Isolated microspore culture in maize (Zea mays L.), production of doubled-haploids via induced androgenesis

Abstract

Whenever we mention the use of doubled haploids (DH), the idea of achieving instant homozygosity immediately comes to mind. The potential uses of DHs, however, reach far beyond practical plant breeding. DH systems are widely useful in plant genome mapping, and in many areas of basic research such as investigations of in vitro embryogenesis and developmental biology. DH lines also may be used to create unique germplasm, for in vitro screening of plants possessing traits such as pathogen and pest resistances, improved quality, and stress tolerance produced either via conventional breeding or transgenic means. Although various approaches including in-situ gynogenesis, in vitro gynogenesis, and in vitro androgenesis have been used to produce maize DH plants, the only potentially efficient approach is in vitro androgenesis, production of haploid/DH through anther/microspore cultures. The procedures and conditions for anther culture have been reviewed extensively elsewhere (Buter, 1997). In isolated microspore cultures, microspores are usually freed from anthers and other tissues by mechanical methods, including macerating the anthers against a stainless steel sieve with a glass rod, chopping anthers with a razor blade, blending anthers in an electric blender or pulverizing anthers with a dispersing tool. Among various isolation techniques, blending seems to be less stressful to the microspores. No conclusive evidence has been available concerning the optimal explant type as well as optimal blending regime. Nevertheless, it is generally agreed that blending should be done in a medium that provides adequate osmolarity and at a relatively low temperature. In addition to anthers, spikelets or tassel segments may be used as explants for microspore isolation. Following the isolation, microspores are usually suspended in induction medium similar to those used in anther culture (Miter, 1997). Embryo-like structures (ELS) may appear 14–21 days after culture initiation, somewhat earlier than after anther culture initiation. Factors affecting isolated microspore cultures include growth conditions for donor plants, pretreatment conditions prior to the isolation, isolation procedures, microspore plating density, concentrations and nature of ingredients used in induction and plant regeneration culture, and physical conditions during the culture process (Zheng et al., 2002). Generally, low yields of embryoids (especially for elite germplasm), difficulty in plant regeneration and low frequencies of chromosome doubling are major limiting factors that have hindered the use of maize microspore culture technology for practical breeding (Biter, 1997). Shipping tassels even overnight has not proved satisfactory. In this Chapter, we describe an efficient system for production of maize DH plants from isolated microspores although further improvements can yet be made. From the onset of the project, our efforts were directed step by step to addressing the major problems in previous systems, i.e. improving the frequency of cells induced into androgenesis, increasing ELS yields, establishing a sound plant regeneration system to produce high frequencies of green doubled haploids. In our research, we combined an inducer chemical formulation (Zheng et al., 2001) with physical stress as developed for wheat microspore culture (Konzak et al., 1999), which acts to switch microspores from their gametophytic to a sporophytic development, to achieve high frequencies of embryogenic microspores. By then optimizing the induction culture medium and conditions, we were able to produce high yields of calli/embryoids and of DH plants. Over 98% of regenerated plants are green, 60–65% of healthy green plants have produced seeds upon sib-pollinations. This system was first established using a sweet corn germplasm ‘Seneca 77’ and has since been found applicable to other types of maize. As a result of our success, a utility patent was applied for, and is pending with United States Patent and Trademark Office.