Technology for Production of Wheat Doubled Haploid via Maize Pollen Induction – an Updated Review

1. Introduction

In many countries, distinctness, uniformity, and stability (DUS) are prerequisites for a new plant variety to obtain protection and registration [1]. In China, there is no exception, and the approval or registration of new varieties, and variety rights protection also require distinctness, uniformity, and stability tests (DUS tests) [2,3]. Distinctness, uniformity, and stability are essential attributes for new wheat varieties. A variety’s uniformity and stability can be estimated by its proportion of homozygous DNA loci and seed purity, the higher ratio of the homozygous DNA loci, the better uniformity and stability of the variety. Varieties with a ratio of homozygous DNA loci less than 90% are poor in uniformity and stability [4]. Seed purity is an important indicator used to evaluate the quality of wheat seeds. The China National Standard (GB 4404.1-2008) [5] stipulates that the purity of wheat foundation seeds should not be less than 99.9%, and the purity of wheat variety seeds should not be less than 99.0%. Therefore, for a wheat line qualified for testing, its ratio of homozygous DNA loci needs to be over 90%, and it has to be advanced to the F₅ or later generations. For wheat lines to reach the level of variety or foundation seeds, the ratio of homozygous DNA loci needs to be over 99.9%, and the generation should reach at least F₁₁. The breeding cycle is thus very long, which certainly limits the wheat seed industry.

Haploid induction and doubled haploid (DH) technology can obtain pure lines with a genotypic homozygosity of 100%, bypassing the need for 6-10 generations of inbreeding through selfing or sib-crossing [6]. This is a significant breakthrough for developing new varieties [7]. This technology requires only one generation to obtain genetically homozygous lines, and will substantially reduce the breeding course [8].

The first naturally occurring haploid plant was discovered in jimson weed in 1922 [9]. Thereafter, using anther and microspore culture techniques, haploid plants of jimson weed and rice were obtained respectively [10,11]. DH technology gradually came into the view of breeders, and varieties of oilseed rape and barley were successively developed [12]. Along with the improved haploid induction and chromosome doubling, doubled haploid technology has been applied in many plant species [13].

DH plants rarely occur in nature but can be induced by in vitro or in vivo treatments [14]. In vitro culture include androgenesis (anther or microspore culture) and gynogenesis (bud, ovary, or unfertilized ovule culture). In vivo methods include induction of parthenogenesis by inactive pollen pollination (ionizing radiation, chemical agents, and high-temperature), and intra- or inter-specific hybridization, also known as the chromosome elimination method [15]. Effectiveness of different methods depends on plant species [13].

In wheat DH breeding, the in vitro anther or microspore culture is limited due to its genotype dependence, time consuming for regeneration, unstable ploidy, and albino seedlings [16,17]. Ionizing radiation and chemical treatments of pollen barely induces haploid and may cause aneuploidy [18]. In contrast, wheat mutants of the pollen-specific phospholipase gene TaMTL can induce wheat to produce haploid seeds [19]. Using the wheat Tamtl mutant, Tang et al. [20] have established a visual screening to identify the haploid seeds. The maize-obligated wheat doubled haploids (mowDH) involves both in vivo and in vitro operations, alien chromosome elimination, and maternal chromosome doubling. The mowDH approach is two to three times more efficient for producing green plantlets than the anther culture [21], shows little or no genotype dependence in wheat, does not require haploid identification, and thus becomes a popular method for producing DH wheat. The chromosome elimination method is well utilized in Yunnan, China, where both wheat and maize can grow in the same season, and several wheat varieties such as Yunmai 110 have been developed [22].

Winter wheat area is about 2.17×10⁷ ha and accounts for 92% of the total wheat acreage in China [23,24]. In the winter wheat region, wheat and maize are rotating crops, thus they do not flower at the same season in nature. Therefore, it is unrealistic to conduct large-scale wheat haploid induction using maize pollen in the winter wheat region. However, newly constructed plant growth facilities can be used to coordinate the flowering time of winter wheat and maize, thus allowing industrialized production of wheat DH via maize pollen grains in the winter wheat region. To improve the mowDH technology and to gain its full potential in the winter wheat region in China, in this paper, we review previous achievements, propose an updated technical scheme for future application, especially in the winter wheat region, and then present outlooks on wheat DH research and application.

