Effects of hormone concentrations on anther cultures and the acquisition of regenerated plants of five awnless triticale genotypes

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The aim of this study was to explore the effects of triticale genotype and the types and ratios of exogenous hormones in the medium on the efficiency of triticale anther culture. Results Anthers of five triticale genotypes were cultured on four different callus induction media and the calli were induced to differentiate into green plants by culture on three different differentiation media. The triticale genotype T8004 showed the best performance in anther culture, with a callus induction rate of 28.64%, a green plantlet differentiation frequency of 33.33%, and a green plantlet production rate of 2.78%. The highest callus induction rates were obtained by culturing anthers on C3 medium, and the highest green plantlet differentiation frequency was obtained by culturing calli on D2 differentiation medium. Flow cytometry analyses showed that 15 of the 20 regenerated plants that grew normally in the field were doubled haploids. The average chromosome doubling success rate was 55.6%. Analyses of agronomic traits showed that the 15 doubled haploid plants reached the standard for awnless triticale, so they are candidate materials for breeding new awnless triticale varieties. Conclusion The anther culture technology of triticale was optimized in this paper, which made it possible to rapidly breed homozygous varieties of awnless triticale. awnless triticale anther culture genotypes hormone concentrations ploidy identification agronomic trait Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Background As a high-quality annual forage grass, triticale (×Triticosecale Wittmack) plays important roles in alleviating the conflict between forage and livestock in pastoral areas, protecting the ecological environment, and promoting the healthy development of grassland agricultural ecosystems [ 1 , 2 ]. At present, most triticale varieties (lines) have awns — bristle-like appendages located on the spikelet. Although awns have roles in increasing the photosynthetic area, improving transpiration, and pest resistance [ 3 , 4 ], they also have some disadvantages. The waxy awn of triticale can puncture the oral cavity of livestock, which can lead to diseases such as pharyngitis, oral ulcers, and submandibular edema [ 5 , 6 ]. When triticale is harvested at the dough stage, the waxy awn can adversely affect the production of high-quality forage products and reduce the palatability of forage [ 7 ]. Awnless triticale does not have these shortcomings. Moreover, many studies have shown that the seed yield of awnless varieties is not lower than that of awned varieties [ 8 , 9 ]. Therefore, it is of great significance to breed awnless triticale lines to improve the palatability of forage and the quality of forage products. In many breeding methods, anther culture is an indispensable rapid breeding technology. It has become a powerful tool for rapid haploid production [ 10 , 11 ]. Varieties with excellent traits are selected as hybrid parents, and homozygous plants are obtained from F1 plants with improved agronomic traits by anther culture followed by chromosome doubling. This doubled-haploid breeding strategy is more efficient than traditional breeding methods [ 12 ]. Homozygotes of triticale with relatively stable traits can be obtained quickly by anther culture. This can save a lot of breeding time and reduce the labor and financial resources required for breeding [ 13 , 14 ]. At present, the anther culture technique is the most widely used method for haploid breeding of triticale. In recent years, researchers have used anther culture technology to produce haploid and doubled haploid plants of various species, most commonly wheat ( Triticum aestivum ) [ 15 ], rice ( Oryza sativa ) [ 16 ], corn ( Zea mays ) [ 17 ], and pepper ( Capsicum annuum ) [ 18 ]. Few studies have focused on the production of doubled haploid triticale. In the few studies that have used triticale in anther culture, the experimental materials were all awned triticale varieties (lines), rather than awnless ones. In the present study, all the experimental materials were awnless triticale lines. The overall goal was to provide a reference for the optimization of anther culture technology for awnless triticale. Among the many factors that affect the efficiency of triticale anther culture, plant genotype is the most important one [ 19 ]. The success rate of anther culture varies greatly among different varieties (genotypes) because of the dependence of anther culture on genotype [ 20 ]. Marciniak et al. [ 21 ] reported that the number of green plants induced to form in anther culture differed significantly among 38 winter triticale genotypes (range, 1.0–16.2/spike) under the same culture conditions. Similarly, there were significant differences in the number of haploid green plants obtained after anther culture of 21 Swedish and Dutch F1 and F2 triticale combinations under the same culture conditions. The average number of haploid green plants ranged from 0.8 to 13.6/spike, and there were significant differences in the number of green plants induced among the lines [ 22 ]. The results of those studies indicate that the triticale genotype significantly affects the efficiency and success rate of anther culture. The type, ratio, and concentrations of exogenous hormones also affect the efficiency of anther culture [ 23 ], and the optimum types, concentrations, and ratios of hormones for anther culture differ among different plants. Kruppa et al. [ 24 ] found that P4mf medium was more suitable than W14mf medium for the production of doubled haploid triticale lines, and the addition of 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (KT) to P4mf medium significantly increased the number of calli and green plants (103.7/anther and 19.7/anther, respectively). Żur et al. [ 25 ] reported that the addition of 0.5 mg/L naphthaleneacetic acid (NAA) and 0.5 mg/L KT to C17 medium significantly increased the callus induction rate of eight triticale varieties, and ‘DH144’ triticale had the highest regeneration rate of green plants (5.4 green seedlings differentiated per 100 calli). Hassawi et al. [ 26 ] tested the effects of seven auxins and four cytokinins in triticale anther culture, and found that the anthers showed the highest callus differentiation rate (13.9%) on medium supplemented with 2,4-D. Özkum and Tipirdamaz [ 27 ] found that the combination of 4.0 mg/L NAA and 1.0 mg/L 6-benzyladenine (BA) in the medium improved the callus regeneration rate of pepper embryoids. Therefore, optimization of hormone concentrations in the medium can improve the success rate in anther culture. We hypothesized that modifying the medium hormone concentration may alter the awnless triticale anther callus induction rate and green plantlet differentiation frequency, which can affect the production of haploid plants that are then treated with colchicine for the doubling of chromosomes to obtain doubled haploid (DH) plants, thereby accelerating the breeding process (Fig. 1 ). In this study, we analyzed the effects of different types and ratios of hormones on the callus induction rate and anther differentiation rate of five different triticale lines in anther culture. The results provide a theoretical basis for improving anther culture and optimizing the regeneration of awnless triticale. 2 Materials and methods 2.1 Site description This study was conducted at the Forage Experimental Station of Gansu Agricultural University (36°03′N, 103°53′E, 1560m above sea level), Lanzhou, China in 2021. The previous crop was high-yield sweet sorghum (Sorghum bicolor) grown under good irrigation conditions. Basic information on the experimental site is shown in Table 1 . Table 1 Basic information of the experimental site Experimental site Mean annual temperature (℃) Frostless period (d) Mean annual rainfall (mm) Soil organic matter (mg/kg) Rapidly available nitrogen (mg/kg) Rapidly available phosphorus (mg/kg) Rapidly available potassium (mg/kg) pH Soil type Forage test station 7.9 171 349.9 2.3 90.05 7.36 172.8 7.35 loessal soil 2.2 Experimental materials The five materials used in this experiment were the new hybrid lines of awnless triticale selected by pedigree method in Gansu Agricultural University, and the line number, name and parents were shown in Table 2 . After a 30-day vernalization, the seeds of 5 awnless triticale lines were planted in the plastic greenhouse experimental base of Gansu Agricultural University (36°03′N, 103°53′E; 1,560 m above sea level), China, in March 2023. A line seeding method was adopted, with row spacing of 20 cm, plant spacing of 5 cm, and a sowing depth of 2 ~ 3 cm. 300 kg·hm-2 of diammonium phosphate was applied before sowing, and 196 kg·hm-2 of urea were applied at the regreening stage and jointing stage, respectively. During the experiment, weeding was carried out, and irrigation was given once at the seedling stage, tillering stage and jointing stage. Sampling began in late May and early June. Table 2 Number, name and parents of five awnless triticale materials Number Name Parents (♀×♂) T8001 T2021-8001 OH2276 (2013) × J9 (2013) T8002 T2021-8002 81S37 (2013) × J35 (2013) T8003 T2021-8003 4009 (2013) × P2 (2013) T8004 T2021-8004 T17 (2013) × J18 (2013) T8005 T2021-8005 89D-8 (2013) × J10 (2013) 2.3 Sampling and pretreatment of young spikes The young spikes of awnless triticale with pollen microspores in the middle and late stages of mononuclear were taken (i.e., spikes of awnless triticale in the field just reached the middle of the flag leaf and the second leaf from the top). The young spikes were cut from about 10 cm below the second leaf from the top, and wrapped into bundles with aluminum foil, and the date and line name were marked. Two anthers from the collected per young spikes were crushed on a glass slide, after which 1 ~ 2 drops of acetic acid magenta dye (Solarbio Inc., Beijing, China) were added and the stained material was examined using the Panthera U microscope (eyepiece 10× and objective 40×; Motic China Group Co., Ltd., Hong Kong, China) to determine whether mononuclear stage had been reached. The incision of the young spikes which was up to the standards by microscopic examination was inserted into a 500 mL beaker filled with 250 mL of tap water, and pretreated at 4°C for 15 days in the dark. 2.4 Spike disinfection and anther inoculation After 15 days of low temperature pretreatment, the flag leaf and the second leaf from the top were cut off. In the biological safety cabinet, disinfected the surface of the test material with 75% alcohol for 1 min, peeled off the young spikes, disinfected with 2% sodium hypochlorite solution for 7 min, rinsed with sterile water for 4 ~ 5 times, and then wiped the surface water of the young spikes with sterile filter paper. Anthers were removed from the sterilized spikes and placed on each of the 4 different CHB induction medium (denoted as C1, C2, C3, C4, respectively). Each dish is inoculated with 40 anthers, and each treatment inoculates 6 ~ 8 Petri dishes (90 mm diameter) (Jingan Inc., Shanghai, China). Based on the hormone content of the optimal CHB induction medium screened by our team in the previous study, the hormone content was further adjusted and refined, and 4 kinds of induction medium were designed, each of which was supplemented with 90 g/L sucrose and 7 g/L agar, respectively, with a pH value of 5.4. The hormone content of 4 induction medium was shown in Table 3 . Table 3 Hormone concentration of induction medium Number Hormone concentration C1 0.5 mg/L 2,4-D + 1.0 mg/L KT C2 1.5 mg/L 2,4-D + 1.5 mg/L KT C3 2.0 mg/L 2,4-D + 1.5 mg/L KT C4 2.5 mg/L 2,4-D + 1.5 mg/L KT 2.4 Induction culture of anther After the inoculation was completed, the petri dish was placed in a incubator (HGZ-250, Yuejin Inc., Shanghai, China) for a 3-day incubation at 32°C in darkness, and then transferred to another incubator for a 60-day incubation at 28°C in darkness. After 8 weeks, the number of callus was counted. 2.5 Differentiation culture of callus Based on the hormone content of the optimal MS differentiation medium screened by our team in the previous study, 3 differentiation medium were designed by adjusting different hormone contents, and each differentiation medium was supplemented with 10 g/L sorbitol, 30 g/L sucrose and 7 g/L agar, respectively, and the pH value was 5.8. The hormone content of the 3 differentiation medium was shown in Table 4 . Awnless triticale lines (T8003, T8004) which produced more callus were selected, and the callus with a diameter of more than 1 mm were transferred to 4 kinds of MS differentiation medium, and 4 calli were placed in each dish, and the petri dishes were placed in an incubator set at 27°C with a 14-h light (3,000 lx): 10-h dark cycle, and the medium was refreshed every 15 days. The number of green plantlets and albino plantlets was counted after 12 weeks. Since T8001, T8002, and T8005 lines induced fewer callus, callus from these three lines were inoculated only on D1 differentiation medium. Table 4 Hormone concentration of differentiation medium Number Hormone concentration D1 1.0 mg/L IAA + 1.0 mg/L 6-BA D2 1.0 mg/L IAA + 1.5 mg/L 6-BA D3 1.0 mg/L IAA + 2.0 mg/L 6-BA 2.6 Plantlets training When the regenerated green plantlets grew to 4 leaves, the parafilm of the culture bottle was removed, and an appropriate amount of tap water was added, and the culture bottle was remained in the incubator set at 27°C with a 14-h light (3,000 lx): 10-h dark cycle for 3 days. After that, the green plantlets were transplanted into plastic pots (10cm × 10cm) (Jiesheng Co., Shenzhen, Guangdong, China) containing nutrient soil (Luneng Co., Tianzhu, Gansu, China). These plastic pots was placed in an artificial climate chamber set at 25°C with a 14-h light : 10-h dark cycle. 2.7 Ploidy Level Analysis of the regeneration green plantlets The ploidy levels of regenerated plants was determined by the CyFlow Ploidy Analyzer (CyFlow Cube 6; Sysmex Co., Hamburg, Germany). Briefly, one leaf were collected from each plant and then placed in 400 µL extraction buffer (CyStain UV Precise P Kit; Sysmex Co., Hamburg, Germany). The leaf was minced for 1 min using a sharp blade and passed through a 30 µm filter to remove cell debris. Next, 1,600 µL DAPI staining solution was added to stain the nuclei prior to the analysis using the CyFlow Ploidy Analyzer (Sysmex Co., Hamburg, Germany). More than 3,000 nuclei were detected per sample. Hexaploid triticale variety ‘Gannong No.3’ (provided by Gansu Agricultural University, Lanzhou, Gansu, China) was used as a control (CK1) to determine whether the regenerated plants were haploid plants. 