6 review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Plant Breeding, 133, 313–320 (2014) © 2014 Blackwell Verlag GmbH doi:10.1111/pbr.12162 Review Review of doubled haploid production in durum and common wheat through wheat maize hybridization Z H I X I A N I U 1, A I X I A N G J I A N G 2, W E S A M A B U H A M M A D 3, A T E N A O L A D Z A D A B B A S A B A D I 3, S T E V E N S X U 1, M O H A M E D M E R G O U M and E L I A S M E L I A S 3,4 United States Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Fargo, ND 58102, USA; Biostatistics Department, Vanderbilt University, Nashville, TN 37232, USA; 3Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA; 4Corresponding author, E-mail: elias.elias@ndsu.edu Received May 15, 2013/Accepted December 21, 2013 Communicated by L Hartl Abstract Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops The procedure for DH production includes two major steps: haploid induction and chromosome doubling In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen Numerous wheat cultivars and mapping populations have been developed using wheat– maize hybridization In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production Key words: doubled haploid production — wide hybridization — chromosome elimination — chromosome doubling — common wheat — durum wheat Doubled haploids (DHs) in plants have complete homozygosity, which can be achieved in one generation from hybrid plants Thus, using the DH method could speed cultivar and population development in crop improvement, genetic manipulation, and plant genome and gene mapping The procedure for DH production includes two major steps: haploid induction and chromosome doubling Chromosome doubling of haploid plants has been routinely and successfully performed using colchicine However, the success and efficiency of haploid induction varies in different crop species The successful utilization of the DH method in most crops to breed commercial cultivars and to develop mapping populations relies highly on an efficient protocol for inducing haploids Haploids in higher plants can occur spontaneously They can also be artificially induced in vivo or in vitro by androgenesis and gynogenesis The first report of the natural occurrence of sporophytic haploids was in the weed species Datura stramonium L (Blakeslee et al 1922) Later, the development of protocols for the production of haploid plants and the techniques of chromosome doubling led to the release of two DH cultivars in 1972 and 1980, respectively: ‘Maris Haplona’ in rapeseed (Brassica napus L.) using microspore culture and ‘Mingo’ in barley (Hordeum vulgare L.) using genome elimination (Thompson 1972, Ho and Jones 1980) In the late 1970s, DH technology developed slowly because of its labour and time-consuming process (Forster et al 2007) But in recent years, technological innovation and the high demand of the end-use applications of DH lines, such as used for marker identification and gene mapping, drew attention to the development of DHs in higher plants (Forster et al 2007) Durum wheat (Triticum turgidum L subsp durum, 2n = 4x = 28 = AABB genomes) and common wheat (Triticum aestivum L., 2n = 6x = 42 = AABBDD genomes) are two important cereal crops for human consumption They are both self-pollinated allopolyploids Previously, the development of durum and common wheat cultivars was mainly implemented using conventional breeding methods, including backcross selection, pedigree selection, bulk selection and single-seed descent (SSD; Mergoum et al 2009) The development of populations used for gene discovery and mapping quantitative trait loci (QTL) also mainly relied on the conventional SSD method These conventional breeding methods require several generations (at least five) of self-pollination to achieve homozygous lines In contrast, the DH breeding method allows wheat breeders to develop ‘completely homozygous lines in one generation from early generation (F1 or F2)’ (Mergoum et al 2009) When the induction of haploids via wheat maize (Zea mays L.) hybridization became successful, the DH method became a popular approach for breeding common wheat cultivars and for developing mapping populations in both durum and common wheat In the past several years, numerous wheat cultivars and mapping populations have been developed worldwide using the DH method In this study, we review the methodologies of the induction of haploids in wheat and durum wheat, the factors which affect the efficiency of DH development via wide hybridization between durum/common wheat and maize and the mechanism of uniparental chromosome elimination during the hybrid embryo development We