2. Origin, Principle, and Advantages of mowDH

Chromosome elimination was first encountered in an interspecific hybridization in tobacco (Nicotiana tabacum × N. sylvestris), resulting in the N. tabacum haploids [25]. Similarly, Gaines and Aase [26] obtained wheat haploids after performing an intergeneric hybridization (T. compactum Host × Aegilops cylindrica L.). Barclay [27] also created wheat haploid plants when using Hordeum bulbosum as pollen donor, a method commonly termed as the bulbosum technique. The H. bulbosum is sensitive to dominant crossability inhibitor genes Kr1 and Kr2, which are located on chromosomes 5B and 5A. However, the bulbosum technique is invalid for wheat genotypes lacking dominant Kr1 and/or Kr2. Later, Laurie and Bennett [28] established an intergeneric system between wheat and maize, representing an early study of mowDH, in which the elimination of maize chromosomes was independent of wheat Kr genes [29,30]. Thus, the mowDH technology became popular, and was often used to produce haploid plants from commercial wheat varieties [31,32,33]. Now, mowDH acts as one of the most practical methods for wheat [34,35,36].

Despite the popularity of mowDH, the underlying mechanism and/or genes causing chromosome elimination are still unclear. There are different hypotheses, including asynchrony in nucleoprotein synthesis [37], asynchronous cell cycles [38], and parent-specific inactivation of centromeres [39]. In Arabidopsis, the centromeric histone H3 (CENH3) regulates the intraspecific genome elimination [40]. Similarly, CENH3 controls the chromosome elimination in the interspecific hybridization of H. vulgare × H. bulbosum [41]. Whether CENH3 accounts for the maize chromosomes elimination in wheat × maize remains to be answered. Chen et al. [42] constitutively expressed the maize CENH3 in wheat ‘Yangmai 158’, however, maize chromosomes were again eliminated in the case of ZmCENH3 overexpressing wheat × maize. Likely, low contents of ZmCENH3 in transgenic wheat may fail to suppress chromosome elimination of maize.

Kapoor et al. [43] compared the haploid plant rate after crossing polyploid wheat (4x and 6x) or hexaploid triticale with maize or Imperata cylindrica. The hexaploid wheat showed higher frequency of embryo and haploid formation than the tetraploid wheat when they were crossed with Imperata, thus the wheat D genome may be prone to trigger chromosome elimination of Imperata. Gurtay et al. [44] also tested the wheat × maize programed haploid production and conducted wheat anther culture to acquire DH plants using spontaneous doubling in androgenesis, which includes the use of ancient, local and modern types of polyploid wheat (4x and 6x). As a result, more haploid embryos were acquired in hexaploid wheat than those in tetraploid wheat, but the plant regeneration was comparable. Apparently, the D genome impacts wheat anther culture and wheat × maize hybridization. Obviously, centromeres, especially those in wheat D genome, play an important role in inducing wheat haploids by eliminating maize chromosomes. However, more studies are needed to understand how wheat D genome functions to eliminate the entire parental chromosomes.

Besides H. bulbosum and maize, other Poaceae species, such as sorghum [45], pearl millet [46], teosinte [47], Tripsacum [48], Job’s tears(Coix lacryma-jobi L.) [49], I. cylindrica [32,50], and Ae. Caudata [51], also induced haploid formation in wheat. In comparison (Table 1), sorghum’s effect highly depends on wheat genotypes, and thus sorghum is not suitable for inducing wheat haploid [52,53]. Surprisingly, according to other studies, pearl millet [53,54,55], teosinte [47,56], and Tripsacum [48,57] can induce wheat haploids with a comparable or higher rate than maize. Only a few studies were conducted with Job’s tears [49] and Ae. Caudata [51], and their applicability for wheat haploid induction needs to be further studied. Although I. cylindrica is a noxious weed [58], I. cylindrica is comparable to maize for inducing haploid embryos in common wheat [50]. But I. cylindrica outperforms maize when used in durum wheat, triticale, or their derivatives [59,60]. In practice, I. cylindrica should be handled with care due to its weedy nature. To gain the first-hand experience, we tested maize hybrid, maize inbred line, sorghum, teosinte, and Job’s tears for inducing wheat haploids. We found that pollen quantity and accessibility was critically countable for developing a large-scale and robust wheat haploid induction, for which maize outperforms all others tested (Figure 1). In addition, a variety of maize genotypes are widely planted in the world with large acreage, which make it possible to screen for ideal genotypes with higher induction rates.

