Abstract. A study was conducted to optimize the pre-treatment conditions in anther culture in an attempt to develop a flexible and efficient system for routine doubled haploid (DH) production in western Canadian barley germplasm. Optimal pre-treatment involved using a high mannitol level at 24^oC. A flexible and rapid system has been established and successfully applied to a wide range of crosses from the Brandon Research Centre breeding program.

Introduction.. Doubled haploidy is becoming an integral part of many breeding programs. In barley, some commercial cultivars have been obtained mainly from the Hordeum bulbosum method (Devaux et al., 1996). Anther or isolated microspore culture has the potential to produce more regenerants. The success is influenced by a number of factors, some of which interact, making it difficult to repeat the results reported in other laboratories (Kasha, 1989). Cold spike pre-treatment has been extensively used in barley anther/microspore culture (Jahne & Lorz, 1995). However, the optimum duration of cold pre-treatment was found to be genotype-dependent, and its beneficial effect could not be shown in some environments (Pickering & Devaux, 1992; Kasha, 1989; Ziauddin et al., 1992). Mannitol solution was found to be an effective alternative, especially when used at high levels (Broughton & Priest, 1997; Hoekstra et al., 1997).

Most of the studies were conducted on highly responsive genotypes, such as cv. Igri. The ability to quickly transfer technologies from such successful genotypes to local promising breeding material involves optimizing a number of factors for any specific research institution and germplasm. This study was designed to optimize the pre-treatment conditions in anther culture in an attempt to develop a flexible and efficient system for routine DH production in western Canadian barley germplasm.

Material and Methods. A preliminary experiment was conducted in 1997 to determine the optimal temperature and mannitol combination, using six hybrids from the barley breeding program at the Agriculture and Agri-Food Canada Research Centre, Brandon, Manitoba. These included five two-row hybrids (BM9647 ‘TR251/CDC Kendall’, BM9651 ‘TR251/BM9126-35’, BM9711 ‘TR253/AC Metcalfe’, BM9713 ‘TR339/AC Metcalfe’, BM9725 ‘BM9240-17//TR251/AC Metcalfe’), and one six-row hybrid (EX640 ‘Q4563-4/BM9220-7’). Plant growth conditions and spike collection were as in Ziauddin et al. (1992). Pre-treatment consisted of plating 100 anthers in mannitol solutions at three different levels (0.3M, 0.7M and 1.0M) and incubating them in the dark for 4 days at 4^oC or 24^oC. Anthers were later transferred to liquid Hunter’s FHG medium (Kasha et al., 1990), and incubated at 27^oC. Induced embryos were transferred to a solid medium and placed at 22^oC under low light (75 mol m^-2 s^-1) for a 16-h photoperiod. Regenerated green plants were transferred to a hormone-free MS medium until transplantation. Number of plants (green and albino) regenerated per 100 transferred embryos and cultured anthers were recorded and subjected to statistical analysis.

A complementary experiment was conducted in 1998 in which 14 crosses, including ten two-row hybrids and four six-row, were utilized for DH production using the best pre-treatment combination identified in the preliminary experiment.

Results and Discussion: Embryogenesis and regeneration of induced embryos/calli were influenced by the genotype, the mannitol level and the temperature (Table 1). Induced embryos/calli became visible about 14 days after culture in the pre-treatment involving the higher mannitol solution, and much later (about 21 days or later) in the other pre-treatments. The genotype response was temperature-dependant, as were mannitol effects. However, ‘genotype x mannitol’ interaction effects were not significant. The genotypic influence on barley anther/microspore culture androgenesis is well documented and the genetic control appears to be rather complex (Kasha et al., 1990; Pickering & Devaux, 1992; Hou et al., 1994). The regeneration of albinos was influenced mainly by the temperature.

Pre-treatment involving high levels of mannitol (0.7 M and 1.0M) at 24^oC showed the best results (Table 1). Similar beneficial effects were also reported by Broughton & Priest (1997) and Walsh & Laufer (1997). However, the advantage of raising the mannitol level higher than 0.37 M could not be demonstrated in some studies (Hoekstra et al., 1997). Combining cold with high levels of mannitol did not translate into increased efficiency, as ‘temperature x mannitol’ interactions were significant in our study.

In the complementary experiment involving 14 crosses, an average of 20 green plants per 100 anthers was obtained using 1.0M mannitol pre-treatment at 24^oC (Table 2). This represents a minimum number of potential regenerants since all induced embryos could not be transferred onto a regeneration medium. The recovery of 150 green plants per cross was considered to be sufficient for our purpose. About 80% of the green plants produced were spontaneously doubled, completely fertile lines. Although a sufficient number of green plants was rapidly and easily obtained, further refinement of the protocol to reduce the occurrence of albinos could make the system even more efficient.

Conclusion: This study was successful in improving the recovery of micropsore-derived doubled haploids by anther culture in both frequency and speed. The system provides enough flexibility for breeding purposes since as few as 5 spikes would suffice to initiate DH production.

Acknowledgement: Financial support from the Western Grains Research Foundation Barley Check-off and the Agriculture and Agri-Food Canada Matching Investment Initiative is gratefully acknowledged.

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