also outline an efficient protocol for DH development in durum and common wheat 314 Methodologies of the Development of Haploids in Wheat and Durum Wheat There are two basic approaches for developing haploids in higher plants: androgenesis (anther or microspore culture) and gynogenesis (ovary or megaspore culture) Production of haploids via anther culture is an in vitro process in which microspore cells with haploid genomes develop to an embryo-like structure on culture medium The embryo-like structure further develops to a haploid plantlet (Jauhar et al 2009) In contrast, haploid induction through ovary or megaspore culture is a megaspore embryogenesis in which unfertilized eggs or other embryo sac cells develop into haploid plantlets by a parthenogenic development process (Yang and Zhou 1982) Production of haploids via anther culture was first reported in Datura innoxia Mill in 1964 (Guha and Maheshwari 1964) The authors reported when anthers of Datura were cultured on Nitsch’s medium containing 15% coconut milk, large number of embryo-like structures were produced Later in 1966, they reported that the embryo-like structures were generated from the pollen grains Since then, anther culture has been widely and successfully utilized for large-scale production of haploids in many crop species because an anther contains a large number of microspores and can be easily collected (see review by Jauhar et al 2009) Haploid induction from ovary culture was first reported in barley in 1976 (San Noeum 1976) Although haploids have been successfully developed from the in vitro culture of unfertilized ovules and ovaries in more than 20 angiosperm species since 1976 (Wu et al 2004), ovary or megaspore culture has not been widely used for large-scale production of haploids in most plant species mainly due to the small number of megaspores in the plant and the difficulty in isolating of the female haploid cells from a plant (Kristof and Imre 1996) In common wheat, haploid plants can be developed through both androgenesis and gynogenesis As in other crops, however, typical gynogenesis through ovary or megaspore culture has not been used for cultivar and population development Only androgenesis by anther culture has been successfully used for practical breeding Many common wheat cultivars around the world were developed through anther culture such as ‘Florin’ (De Buyser et al 1987), ‘McKenzie’ (Graf et al 2003) and ‘AC Andrew’ (Sadasivaiah et al 2004) Anther or microspore culture in wheat has some obstacles for haploid production, such as high rates of albinism, low response of some genotypes, and long periods of the inducing and regenerating process, which could cause detrimental gametoclonal variation and mixed-ploidy plants (Andersen et al 1987, Chu and Hill 1988, Cooper and Griffin 1988, Agache et al 1989, Baenziger et al 1989, Simmonds 1989, Tuvesson et al 1989, Kisana et al 1993, Ekiz and Konzak 1994, Liu et al 2002) Therefore, a more efficient procedure than anther culture, such as wide hybridization followed by chromosome elimination, becomes necessary to meet with the needs of wheat breeding Haploid production through wide hybridization is considered gynogenesis because the haploids developed from this procedure contain the maternal haploid genome This procedure was first discovered in crosses between barley (H vulgare L.) and Bulbous barley (Hordeum bulbosum L.) In that case, the genome of Bulbous barley was eliminated during the hybrid seed development, and the barley haploid plants were obtained (Kasha and Kao 1970) Further, the H bulbosum method was applied to successfully induce wheat haploids (Barclay 1975, Pickering and Morgan 1985) Because Bulbous barley is Z NIU, A JIANG, W ABU HAMMAD et al sensitive to the dominant crossability inhibitor genes Kr1 and Kr2, located on chromosomes 5B and 5A of most wheat cultivars, wheat haploid production through wheat–Bulbous barley hybridization is limited only to the crossable wheat genotypes Therefore, a new wide hybridization system between wheat and maize was established in 1986 (Laurie and Bennett 1986); the modification of this technique enabled the production of haploid plants from many commercial wheat cultivars and hybrids (Laurie and Bennett 1988, Suenaga and Nakajima 1989, Laurie et al 1990, Laurie and Reymondie 1991, Comeau et al 1992, Riera-Lizarazu et al 1992, Kisana et al 1993, Campbell et al 2000, Jauhar et al 2009) Lein (1943) reported the effect of the two genes Kr1 and Kr2 on the crossability of wheat with rye (Secale cereal L.) In 1970, chromosome 5D was reported to carry the Kr3 gene (Krowlow 1970) The location of the Kr1 on chromosome 5B, Kr2 