2.8 Chromosome Doubling, plantlets Training, and Transplanting When the green plantlets had three tillers (or were approximately 12 cm tall), they were removed from the plastic pot, rinsed, and then their roots were soaked in a solution containing 0.1% colchicine, 2% dimethyl sulfoxide, and 0.05% Tween-20 for 4.5 h chromosome doubling step. The roots were rinsed by tap water for 2 h and then the green plantlets were replanted in plastic pots and incubated in the artificial climate chamber for 3 ~ 5 weeks to resume growth. They were subsequently transferred to a field. 2.9 Ploidy Level Analysis Flow cytometry was used to analyze the ploidy of plantlets that grew normally in the field (before the jointing stage). The analysis method is the same as 1.8. The octaploid tritcale variety ‘Jinsong 49’ (provided by the Hebei Academy of Agriculture and Forestry Science, Shijiazhuang, Hebei, China) was used as a control (CK2) to determine whether the regenerated seedlings were double haploid plants. 2.10 Agronomic Trait Analysis The following traits of the regenerated plants were examined at the flowering stage: plant height and number of branch per plant. The following spike parameters were examined at the dough stage: tip awn length, side awn length, spike length, number of spikelets, number of grains per spike, and grain weight per spike. T2023-8003 was used as the control group (CK3). 2.11 Statistical Analysis Each petri dish with inoculated anthers was taken as a replicate, and 6 ~ 8 replicates are treated with each treatment. In callus induction trials, there are at least 3 replicates per treatment. The analysis of variance was performed by SPSS 20.0 software. If significant differences were detected, Duncan’s multiple comparison test was performed to compare the differences. CIR (%) = Number of calli/Number of anthers used for the inoculation × 100 DFG (%) = Number of green plantlets/Number of calli × 100 DFA (%) = Number of albino plantlets/Number of calli × 100 PRG (%) = Number of green plantlets/Number of anthers used for the inoculation × 100 PRA (%) = Number of albino plantlets/Number of anthers used for the inoculation × 100 PRR (%) = Total numbers of plantlets/Number of anthers used for the inoculation × 100 3 Results 3.1 Microscopic Examination of Anthers The young spikes of five triticale lines were observed under a light microscope (Fig. 2 ). In all the lines, most of the microspores were in the late uninucleate stage, which is the optimum stage for anther culture (Fig. 2 a,b). 3.2 Analysis of Variance Table 1 showed that the significant or extremely significant differences were detected for triticale line, except for the DFG, PRG and PRA. The significant or extremely significant differences were detected for hormone concentration of induction / differentiation medium, except for the DFA, PRA and PRR, and the significant or extremely significant differences were detected for the interactive effects of triticale line and hormone concentration of induction / differentiation medium, except for the PRA. The multiple comparison test was completed for the above parameters with significant or extremely significant differences (Table 5 ). Table 5 Variance analysis of induction and differentiation indicators of awnless triticale anther culture Variation F Value CIR (%) DFG (%) DFA (%) PRG (%) PRA (%) PRR (%) Triticale line 7.13** 2.88* 1.90 2.21 0.99 3.49* Hormone concentration of induction / differentiation medium 4.00* 15.48** 1.13 4.43* 1.13 1.67 Triticale line × hormone concentration of induction / differentiation medium 31.52** 8.27** 3.15* 3.00* 1.60 2.71* Note: CIR: Callus induction rate. DFG: Green plantlet differentiation frequency; DFA: Albino plantlet differentiation frequency. PRG: Green plantlet production; PRA: Albino plantlet production; PRR: Plant regeneration rate; * indicates significant differences at 0.05 level; ** indicates extremely significant differences at 0.01 level, the same as below. 3.3 Effects of triticale genotype and hormone concentrations in the medium on callus induction 3.3.1 Differences in callus induction rate among five triticale lines There were significant differences in the average callus induction rate (CIR) among the various triticale lines (Fig. 3 ). The average CIR was highest in T8004 (16.67%), followed by T8003 (9.79%), and these rates were significantly higher than those of T8001 and T8002 (P < 0.05). The average CIR was lowest in T8001 at 6.25%, which was significantly lower than those of T8003 and T8004 (P < 0.05). These findings show that the average CIR differed significantly among the five triticale lines. 3.3.2 Differences in CIR among media with different hormone concentrations As the 2,4-D concentration in the medium increased, the average CIR of different triticale lines first increased and then decreased. The average CIR was highest on C3 medium (14.17%), followed by C4 medium (12.83%), significantly higher than that on C1 medium, but not significantly different from that on C2 medium. The average CIR was lowest on C1 medium (7.50%), which was significantly lower than those on C3 and C4 media. Thus, the highest average CIR was achieved on C3 medium (Fig. 4 ). 3.3.3 Significance of genotype × hormone concentration interaction for CIR The interaction between genotype and hormone concentration was significant for the CIR (Fig. 5 ). For the genotypes T8001, T8004, and T8005, the highest CIRs were achieved on C3 medium (7.50%, 25.83%, and 13.33%, respectively), and these rates were significantly higher than those on other media. The CIR of T8002 was significantly higher on C2 medium (13.33%) than on other media, and the CIR of T8003 was significantly higher on C4 medium (21.67%) than on other media. These results indicate that the different triticale lines required different hormone concentrations to achieve the highest CIRs (Fig. 5 ). In terms of hormone concentrations, the highest CIR on C1 medium was for line T8004; the highest CIR on C2 medium was for line T8002; and both of those CIRs were higher or significantly higher than those of the other genotypes on those media. The highest CIRs on C3 and C4 media were for lines T8004 and T8003, respectively, and their CIRs were higher or significantly higher than those of other genotypes on those media. These results indicate that different hormone concentrations were required for optimum callus differentiation of the five triticale lines (Fig. 5 ). 3.4 Differentiation of triticale calli obtained in anther culture 3.4.1 Differences in differentiation frequency, plantlet production, and regeneration rate among triticale lines The number of calli produced in anther culture differed markedly among the different triticale lines. Large numbers of calli were produced by the T8003 and T8004 lines, whereas fewer were produced by the T8001, T8002 and T8005 lines. To compare the effects of different hormone concentrations on callus differentiation, the calli produced by the T8003 and T8004 lines were cultured on D1, D2 and D3 differentiation media, and the calli produced by T8001, T8002, and T8005 were cultured on D1 differentiation medium. As shown in Table 6 , there were significant differences in the green plantlet differentiation frequency (DFG) and plant regeneration rate (PRR) of the triticale lines among the three media containing different hormone concentrations. The highest average DFG was in T8005 (20.83%), and this was significantly higher than that of T8002 (P < 0.05), but not significantly different from those of T8001, T8003, and T8005. The highest average PRR was in T8001 (2.92%), significantly higher than that of T8002 (P < 0.05), but not significantly different from those of T8003, T8004 and T8005 (Table 6 ). Table 6 Differences of the average DFG and PRR in triticale lines for different hormone concentrations. Triticale line DFG (%) PRR (%) T8001 15.28 ± 1.39a 2.92 ± 0.42a T8002 0.00 ± 0.00b 0.83 ± 0.42b T8003 16.67 ± 2.69a 1.67 ± 0.42ab T8004 19.44 ± 4.05a 1.81 ± 0.30ab T8005 20.83 ± 4.17a 2.50 ± 0.27a Notes: DFG: green plantlet differentiation frequency; PRR: plant regeneration rate. 3.4.2 Differences in differentiation frequency, plantlet production among three different differentiation media As shown in Table 7 , there were significant differences in the average DFG and PRG of the triticale lines among the three differentiation media with different hormone concentrations. The highest average DFG and PRG (29.17% and 2.43%, respectively) were achieved on D2 medium, and were significantly higher than their respective values on D1 and D3 media. These results show that the highest DFG and PRG were achieved on D2 medium, so this medium was most conducive to obtaining green plantlets from calli produced in anther culture. Table 7 Differences of the average DFG and PRG in hormone concentrations for different triticale line Hormone concentration DFG (%) PRG (%) D1 9.72 ± 2.02c 1.08 ± 0.24b D2 29.17 ± 2.64a 2.43 ± 0.5a D3 18.75 ± 2.8b 1.39 ± 0.23b Notes: DFG: Green plantlet differentiation frequency; PRG: Green plantlet production. 3.4.3 Effects of the genotype × hormone interaction in terms of differentiation frequency, plantlet production, and plant regeneration rate The effect of the genotype × hormone concentration interaction was significant for DFG and DFA (P < 0.05). The calli of T8003 and T8004 were cultured on D1, D2, and D3 media, and as the 6-BA concentration in the medium increased, the DFG of T8003 and T8004 showed a trend of first increasing and then decreasing. The DFG of T8003 and T8004 were highest on D2 medium (25.00% and 33.33%, respectively), and these values were higher or significantly higher than those on D1 and D3 media. The DFA of T8003 and T8004 were lowest on D2 medium (0.00% and 4.17%, respectively), and these values were lower or significantly lower than those of the same genotypes on D1 and D3 media. These results show that D2 medium was the most conducive to obtaining green plantlets of the triticale lines T8003 and T8004 (Table 8 ). When the calli of the five triticale lines were cultured on D1 differentiation medium, there were significant differences in DFG and DFA among the five lines (P < 0.05). The DFG of T8005 was the highest (16.67%), which was significantly higher than that of T8002, but not significantly different the other triticale lines. The T8001 genotypes had the second-highest DFG (15.28%). The calli of T8002 did not differentiate into green plants so its DFG was 0.00%. The T8005 genotype had the lowest DFA (0.00%), followed by T8003 (4.17%), whereas T8004 had the highest DFA (20.83%), which was significantly higher than those of the other genotypes. In summary, there were significant differences in the differentiation rate of calli among different genotypes of triticale on the same medium, and this was related to genotype (Table 8 ). Table 8 The effect of the interaction of triticale line and hormone concentration on DFG and DFA Triticale line HC (mg/L) NC NGP NAP DFG (%) DFA (%) T8001 D1 32 5 2 15.28 ± 1.39bc 5.55 ± 2.78b T8002 D1 24 0 2 0.00 ± 0.00d 8.33 ± 4.17b T8005 D1 24 4 0 16.67 ± 4.17bc 0.00 ± 0.00b T8003 D1 20 2 1 8.33 ± 4.17cd 4.17 ± 4.17b D2 20 5 0 25.00 ± 0.00ab 0.00 ± 0.00b D3 20 3 2 16.67 ± 4.17bc 8.33 ± 4.17b T8004 D1 20 2 4 8.33 ± 4.17cd 20.83 ± 4.17a D2 20 7 1 33.33 ± 4.17a 4.17 ± 4.17b D3 20 4 1 20.83 ± 4.17b 4.17 ± 4.17b Note: HC: hormone concentration; NC: Number of callus; NGP: Number of green plantlet; NAP: Number of albino plantlet. As shown in Table 5 , the PRG and PRR of triticale plants differed significantly among the three media with different hormone concentrations (P < 0.05). When the calli of T8003 and T8004 were cultured on D1, D2, and D3 differentiation media, the highest PRG for the calli of both T8003 and T8004 was on D2 medium (2.08% and 2.78%, respectively). The PRG of T8003 and T8004 calli on D2 medium was higher or significantly higher than those on D1 and D3 media, and the PRA of T8003 and T8004 calli was lower on D2 medium than on D1 and D3 media. These results show that D2 differentiation medium was more suitable than the D1 and D3 media for callus differentiation of the T8003 and T8004 lines (Table 9 ). When all five triticale lines were cultured on D1 differentiation medium, the PRG, PRA, and PRR differed significantly among the five lines. T8001 had the highest PRG (2.08%), followed by T8005. The T8002 genotype had the lowest PRG (0.00%). T8005 had the lowest PRA (0.00%), which was lower or significantly lower than that of other genotype, while T8004 had the highest PRA (1.53%), which was higher or significantly higher than that of other genotype. In terms of PRR, T8001 had the highest PRR (2.92%), followed by T8004 (2.36%), whereas T8002 had the lowest PRR (0.83%), which was significantly lower than those of the other genotypes (Table 9 ). These results indicate that there were significant differences in the PRG and PRA among different triticale lines (genotypes) under the same culture conditions (Table 9 ). Table 9 The effect of the interaction of triticale line and hormone concentration on plantlet production and plant regeneration rate of triticale Triticale line HC (mg/L) NA NGP NAP PRG (%) PRA (%) PRR (%) T8001 D1 240 5 2 2.08 ± 0.42ab 0.83 ± 0.42ab 2.92 ± 0.42ab T8002 D1 240 0 2 0.00 ± 0.00c 0.83 ± 0.42ab 0.83 ± 0.42d T8005 D1 240 4 0 1.67 ± 0.42ab 0.00 ± 0.00b 1.67 ± 0.42bcd T8003 D1 240 2 1 0.83 ± 0.42bc 0.42 ± 0.42ab 1.25 ± 0.72cd D2 240 5 0 2.08 ± 0.42ab 0.00 ± 0.00b 2.08 ± 0.42abcd D3 240 3 2 1.25 ± 0.00abc 0.83 ± 0.42ab 2.08 ± 0.42abcd T8004 D1 280 2 4 0.83 ± 0.42bc 1.53 ± 0.50a 2.36 ± 0.14abc D2 280 7 1 2.78 ± 0.97a 0.42 ± 0.42ab 3.19 ± 0.56a D3 280 4 1 1.53 ± 0.50abc 0.42 ± 0.42ab 1.94 ± 0.37abcd Note: NC: Number of callus. 3.5 Identification of the ploidy level of regenerated plants During the process of anther culture, pollen microspores are induced to form callus, which then differentiates into green plants. The cells of the anther wall, anther septum, and anther filament may also dedifferentiate to form callus, which can also differentiate into green plants. The green plants induced from pollen microspores are haploid (triploid in this experiment), and the green plants induced from the cells of the anther wall, anther septum, and filament are diploid (hexaploid in this experiment). Therefore, we analyzed the ploidy levels of the green plants obtained in anther culture by flow cytometry to distinguish between the haploids and diploids. In this experiment, the control ‘Gannon No.3’ triticale (CK1) was hexaploid (2n = 6x = 42), and T8001, T8002, T8003, T8004, and T8005 were also hexaploids (2n = 6x = 42). As shown in Fig. 5 , the position of the fluorescence peak in the control ‘Gannon No.3’ triticale (CK1) was at 21000–27000 (Fig. 6 a). Therefore, when analyzing the green plants obtained in anther culture, a fluorescence peak at half of this value indicated that the plant was haploid. The fluorescence peak positions of green plants induced from the T8001, T8002, T8003, T8004, and T8005 lines were at 9000–15000, approximately half of that in the control group, indicating that the induced green plants were haploids (Fig. 6 b). Note (a): ‘Gannong No. 3’ triticale (CK 1 ); (b): haploid regenerated plants. 