Doubled Haploids in Genetics and Plant Breeding

3 Doubled Haploids in Genetics and Plant Breeding* Brian P. Forster and WjJJiam T.B. Th omas Scottish Crop Research Institute, Invergowrie , Dundee, DD2 SDA, Scotland , UK I. INTRODUCTION II. DOUBLED HAPLOID TECHNOLOGY III. DOUBLED HAPLOID POPULATIONS IN GENETICS A. ClAssic Studies B. Early Studies on Doub led Hnploi d Populations C. Bulked Segregant Analysis D. Gent:ti c Maps E. Mflpping of Quantitative Traits F. Genomics I V. DOUBLED H A PLOIDS IN BREEDING A. Early Breedi ng Att e mpts B. El ite CrOSSing C. Comparison of Breeding Methods D. Cui ti vtl r Releases E. Back cross Co nvers ion V. PROSPECTS

LITERATU RE CITED

I. INTRODUCTION

Doubled h aploids (DHs) are produced from haplOids. Haploid cell s occur naturally in the gametophytic phases of higher p lants, in their ovnles and pollen. It is the cells of th ese tissues that are the ma in targets for doubled haploidy. By manipulating the environment of the ga metic cells it is possible to divert development to produce embryos rather than mature poll en grains or ovules. The microspores of developing pollen grains are 1< Ac knowledgme nts : The Scottish Crop Research Institute rece ives gran t-in -aid from the Scottish Executive Env ironment and Rura l A ffairs Departm en t. The authors are nlso gratefu l to membe rs of the EU COST Action 85 1 "Gametic cells and molecu lar breed ing for crop improvement" for valua ble comment.

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 04 71666939 © 2005 John Wiley & Sons, Inc. 57

58

B. FORSTER AND W. THOMAS

of particular interest since plants generally produce pollen to excess. Microspore embryogenesis therefore has the potential to generate several hundred DH plants from a single anther. Induced or spontaneous chro­ mosome doubling of haploid embryogenic cells produces doubled hap­ loid plants. These are completely homozygous, true breeding, and consequently of great importance in genetics and plant breeding. The relevance of doubled haploids (DHs) to plant breeding has increased markedly in recent years owing to the expansion of application to over 200 species and the development of more reliable protocols. Doubled haploids can be exploited not only to produce new cultivars, but also to construct genetic maps, locate genes of agronomic and economic importance, iden­ tify markers for trait selection, and to increase plant breeding efficiency. The benefits accrue from the efficiency of DH technology in producing com­ pletely homozygous lines, vital resources in plant breeding and genetics. The potential of doubled haploidy has been the subject of various reviews (Choo et al. 1985; Dunwell 1985; Kasha et al. 1989; Pickering and Devaux 1992; Touraev et al. 2001; Maluszynski et al. 2003a). The recent rapid expansion of doubled haploid technology, which now includes over 200 plant species (Maluszynski et al. 2003b) sets the stage for greater DH deployment in a range of species and disciplines. We describe the exploitation of DHs from classic genetic studies to contemporary plant breeding and attempt to gauge current and future impact.

II. DOUBLED HAPLOID TECHNOLOGY

Doubled haploids can be produced via in vivo and in vitro systems. Hap­ loid embryos are produced in vivo by parthenogenesis, pseudogamy, or chromosome elimination after wide crossing. The haploid embryo is res­ cued, cultured, and chromosome-doubling produces doubled haploids. The in vitro methods incllide gynogenesis (ovary and flower culture) and androgenesis (anther and microspore culture). Methods can be complex and exacting. Critical stages include: growing conditions, pre-treatment of floral parts (before and after collection of material for culture), phys­ iology of donor plants, genotype, media composition, incubation, regen­ eration, and chromosome doubling (Kasha and Reinbergs 1981; Pickering and Devaux 1992; Jain et al. 1996). Androgenesis, where avail­ able, is the preferred method (Sopory and Munshi 1996). In 1922, Blakeslee et al. reported the finding of a haploid plant in Jim­ son weed, Datura stramonium. It took another 40 years to develop labo­ ratory methods for the routine production of Datura haploids via embryogenesis in cultured anthers (Guha and Maheswari 1964, 1966). This pioneering work led to the testing of many species, but responses to the protocols used have been extremely varied across the range of

