anther culture methods have the potentypes differed in responsiveness to embryogenesis and green tial to produce more than a thousand haploid plants per plant regeneration on the basis of previous anther culture cultured anther; all other methods are limited to one experiments. One to three plants per pot (20 by 25 cm in diameter) were grown in a greenhouse controlled at 27 2 C, at a light regime of 17 h light and 7 h dark. Winter wheat Northwest Plant Breeding Company, 2001 Country Club Road, Pullman, WA 99163. Received 10 April 2001.*Corresponding author Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2-HNA, 2-hy([email protected]). droxynicotinic acid; BAP, 6-benzylaminopurine; DH, doubled haploid; NPB, Northwest Plant Breeding Company. Published in Crop Sci. 42:686–692 (2002).