3.6 Chromosome doubling of haploid plants Chromosome doubling was induced in the identified haploid plants using colchicine solution. The plant roots were soaked in 0.1% colchicine solution for 4.5 h to induce chromosome doubling, then rinsed with tap water for 2 h and replanted in plastic pots. As shown in Table 6 , a total of 23 plants survived after root soaking, with survival rates ranging from 75.0–100% among the five genotypes. The average survival rate across the five lines was 85.2%. The survival rate of T8005 was 100%, whereas 1, 1, and 2 plants of T8001, T8003, and T8004, respectively, did not survive. The survival rates of the T8001, T8003, and T8004 lines were 75.0%, 90.0%, and 81.8%, respectively (Table 10 ). Table 10 The number of survival green plantlet after chromosome doubling and Survival rate after chromosome doubling Triticale line TNGP NHGP NSGPCD SR/% T8001 5 4 3 75.0 T8002 0 0 0 0.0 T8003 10 10 9 90.0 T8004 13 11 9 81.8 T8005 4 2 2 100.0 Total 32 27 23 85.2 Note: TNGP: The total number of green plantlet induced; NHGP: The number of haploid green plantlet; NSGPCD: The number of survival green plantlet after chromosome doubling; SR: Survival rate. 3.7 Identification of doubled haploid regenerated plants The ploidy level of regenerated plants that grew normally to the booting stage was identified by flow cytometry. CK2 was the octaploid ‘Jinsong 49’ triticale (2n = 8x = 56) (Fig. 7 a), with a fluorescence peak at 21000–27000. The regenerated plants with fluorescence peaks at three-quarters of that of CK2 were identified as haploid. All of the measured regenerated plants had fluorescence peaks at 16000–21000 (i.e., three-quarters of CK2), so they were identified as doubled haploid (Fig. 7 b). Of the 23 plants transplanted into the field, 20 survived. Of those, 15 plants were doubled haploid plants and five were mixoploid. The chromosome doubling success rate ranged from 25.0–63.6% among the five lines, and the average chromosome doubling success rate was 55.6%. T8004 had the highest chromosomal doubling success rate (63.6%) and T8001 had the lowest (25.0%). This difference may be related to genotype. The above results show that the chromosomal doubling success rate differed among the five genotypes of triticale (Table 11 ). Table 11 Ploidy changes and success rate of chromosome doubling in the number of regenerated plantlet after chromosome doubling Triticale line NHGP NGPT NSGP Double haploid plant MOPP SRCD/% T8001 4 3 3 2 1 25.0 T8002 0 0 0 0 0 0.0 T8003 10 9 8 5 3 30.0 T8004 11 9 8 7 1 63.6 T8005 2 2 1 1 0 50.0 Total 27 23 20 15 5 55.6 Note: NHGP: The number of haploid green plantlet; NGPT: The number of green plantlet transplanted in field; NSGP: The number of survival green plantlet; MOPP: Mixoploidy and other ploidy plants; SRCD: Success rate of chromosome doubling. 3.8 Investigation of agronomic traits in the field of DH0 generation plants The calli that formed in anther culture differentiated to form haploid green plants, which were transplanted into the field after chromosome doubling. The agronomic traits of the doubled haploid regenerated plants that grew to the flowering stage in the field were measured (Fig. 8 ). To reduce the influence of tissue culture on plants, the traits of the DH1 generation population were measured in this experiment. As shown in Table 12 , the plant height of the 11 triticale plants was 126.8–153.7 cm, and plant height of 4 plants was significantly higher than that of CK3 (T2021-8003). The tip awn and side awn of the 11 regenerated triticale plants were extremely short, both with lengths shorter than 5.0 mm, which reached the standard of awnless triticale. Thus, these 11 plants were identified as potential materials for further breeding of awnless varieties (Fig. 9 ). In terms of the panicle traits, spikelet number of 4 plants was higher than that of CK3 and number of grains per spike of 2 plants was higher than that of CK3, while all the plants had grain weight per spike that were lower than or significantly lower than those of CK3. These results indicate that, DH1 generation plants were lower than those of mature lines in terms of spike traits, especially grain weight per spike. Table 12 The agronomic traits of the 11 DH1 generation double haploid plants Number Plant height/cm Tip awn/mm Side awn/mm Spike length/cm Number of spikelet Number of grains per spike Grain weight per spike/g CK 142.5 ± 0.9d 3.51 ± 0.3cd 2.75 ± 0.2abc 12.5 ± 0.3a 30.3 ± 0.3cd 42.7 ± 1.5b 1.99 ± 0.12a Z23-1 126.8 ± 0.8g 3.6 ± 0.1bcd 2.74 ± 0.2abc 11.2 ± 0.2de 31.0 ± 1.0bc 43.0 ± 1.0b 1.50 ± 0.07fg Z23-2 134.1 ± 0.7ef 3.24 ± 0.1d 3.07 ± 0.1abc 11.7 ± 0.3bcd 34.7 ± 0.7a 49.7 ± 1.5a 1.67 ± 0.08bc Z23-3 151.7 ± 1.3ab 4.26 ± 0.1ab 3.89 ± 0.4a 12.3 ± 0.1ab 33.3 ± 0.7ab 41.0 ± 1.2bc 1.79 ± 0.10cde Z23-4 135.8 ± 1.2ef 3.49 ± 0.3cd 2.63 ± 0.5bc 10.6 ± 0.3e 26.7 ± 0.7e 28.7 ± 1.3e 1.40 ± 0.03de Z23-5 144.7 ± 0.8cd 4.26 ± 0.2ab 2.02 ± 0.5c 11.5 ± 0.3cd 28.0 ± 1.2de 32.3 ± 1.5de 1.19 ± 0.05bcd Z23-6 133.0 ± 1.2f 3.68 ± 0.2bcd 2.07 ± 0.1c 9.5 ± 0.1f 28.7 ± 0.7cde 35.0 ± 1.7d 1.35 ± 0.05ab Z23-7 153.7 ± 0.7a 2.94 ± 0.5d 3.96 ± 0.1a 12.3 ± 0.1ab 33.3 ± 0.7ab 44.0 ± 2.1b 1.87 ± 0.04efg Z23-8 150.3 ± 0.7b 3.39 ± 0.2cd 2.63 ± 0.5bc 12.0 ± 0.2abc 30.7 ± 1.3cd 37.0 ± 1.5cd 1.48 ± 0.07g Z23-9 146.5 ± 0.9c 4.50 ± 0.2a 2.96 ± 0.4abc 11.7 ± 0.1bcd 29.3 ± 0.7cd 34.0 ± 1.0d 1.26 ± 0.06efg Z23-10 137.0 ± 1.2e 4.11 ± 0.1abc 3.87 ± 0.1a 10.7 ± 0.3e 30.7 ± 0.7cd 46.0 ± 2.6ab 1.73 ± 0.07ab Z23-11 144.3 ± 1.6cd 4.73 ± 0.2a 3.79 ± 0.6ab 11.0 ± 0.2de 28.7 ± 0.7cde 37.0 ± 1.5cd 1.55 ± 0.06def 4 Discussion 4.1 Effect of genotype on callus induction rate and green plantlet differentiation frequency The efficiency of anther culture is affected by various factors, among which the genotype of the plant is the most important [ 19 ]. Even when the culture conditions are exactly the same, the efficiency of anther culture differs among different plant genotypes [ 20 ]. High-response and low-response genotypes show quite different responses in anther culture [ 28 ]. For example, Żur et al. [ 25 ] detected a significant difference in the number of calli produced in anther culture between four ‘responsive’ triticale lines and four ‘recalcitrant’ triticale lines. Under the same culture conditions, the number of calli produced reached 60–125 per 100 anthers in the four ‘responsive’ lines, but only 5–12 per 100 anthers in the four ‘recalcitrant’ lines, demonstrating large differences in the CIR between ‘responsive’ and ‘recalcitrant’ lines. Sun et al. [ 29 ] used six genotypes of winter triticale in anther culture, and obtained CIRs ranging from 2.12% (in the winter triticale variety H311) to 14.88% (in the winter triticale variety H11). These findings highlight the importance of genotype in anther culture of triticale. In anther culture of two winter wheat varieties, the high-response wheat genotype ‘Svilena’ produced an average of 179.9 calli per 100 anthers, whereas the low-response wheat genotype ‘Berengar’ produced only 4.8 calli per 100 anthers. In anther cultures of oat [ 31 ], eggplant [ 32 ], and pepper [ 33 ], genotype also significantly affected the efficiency of callus formation. In this study, under the same induction conditions (C3 medium), the CIR varied widely among the five genotypes of triticale (range, 5.00–25.83%). The CIR was highest in T8003 (25.83%) and lowest in T8002 (5.00%) and was significantly different between these two lines. The results are consistent with those of Żur et al. [ 25 ] and Sun [ 29 ], and illustrate the importance of genotype in the success of anther culture. In this study, the CIR of five winter triticale lines ranged from 5.00–25.83%; higher than the rates reported by Sun (2.12–14.88%) and those of the four ‘recalcitrant’ lines reported by Żur et al. (5–12%), but lower than those of the four ‘responsive’ triticale lines reported by Żur et al. (60–125%). Therefore, the next step should be to find high-response genotypes of awnless triticale. As well as affecting the CIR, the genotype of the materials also affects the DFG of triticale. Immonen and Robinson [ 34 ] studied the regeneration ability of 10 genotypes of winter triticale from Canada and Belarus. They found that after 2 weeks of cold treatment, each genotype of triticale produced green plantlets, but there was a significant difference in the DFG (0.0–26.7%) among the 10 genotypes. The DFG was highest in the ‘Paljus’ line (26.7%), followed by ‘LP4496.5.92’ (24.7%), and lowest in the ‘Modul’ line (3.1%). Ślusarkiewicz-Janzina and Ponitka (2003) [ 35 ] used seven triticale genotypes and obtained DFGs ranging from 0.4–7.2%. Among those seven genotypes, ‘19Sz’ showed the highest DFG (7.2%), followed by ‘4Sz’ (4.3%), and ‘16Ch’ had the lowest DFG (0.4%). The results of those studies clearly show that genotype has a significant effect on the DFG. In the present study, when the five genotypes were cultured on the same induction medium (D2), T8004 had the highest DFG (33.3%), followed by T8003 (25.0%), significantly higher than the highest DFG obtained in the study of Ślusarkiewicz-Jarzina and Ponitka [ 35 ]. The DFG of T8004 was also significantly higher than that reported by Immonen and Robinson [ 34 ], and this was related to the triticale genotype. Our results indicate that T8004 and T8003 have strong differentiation abilities and produce high-quality green plantlets. Therefore, both of these lines are suitable materials for further research on anther culture of triticale. 4.2 Effect of hormone concentration ratio on callus induction rate and green plantlet differentiation frequency In anther culture, hormones are important factors for callus induction and callus differentiation, and the type and concentration of exogenous hormones added to the culture medium will have an important impact on the induction and proliferation of calli or embryoids [ 35 ]. Auxins such as 2,4-D, indole acetic acid (IAA), indole-3-butyric acid (IBA), and NAA, and cytokinins such as 6-BA, KT, and ZT (zeatin) are commonly used hormones [ 36 , 37 ]. In triticale anther culture, the auxin 2,4-D plays an important role in inducing callus formation, but its effects are weaker if it is used alone [ 38 ]. Although the addition of only 2,4-D to MS medium induced callus formation in red clover, few calli were produced and their quality was poor, and this was not conducive to the differentiation of buds and roots in the later stages [ 39 ]. Similar findings have been reported for wheat in anther culture [ 40 ]. Therefore, 2,4-D should be used with a cytokinin at an appropriate concentration to induce the formation of abundant high-quality calli. The combination of 2,4-D and KT is often used to induce callus from plant materials. Sun [ 29 ] conducted triticale anther culture with different types and ratios of hormones and obtained the highest CIR (14.88%) on medium containing 2.0 mg/L 2,4-D + 1.0 mg/L KT. Żur et al. [ 25 ] found that C17 medium containing 2.0 mg/L 2,4-D + 0.5 mg/L KT was the most suitable for inducing calli from four triticale lines (‘DH19’, ‘DH72’, ‘DH119’, and ‘DH144’), obtaining CIRs of up to 12.0%. The types and ratios of exogenous hormones have also been shown to affect the CIR in anther cultures of other crops. For example, Barroso et al. [ 41 ] conducted anther culture using eight varieties of capsicum (G3–G10) and obtained CIRs of 1.0–8.6%. The highest CIR of 8.6% (in the G3 genotype), was obtained by culturing the anthers on C medium supplemented with 22.6 µm 2,4-D + 23.25 µm KT. In this study, the anthers of five genotypes of triticale were cultured on C3 induction medium. The T8001, T8004, and T8005 lines had the highest CIRs. The highest CIR (25.83% in T8004) was higher than those reported by Sun [ 29 ], Żur et al. [ 25 ], and Barroso et al [ 41 ]. These results indicate that C3 induction medium is suitable for callus induction from awnless triticale lines. Compared with callus induction, callus differentiation requires different types and concentrations of hormones. The combination of IAA and 6-BA is commonly used to induce triticale calli to differentiate into plantlets. Li [ 42 ] obtained the highest DFG (25.0%) by culturing calli of triticale ‘Gannong No.1’ and ‘Shida No.1’ on MS medium containing 1.0 mg/L IAA + 1.0 mg/L 6-BA. González and Jouve [ 43 ] found that the calli of 10 winter triticale lines cultured on RG1 medium containing 1.0 mg/L IAA + 0.4 mg/L NAA had an average DFG of 7.77%, while the calli cultured on RG3 medium containing on 0.2 mg/L NAA + 0.5 mg/L 6-BA had an average DFG of 6.67%. In the present study, the calli of T8003 and T8004 had the highest DFGs (25.00% and 33.33%, respectively) on D2 medium containing 1.0 mg/L IAA + 1.5 mg/L 6-BA. These rates are higher than those reported by Li [ 42 ], and González and Jouve [ 43 ]. As the concentration of 6-BA in the medium increased, the DFG increased first and then decreased. This may be because 6-BA can promote bud formation, so appropriately increasing the 6-BA concentration was conducive to bud differentiation. Therefore, D2 medium was more conducive to DFG in anther cultures of T8003 and T8004. 4.3 Effect of the concentration of colchicine solution and soaking time on the success of chromosome doubling Colchicine treatment can improve the probability of polyploidy and increase the polyploidy induction rate of regenerated plants, but it can also negatively affect the plantlets, causing wilting and reducing the survival rate [ 44 ]. Colchicine disrupts spindle formation by inhibiting the formation of spindle filaments. This means that the replicated chromosomes cannot be pulled towards the poles and separate, resulting in the doubling of chromosomes [ 45 ]. Different concentrations of colchicine solution and different soaking times significantly affect the success rate of chromosome doubling of regenerated plants. Slusarkiewicz-Jarzina et al. [ 46 ] soaked the roots of 215 triticale haploid regenerated plants in 4% colchicine solution for 6 h (at 25°C in light conditions), and obtained 128 doubled haploid plants, i.e., a chromosome doubling efficiency of 59.9%. In other crops, maize plantlets showed a chromosome doubling rate of 31.83% when their roots were soaked in 0.10% colchicine for 5.0 h, and a lower chromosome doubling rate (27.67%) when the roots were soaked with 0.70% colchicine for 5.0 h [ 47 ]. In this experiment, chromosome doubling of the regenerated plants was induced by soaking the roots in 0.1% colchicine solution for 4.5 h, and the average success rate of chromosome doubling was 55.6%. The highest chromosome doubling success rate was in T8004 (63.6%), higher than those reported by Slusarkiewicz-Jarzina et al. [ 46 ] and Chaikam et al. [ 47 ]. The toxic effects of colchicine can lead to a high mortality rate of haploids, and this can severely reduce the efficiency of the anther culture process [ 45 ]. In our study, the survival rate of green plantlets whose roots were soaked in 0.1% colchicine solution for 5.0 h was 82.7%; but was higher (85.2%) for plantlets whose roots were soaked in 0.1% colchicine solution for 4.5 h. The chromosome doubling efficiency of the five triticale genotypes in this study ranged from 25.0–63.6%, which shows that the genotype also affected the efficiency of chromosome doubling, consistent with the findings of Ragot and Steen [ 47 ]. The concentration of the colchicine solution and the soaking time significantly affected the chromosome doubling efficiency and also affected the survival rate of the regenerated plantlets. 