Microspore Culture in Wheat (Triticum Aestivum) — Doubled Haploid Production Via Induced Embryogenesis
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Plant Cell. Tissue and Organ Culture 73: 213-230, 2003. 213 2003 Kiuwer Academic Publishers. Printed in the Netherlands. Review of Plant Biotechnology and Applied Genetics Microspore culture in wheat (Triticum aestivum) — doubled haploid production via induced embryogenesis Ming Y. Zheng Department of Biology, Gordon College, 255 Grapevine Road, Wenham, MA 01984, USA (Fax: + 1-978-867-4666; E-mail: [email protected]) Received 12 July 2002: accepted in revised form 14 January 2003 Key words: androgenesis, cellular mechanisms, embryoids, genetic control, molecular and biochemical mechanisms, plant regeneration, somatic embryogenesis Abstract The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids ‘germinate’ to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research. Abbreviations: ABA − abscisic acid; DH − doubled haploid; HSP − heat shock protein; OVCM − ovary- conditioned medium; PGR − plant growth regulator; PPB − preprophase band Introduction Embryoids ‘germinate’ to produce either haploid or doubled haploid plants (DHs). This phenomenon, an Angiosperms exhibit life cycles of alternating generations excellent example of plant cell totipotency, is defined as between a haploid gametophyte and a diploid sporophyte. androgenesis or microspore embryogenesis, two terms that In the gametophyte phase, microspore mother cells in are often used interchangeably. The production of DHs anther locules undergo meiosis and mitosis that result in through androgenesis is a proven method to obtain the formation of male gametophytes or pollen grains. homozygous individuals in a single step. Thus, the method Prompted by certain environmental cues, immature pollen is useful in crop improvement, genetic manipulation, and (microspores) can be switched from gametophytic to in many areas of basic research related to plant sporophytic development, leading to the formation of developmental biology. The attainment of homozygosity ‘pseudoembryos’ commonly known as embryoids in one generation helps reduce the numerous cycles of (Reynolds, 1997; Touraev et al., 2001; Zheng et al., 2001). inbreeding necessary in conventional pure line breeding systems. With an efficient plant regeneration system, 214 gametic cells are also preferred targets for genetic Jähne and Lörz, 1995; Cordewener et al., 1995a; transformation and transgenic studies. DHs are frequently Reynolds, 1997; Touraev et al., 2001). Although wheat used in plant genome mapping. The direct access to microspore embryogenesis is the focal topic, reference to single-cells and the formation of true emhryoids makes the some aspects of anther/microspore cultures in other microspore culture an ideal system for studies of species is inevitable due to the similarity in fundamental embryogenesis and other aspects of plant developmental mechanisms of androgenesis. biology (Vicente et al., 1991; Reynolds, 1997; Touraev et al., 2001). Although the first success in regeneration of wheat Culture of microspores via induced (Triticum aestivun) plants through anther culture was embryogenesis achieved in the early 1970s (Research Group 303, 1972; Chu et al., 1973; Ouyang et al., 1973; Picard and De The first success in culturing microspores free of the Buyser, 1973; Craig, 1974), progress was slow. The early surrounding anther tissue was reported by Nitsch and culture methods primarily relied upon the natural Norreel (1973) in Datura innoxia. This and other early occurrence of embryogenic microspores within certain systems, however, are not strictly isolated microspore genotypes, hence were inefficient, yielding only a fraction culture as a pre-culture period for anthers or flower parts of the plants easily obtained today. In addition, these early is employed prior to the release of microspores. Following systems mainly used agar-solidified media, and rather the pre-culture period, microspores are either freed high levels of PGRs for callus induction. As a result, mechanically through homogenization (Lichter, 1982) or plants were mostly obtained through differentiation and shed from anthers into liquid medium (Ziauddin et al., regeneration of calli. The ratios of green vs. albino plants 1990). The mechanical isolation and subsequent culture of often were disappointingly low, and the high levels of microspores for plant regeneration in wheat is a more PGRs induced chromosome aberrations and undesirable recent event (Mejza et al., 1993; Gustafson et al., 1995). somatic variations as well. Once liquid media were In spite of low efficiencies, these two groups reported first employed in culture, the yields of calli/embryoids were success in culturing wheat microspores isolated from markedly improved and the regenerated plants were more mechanical blending. Since then, the culture systems for stable due to lowered PGR levels (Chuang et al., 1978; isolated wheat microspores have been improved to the Ouyang et al.. 1978). These liquid media included an practical level. Not only have the culture efficiencies been extract from boiled potatoes. While not readily definable, drastically improved, but also the germplasm range on the media worked well and were effectively used to study which microspore culture can be employed successfully and identify other factors that affect the responses of has been largely expanded. A reasonable number of DHs wheat anthers to in vitro culture manipulations. can be obtained from genotypes previously described as recalcitrant (Konzak et al., 1999; Zheng et al., 2001; Liu et As with other small grain cereals, DR production in wheat al., 2002). In these systems, microspores isolated from has advanced rapidly in recent years through isolated numerous genotypes are capable of producing adequate microspore cultures (Hu et al., 1995; Touraev et al., l996b, DHs for use in wheat breeding. The successes can be 2001; Hu and Kasha, 1997; Konzak et al., 1999; Zheng et attributed to a better understanding of androgenesis, more al., 2001, 2002b; Liu et al., 2002). The increase in effective manipulation of microspores for embryogenesis, efficiency was made possible through the direct access to and optimization of induction culture for embryoid and artificial manipulation of individual microspores and production (Table 1). subsequent success in plant production. This review provides a historical overview on wheat androgenesis and summarizes some recent progress in wheat microspore Growth and harvest of donor plants culture systems as well as our current understanding of the genetic, cellular, biochemical and molecular events It is widely known that growth conditions influence the associated with microspore embryo-genesis. Reviews on androgenic response by affecting the vigor and quality of other aspects of wheat anther/microspore culture can be donor plants (Kasha et al., 1990; Jähne and Lörz, 1995). found elsewhere (Kasha et al., 1990; Dunwell, 1992; The main factors include light intensity and quality, nutrition, photoperiod, and temperature. 215 Table I. Highlights of isolated wheat microspore culture processes Stage Manipulation Key events/parameters End product Pretreatment High (33 °C)/low (4 °C) (Induction of temperature, starvation (sugar, Viable microspores (some characterized by I embryogenic N), and chemical, alone or in ‘fibrillar’ cytoplasm or ‘star-like’ structure) potential) combination Mechanical blending (speed and Isolation and length); filtration and density Homogeneous population of embryogenic II purification centrifugation of 0.3 M mannitol microspores over 0.58 M maltose 9% maltose; PGR balance Induction (2,4-D, PAA, kinetin); ovaries culture Low rate of embryogenic abortion: large Ill or OVCM; temperature 26−28 °C; (embryoid number of mature embryoids osmolarity 300−320 mOsmole development) -1 kg H2O Plant recovery 190−2 semi-solid medium; IV (embryoid 0.088 M sucrose; PGR-free; Production of green plants germination) light supplement In wheat, lower temperatures (12−18 °C) have positive Pretreatment — transition to sporophytic development effects on culture response (Simmonds, 1989; Ziauddin et al., 1992). However, donor plants grown in a green- Although a number of procedures or systems have been house with reasonable control of temperature and light used in