on chromosome 5A and Kr3 on chromosome 5D was confirmed using monosomic analysis of a Chinese common wheat landrace J-11 (Zheng et al 1992) In 1986, Yen et al reported the presence of the Kr4 gene in the Chinese landrace Sichuan White Wheat Complex Zheng et al (1992) confirmed the location of Kr4 on chromosome 1A and reported its effect on the crossability of wheat with rye is stronger than Kr2, but weaker than Kr1 In durum wheat, Kr1 and Kr2 not affect the crossability with maize which makes the production of DH through maize pollination a viable and efficient method (Almouslem et al 1998) In addition to H bulbosum and maize, several other plant species, including pearl millet [Pennisetum glaucum (L.) R Br.] (Inagaki and Mujeeb-Kazi 1995), sorghum [Sorghum bicolor (L.) Moench] (Maluszynski 2003) and cogongrass [Imperata cylindrica (L.) P Beauv.] (Chaudhary et al 2005), have been investigated for their ability to induce wheat haploids through wide hybridization For pearl millet and sorghum, there was a strong wheat genotypic barrier for embryo formation (Inagaki and Mujeeb-Kazi 1995, Maluszynski 2003) The wheat cogongrass hybridization was reported to have an equal efficiency of haploid embryo formation as the wheat maize hybridization system (Chaudhary et al 2005) But, cogongrass is a noxious weed and is prohibited from being introduced in certain areas of North America More recently, Ravi and Chan (2010) reported a centromeremediated genome elimination procedure for producing haploids in Arabidopsis thaliana (L.) Heynh by manipulating a centromere-specific histone CENH3 In this procedure, cenh3 null mutants used as either male or female were hybridized to normal plants, and the chromosomes entering the zygote from the mutant were eliminated during the hybrid embryo development (Ravi and Chan 2010) The centromere-mediated genome elimination directly produced hybrid seeds which contain chromosomes only from the normal parent used as either male or female, and this differs from the wide hybridization method which produces haploids having chromosomes only from the female parents Because this procedure does not involve tissue culture and wide hybridization, it may provide a more efficient production of haploids in wheat and other crops However, the procedure has not been used in wheat yet; even the feasibility for manipulation of the histone protein CENH3 in wheat has not been investigated Therefore, the wheat maize hybridization system is currently the most practical way to develop DHs in durum and common wheat (Laurie and Bennett 1988, Islam and Shepherd 1994, Pienaar and Lesch 1994, Inagaki et al 1998, Campbell et al 2000) Durum doubled haploid production Factors Affecting Efficiency of DH Production in Wide Hybridization between Wheat/Durum Wheat and Maize Although maize is relatively insensitive to the action of wheat Kr genes (Laurie and Bennett 1987, Ohkawa et al 1992, Inagaki and Mujeeb-Kazi 1995, Li et al 1996), many other factors affect the efficiency of DH production in the durum/common wheat maize system These factors include the wheat and maize genotypes; position of the spikelet in the flower; temperature; light intensity and photoperiod during the plant growth period; the type and concentration of plant growth regulators (PGRs) applied after pollination; the biochemical elements added to the rescue media, the concentration of colchicine used for chromosome doubling; and the plant growth stage, duration and temperature when the colchicine is applied (Laurie and Bennett 1989, Suenaga and Nakajima 1989, O’Donoughue and Bennett 1994, Pienaar and Lesch 1994, Inagaki and Bohorova 1995, Inagaki and Mujeeb-Kazi 1996, Morshedi et al 1996, Pienaar et al 1996, Wedzony and Van Lammeren 1996, Campbell et al 1998) Wheat and Maize Genotypes The effects of wheat and maize genotypes on the frequency of haploid embryo formation and haploid plant production were investigated in numerous studies, but the results are still controversial Suenaga and Nakajima (1989) reported that when four Japanese wheat cultivars were pollinated with five maize genotypes, the frequency of embryo formation was only affected by the maize genotypes, not by the wheat cultivars But several other studies showed that wheat genotype significantly affects the percentages of the embryos per pollinated florets (Inagaki and Tahir 1990, Laurie and Reymondie 1991, Martins-Lopes et al 2001, Niroula and Thapa 2009) Lefebvre and Devaux (1996) demonstrated that the haploid production efficiency was affected by both wheat and maize genotypes based on a study of 18 wheat F1 hybrids crossed with five maize genotypes using a replicated block