Anther culture for doubled haploid production in barley (Hordeum vulgare L.)

Abstract

In barley, haploids can be produced both from female and male gametophytes, through a number of techniques, including: chromosome elimination following inter-specific crosses with Hordeum bulbosum, androgenesis using in vitro culture of anthers or isolated microspores, and gynogenesis using ovule culture in vitro. Among these systems, only two have been developed for large-scale production in barley: androgenesis including anther and microspore culture, and wide hybridisation followed by chromosome elimination. The isolated microspore culture system, often referred to as microspore or pollen embryogenesis, provides the most efficient and uniform route to mass scale production of haploid plants. It is, however, technically demanding and requires controlled environment for donor plant growth. Using anther culture and the protocol outlined below, we were able to produce green regenerants from donor plants grown in a greenhouse or in field conditions, for a wide range of barley genotypes. Among many factors, composition of the induction medium has been found to be important or even critical for androgenic response. In barley anther culture, major improvements have been achieved by replacement of sucrose by maltose as a carbon source, the balance of organic and inorganic nitrogen compounds and the replacement of agar by Ficoll or agarose in the induction medium. In the presented protocol, liquid induction medium BAC3 (Szarejko and Kasha, 1991; Cai et al., 1992) supplemented with Ficoll 400 (a high molecular weight sucrose polymer which increases medium density) gave a higher androgenic response than other media tested, due to better embryo formation. We have routinely used BAC3 medium (Szarejko and Kasha, 1991; Cai et al.,1992) for anther culture of barley varieties, hybrids, mutants and other breeding materials.