5 Conclusions Five triticale genotypes showed significantly different responses in anther culture in terms of callus induction rate, green plant differentiation rate, and plant survival rate. Among the five tested triticale lines, T8004 showed the best response in anther culture. The highest number of calli was obtained on C3 induction medium, and the largest number of high-quality plantlets was obtained on D2 differentiation medium. The different genotypes of triticale required different hormone concentrations to achieve the highest callus induction rates. Chromosome doubling of the triticale haploid plants was induced by soaking the roots in 0.1% colchicine solution for 4.5 h. The average survival rate of plantlets was 85.2%, and the average success rate of chromosomal doubling of plantlets was 55.6%. Appropriately reducing the colchicine treatment time to induce chromosome doubling improved the survival rate of green plantlets. The 15 regenerated plants all reached the standard of awnless triticale, so they have potential applications as materials for breeding new awnless triticale varieties. Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid KT 6-Furfurylaminopurine DAPI 4',6-Diamidino-2-phenylindole dihydrochloride ABA Abscisic acid IAA Indoleacetic acid 6-BA N6-benzyladenine CIR Callus induction rate DFG Green plantlet differentiation frequency DFA Albino plantlet differentiation frequency PRG Green plantlet production PRA Albino plantlet production PRR Plant regeneration rate DH double haploid Declarations Author contributions WD and JM designed the experiment. JM, FZ and YZ performed the experiment and data analysis. JM wrote the manuscript. XT and WD helped to revise the manuscript. All authors reviewed and approved the manuscript. Funding This study was supported by the National Natural Science Foundation (32260339), Industry Supporting Program (2022CYZC-49) and Key Projects (21ZD4NA012) of Gansu Province, and Major Science and Technology Project of Tibet (XZ202101ZD003N), China, and Major Project of Agri-cultural Biological Breeding (2023ZD0402605-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments: We wish to thank Dr. Nan Xie (Hebei Academy of Agriculture and Forestry Science) and Dr. Xiaohu Lin and Dr. Han Li (Henbei Normal University of Science and Technology) for providing octoploid triticale seeds, and we also appreciate Liwen Bianji (Edanz) and Mr. Jennifer Smith for editing the English text of a draft of this manuscript. Data availability The datasets supporting the conclusions of this article are included within the article. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References McGoverin CM, Snyders F, Muller N, Botes W, Fox G, Manley M. A review of triticale uses and the effect of growth environment on grain quality. J. Sci. Food Agric. 2011;91,1155-65. https://doiorg/101002/jsfa4338. 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Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal Res. Commun. 2003;31,289-296. https://doiorg/101079/97808519965300447. Weigt D, Niemann J, Siatkowski I, Zyprych-Walczak J, Olejnik P, Kurasiak-Popowska D. Effect of zearalenone and hormone regulators on microspore embryogenesis in anther culture of wheat. Plants (Basel). 2019;8,487. https://doiorg/103390/plants8110487. Szechyńska-Hebda M, Skrzypek E, Dąbrowska G, Biesaga-Kościelniak J, Filek M, Wędzony M. The role of oxidative stress induced by growth regulators in the regeneration process of wheat. Acta. Physiol. Plant. 2007;29,327–337. https://doiorg/101007/s11738-007-0042-5. Birsin MA, Ozgen MA. Comparison of callus induction and plant regeneration from different embryo explants of triticale (× Triticosecale Wittmack). Cell Mol. Biol. 2004;9,353-361. https://doiorg/101007/s00018-003-3367-2. Kubes J, Skalicky M, Tumova L, Martin J, Hejnak V, Martinkova J. Vanadium elicitation of Trifolium pratense L. cell culture and possible pathways of produced isoflavones transport across the plasma membrane. Plant Cell. Rep. 2019;38,657-671. https://doiorg/101007/s00299-019-02397-y. Zheng MY, Konzak CF. Effect of 24-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat ( Triticum aestivum L). Plant Cell. Rep. 1999;19,69–73. https://doiorg/101007/s002990050712. Barroso PA, Rêgo MM, Rêgo ER, Soares WS. Embryogenesis in the anthers of different ornamental pepper ( Capsicum annuum L) genotypes. Genet. Mol. Res. 2015;14,13349-63. https://doiorg/104238/2015October2632. Li X. Studies on the anther culture and embryo rescue technology of triticale. Gansu Agricultural University, Lanzhaou, China; 2017. P.25-27. González JM, Jouve N. Improvement of anther culture media for haploid production in triticale. Cereal Res. Commun. 2000;28,65-72. https://doiorg/101007/s100870050020. Głowacka K, Jeżowski S, Kaczmarek Z. In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterisation of induced polyploids in two Miscanthus species. Ind. Crop Prod. 2010;32,88–96. https://doiorg/101016/jindcrop201003009. Broughton S, Castello M, Liu L. The effect of caffeine and trifluralin on chromosome doubling in wheat anther culture. Plants (Basel). 2020;9,105. https://doiorg/103390/plants9010105. Slusarkiewicz-Jarzina A, Ponitka A. Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal. Res. Commun. 2003;31,289–296. https://doiorg/101079/97808519965300447. Chaikam V, Gowda M, Martinez L, Ochieng J, Omar HA, Prasanna BM. Improving the efficiency of colchicine-based chromosomal doubling of maize haploids. Plants (Basel). 2020;9,459. https://doiorg/103390/plants9040459. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Jan, 2025 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 13 Nov, 2024 Reviews received at journal 11 Nov, 2024 Reviews received at journal 06 Nov, 2024 Reviews received at journal 05 Nov, 2024 Reviews received at journal 28 Oct, 2024 Reviews received at journal 27 Oct, 2024 Reviewers agreed at journal 26 Oct, 2024 Reviewers agreed at journal 22 Oct, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers agreed at journal 07 Oct, 2024 Reviewers agreed at journal 06 Oct, 2024 Reviewers invited by journal 04 Oct, 2024 Editor assigned by journal 03 Oct, 2024 Submission checks completed at journal 03 Oct, 2024 First submitted to journal 29 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5176133","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377848647,"identity":"9f7c09c2-06af-482c-9a3e-e0369fa04375","order_by":0,"name":"Jun ma","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"ma","suffix":""},{"id":377848649,"identity":"379af30b-9ffd-478d-b9ff-a319bea191d5","order_by":1,"name":"Fangyuan Zhao","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fangyuan","middleName":"","lastName":"Zhao","suffix":""},{"id":377848651,"identity":"a1e7452a-7284-4cd6-85a4-c0556604a260","order_by":2,"name":"Yinxia Zhang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yinxia","middleName":"","lastName":"Zhang","suffix":""},{"id":377848655,"identity":"b48f4e45-ea3e-4522-a928-1385499627cd","order_by":3,"name":"Xinhui Tian","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinhui","middleName":"","lastName":"Tian","suffix":""},{"id":377848658,"identity":"05eba95e-3e54-48ff-b8cb-6f89098d0f77","order_by":4,"name":"Wenhua Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYDCCAwxsDB+gbAmitTDOIFkLMw9JWvhu5Jg9tvlVJ29wgPngbR4GuzyCWiRv5Jgb5/YdNtxwgC3ZmochuZigFoMbudukc3sOMG44wGMmzcNwILGBKC2WPXX2Gw7wfyNBC8MP5kSgLWzEaZE88/6bZG/D4eSZh9mMLecYJBPWwnc8LU3ix586277jzQ9vvKmwI6wFDBjbgAQz2J1EqQeBP0SrHAWjYBSMgpEIAIusPfVm3qreAAAAAElFTkSuQmCC","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Wenhua","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2024-09-29 17:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5176133/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5176133/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-024-01322-z","type":"published","date":"2025-01-07T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71537835,"identity":"ac0315a6-e736-4557-8009-44121c20e312","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4000345,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the collection of anthers, inoculation of induction medium, induction of callus, differentiation of plantlets, cultivation of plantlets after chromosome doubling, and plant growth in a field.\u003c/p\u003e","description":"","filename":"Figure1Schematicdiagram.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/314e8d84d4f03eb5779350db.png"},{"id":71537837,"identity":"82bbda34-b6f7-47e5-b88e-927ad27821bb","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3339975,"visible":true,"origin":"","legend":"\u003cp\u003eMicrographs of triticale microspores at late uninucleate stage. (a) Microspores of T8003 line at 400× magnification. (b) Microspores of T8003 line at 800× magnification.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/a35b9c424a070d39f788f6d1.png"},{"id":71539232,"identity":"9ed14620-c622-4f45-a726-c7f806ec0567","added_by":"auto","created_at":"2024-12-16 14:25:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77230,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences of the average callus induction rate in triticale lines for different hormone concentrations.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/5b011d87be1b68006ba11523.png"},{"id":71537832,"identity":"5add7e38-2541-4674-a324-2a18e9a8dafa","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77638,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences of the average callus induction rate in hormone concentrations for different triticale line\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/24db6100b7ff43342937f1aa.png"},{"id":71537836,"identity":"d22df295-1a98-4611-8610-879fadd5401c","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107337,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the interaction between triticale line and hormone concentration on callus induction rate\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/fcaf8712f0797fb4d16a9b03.png"},{"id":71537833,"identity":"db681a99-3cd1-4dc0-84bb-382037553eb3","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59174,"visible":true,"origin":"","legend":"\u003cp\u003eResults of ploidy identification of triticale regenerated plantlet\u003c/p\u003e\n\u003cp\u003eNote: (a): ‘Gannong No. 3’ triticale (CK\u003csub\u003e1\u003c/sub\u003e); (b): haploid regenerated plants.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/54fa8fe3d973a0d5989f97d1.png"},{"id":71537841,"identity":"abe21fad-4ad9-4cee-855b-c77240ee0f2c","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70956,"visible":true,"origin":"","legend":"\u003cp\u003eResults of ploidy identification of ‘Jinsong No.49’(CK2) (a) and double haploid plantlet (b)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/64a5d90e5dc2e7dacb7039fa.png"},{"id":71537839,"identity":"495fcd84-b59e-4a39-9c2b-08166056ef89","added_by":"auto","created_at":"2024-12-16 14:17:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":348661,"visible":true,"origin":"","legend":"\u003cp\u003eProduction of doubled haploid triticale plants. a: The microspore mononuclear border stage; b: Collected triticale young spikes; c: Induction medium inoculated with anthers; d: Callus; e: Differentiated green plantlet and albino plantlet; f: Growing green plantlet; g: open the culture bottle and train seedling for 3 days; h: Green plantlets before chromosome doubling; i: Green plantlets after chromosome doubling. j: Green plantlets grew for 2 weeks after chromosome doubling; k: Green plantlets grew in a greenhouse.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/5e3da1e8d9188cc7e2834089.png"},{"id":71539233,"identity":"0a6181ca-1a1b-41b1-8dfc-43ae3e5a688e","added_by":"auto","created_at":"2024-12-16 14:25:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3365065,"visible":true,"origin":"","legend":"\u003cp\u003eSpike of triticale plant Z23-8(a) and Z23-11(b)\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/738729cca819a866974c83aa.png"},{"id":73694184,"identity":"8b3e5b2b-cf94-41b7-a8f1-fc0653eeb050","added_by":"auto","created_at":"2025-01-13 16:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17507465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5176133/v1/76d88521-9d33-457f-836d-959c7990e7d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of hormone concentrations on anther cultures and the acquisition of regenerated plants of five awnless triticale genotypes","fulltext":[{"header":"1 Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs a high-quality annual forage grass, triticale (\u0026times;Triticosecale Wittmack) plays important roles in alleviating the conflict between forage and livestock in pastoral areas, protecting the ecological environment, and promoting the healthy development of grassland agricultural ecosystems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At present, most triticale varieties (lines) have awns \u0026mdash; bristle-like appendages located on the spikelet. Although awns have roles in increasing the photosynthetic area, improving transpiration, and pest resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], they also have some disadvantages. The waxy awn of triticale can puncture the oral cavity of livestock, which can lead to diseases such as pharyngitis, oral ulcers, and submandibular edema [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. When triticale is harvested at the dough stage, the waxy awn can adversely affect the production of high-quality forage products and reduce the palatability of forage [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Awnless triticale does not have these shortcomings. Moreover, many studies have shown that the seed yield of awnless varieties is not lower than that of awned varieties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is of great significance to breed awnless triticale lines to improve the palatability of forage and the quality of forage products.