design They reported that the interaction between the parental genotypes was significant for the number of haploid embryos/100 florets The efficiency of haploid induction in common wheat is usually higher than durum wheat using wide hybridization with maize High ploidy level and the D genome of common wheat may play an important role in DH production Do gramaciAltuntepe and Jauhar (2001) pollinated durum wheat ‘Langdon’, 14 Langdon D-genome disomic substitution lines (Joppa and Williams 1988, Li et al 2006) and Langdon ph1b mutant with maize pollen and found that the efficiency of haploid production varied among different substitution lines The most efficient line was the 5D (5B) substitution, indicating that substituting chromosome 5D for 5B improves the efficiency of haploid production in durum wheat Almouslem et al (1998) reported clear genotypic differences of maternal durum genotypes used in the production of DH plants The Position of the Spikelet in the Flower and the Timing for Pollination Wheat florets flower in order starting from the middle florets It usually takes about days for all the florets on a spike to finish flowering The fertilization frequencies depend on the stage of the floret, and the best results occurred when the stigma was at 315 the feathery stage (Laurie and Bennett 1989) The position of the spikelet significantly affected the ratio of embryo formation, and the highest values were obtained in the middle position of the spikes (Martins-Lopes et al 2001), while another study demonstrated that the fertilization rate was not affected by spikelet position within a certain time of pollination (Bitsch et al 1998) Pollination with maize at a time close to anthesis not only results in a better crossability, but also in a good embryo quality, which determines the germinating rate of the embryos Environmental Elements: Temperature, Light and Photoperiod Both temperature and light intensity significantly influenced the frequency of haploid embryo recovery, with light intensity having a greater effect Light intensity at 1000 lmol/m2/s radiance at 22/17°C (day/night) resulted in a high frequency of haploid embryos (Campbell et al 1998) Light intensity may affect pollen tube growth, the predetermination step for successful fertilization (Campbell et al 2001) The efficiency of DH production in the wheat maize system is also affected by the time of year when the crosses are made, for example fall (August–December) vs spring (January–April); the higher efficiency was usually obtained in the spring (Pienaar and Lesch 1994, Pienaar et al 1996, Campbell et al 2000) This could be a contribution of the longer photoperiod and stronger light intensity in the spring, which affects the female plant vigour, the fertilization ability of the egg cells, the pollen tube growth in the female plants and the viability of the hybrid seeds The environment influenced wheat embryo survival and pollen tube growth in a genotypically dependent manner (O’Donoughue and Bennett 1994, Campbell et al 2001) Campbell et al (2001) reported that when two wheat cultivars, ‘Karamu’ and ‘Kotuku’, were crossed with the same maize genotype at two irradiance levels (250 or 750 lmol/m2/s, PAR), pollen tube growth was significantly affected by light intensity in ‘Karamu’, but not in ‘Kotuku’ O’Donoughue and Bennett (1994) showed that the cultivar ‘Rampton Rivet’ had significantly better embryo recovery in a 20°C growth room than in an unheated glasshouse, whereas ‘Wakona’ and ‘Chinese Spring’ were unaffected Postpollination Treatment There are two barriers to wheat DH production via maize hybridization One is the low ratio of embryo/embryoless caryopses produced, and the other is the absence of endosperm in the hybrid seeds (Zenkteler and Nitzsche 1984, Laurie and Bennett 1986, 1987), which results in embryo death when they are left to develop in plants (Laurie and Bennett 1988) Postpollination treatments reported in the literature (Suenaga and Nakajima 1989, Laurie et al 1990, Laurie and Reymondie 1991) include (i) immediately culturing the pollinated spikelets for weeks (Laurie and Bennett 1988), (ii) consistently applying 0.5 mg/l dichlorophenoxyacetic acid (2,4-D) to the pollinated spikes for 2–3 weeks, (iii) injecting or spraying 100 mg/l 2,4-D to the internode and/or to the spikelets of the pollinated spikes once or twice (Matzk and Mahn 1994), (iv) applying a solution of a combination of an auxin [picloram (4-amino-3,5,6-trichloropicolinic acid), 2,4-D, or 2,4,5-T (2,4,5-trichlorophenoxyacetic acid)] with 6-benzylaminopurine (6-BA) or with a combination of 2,4-D and gibberellic acid (GA3) in the florets at 24–30 h