3. Research Progress of the mowDH Technology

Main steps in mowDH are wheat and maize planting, wheat emasculation, maize pollen pollination, hormone treatment, embryo rescue, doubling treatment, and DH plant harvesting (Figure 2). Along the process, numerous factors control the ending wheat DH rate, which is actually attributed to pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF), haploid formation frequency (HFF), and haploid doubling frequency [59,60,64,65,66]. However, the inheritance of PFF, EFF, and HRF is independent [61,67], and they can ultimately be reflected in the embryo rate (obtained wheat haploid embryos after crossing and hormone treatment), the plantlet rate (obtained wheat haploid plantlets after haploid embryo rescue), and the doubling rate (obtained wheat DH plantlets after chromosome doubling). Over the years, the mowDH technology has been gradually upgraded, and now offers a powerful tool for wheat breeding.

3.1. Genotype Effects

The mowDH technology functions independent of wheat Kr1 and Kr2 genes, behaves superior to the bulbosum technique [68,69], and thus is reliable and widely used in wheat. Over the years, there is a non-stop passion to understand how wheat and/or maize genotypes affect the efficiency of mowDH, and complex conclusions have been drawn.

For maize, most researchers believe that the maize genotype has significant influence on mowDH [13,60,61,64,70,71,72], partially through modulating EFF and HRF. Niroula et al. [34] proposes using more responsive maize genotypes to enhance wheat DH production. Specific genotype accounts for haploid embryo induction or embryo regeneration, respectively [64]. In another case, the anther culture-responsive F₁ hybrids of hexaploid wheat were tested with three sweet corns ‘Baron’, ‘Challenger’, and ‘Merit’, of which Challenger had the highest haploid embryo rate (3.5%), but not for the plantlet regeneration. Surprisingly, the use of pollen mixture of multiple sweet corn genotypes enhanced haploid plantlet regeneration [72].

For wheat itself, an early study failed to show the genotype effect on mowDH [70]. In contrast, Verma et al. [64] proved that wheat genotypes significantly influenced PFF and EFF, but not as good as those from the maize side. Today, wheat genotypes are primarily counted towards affecting mowDH [61,67,71,73]. When both winter and/or spring wheat were considered, winter wheat (winter parents and winter × winter F₁s) performed better than the non-winter wheat (spring parents, spring × spring F₁s, and winter × spring F₁s) towards embryo formation. However, the winter × spring F₁s performed the best in acquiring regenerated plantlets [67].

To study the interaction between wheat and maize, Singh et al. [71] compared winter wheat, spring wheat, and their F₁s in conjunction with specific maize. There was significant interaction on embryo formation and regeneration of plantlets; the wheat × maize interaction for embryo formation and regeneration was due to non-additive gene action. In addition, the DNA heterozygosity in wheat and maize genotypes improved the haploid induction rate. Dhiman et al. [61] further demonstrated the overall contribution of the maize induction line to embryo formation and regeneration was the highest, followed by the wheat line × maize induction line interaction.

Collectively, genotypes of wheat, maize, and their interaction all play roles in mowDH. In future, more maize genotypes should be tested in conjunction with target wheat genotypes, which is designed to acquire specific maize and/or their interaction with specific wheat in conferring excellent EFF and HRF, and they will be applied to advance the mowDH technology.

