The main steps in the Wheat × Maize System are wheat and maize planting, wheat emasculation, maize pollen pollination, hormone treatment, embryo rescue, doubling treatment, and DH plant harvesting (Figure 1). 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 ^{[1][2][3][4][5]}[59,60,64,65,66]. However, the inheritance of PFF, EFF, and HRF is independent ^[6][7][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 Wheat × Maize System has been gradually upgraded and now offers a powerful tool for research on wheat genetics and breeding. This includes applications such as molecular cytogenetic characterization ^[8][68], the development of thermophoto sensitive genic male sterile lines ^[9][36], QTL mapping ^[10][69], and so on.

The Wheat × Maize System functions independent of wheat Kr1 and Kr2 genes, behaves superior to the bulbosum technique ^[11][12][70,71], and thus is reliable and widely used in wheat. Over the years, there has been a nonstop passion for understanding how wheat and/or maize genotypes affect the efficiency of the Wheat × Maize System, and complex conclusions have been drawn.

For maize, most researchers believe that the maize genotype has a significant influence on the Wheat × Maize System ^{[2][3][6][13][14][15][16]}[13,60,61,64,72,73,74], partially through modulating EFF and HRF. Niroula et al. ^[17][34] propose using more responsive maize genotypes to enhance wheat DH production. Specific genotypes account for haploid embryo induction and embryo regeneration, respectively ^[3][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 a pollen mixture of multiple sweet corn genotypes enhanced haploid plantlet regeneration ^[16][74].

For wheat itself, an early study failed to show the genotype effect on the Wheat × Maize System ^[14][72]. In contrast, Verma et al. ^[3][64] proved that wheat genotypes significantly influenced PFF and EFF but were not as good as those from the maize side. Today, wheat genotypes are primarily counted towards affecting the Wheat × Maize System ^{[6][7][15][18]}[61,67,73,75]. When both winter and/or spring wheat were considered, the winter wheat (winter parents and winter × winter F₁s) performed better than the nonwinter 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 ^[7][67].

To study the interaction between wheat and maize, Singh et al. ^[15][73] 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 nonadditive gene action. In addition, the DNA heterozygosity in wheat and maize genotypes improved the haploid induction rate. Dhiman et al. ^[6][61] further demonstrated that 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 and maize and their interaction all play roles in the Wheat × Maize System. In the future, more maize genotypes should be tested in conjunction with target wheat genotypes, which are 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 Wheat × Maize System. What researchers need to do is to extensively screen a multitude of maize hybrid varieties against various wheat genotypes, selecting several maize varieties with high EFF and HRF. These maize varieties will then be cultivated in a greenhouse for pollen collection. The collected pollens will be thoroughly mixed. The mixed pollens will be used to pollinate the emasculated wheat spikes.

Gu et al. ^[19][76] achieved a haploid embryo rate of 31.6% in the Wheat × Maize System 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 °C in light, 16–17 °C in dark, and an ambient humidity of 70%. However, the haploid embryo rate was only 9.6% using plants from fields. Khan et al. ^[20][77] further conducted an in vitro culture of 25 hexaploid wheat genotypes from fields using 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 to 26 °C, pollination at 24 h postemasculation, a light intensity of 10,000 Lux (140 μmol/m²/s), and a relative humidity of 60–65% at 20–22 °C. Khan et al. ^[21][78] investigated the haploid induction rate between wheat F₁s and Z. mays/I. cylindrica under different conditions. The DH production rate of the F₁s in the greenhouse was considerably higher than that of the F₁s in the field. Thus, the growing condition of both wheat and maize plays a pivotal role in the Wheat × Maize System, and optimal environmental factors can be drawn for an improved Wheat × Maize System. The environmental factors proposed by Khan et al. ^[20][77] can serve as a reference for technical improvement.

In the laboratory, wheat breeding lines (≥F3 generation) and hybrid maize varieties are grown in environmentally controlled facilities: wheat under 20–24 °C, day/night of 20 h/4 h, light intensity > 420 μmol/m²/s, and humidity 60–65%; maize under 22–24 °C, day/night of 12 h/12 h, light intensity > 140 μmol/m²/s, and humidity 55–60%. These specific environmental parameters have led to a nearly 100% pseudoseed formation frequency (PFF) with the well-filled seeds.