after pollination (Pienaar et al 1997, Singh et al 2001) and (v) applying a solution of dicamba (3,6-dichloro-o-anisic acid) or 316 ZEN (zearalenone) after pollination (Pienaar et al 1997, Biesaga-Koscielniak et al 2003, Garcıa-llamas et al 2004) Among these treatments, treatment may induce high rates of regenerable embryos, while treatment may stimulate the growth of haploid embryos but result in severely deformed embryos, with a low capability to germinate in vitro Wedzony and Van Lammeren (1996) investigated pollen tube growth and embryogenesis after 2,4-D treatment and demonstrated that 2,4-D increased the number of the pollen tubes that reached the micropyle and multiplied the number of sperm cells in the pollen tube, thus increasing successful intergeneric fertilization We evaluated the effect of 2,4-D, GA3, and pH and their interactions on the efficiency of haploid production in durum wheat using the second-order response surface model (Myers et al 1986) and found that the maximum number of haploid embryos/100 ovaries (E/O) can be reached when 2,4-D is 213.05 mg/l at a pH value of 10.36 without GA3 (E M Elias and Z Niu, unpublished data) Almouslem et al (1998) also studied the effect of four postpollination treatments on DH production in durum wheat They were able to produce 142 haploid plants using 10 durum genotypes, three commercial maize pollinators, and a postpollination combination of 2,4-D and AgNO3 Embryo Rescue The elements of the rescue medium affect embryo germination Germination efficiency as affected by MS (Murashige and Skoog 1962), ½ MS (half strength MS) and B5 (Gamborg et al 1968) media has been extensively tested (Suenaga and Nakajima 1989, Comeau et al 1992, Cherkaoui et al 2000, Dogramaci-Altuntepe and Jauhar 2001) Cherkaoui et al (2000) rescued excised embryos from 10 durum wheat cultivars crossed with eight maize genotypes on MS, ½ MS and B5 media and found that B5 and ½ MS media were more efficient than MS The concentration of sucrose supplemented in the media is the major element affecting the germination of the rescued embryos In our laboratory, we tested MS basal media supplemented with five different sucrose concentrations (0, 20, 50, 80 and 100.0 g/l) and found the maximum embryo germination rate was obtained at 50 g/l We also tested embryo culture by the transplanted nurse endosperm method, in which the excised embryos were placed on the 20-day-old seed endosperm tissue and then cultured on the MS medium This method was more efficient for the small embryos, but was more labour- and time-consuming compared with the regular embryo rescue method Thus, it is more efficient to use both methods for the different size of embryos rescued, that is, the big embryos are directly cultured on MS medium, and the small embryos are cultured with transplanted nurse endosperm Another factor that is important in embryo rescue is knowing whether the seed has an embryo To save time and energy, only seeds that are known to have embryos need to be rescued because, on average, only one-third of the seed carry embryos Bains et al (1998) developed a simple technique to distinguish between seed with and without embryos They were able to detect 97.8% of the seed that contained embryos by placing immature seeds from wheat maize crosses on a transparent surface illuminated from above Colchicine Treatment A successful chromosome-doubling process is essential for the production of homozygous plants after haploid plants are derived Z NIU, A JIANG, W ABU HAMMAD et al from wheat or durum wheat hybridization with maize Different doubling agents have been studied, such as caffeine (Thomas et al 1997), nitrous oxide (Hansen et al 1988), antimicrotubule herbicides trifluralin or amiprophos-methyl (APM; Hansen and Andersen 1998); but the most commonly used chemical agent for chromosome doubling is colchicine, which disrupts mitosis by inhibiting formation of spindle fibres and disturbing normal polar chromosomal migration, resulting in chromosome doubling (Jensen 1974) Many factors affect the chromosome-doubling process, such as colchicine concentration, addition of other synthetic compounds, treatment temperature and length, the development stage of plants and growing conditions after colchicine treatment Colchicine treatment can be applied at different stages, from postpollination of the female plants to the tillering stages of the haploid plants (Jensen 1974, Thiebaut et al 1979, Inagaki 1985, Sood et al 2003) In cereal crops, such as wheat and barley, colchicine treatment is normally recommended at the 3- to 4-tiller stage for 5–8 h by submerging the whole root system in a colchicine solution