\u003c/p\u003e \u003cp\u003eIn many breeding methods, anther culture is an indispensable rapid breeding technology. It has become a powerful tool for rapid haploid production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Varieties with excellent traits are selected as hybrid parents, and homozygous plants are obtained from F1 plants with improved agronomic traits by anther culture followed by chromosome doubling. This doubled-haploid breeding strategy is more efficient than traditional breeding methods [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Homozygotes of triticale with relatively stable traits can be obtained quickly by anther culture. This can save a lot of breeding time and reduce the labor and financial resources required for breeding [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. At present, the anther culture technique is the most widely used method for haploid breeding of triticale. In recent years, researchers have used anther culture technology to produce haploid and doubled haploid plants of various species, most commonly wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], rice (\u003cem\u003eOryza sativa\u003c/em\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], corn (\u003cem\u003eZea mays\u003c/em\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Few studies have focused on the production of doubled haploid triticale. In the few studies that have used triticale in anther culture, the experimental materials were all awned triticale varieties (lines), rather than awnless ones. In the present study, all the experimental materials were awnless triticale lines. The overall goal was to provide a reference for the optimization of anther culture technology for awnless triticale.\u003c/p\u003e \u003cp\u003eAmong the many factors that affect the efficiency of triticale anther culture, plant genotype is the most important one [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The success rate of anther culture varies greatly among different varieties (genotypes) because of the dependence of anther culture on genotype [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Marciniak et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] reported that the number of green plants induced to form in anther culture differed significantly among 38 winter triticale genotypes (range, 1.0\u0026ndash;16.2/spike) under the same culture conditions. Similarly, there were significant differences in the number of haploid green plants obtained after anther culture of 21 Swedish and Dutch F1 and F2 triticale combinations under the same culture conditions. The average number of haploid green plants ranged from 0.8 to 13.6/spike, and there were significant differences in the number of green plants induced among the lines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The results of those studies indicate that the triticale genotype significantly affects the efficiency and success rate of anther culture. The type, ratio, and concentrations of exogenous hormones also affect the efficiency of anther culture [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and the optimum types, concentrations, and ratios of hormones for anther culture differ among different plants. Kruppa et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] found that P4mf medium was more suitable than W14mf medium for the production of doubled haploid triticale lines, and the addition of 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (KT) to P4mf medium significantly increased the number of calli and green plants (103.7/anther and 19.7/anther, respectively). Żur et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] reported that the addition of 0.5 mg/L naphthaleneacetic acid (NAA) and 0.5 mg/L KT to C17 medium significantly increased the callus induction rate of eight triticale varieties, and \u0026lsquo;DH144\u0026rsquo; triticale had the highest regeneration rate of green plants (5.4 green seedlings differentiated per 100 calli). Hassawi et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] tested the effects of seven auxins and four cytokinins in triticale anther culture, and found that the anthers showed the highest callus differentiation rate (13.9%) on medium supplemented with 2,4-D. \u0026Ouml;zkum and Tipirdamaz [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] found that the combination of 4.0 mg/L NAA and 1.0 mg/L 6-benzyladenine (BA) in the medium improved the callus regeneration rate of pepper embryoids. Therefore, optimization of hormone concentrations in the medium can improve the success rate in anther culture. We hypothesized that modifying the medium hormone concentration may alter the awnless triticale anther callus induction rate and green plantlet differentiation frequency, which can affect the production of haploid plants that are then treated with colchicine for the doubling of chromosomes to obtain doubled haploid (DH) plants, thereby accelerating the breeding process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this study, we analyzed the effects of different types and ratios of hormones on the callus induction rate and anther differentiation rate of five different triticale lines in anther culture. The results provide a theoretical basis for improving anther culture and optimizing the regeneration of awnless triticale.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Site description\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study was conducted at the Forage Experimental Station of Gansu Agricultural University (36\u0026deg;03\u0026prime;N, 103\u0026deg;53\u0026prime;E, 1560m above sea level), Lanzhou, China in 2021. The previous crop was high-yield sweet sorghum (Sorghum bicolor) grown under good irrigation conditions. Basic information on the experimental site is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic information of the experimental site\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean annual temperature (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrostless period (d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean annual rainfall (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSoil organic matter (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRapidly available nitrogen (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRapidly available phosphorus (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRapidly available potassium (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSoil type\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForage test station\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e349.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e90.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e172.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eloessal soil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe five materials used in this experiment were the new hybrid lines of awnless triticale selected by pedigree method in Gansu Agricultural University, and the line number, name and parents were shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. After a 30-day vernalization, the seeds of 5 awnless triticale lines were planted in the plastic greenhouse experimental base of Gansu Agricultural University (36\u0026deg;03\u0026prime;N, 103\u0026deg;53\u0026prime;E; 1,560 m above sea level), China, in March 2023. A line seeding method was adopted, with row spacing of 20 cm, plant spacing of 5 cm, and a sowing depth of 2\u0026thinsp;~\u0026thinsp;3 cm. 300 kg\u0026middot;hm-2 of diammonium phosphate was applied before sowing, and 196 kg\u0026middot;hm-2 of urea were applied at the regreening stage and jointing stage, respectively. During the experiment, weeding was carried out, and irrigation was given once at the seedling stage, tillering stage and jointing stage. Sampling began in late May and early June.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber, name and parents of five awnless triticale materials\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParents (♀\u0026times;♂)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2021-8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOH2276 (2013) \u0026times; J9 (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2021-8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81S37 (2013) \u0026times; J35 (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2021-8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4009 (2013) \u0026times; P2 (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2021-8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT17 (2013) \u0026times; J18 (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2021-8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89D-8 (2013) \u0026times; J10 (2013)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sampling and pretreatment of young spikes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe young spikes of awnless triticale with pollen microspores in the middle and late stages of mononuclear were taken (i.e., spikes of awnless triticale in the field just reached the middle of the flag leaf and the second leaf from the top). The young spikes were cut from about 10 cm below the second leaf from the top, and wrapped into bundles with aluminum foil, and the date and line name were marked. Two anthers from the collected per young spikes were crushed on a glass slide, after which 1\u0026thinsp;~\u0026thinsp;2 drops of acetic acid magenta dye (Solarbio Inc., Beijing, China) were added and the stained material was examined using the Panthera U microscope (eyepiece 10\u0026times; and objective 40\u0026times;; Motic China Group Co., Ltd., Hong Kong, China) to determine whether mononuclear stage had been reached. The incision of the young spikes which was up to the standards by microscopic examination was inserted into a 500 mL beaker filled with 250 mL of tap water, and pretreated at 4\u0026deg;C for 15 days in the dark.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Spike disinfection and anther inoculation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter 15 days of low temperature pretreatment, the flag leaf and the second leaf from the top were cut off. In the biological safety cabinet, disinfected the surface of the test material with 75% alcohol for 1 min, peeled off the young spikes, disinfected with 2% sodium hypochlorite solution for 7 min, rinsed with sterile water for 4\u0026thinsp;~\u0026thinsp;5 times, and then wiped the surface water of the young spikes with sterile filter paper. Anthers were removed from the sterilized spikes and placed on each of the 4 different CHB induction medium (denoted as C1, C2, C3, C4, respectively). Each dish is inoculated with 40 anthers, and each treatment inoculates 6\u0026thinsp;~\u0026thinsp;8 Petri dishes (90 mm diameter) (Jingan Inc., Shanghai, China).\u003c/p\u003e \u003cp\u003eBased on the hormone content of the optimal CHB induction medium screened by our team in the previous study, the hormone content was further adjusted and refined, and 4 kinds of induction medium were designed, each of which was supplemented with 90 g/L sucrose and 7 g/L agar, respectively, with a pH value of 5.4. The hormone content of 4 induction medium was shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHormone concentration of induction medium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHormone concentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;1.0 mg/L KT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;1.5 mg/L KT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;1.5 mg/L KT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;1.5 mg/L KT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Induction culture of anther\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter the inoculation was completed, the petri dish was placed in a incubator (HGZ-250, Yuejin Inc., Shanghai, China) for a 3-day incubation at 32\u0026deg;C in darkness, and then transferred to another incubator for a 60-day incubation at 28\u0026deg;C in darkness. After 8 weeks, the number of callus was counted.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Differentiation culture of callus\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on the hormone content of the optimal MS differentiation medium screened by our team in the previous study, 3 differentiation medium were designed by adjusting different hormone contents, and each differentiation medium was supplemented with 10 g/L sorbitol, 30 g/L sucrose and 7 g/L agar, respectively, and the pH value was 5.8. The hormone content of the 3 differentiation medium was shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAwnless triticale lines (T8003, T8004) which produced more callus were selected, and the callus with a diameter of more than 1 mm were transferred to 4 kinds of MS differentiation medium, and 4 calli were placed in each dish, and the petri dishes were placed in an incubator set at 27\u0026deg;C with a 14-h light (3,000 lx): 10-h dark cycle, and the medium was refreshed every 15 days. The number of green plantlets and albino plantlets was counted after 12 weeks. Since T8001, T8002, and T8005 lines induced fewer callus, callus from these three lines were inoculated only on D1 differentiation medium.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHormone concentration of differentiation medium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHormone concentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;1.0 mg/L 6-BA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;1.5 mg/L 6-BA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;2.0 mg/L 6-BA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plantlets training\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhen the regenerated green plantlets grew to 4 leaves, the parafilm of the culture bottle was removed, and an appropriate amount of tap water was added, and the culture bottle was remained in the incubator set at 27\u0026deg;C with a 14-h light (3,000 lx): 10-h dark cycle for 3 days. After that, the green plantlets were transplanted into plastic pots (10cm \u0026times; 10cm) (Jiesheng Co., Shenzhen, Guangdong, China) containing nutrient soil (Luneng Co., Tianzhu, Gansu, China). These plastic pots was placed in an artificial climate chamber set at 25\u0026deg;C with a 14-h light : 10-h dark cycle.