Protocol for wheat (Triticum aestivum L.) anther culture

Abstract

After many years of different experiments around the world on haploid production different methods have become available to the breeders. In Szeged, in vitro androgenesis via anther culture is one of the most efficient systems of homozygous line production. It can be widely applied in wheat and could make a significant contribution to the breeding of new varieties by saving time and increasing selection efficiency via doubled haploid (DH) lines. During the development of the doubled haploid production system into a routine technique, many problems have been solved (induction medium, genotype dependence, carbohydrate source, breeding applications, etc.). Induction medium is one of the most important factors for the induction of androgenesis and the subsequent development of embryoids and plantlets. The potato (P-4) medium published by Ouyang et al. (1983), seems to be the most effective haploid induction medium, after our minor modifications (P-4mf, Table 2.9-1). The amino acids glutamine and asparagine have been shown to enhance androgenesis and they can partially replace the potato extract in potato medium. Serine has reputedly a positive effect on the anther wall in Nicotiana anther culture. Marsolais et al. (1986) and others, have described an efficient method of green plant production by anther culture of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) using Ficoll in the media. Ficoll prevents anthers from sinking into the medium and avoids the presence of inhibitors from agar leading to improved androgenic response. Its application has opened up a new chapter in wheat anther culture. Among the conditions and variables used in anther culture system, incubation temperature and stage of microspore development are important factors. Cold pretreatment breaks down normal microspore development (gametophytic pathway) and increases the frequency of symmetrical mitoses, which causes a further increase in the appearance of spontaneous doubled haploid formation. Short heat shock treatments at the initiation of culture are also effective. The replacement of sucrose by other sugars, notably by maltose, enhances the success of anther culture. Experiments on cereals have generally shown an increase in embryo induction or an improvement in embryo development and green plant regeneration by the use of maltose instead of sucrose (Orshinsky et al., 1990).