3.2. Environmental Factors

Gu et al. [74] achieved a haploid embryo rate of 31.6% in mowDH when cut plants were in vitro cultured in a nutrient solution (40 g/L sucrose, 10 mg/L silver nitrate, 3 g/L calcium phosphate, and 8 ml/L sulfurous acid) under controlled conditions of 22-23 ℃ in light, 16-17 ℃ in dark and an ambient humidity of 70%. However, the haploid embryo rate was only 9.6% using plants from fields. Khan et al. [75] further conducted in vitro culture of 25 hexaploid wheat genotypes from fields used a tillering medium containing 100 mg/L 2,4-D, 40 g/L Sucrose, and 8 ml/L Sulfurous acid. They analyzed the controlled factors such as temperature during pollen collection, time of pollination, light intensity, and relative humidity towards haploid seed formation. As a result, the optimal factors are maize pollen from 21-26 ℃, pollination at 24 hours post emasculation, a light intensity of 10,000 Lux, and a relative humidity of 60-65% at 20-22 ℃. Khan et al. [76] investigated the haploid induction rate between wheat F₁s and Z. mays/I. cylindrica under different conditions. The DH production rate of the F₁s in greenhouse was considerably higher than those of the F₁s in field. Thus, the growing condition of both wheat and maize plays a pivotal role in mowDH, and optimal environmental factors can be drawn for an improved mowDH. The environmental factors proposed by Khan et al. [75] can serve as a reference for technical improvement.

3.3. Treatment of Wheat Spikes and Timing of Pollination

Growth condition, or controlled environment, is preferred for conducting mowDH. However, due to limited space of any environmentally controlled facility, immature wheat spikes were harvested during heading and then subjected to in vitro culture [74,75,77]. Today, modern and spacious greenhouses are readily accessible, which allows to maintain enough wheat and maize plants continuously throughout the year. Therefore, it is not necessary to in vitro culture wheat immature spikes. According to Laurie [29], any accountable pollination is based on the wheat floret status, those with a feathery stigma being best. Martins-Lopes et al. [78] studied the spikelet’s position effect on wheat × maize compatibility and found more success with middle spikelets. Thus, maize pollen should be applied to middle spikelets with a feathery stigma in order to obtain more haploid embryos under the controlled conditions.

3.4. Hormone Treatment

Phytohormone treatment post the wheat × maize pollination is crucial for haploid production. The applied hormones promote ovary growth and survival rate of haploid embryos, from which haploid embryo rescue on media becomes more practical and effective [31,45,79]. To improve mowDH, a verity of hormones were tested, including 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, picloram, indole-3-acetic acid (IAA), phenylacetic acid (PAA), silver nitrate, 1-naphthaleneacetic acid (NAA), kinetin, 6-benzyladenine (BA) and zearalenone [80]. Among them, 2,4-D is widely used to control organ regeneration and callus induction. The 2,4-D also regulates early and post-embryogenic plant development involving both somatic and zygotic embryogenesis [81].

When applying a hormone in mowDH, the dosage, timing and methodology of it should be determined. At 100 ppm, 2, 4-D effectively induces haploid embryos in hexaploid wheat [70,82,83]. At 250 ppm, 2, 4-D effectively promotes haploid production in tetraploid wheat [79]. Kaushik et al. [82] tested different application methods of 2,4-D, including spray, tiller injection, dipping, and spikelet culture, of which only the spikelet culture method behaved well in recovering embryos. Despite this, we adopted the spraying method because of its simplicity and high efficiency in our hand; and we have acquired an average embryo rate of 12.9%.

3.5. Embryo Rescue

During the mowDH process, maize chromosomes are eliminated not only in embryo cells, but also in endosperm cells, which will cause seed abortion [50,75,84,85]. Therefore, wheat haploid embryos must be rescued by tissue culture to generate haploid plantlet. In practice, wheat embryo rescue is highly dependent on the plant regeneration media. Among B5, MS, and ½ MS tested, Cherkaoui et al. [86] found that B5 and ½ MS were superior to MS in obtaining young embryos for the tetraploid wheat × maize hybridizations. The supplement of putrescine and spermidine, each in 0.5 mg/L in the embryo rescue medium, SM (Standard Medium), resulted in 69.3% regeneration rate of wheat plantlets, but only 33.5% regeneration in the control group [87]. Most tests are needed with how to supply putrescine and spermidine in B5 and/or ½ MS medium.