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Ploidy Level Analysis of the regeneration green plantlets\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe ploidy levels of regenerated plants was determined by the CyFlow Ploidy Analyzer (CyFlow Cube 6; Sysmex Co., Hamburg, Germany). Briefly, one leaf were collected from each plant and then placed in 400 \u0026micro;L extraction buffer (CyStain UV Precise P Kit; Sysmex Co., Hamburg, Germany). The leaf was minced for 1 min using a sharp blade and passed through a 30 \u0026micro;m filter to remove cell debris. Next, 1,600 \u0026micro;L DAPI staining solution was added to stain the nuclei prior to the analysis using the CyFlow Ploidy Analyzer (Sysmex Co., Hamburg, Germany). More than 3,000 nuclei were detected per sample. Hexaploid triticale variety \u0026lsquo;Gannong No.3\u0026rsquo; (provided by Gansu Agricultural University, Lanzhou, Gansu, China) was used as a control (CK1) to determine whether the regenerated plants were haploid plants.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Chromosome Doubling, plantlets Training, and Transplanting\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhen the green plantlets had three tillers (or were approximately 12 cm tall), they were removed from the plastic pot, rinsed, and then their roots were soaked in a solution containing 0.1% colchicine, 2% dimethyl sulfoxide, and 0.05% Tween-20 for 4.5 h chromosome doubling step. The roots were rinsed by tap water for 2 h and then the green plantlets were replanted in plastic pots and incubated in the artificial climate chamber for 3\u0026thinsp;~\u0026thinsp;5 weeks to resume growth. They were subsequently transferred to a field.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Ploidy Level Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlow cytometry was used to analyze the ploidy of plantlets that grew normally in the field (before the jointing stage). The analysis method is the same as 1.8. The octaploid tritcale variety \u0026lsquo;Jinsong 49\u0026rsquo; (provided by the Hebei Academy of Agriculture and Forestry Science, Shijiazhuang, Hebei, China) was used as a control (CK2) to determine whether the regenerated seedlings were double haploid plants.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Agronomic Trait Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe following traits of the regenerated plants were examined at the flowering stage: plant height and number of branch per plant. The following spike parameters were examined at the dough stage: tip awn length, side awn length, spike length, number of spikelets, number of grains per spike, and grain weight per spike. T2023-8003 was used as the control group (CK3).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach petri dish with inoculated anthers was taken as a replicate, and 6\u0026thinsp;~\u0026thinsp;8 replicates are treated with each treatment. In callus induction trials, there are at least 3 replicates per treatment. The analysis of variance was performed by SPSS 20.0 software. If significant differences were detected, Duncan\u0026rsquo;s multiple comparison test was performed to compare the differences.\u003c/p\u003e \u003cp\u003eCIR (%)\u0026thinsp;=\u0026thinsp;Number of calli/Number of anthers used for the inoculation \u0026times; 100\u003c/p\u003e \u003cp\u003eDFG (%)\u0026thinsp;=\u0026thinsp;Number of green plantlets/Number of calli \u0026times; 100\u003c/p\u003e \u003cp\u003eDFA (%)\u0026thinsp;=\u0026thinsp;Number of albino plantlets/Number of calli \u0026times; 100\u003c/p\u003e \u003cp\u003ePRG (%)\u0026thinsp;=\u0026thinsp;Number of green plantlets/Number of anthers used for the inoculation \u0026times; 100\u003c/p\u003e \u003cp\u003ePRA (%)\u0026thinsp;=\u0026thinsp;Number of albino plantlets/Number of anthers used for the inoculation \u0026times; 100\u003c/p\u003e \u003cp\u003ePRR (%)\u0026thinsp;=\u0026thinsp;Total numbers of plantlets/Number of anthers used for the inoculation \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Microscopic Examination of Anthers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe young spikes of five triticale lines were observed under a light microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In all the lines, most of the microspores were in the late uninucleate stage, which is the optimum stage for anther culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Analysis of Variance\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed that the significant or extremely significant differences were detected for triticale line, except for the DFG, PRG and PRA. The significant or extremely significant differences were detected for hormone concentration of induction / differentiation medium, except for the DFA, PRA and PRR, and the significant or extremely significant differences were detected for the interactive effects of triticale line and hormone concentration of induction / differentiation medium, except for the PRA. The multiple comparison test was completed for the above parameters with significant or extremely significant differences (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVariance analysis of induction and differentiation indicators of awnless triticale anther culture\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eF Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCIR (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDFG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDFA (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePRA (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePRR (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.13**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.88*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.49*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormone concentration of induction / differentiation medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.48**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.43*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line \u0026times; hormone concentration of induction / differentiation medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.52**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.27**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.15*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.71*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: CIR: Callus induction rate. DFG: Green plantlet differentiation frequency; DFA: Albino plantlet differentiation frequency. PRG: Green plantlet production; PRA: Albino plantlet production; PRR: Plant regeneration rate; * indicates significant differences at 0.05 level; ** indicates extremely significant differences at 0.01 level, the same as below.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of triticale genotype and hormone concentrations in the medium on callus induction\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Differences in callus induction rate among five triticale lines\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThere were significant differences in the average callus induction rate (CIR) among the various triticale lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The average CIR was highest in T8004 (16.67%), followed by T8003 (9.79%), and these rates were significantly higher than those of T8001 and T8002 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The average CIR was lowest in T8001 at 6.25%, which was significantly lower than those of T8003 and T8004 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings show that the average CIR differed significantly among the five triticale lines.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Differences in CIR among media with different hormone concentrations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs the 2,4-D concentration in the medium increased, the average CIR of different triticale lines first increased and then decreased. The average CIR was highest on C3 medium (14.17%), followed by C4 medium (12.83%), significantly higher than that on C1 medium, but not significantly different from that on C2 medium. The average CIR was lowest on C1 medium (7.50%), which was significantly lower than those on C3 and C4 media. Thus, the highest average CIR was achieved on C3 medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Significance of genotype \u0026times; hormone concentration interaction for CIR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe interaction between genotype and hormone concentration was significant for the CIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For the genotypes T8001, T8004, and T8005, the highest CIRs were achieved on C3 medium (7.50%, 25.83%, and 13.33%, respectively), and these rates were significantly higher than those on other media. The CIR of T8002 was significantly higher on C2 medium (13.33%) than on other media, and the CIR of T8003 was significantly higher on C4 medium (21.67%) than on other media. These results indicate that the different triticale lines required different hormone concentrations to achieve the highest CIRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn terms of hormone concentrations, the highest CIR on C1 medium was for line T8004; the highest CIR on C2 medium was for line T8002; and both of those CIRs were higher or significantly higher than those of the other genotypes on those media. The highest CIRs on C3 and C4 media were for lines T8004 and T8003, respectively, and their CIRs were higher or significantly higher than those of other genotypes on those media. These results indicate that different hormone concentrations were required for optimum callus differentiation of the five triticale lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Differentiation of triticale calli obtained in anther culture\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Differences in differentiation frequency, plantlet production, and regeneration rate among triticale lines\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe number of calli produced in anther culture differed markedly among the different triticale lines. Large numbers of calli were produced by the T8003 and T8004 lines, whereas fewer were produced by the T8001, T8002 and T8005 lines. To compare the effects of different hormone concentrations on callus differentiation, the calli produced by the T8003 and T8004 lines were cultured on D1, D2 and D3 differentiation media, and the calli produced by T8001, T8002, and T8005 were cultured on D1 differentiation medium. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, there were significant differences in the green plantlet differentiation frequency (DFG) and plant regeneration rate (PRR) of the triticale lines among the three media containing different hormone concentrations. The highest average DFG was in T8005 (20.83%), and this was significantly higher than that of T8002 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not significantly different from those of T8001, T8003, and T8005. The highest average PRR was in T8001 (2.92%), significantly higher than that of T8002 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not significantly different from those of T8003, T8004 and T8005 (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferences of the average DFG and PRR in triticale lines for different hormone concentrations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDFG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRR (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNotes: DFG: green plantlet differentiation frequency; PRR: plant regeneration rate.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Differences in differentiation frequency, plantlet production among three different differentiation media\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, there were significant differences in the average DFG and PRG of the triticale lines among the three differentiation media with different hormone concentrations. The highest average DFG and PRG (29.17% and 2.43%, respectively) were achieved on D2 medium, and were significantly higher than their respective values on D1 and D3 media. These results show that the highest DFG and PRG were achieved on D2 medium, so this medium was most conducive to obtaining green plantlets from calli produced in anther culture.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferences of the average DFG and PRG in hormone concentrations for different triticale line\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormone concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDFG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRG (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNotes: DFG: Green plantlet differentiation frequency; PRG: Green plantlet production.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003e3.4.3 Effects of the genotype \u0026times; hormone interaction in terms of differentiation frequency, plantlet production, and plant regeneration rate\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effect of the genotype \u0026times; hormone concentration interaction was significant for DFG and DFA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The calli of T8003 and T8004 were cultured on D1, D2, and D3 media, and as the 6-BA concentration in the medium increased, the DFG of T8003 and T8004 showed a trend of first increasing and then decreasing. The DFG of T8003 and T8004 were highest on D2 medium (25.00% and 33.33%, respectively), and these values were higher or significantly higher than those on D1 and D3 media. The DFA of T8003 and T8004 were lowest on D2 medium (0.00% and 4.17%, respectively), and these values were lower or significantly lower than those of the same genotypes on D1 and D3 media. These results show that D2 medium was the most conducive to obtaining green plantlets of the triticale lines T8003 and T8004 (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen the calli of the five triticale lines were cultured on D1 differentiation medium, there were significant differences in DFG and DFA among the five lines (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The DFG of T8005 was the highest (16.67%), which was significantly higher than that of T8002, but not significantly different the other triticale lines. The T8001 genotypes had the second-highest DFG (15.28%). The calli of T8002 did not differentiate into green plants so its DFG was 0.00%. The T8005 genotype had the lowest DFA (0.00%), followed by T8003 (4.17%), whereas T8004 had the highest DFA (20.83%), which was significantly higher than those of the other genotypes. In summary, there were significant differences in the differentiation rate of calli among different genotypes of triticale on the same medium, and this was related to genotype (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of the interaction of triticale line and hormone concentration on DFG and DFA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDFG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDFA (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: HC: hormone concentration; NC: Number of callus; NGP: Number of green plantlet; NAP: Number of albino plantlet.