Growth condition, or controlled environment, is preferred for conducting the Wheat × Maize System. However, due to the limited space of any environmentally controlled facility, immature wheat spikes were harvested during heading and then subjected to in vitro culture ^[19][20][22][76,77,79]. 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 ^[23][29], any accountable pollination is based on the wheat floret status, those with a feathery stigma being best. Martins-Lopes et al. ^[24][80] 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 controlled conditions.

Phytohormone treatment post 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 ^[25][26][27][31,45,81]. To improve the Wheat × Maize System, a variety 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 ^[28][82]. Among them, 2,4-D is widely used to control organ regeneration and callus induction. The 2,4-D also regulates early and postembryogenic plant development involving both somatic and zygotic embryogenesis ^[29][83].

When applying a hormone in the Wheat × Maize System, the dosage, timing, and methodology of it should be determined. At 100 ppm, 2, 4-D effectively induces haploid embryos in hexaploid wheat ^[14][30][31][72,84,85]. At 250 ppm, 2, 4-D effectively promotes haploid production in tetraploid wheat ^[27][81]. Kaushik et al. ^[30][84] 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. 

During the Wheat × Maize System process, maize chromosomes are eliminated not only in embryo cells but also in endosperm cells, which will cause seed abortion ^{[20][32][33][34]}[50,77,86,87]. Therefore, wheat haploid embryos must be rescued by tissue culture to generate haploid plantlets. In practice, wheat embryo rescue is highly dependent on the plant regeneration media. Among B5, MS, and ½ MS tested, Cherkaoui et al. ^[35][88] 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 a 69.3% regeneration rate of wheat plantlets but only 33.5% regeneration in the control group (SM) ^[36][89]. Most tests are needed with how to supply putrescine and spermidine in B5 and/or ½ MS medium.

During this phase, researchers' methodology entails using a clean bench where haploid embryos are carefully isolated from sterilized immature seeds under a dissection microscope. The isolated embryos are then plated on ½ MS medium (½ MS + 20 g/L sucrose + 2.4 g/L plant agar, pH 5.8). These embryos are cultured under controlled conditions at a temperature of 20–24 °C with a 16 h light/8 h dark photoperiod and a light intensity of 67.2 μmol/m²/s. 

Wheat haploid plants obtained through the Wheat × Maize System naturally remain undoubled ^[37][90]. Chromosome doubling is essential to acquire homozygous and stable diploid plants. Antimitotic compounds are selected to double plant chromosomes ^[38][91], for which colchicine is the most 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 ^[39][92] and poor grain setting in DH plants (Figure 2). Colchicine treatment is partially lethal to plant haploids; thus, it 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 ^[40][93] trimmed roots by keeping 2–3 cm on haploid plantlets and soaked the trimmed roots in 0.1% colchicine (with 2% dimethyl sulfoxide/DMSO and fifteen Tween-20 drops per liter) at 20 °C for 5 h. At the 2–3 tiller stage, the colchicine application resulted in a 95.6% doubling rate. Khan et al. ^[41][94] treated haploids with 3–5 tillers in 0.1–0.2% colchicine for 3 h and provided continuous air flow in the solution. Niu et al. ^[42][95] also supplied air during colchicine treatment at 14–16 °C and achieved over 90% survival and chromosome doubling among the treated wheat plantlets. Sharma et al. ^[43][96] also studied the in vitro effect of colchicine. The wheat DH production was enhanced after four hours of treatment with 0.075% colchicine in hexaploid wheat and 0.15% colchicine in tetraploid wheat. 

All in all, many studies have been conducted on the Wheat × Maize System in recent years. Genotypes of wheat and maize and their interaction all play roles in the wheat DH line production via maize pollen induction. Wheat breeding materials and hybrid maize varieties should be grown in environmentally controlled conditions for a high efficiency of DH line production. Optimization of the procedures, including treatment of wheat spikes and timing of pollination, hormone treatment, embryo rescue, and doubling treatment, could further enhance the efficiency of wheat DH line production in the Wheat × Maize System.