3.6. Doubling Treatment

Wheat haploid plants obtained through the mowDH technology naturally remain undoubled [88]. Chromosome doubling is essential to acquire homozygous and stable diploid plants. Antimitotic compounds are selected to double plant chromosomes [89], for which colchicine is the mostly applied agent. Colchicine inhibits spindle function during mitosis and stops the polar segregation of sister chromatids, ending with a doubled nucleus. In the process, chromosome-doubled chimera sectors are formed, which leads to partial fertility [90] and poor grain-setting in DH plants (Figure 3). Colchicine treatment is partially lethal to plant haploids, thus only results in a low frequency of doubled haploids. It is necessary to optimize the dosage, processing time, and plant stages for an effective colchicine treatment, particularly when dealing with new plant genotypes.

Inagaki [91] trimmed roots by keeping 2-3 cm on haploid plantlets, soaked the trimmed roots in 0.1% colchicine (with 2% dimethyl sulfoxide/DMSO and fifteen Tween-20 drops per liter) at 20 ℃ for 5 hours. At the 2-3 tiller stage, the colchicine application resulted in 95.6% doubling rate. Khan et al. [92] treated haploids with 3-5 tillers in 0.1-0.2% colchicine for 3 hours and provided continuous air flow in the solution. Niu et al. [93] also supplied air during colchicine treatment at 14-16 ℃, and achieved over 90% survival and chromosome doubling among the treated wheat plantlets. Sharma et al. [94] also studied the in vitro effect of colchicine. The wheat DH production was enhanced after four hours’ treatment with 0.075% colchicine in hexaploid wheat and 0.15% colchicine in tetraploid wheat. However, in our cases, haploid plantlets at the 2-3 tiller stage were treated in 0.05% colchicine for 16 hours, resulting in over 90% survival and chromosome doubling rates.

4. Stability of Doubled Haploids

DH stability is crucial for agronomy, breeding, and research. In mowDH, maize chromosomes disappear during early embryo cell divisions [46] or later in chromosome doubling [95]. The genetic stability is then established after doubling of wheat haploid chromosomes. Using six glutenin loci of the mowDH decedents, Kammholz et al. [96] proved their stable inheritability across generations. Furthermore, Chen et al. [97] and Brazauskas [98] demonstrated the predominant genetic stability from the wheat side, and there was little or no maize DNA in the mowDH decedents.

However, colchicine also causes chromosomal aberrations, such as aneuploidy [99,100,101]. Suenaga and Nakajima [102] evaluated 110 wheat DH lines, of which 15 DH lines exhibited irregular phenotypes, such as dwarfism, poor seed setting, spike variation, and leaf stripes. Likely, they were caused by colchicine treatment. Shrestha et al. [103] studied two wheat DH populations and found many chromosomal aberrations including duplication, deletion, translocation, and aneuploidy, which were likely caused by unusual chemical exposure during haploid induction and chromosome doubling.

Apparently, the stability of DH lines is based on both haploid embryo induction and colchicine doubling. In mowDH, maize DNA barley remains in haploid embryos, thus the chromosomal aberrations in the resulted wheat DH lines are considerably caused by colchicine treatment. In our study, however, there is no significant variation in spike traits and leaf morphology (Figure 4).

5. An Optimized mowDH Procedure for Winter Wheat

Considering others and our experiences, we here propose an updated mowDH protocol for winter wheat in China (Figure 2).

(1)

Wheat breeding lines (≥ F₃ generation) and hybrid maize varieties are maintained in environmentally controlled facilities: wheat under 20-24 ℃, day/night of 20h/4h, light intensity > 30000 lux, and humidity 60-65%; maize under 22-24 ℃, day/night of 12h/12h, light intensity > 10000 lux, and humidity 55-60%. A set of ten maize seeds are sown once a week, to continuously supply pollen grains. Winter or semi-winter wheat is vernalized under 4 ℃ for 40 days before shifting to regular growth.

(2)

When heading, wheat spikes are manually emasculated.

(3)

Mature pollen grains are collected from maize tassel around 10 am, and are applied with a brush to the emasculated wheat florets at the feathery pistil stage. The pollination time is recorded.