3. DOUBLED HAPLOIDS IN GENETICS AND PLANT BREEDING

59

species. Tobacco, rapeseed, and barley are currently among the most responsive species, and although these are considered model species for DH production, response is further dependent on genotype. Legumes remain a particularly difficult group. There has been a progressive trend in reclassification from recalcitrant to responsive to highly responsive. Barley, for example, was considered recalcitrant to androgenetic proto­ cols before the patented incorporation of maltose in anther/micros pore medium (Hunter 1987), which paved the way to large-scale commercial application. A typical scheme for barley anther culture is given in Fig. 3.1. Legumes have been a notoriously difficult group, but in 1994 Ormerod and Caligari reported successful anther and micros pore culture of lupin. Potato moved from recalcitrant to responsive in anther culture (Rokka et al. 1996) and, more recently, improved microspore culture methods have been described for wheat (Liu et al. 2002). Problems in doubled haploid production vary among species. For most species androgenesis is the pre­ ferred route, but for some (e.g., onion), the only practical method is gyno­ genesis. For large trees there is the practical problem of controlling the donor plant environment and materials for culture (flower buds) are restricted to outdoor collections during spring months. A frequent prob­ lem in cereals and other grasses is genotype dependency; many genotypes regenerate high frequencies of albino plants from cultured tissues (e.g., Holme et al. 1999). The production of doubled haploids using wide cross­ ing can circumvent many of these problems. In barley (Hordeum vulgare), haploids can be produced by wide crossing with the related species H. bulbosum , fertilization is effected , but during the early stages of seed development the H. bulbosum chromosomes are eliminated leaving a haploid embryo. The embryos can be rescued and cultured, and plants derived from them artificially doubled (Devaux 2003; Hayes et al. 2003). The bulbosum technique has been very successful in producing DH cul­ tivars (the majority of barley cultivars , 74/115, listed in the web-site http://www.scri.sari.ac. uklassoc/costB51 IDefault.htm have been pro­ duced by this method). Wide crossing is also common in haploid/dou­ bled haploid production in bread wheat (Inagaki 2003), macaroni wheat (Jauhar 2003), and triticale (W'i!dzony 2003); in these protocols maize is commonly used as a pollinator. Pollination with maize is also success­ ful in oat haploid production (Rines 2003). There are, however, disad­ vantages in wide crossing: it is inefficient when compared with a responsive anther/microspore method, more crossing is required, spe­ cialized growing conditions are needed to grow both the target (female) and inducer (pollinator) species, haploid embryos must be rescued and cultured before they die, and at some stage artificial chromosome dou­ bling (often involving toxic chemicals) is required. Table 3.1 lists species in which doubled haploidy is an active area in European research and breeding. We have tried to order the species

B. FORSTER AND W. THOMAS

60

Doubled haploid production in a barley cross via anther culture

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2

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.

3

,,'

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I

Fig.3.1. Doubled haploid production in barley via anther culture. There are four basic components in the production of a barley DH population. A. Crossing: typically the DH population is derived from a cross; this can be done at any generation. Crossing normally consists of emasculation of the female parent (1). selection and preparation of a male par­ ent (dehiscing ear, 2). controlled pollination (3). and protection (4). B. Donor plant pro­ duction: In order to save time, developing seed from crosses can be extracted from ears [1) and immature embryos excised and cultured (2). The progeny is grown out in controlled environment conditions [3) that maximize the harvest of anthers containing responsive microspores. Spikes are harvested when developing microspores are at the un i-nucleate stage (4). C. Anther culture: anthers (or ears) are pre-treated by either cold or starvation treatments (1) and then transferred to specialized media to induce embryogenesis of the microspores (2). The green shoots that arise are transferred to another medium to promote root growth (3). D. Bulking of DH lines: rooted plantlets are transferred to pots, usually in a glasshouse (1), where they are grown on to maturity. In barley, 60-90% of plants are spon­ taneously doubled and the production of excess plants precludes the need for chemical­ induced doubling, though this is required in other species. Sufficient seed can be produced in the glasshouse for field trial evaluation in plots in the next generation (2).

....

0">

(Allium cepa)

Onion

(Cucumis sativus)

Cucumber

(Capsicum annuum)

Pepper

(Oryza sativa)

Rice

(Solanum melongena)

Eggplant

(x Triticosecale)

Triticale

(Zea mays)

Maize

(Triticum aestivum)

Wheat

(Hordeum vulgare)

Barley

(Brassica oleracea)

Cauliflower, broccoli , Brussels sprouts . cabbage, etc.

(Brassica napus)

Rapeseed

(Nicotiana tabacum)

Tobacco

Flower culture, gynogenesis

Gynogenesis

Anther culture. haploid somaclone Anther culture

Anther culture, microspore culture. wide crossing Anther culture

Anther culture, microspore culture , wide crossing Anther culture, microspore culture, wide crossing Inducer lines

Microspore culture, anther culture Microspore culture. anther culture Microspore culture. anther culture

Common methods

>1.000

>1 ,000

>1,000

>1,000

>5,000

>10,000

>10.000

>10 ,000

>10.000

>10 ,000

>10 ,000

>10.000

Production capacity (no. plants produced)

Current status of doubled haploid production in some target species.

Species Common name (Latin name)

Table 3.1.

No

Yes. F, hybrids

Yes , F, hybrids

Yes

Yes, F, hybrids

?

?

Yes

Yes

Yes

Yes

Yes

Cultivars developed via doubled haploidy

(continued)

Genotype. inbreeding depression. low fertility, chromosome doubling

Genotype, inbreeding depression

Genotype , embryo germination

Genotype

Genotype. albinism, chromosome doubling Chromosome doubling. genotype

Genotype, albinism, laboratory specificity Genotype, chromosome doubling, albinism Chromosome doubling

Genotype , germination of DH embryos. polyploids produced Genotype , germination of DH embryos , polyploids produced

Genotype

Major problems

N

Ol

Continued

(Populus spp.)

Aspen

(Quercus suber)

Cork Oak

(Malus spp.)

Apple

(Linum usitatissimum)

Citrus/clementine (Citrus spp.) Flax

(Asparagus o/ficinaJis)

Asparagus

(Beta vulgaris)

Sugar beet

(Solanum tuberasum)

Potato

(LaJium perenne)

Ryegrass

(Avena sativa)

Oat

(Secale cereale)

Rye

(Brassica. carinata)

Ethiopian mustard

(Brassica juncea)

Mustard

(Brassica rapa)

Turnip rape

Species Common name (Latin name)

Table 3.1.

Anther culture

Anther culture, microspore culture Anther culture , microspore culture. in situ Anther culture

Anther culture, crossed with supermale Anther culture, gynogenesis

Gynogenesis

Anther culture, wide crossing

Anther culture

Anther culture

Microspore culture. anther culture Microspore culture. anther culture Microspore culture. anther culture Anther culture

Common methods

?

?

10-100

?

>200

?

?