containing 0.1% colchicine, 2% dimethyl sulfoxide (DMSO), 0.3 ml/l Tween 20 and 10 mg/l of gibberellic acid (GA3; Jensen 1974, Thiebaut et al 1979, Inagaki 1985); and the doubling rate can reach 95.6% (Inagaki 1985) The colchicine treatment can be applied before the seedling stage of haploid plants by adding 0.5% colchicine to the rescue medium for 48 h The colchicine solution (1% colchicine with 100 ppm 2,4-D) can also be injected into the uppermost internode of pollinated spikes at 48 and 72 h after pollination, and the chromosome-doubling rate varies from 33% to 100% (Sood et al 2003) We found that the rate of embryo germination would be greatly reduced when adding colchicine to the rescue media or injecting colchicine to the internodes after pollination (unpublished data) The higher colchicine concentration increases the doubling rate, but it also results in deformed plants, low survival rate and increased cost The optimal colchicine treatment should have a high rate of embryo germination and a high plant survival rate, with a high rate of chromosome doubling For wheat haploid chromosome doubling, we treated haploid plants at the 2- to 3tiller stage with a solution containing 0.45 g/l colchicine, 20 ml/l DMSO, 100 mg/l GA3, 0.3 ml/l Tween 20 with a pH 5.5 for 6– h at 18–20°C in the dark During the treatment, a gentle air flow into the colchicine solution supplied the roots with oxygen After treatment, the plants were rinsed with running water overnight, transferred to soil and kept in a growth chamber at 14– 16°C under 16/8 h day/night until the new tillers emerged The plants were then moved to normal growing conditions in a greenhouse The plant survival rate could reach 99% and the chromosome-doubling rate could reach 96–98% (unpublished data) Mechanism of Uniparental Chromosome Elimination in the Wide Hybridization System Various theories have been proposed to explain the mechanism of uniparental chromosome elimination in the wide hybridization system However, there is no conclusive explanation for the actual process Chromosome elimination could be caused by the difference in timing of mitotic processes (Gupta 1969), the genomic balance (Kasha and Kao 1970) and/or the failure of the chromosome to initiate or to complete either congregation at metaphase or migration to the poles at anaphase (Bennett et al 1976) In the barley-H bulbosum system, the genetic factors controlling genomic balance are located on barley chromosomes Durum doubled haploid production 2H and 3H (Ho and Kasha 1975), and a gene controlling incompatibility between barley and H bulbosum was located on barley chromosome 5H (Pickering 1983) Orton and Tai (1977) proposed that the spindle organizer mechanism played a role in chromosome elimination They indicated that if the parental spindle organizers are genetically similar, normal chromosome behaviour will result; but, if the spindle organizers are genetically dissimilar, independent function occurs even within the same protoplast of an offspring Each spindle organizer will then migrate and establish itself as a pole and attract its own chromosome The net results will be multipolar cell division and the separation of different genomes into different daughter cells (Orton and Tai 1977) Sanie et al (2011) studied the role of the centromere-specific histone H3 variant (CENH3) in the process of uniparental chromosome elimination in crosses of H vulgare Hordeum bulbosum Four conclusions were drawn from their study: centromere inactivity of H bulbosum chromosomes triggers uniparental chromosome elimination; centromeric loss of CENH3 protein causes centromere inactivity; not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present; and ‘diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis’ During hybrid embryo development in wheat pearl millet crosses, all pearl millet chromosomes occupied peripheral interphase positions and were randomly eliminated between six and 23 days after pollination (Gernand et al 2005) In hybrid embryos between wheat and maize, the maize chromosomes to be eliminated were peripherally located on the metaphase plates and lagged behind the wheat chromosomes at anaphase (Laurie and Bennett 1989) However, in the barley H bulbosum system, the H bulbosum chromosomes did not directly undergo the distinct peripheral localization process, but they went through the nuclear extrusion and budding process (Kim et al 2002) In the wheat pearl millet system, Gernand et al (2005) observed that the heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin were similar to the events of programmed cell death (Fukuda 2000) So, the plants could conserve the ability to