(4)

At 24 hours post pollination, 2,4-D (100 ppm) is sprayed on top of the pollinated wheat florets.

(5)

At 15 days post pollination, manually pollinated spikes and any immature seeds born are harvested.

(6)

Immature seeds are sterilized in 70% alcohol for 1 minute, and in 15% sodium hypochlorite for 20 minutes, and then rinsed with sterile water five times.

(7)

Within a clean hood, haploid embryos are isolated from the sterilized immature seeds using a dissection microscope, and isolated haploid embryos are then planted on ½ MS medium (½ MS + 20 g/L sucrose + 2.4 g/L plant agar, pH 5.8).

(8)

Immature embryos are cultured under 20-24 °C, 16h light / 8h dark, and a light intensity of 4800 Lux. Any plantlets with shoots and roots are transferred into culture bottles (150 ml) and are cultured on ½ MS medium under 20-24 °C, 16h light / 8h dark, and a light intensity of 4800 Lux.

(9)

At 3-leaf stage, the culture bottle is left open for 24 hours. Haploid plantlets are then transplanted into small pots with Pindstrup substrate (PH 5.5, Pindstrup Mosebrug A/S, Denmark) for cultivation.

(10)

Haploid plantlets with 2-3 tillers are taken out of the small pots. Extra roots are trimmed to keep only 2-3 cm on plantlets. Haploid plantlets with trimmed roots are then soaked in colchicine solution (0.05%) for 16 hours.

(11)

After colchicine treatment, plantlets are rinsed with running water for 30 minutes, and then transplanted into small pots with Pindstrup substrate (PH 5.5).

(12)

When new tillers emerge, plantlets are then vernalized at 4 ℃ for 40 days, and then transplanted into growth pots with the seedling substrate (Jinan Fengyuan Agricultural Technology Co., China). Plants are maintained until mature.

With this updated procedure, we have provided excellent service to satisfy the breeding and research need in Spring Valley Agriscience Co. In the second half of 2023, for mowDH, we achieved an average embryo rate of 12.9%, an average plantlet rate of 51.8%, and an average doubling rate of 86.0%.

6. Conclusions and Prospects

In recent years, the genome editing was used to develop novel wheat DH technologies, for example the TaMTL-based maternal haploid induction [19] and the TaCENH3α-based paternal haploid induction [104]. This indeed opens a new path to upgrade wheat DH technologies, but its application to the industrialized production of wheat DH lines still requires time for verification.

Wheat DH technologies, including anther culture, microspore culture, and mowDH have been widely applied to speed up research and breeding. The fast growth in research and facility allows a full exploration of mowDH, which in future might become a high throughput system for wheat DH lines. Nevertheless, there is still considerable room for improvement of the mowDH system. Many factors including genotype, environment, pollination and hormone treatment, embryo rescue, and doubling treatment methods can influence the production efficiency. Thus, it is necessary to improve and optimize the mowDH system around these factors. Screening the best maize induction lines (varieties) for specific types of wheat, and identifying the optimal growth environment conditions, pollination times, hormone treatments, embryo rescue, and doubling treatment plans could further improve the DH production efficiency. Moreover, the exact mechanism or gene for chromosome elimination in this technique is still unclear and needs further study. This will provide guidance for continuous improvement of the mowDH system and extend its application to other crops.

In future, with the advancement of technology and in-depth research, the mowDH system will be further developed and applied. Firstly, the continuous optimization of DH line production techniques, driven by ongoing in-depth research, will render the DH line production process more efficient and stable. Secondly, this technique in conjunction with greenhouse generation advancement technology and modern biotechnology such as marker-assisted selection, gene editing, mutation induction, transgene, genomic selection, etc., will further improve selection efficiency and accuracy, shorten breeding cycles, greatly enhance the efficiency of genetic improvement, and provide more possibilities for wheat genetics and breeding. Lastly, the improvement of equipment and facilities, and the establishment of an industry-scale production procedure for mowDH will meet the demands of scientific research on wheat genetics and breeding and wheat production. In conclusion, the mowDH system possesses tremendous potential for development and will play as a routine technique a more significant role in research on wheat genetics and breeding.