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the PRG and PRR of triticale plants differed significantly among the three media with different hormone concentrations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When the calli of T8003 and T8004 were cultured on D1, D2, and D3 differentiation media, the highest PRG for the calli of both T8003 and T8004 was on D2 medium (2.08% and 2.78%, respectively). The PRG of T8003 and T8004 calli on D2 medium was higher or significantly higher than those on D1 and D3 media, and the PRA of T8003 and T8004 calli was lower on D2 medium than on D1 and D3 media. These results show that D2 differentiation medium was more suitable than the D1 and D3 media for callus differentiation of the T8003 and T8004 lines (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen all five triticale lines were cultured on D1 differentiation medium, the PRG, PRA, and PRR differed significantly among the five lines. T8001 had the highest PRG (2.08%), followed by T8005. The T8002 genotype had the lowest PRG (0.00%). T8005 had the lowest PRA (0.00%), which was lower or significantly lower than that of other genotype, while T8004 had the highest PRA (1.53%), which was higher or significantly higher than that of other genotype. In terms of PRR, T8001 had the highest PRR (2.92%), followed by T8004 (2.36%), whereas T8002 had the lowest PRR (0.83%), which was significantly lower than those of the other genotypes (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These results indicate that there were significant differences in the PRG and PRA among different triticale lines (genotypes) under the same culture conditions (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of the interaction of triticale line and hormone concentration on plantlet production and plant regeneration rate of triticale\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePRG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePRA (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePRR (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42bcd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72cd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42abcd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42abcd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14abc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37abcd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: NC: Number of callus.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Identification of the ploidy level of regenerated plants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDuring the process of anther culture, pollen microspores are induced to form callus, which then differentiates into green plants. The cells of the anther wall, anther septum, and anther filament may also dedifferentiate to form callus, which can also differentiate into green plants. The green plants induced from pollen microspores are haploid (triploid in this experiment), and the green plants induced from the cells of the anther wall, anther septum, and filament are diploid (hexaploid in this experiment). Therefore, we analyzed the ploidy levels of the green plants obtained in anther culture by flow cytometry to distinguish between the haploids and diploids.\u003c/p\u003e \u003cp\u003eIn this experiment, the control \u0026lsquo;Gannon No.3\u0026rsquo; triticale (CK1) was hexaploid (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42), and T8001, T8002, T8003, T8004, and T8005 were also hexaploids (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the position of the fluorescence peak in the control \u0026lsquo;Gannon No.3\u0026rsquo; triticale (CK1) was at 21000\u0026ndash;27000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Therefore, when analyzing the green plants obtained in anther culture, a fluorescence peak at half of this value indicated that the plant was haploid. The fluorescence peak positions of green plants induced from the T8001, T8002, T8003, T8004, and T8005 lines were at 9000\u0026ndash;15000, approximately half of that in the control group, indicating that the induced green plants were haploids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003e(a): \u0026lsquo;Gannong No. 3\u0026rsquo; triticale (CK\u003csub\u003e1\u003c/sub\u003e); (b): haploid regenerated plants.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Chromosome doubling of haploid plants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eChromosome doubling was induced in the identified haploid plants using colchicine solution. The plant roots were soaked in 0.1% colchicine solution for 4.5 h to induce chromosome doubling, then rinsed with tap water for 2 h and replanted in plastic pots. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a total of 23 plants survived after root soaking, with survival rates ranging from 75.0\u0026ndash;100% among the five genotypes. The average survival rate across the five lines was 85.2%. The survival rate of T8005 was 100%, whereas 1, 1, and 2 plants of T8001, T8003, and T8004, respectively, did not survive. The survival rates of the T8001, T8003, and T8004 lines were 75.0%, 90.0%, and 81.8%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe number of survival green plantlet after chromosome doubling and Survival rate after chromosome doubling\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTNGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNHGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNSGPCD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSR/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e81.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: TNGP: The total number of green plantlet induced; NHGP: The number of haploid green plantlet; NSGPCD: The number of survival green plantlet after chromosome doubling; SR: Survival rate.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Identification of doubled haploid regenerated plants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe ploidy level of regenerated plants that grew normally to the booting stage was identified by flow cytometry. CK2 was the octaploid \u0026lsquo;Jinsong 49\u0026rsquo; triticale (2n\u0026thinsp;=\u0026thinsp;8x\u0026thinsp;=\u0026thinsp;56) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), with a fluorescence peak at 21000\u0026ndash;27000. The regenerated plants with fluorescence peaks at three-quarters of that of CK2 were identified as haploid. All of the measured regenerated plants had fluorescence peaks at 16000\u0026ndash;21000 (i.e., three-quarters of CK2), so they were identified as doubled haploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOf the 23 plants transplanted into the field, 20 survived. Of those, 15 plants were doubled haploid plants and five were mixoploid. The chromosome doubling success rate ranged from 25.0\u0026ndash;63.6% among the five lines, and the average chromosome doubling success rate was 55.6%. T8004 had the highest chromosomal doubling success rate (63.6%) and T8001 had the lowest (25.0%). This difference may be related to genotype. The above results show that the chromosomal doubling success rate differed among the five genotypes of triticale (Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePloidy changes and success rate of chromosome doubling in the number of regenerated plantlet after chromosome doubling\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNHGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNGPT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNSGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDouble haploid plant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMOPP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSRCD/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e63.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e55.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: NHGP: The number of haploid green plantlet; NGPT: The number of green plantlet transplanted in field; NSGP: The number of survival green plantlet; MOPP: Mixoploidy and other ploidy plants; SRCD: Success rate of chromosome doubling.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Investigation of agronomic traits in the field of DH0 generation plants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe calli that formed in anther culture differentiated to form haploid green plants, which were transplanted into the field after chromosome doubling. The agronomic traits of the doubled haploid regenerated plants that grew to the flowering stage in the field were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). To reduce the influence of tissue culture on plants, the traits of the DH1 generation population were measured in this experiment. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, the plant height of the 11 triticale plants was 126.8\u0026ndash;153.7 cm, and plant height of 4 plants was significantly higher than that of CK3 (T2021-8003). The tip awn and side awn of the 11 regenerated triticale plants were extremely short, both with lengths shorter than 5.0 mm, which reached the standard of awnless triticale. Thus, these 11 plants were identified as potential materials for further breeding of awnless varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In terms of the panicle traits, spikelet number of 4 plants was higher than that of CK3 and number of grains per spike of 2 plants was higher than that of CK3, while all the plants had grain weight per spike that were lower than or significantly lower than those of CK3. These results indicate that, DH1 generation plants were lower than those of mature lines in terms of spike traits, especially grain weight per spike.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab12\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 12\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe agronomic traits of the 11 DH1 generation double haploid plants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlant height/cm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTip awn/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSide awn/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpike length/cm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNumber of spikelet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber of grains per spike\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGrain weight per spike/g\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e142.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e126.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e43.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07fg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e134.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10cde\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e135.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e144.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05bcd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e133.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04efg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e146.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06efg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e137.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ23-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e144.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06def\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effect of genotype on callus induction rate and green plantlet differentiation frequency\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe efficiency of anther culture is affected by various factors, among which the genotype of the plant is the most important [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Even when the culture conditions are exactly the same, the efficiency of anther culture differs among different plant genotypes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. High-response and low-response genotypes show quite different responses in anther culture [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For example, Żur et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] detected a significant difference in the number of calli produced in anther culture between four \u0026lsquo;responsive\u0026rsquo; triticale lines and four \u0026lsquo;recalcitrant\u0026rsquo; triticale lines. Under the same culture conditions, the number of calli produced reached 60\u0026ndash;125 per 100 anthers in the four \u0026lsquo;responsive\u0026rsquo; lines, but only 5\u0026ndash;12 per 100 anthers in the four \u0026lsquo;recalcitrant\u0026rsquo; lines, demonstrating large differences in the CIR between \u0026lsquo;responsive\u0026rsquo; and \u0026lsquo;recalcitrant\u0026rsquo; lines. Sun et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] used six genotypes of winter triticale in anther culture, and obtained CIRs ranging from 2.12% (in the winter triticale variety H311) to 14.88% (in the winter triticale variety H11). These findings highlight the importance of genotype in anther culture of triticale. In anther culture of two winter wheat varieties, the high-response wheat genotype \u0026lsquo;Svilena\u0026rsquo; produced an average of 179.9 calli per 100 anthers, whereas the low-response wheat genotype \u0026lsquo;Berengar\u0026rsquo; produced only 4.8 calli per 100 anthers. In anther cultures of oat [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], eggplant [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and pepper [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], genotype also significantly affected the efficiency of callus formation. In this study, under the same induction conditions (C3 medium), the CIR varied widely among the five genotypes of triticale (range, 5.00\u0026ndash;25.83%). The CIR was highest in T8003 (25.83%) and lowest in T8002 (5.00%) and was significantly different between these two lines. The results are consistent with those of Żur et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Sun [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and illustrate the importance of genotype in the success of anther culture. In this study, the CIR of five winter triticale lines ranged from 5.00\u0026ndash;25.83%; higher than the rates reported by Sun (2.12\u0026ndash;14.88%) and those of the four \u0026lsquo;recalcitrant\u0026rsquo; lines reported by Żur et al. (5\u0026ndash;12%), but lower than those of the four \u0026lsquo;responsive\u0026rsquo; triticale lines reported by Żur et al. (60\u0026ndash;125%). Therefore, the next step should be to find high-response genotypes of awnless triticale.