distinguish host chromatin/DNA from foreign chromatin/DNA and eliminate the alien genome from the host genome Specific chromatin topology and posttranslational histone modifications might be the key for the recognition and subsequent elimination of DNA, which might result in endonuclease activation and genome-specific fragmentation (Houben et al 2011) Outlines of a Highly Efficient Approach to Develop DHs in Wheat and Durum Wheat Based on the factors affecting DH production via wheat maize hybridization, we optimized the major factors to improve efficiency of DH production and developed a highly efficient protocol, which is outlined as follow: Plant sweet corn (hybrid cultivar ‘Early Sunglow’) seed 10– 14 days earlier than durum/common wheat F1 hybrids Plant corn for 5–6 times with an interval of 5–7 days Plant durum/common wheat F1 seeds at the same time as the third corn planting During the durum/common wheat heading stage, emasculate the durum/common wheat spikes At 2–3 days after emasculation, collect fresh corn pollen (light yellow colour), and pollinate emasculated durum/ common wheat spikes 317 At 24 h after pollination, spray pollinated spikes with 2.4-D solution (213.05 mg/l, pH = 10.36) At 14–16 days after treatment with 2.4-D, cut the spikes from the plants, and remove the seeds from the spikes for embryo rescue Sterilize the seeds with 70% ethanol for and then 20% Clorox (commercial solution) for 15 min, and rinse three times with sterilized distilled H2O Aseptically excise the embryos, and culture the big embryos directly on the MS basal medium with 50 g/l sucrose and 8% agar in Petri dishes Culture the small embryos on the MS media with transplanted nursing endosperm Keep the Petri dishes with embryos at room temperature (20–24°C) in the dark for 1–2 weeks After the embryos germinate, transfer the small seedlings to test tubes or jars containing ½ MS media with 30 g/l sucrose and g/l agar Keep them at 20–24°C, 16/8 (light/dark) h for about weeks 10 When the plants grow up to 5–6 cm, transfer them to 4-inch clay pots filled with soil and fertilize with slow-release fertilizer Keep them in the growth chamber at 18–20°C 16/8 (day/night) h photoperiod 11 When plants have grown to the 2–3 tiller stage, dig the plants from clay pots, wash the roots thoroughly and immerse the roots and crown parts of the plants in colchicine solution: colchicine (0.45 g/l) + DMSO (20 ml/l) + GA3 (100 mg/l) + Tween 80 (0.3 ml/l), pH = 5.5, at 20–22°C, dark for h During the treatment, provide a gentle air flow into the colchicine solution 12 After the colchicine treatment, rinse the plants with running water overnight, and transfer them back to clay pots filled with soil and fertilized with slow-release fertilizer 13 Keep the plants in a growth chamber (16/8 day/night photoperiod at 14–16°C) for about weeks until plants recover (new tillers grow out) and then move the plants to the greenhouse (16/8 day/night photoperiod) at 20–24°C until maturity We observed that air flow during the colchicine treatment and the low temperature (14–16°C) condition after treatment greatly improve the survival of the colchicine-treated plants Using this procedure, rates of survival and chromosome doubling of the treated haploid plants were over 90% This protocol has been successfully used to develop mapping populations and adapted germplasm in our wheat germplasm enhancement programme and durum wheat breeding programme (Chu et al 2008a,b, 2009, 2010a,b, 2011a,b, Gu et al 2010) Twelve populations each consisting of over 120 DH lines have been developed to identify and map various genes for important agronomic traits such as gluten strength, seed dormancy (Gu et al 2010), growth habit (Chu et al 2011b) and resistance to various wheat diseases such as tan spot (Chu et al 2008b, 2009, 2010a), leaf rust (Chu et al 2009), Stagonospora nodorum blotch (Chu et al 2010b, Friesen et al 2012), Fusarium head blight (Chu et al 2011a) and crown rust (Niu et al 2013) In addition, we have provided this protocol to numerous wheat genetics and breeding research programmes Acknowledgements Authors thank Stan S Stancyk for the technical support during the development of the protocols and to Daryl L Klindworth for revising the manuscript 318 References Agache, S., B Bachelier, J De Buyser, Y Henry, and J W Snape, 1989: Genetic analysis of anther culture response in wheat using aneuploid, chromosome substitution and translocation lines Theor Appl Genet 77, 7—11 Almouslem, A B., P P Jauhar, T S Peterson, V R Bommineni, and M B Rao, 1998: Haploid durum wheat production via hybridization with maize Crop 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