\u003c/p\u003e \u003cp\u003eAs well as affecting the CIR, the genotype of the materials also affects the DFG of triticale. Immonen and Robinson [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] studied the regeneration ability of 10 genotypes of winter triticale from Canada and Belarus. They found that after 2 weeks of cold treatment, each genotype of triticale produced green plantlets, but there was a significant difference in the DFG (0.0\u0026ndash;26.7%) among the 10 genotypes. The DFG was highest in the \u0026lsquo;Paljus\u0026rsquo; line (26.7%), followed by \u0026lsquo;LP4496.5.92\u0026rsquo; (24.7%), and lowest in the \u0026lsquo;Modul\u0026rsquo; line (3.1%). Ślusarkiewicz-Janzina and Ponitka (2003) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] used seven triticale genotypes and obtained DFGs ranging from 0.4\u0026ndash;7.2%. Among those seven genotypes, \u0026lsquo;19Sz\u0026rsquo; showed the highest DFG (7.2%), followed by \u0026lsquo;4Sz\u0026rsquo; (4.3%), and \u0026lsquo;16Ch\u0026rsquo; had the lowest DFG (0.4%). The results of those studies clearly show that genotype has a significant effect on the DFG. In the present study, when the five genotypes were cultured on the same induction medium (D2), T8004 had the highest DFG (33.3%), followed by T8003 (25.0%), significantly higher than the highest DFG obtained in the study of Ślusarkiewicz-Jarzina and Ponitka [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The DFG of T8004 was also significantly higher than that reported by Immonen and Robinson [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and this was related to the triticale genotype. Our results indicate that T8004 and T8003 have strong differentiation abilities and produce high-quality green plantlets. Therefore, both of these lines are suitable materials for further research on anther culture of triticale.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effect of hormone concentration ratio on callus induction rate and green plantlet differentiation frequency\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn anther culture, hormones are important factors for callus induction and callus differentiation, and the type and concentration of exogenous hormones added to the culture medium will have an important impact on the induction and proliferation of calli or embryoids [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Auxins such as 2,4-D, indole acetic acid (IAA), indole-3-butyric acid (IBA), and NAA, and cytokinins such as 6-BA, KT, and ZT (zeatin) are commonly used hormones [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In triticale anther culture, the auxin 2,4-D plays an important role in inducing callus formation, but its effects are weaker if it is used alone [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although the addition of only 2,4-D to MS medium induced callus formation in red clover, few calli were produced and their quality was poor, and this was not conducive to the differentiation of buds and roots in the later stages [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similar findings have been reported for wheat in anther culture [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, 2,4-D should be used with a cytokinin at an appropriate concentration to induce the formation of abundant high-quality calli.\u003c/p\u003e \u003cp\u003eThe combination of 2,4-D and KT is often used to induce callus from plant materials. Sun [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] conducted triticale anther culture with different types and ratios of hormones and obtained the highest CIR (14.88%) on medium containing 2.0 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;1.0 mg/L KT. Żur et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] found that C17 medium containing 2.0 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;0.5 mg/L KT was the most suitable for inducing calli from four triticale lines (\u0026lsquo;DH19\u0026rsquo;, \u0026lsquo;DH72\u0026rsquo;, \u0026lsquo;DH119\u0026rsquo;, and \u0026lsquo;DH144\u0026rsquo;), obtaining CIRs of up to 12.0%. The types and ratios of exogenous hormones have also been shown to affect the CIR in anther cultures of other crops. For example, Barroso et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] conducted anther culture using eight varieties of capsicum (G3\u0026ndash;G10) and obtained CIRs of 1.0\u0026ndash;8.6%. The highest CIR of 8.6% (in the G3 genotype), was obtained by culturing the anthers on C medium supplemented with 22.6 \u0026micro;m 2,4-D\u0026thinsp;+\u0026thinsp;23.25 \u0026micro;m KT. In this study, the anthers of five genotypes of triticale were cultured on C3 induction medium. The T8001, T8004, and T8005 lines had the highest CIRs. The highest CIR (25.83% in T8004) was higher than those reported by Sun [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Żur et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and Barroso et al [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These results indicate that C3 induction medium is suitable for callus induction from awnless triticale lines.\u003c/p\u003e \u003cp\u003eCompared with callus induction, callus differentiation requires different types and concentrations of hormones. The combination of IAA and 6-BA is commonly used to induce triticale calli to differentiate into plantlets. Li [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] obtained the highest DFG (25.0%) by culturing calli of triticale \u0026lsquo;Gannong No.1\u0026rsquo; and \u0026lsquo;Shida No.1\u0026rsquo; on MS medium containing 1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;1.0 mg/L 6-BA. Gonz\u0026aacute;lez and Jouve [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] found that the calli of 10 winter triticale lines cultured on RG1 medium containing 1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;0.4 mg/L NAA had an average DFG of 7.77%, while the calli cultured on RG3 medium containing on 0.2 mg/L NAA\u0026thinsp;+\u0026thinsp;0.5 mg/L 6-BA had an average DFG of 6.67%. In the present study, the calli of T8003 and T8004 had the highest DFGs (25.00% and 33.33%, respectively) on D2 medium containing 1.0 mg/L IAA\u0026thinsp;+\u0026thinsp;1.5 mg/L 6-BA. These rates are higher than those reported by Li [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and Gonz\u0026aacute;lez and Jouve [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As the concentration of 6-BA in the medium increased, the DFG increased first and then decreased. This may be because 6-BA can promote bud formation, so appropriately increasing the 6-BA concentration was conducive to bud differentiation. Therefore, D2 medium was more conducive to DFG in anther cultures of T8003 and T8004.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.3 Effect of the concentration of colchicine solution and soaking time on the success of chromosome doubling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eColchicine treatment can improve the probability of polyploidy and increase the polyploidy induction rate of regenerated plants, but it can also negatively affect the plantlets, causing wilting and reducing the survival rate [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Colchicine disrupts spindle formation by inhibiting the formation of spindle filaments. This means that the replicated chromosomes cannot be pulled towards the poles and separate, resulting in the doubling of chromosomes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Different concentrations of colchicine solution and different soaking times significantly affect the success rate of chromosome doubling of regenerated plants. Slusarkiewicz-Jarzina et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] soaked the roots of 215 triticale haploid regenerated plants in 4% colchicine solution for 6 h (at 25\u0026deg;C in light conditions), and obtained 128 doubled haploid plants, i.e., a chromosome doubling efficiency of 59.9%. In other crops, maize plantlets showed a chromosome doubling rate of 31.83% when their roots were soaked in 0.10% colchicine for 5.0 h, and a lower chromosome doubling rate (27.67%) when the roots were soaked with 0.70% colchicine for 5.0 h [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In this experiment, chromosome doubling of the regenerated plants was induced by soaking the roots in 0.1% colchicine solution for 4.5 h, and the average success rate of chromosome doubling was 55.6%. The highest chromosome doubling success rate was in T8004 (63.6%), higher than those reported by Slusarkiewicz-Jarzina et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and Chaikam et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The toxic effects of colchicine can lead to a high mortality rate of haploids, and this can severely reduce the efficiency of the anther culture process [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In our study, the survival rate of green plantlets whose roots were soaked in 0.1% colchicine solution for 5.0 h was 82.7%; but was higher (85.2%) for plantlets whose roots were soaked in 0.1% colchicine solution for 4.5 h. The chromosome doubling efficiency of the five triticale genotypes in this study ranged from 25.0\u0026ndash;63.6%, which shows that the genotype also affected the efficiency of chromosome doubling, consistent with the findings of Ragot and Steen [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The concentration of the colchicine solution and the soaking time significantly affected the chromosome doubling efficiency and also affected the survival rate of the regenerated plantlets.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFive triticale genotypes showed significantly different responses in anther culture in terms of callus induction rate, green plant differentiation rate, and plant survival rate. Among the five tested triticale lines, T8004 showed the best response in anther culture. The highest number of calli was obtained on C3 induction medium, and the largest number of high-quality plantlets was obtained on D2 differentiation medium. The different genotypes of triticale required different hormone concentrations to achieve the highest callus induction rates. Chromosome doubling of the triticale haploid plants was induced by soaking the roots in 0.1% colchicine solution for 4.5 h. The average survival rate of plantlets was 85.2%, and the average success rate of chromosomal doubling of plantlets was 55.6%. Appropriately reducing the colchicine treatment time to induce chromosome doubling improved the survival rate of green plantlets. The 15 regenerated plants all reached the standard of awnless triticale, so they have potential applications as materials for breeding new awnless triticale varieties.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2,4-D \u0026nbsp; \u0026nbsp; 2,4-Dichlorophenoxyacetic acid\u003c/p\u003e\n\u003cp\u003eKT \u0026nbsp; \u0026nbsp; \u0026nbsp; 6-Furfurylaminopurine\u003c/p\u003e\n\u003cp\u003eDAPI \u0026nbsp; \u0026nbsp;4\u0026apos;,6-Diamidino-2-phenylindole dihydrochloride\u003c/p\u003e\n\u003cp\u003eABA \u0026nbsp; \u0026nbsp;Abscisic acid\u003c/p\u003e\n\u003cp\u003eIAA \u0026nbsp; \u0026nbsp; \u0026nbsp;Indoleacetic acid\u003c/p\u003e\n\u003cp\u003e6-BA \u0026nbsp; \u0026nbsp; N6-benzyladenine\u003c/p\u003e\n\u003cp\u003eCIR \u0026nbsp; \u0026nbsp;Callus induction rate\u003c/p\u003e\n\u003cp\u003eDFG \u0026nbsp; \u0026nbsp;Green plantlet differentiation frequency\u003c/p\u003e\n\u003cp\u003eDFA \u0026nbsp; \u0026nbsp;Albino plantlet differentiation frequency\u003c/p\u003e\n\u003cp\u003ePRG \u0026nbsp; \u0026nbsp;Green plantlet production\u003c/p\u003e\n\u003cp\u003ePRA \u0026nbsp; \u0026nbsp;Albino plantlet production\u003c/p\u003e\n\u003cp\u003ePRR \u0026nbsp; \u0026nbsp;Plant regeneration rate\u003c/p\u003e\n\u003cp\u003eDH \u0026nbsp; \u0026nbsp; \u0026nbsp;double haploid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWD and JM designed the experiment. JM, FZ and YZ performed the experiment and data analysis. JM wrote the manuscript. XT and WD helped to revise the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation (32260339), Industry Supporting Program (2022CYZC-49) and Key Projects (21ZD4NA012) of Gansu Province, and Major Science and Technology Project of Tibet (XZ202101ZD003N), China, and Major Project of Agri-cultural Biological Breeding (2023ZD0402605-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We wish to thank Dr. Nan Xie (Hebei Academy of Agriculture and Forestry Science) and Dr. Xiaohu Lin and Dr. Han Li (Henbei Normal University of Science and Technology) for providing octoploid triticale seeds, and we also appreciate Liwen Bianji (Edanz) and Mr. Jennifer Smith for editing the English text of a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcGoverin CM, Snyders F, Muller N, Botes W, Fox G, Manley M. 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Plants (Basel). 2020;9,105. https://doiorg/103390/plants9010105.\u003c/li\u003e\n\u003cli\u003eSlusarkiewicz-Jarzina A, Ponitka A. Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal. Res. Commun. 2003;31,289\u0026ndash;296. https://doiorg/101079/97808519965300447.\u003c/li\u003e\n\u003cli\u003eChaikam V, Gowda M, Martinez L, Ochieng J, Omar HA, Prasanna BM. Improving the efficiency of colchicine-based chromosomal doubling of maize haploids. Plants (Basel). 2020;9,459. https://doiorg/103390/plants9040459.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"awnless triticale, anther culture, genotypes, hormone concentrations, ploidy identification, agronomic trait","lastPublishedDoi":"10.21203/rs.3.rs-5176133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5176133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rapid production of doubled haploids by anther culture technology is an important breeding method for awnless triticale. The aim of this study was to explore the effects of triticale genotype and the types and ratios of exogenous hormones in the medium on the efficiency of triticale anther culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnthers of five triticale genotypes were cultured on four different callus induction media and the calli were induced to differentiate into green plants by culture on three different differentiation media. The triticale genotype T8004 showed the best performance in anther culture, with a callus induction rate of 28.64%, a green plantlet differentiation frequency of 33.33%, and a green plantlet production rate of 2.78%. The highest callus induction rates were obtained by culturing anthers on C3 medium, and the highest green plantlet differentiation frequency was obtained by culturing calli on D2 differentiation medium. Flow cytometry analyses showed that 15 of the 20 regenerated plants that grew normally in the field were doubled haploids. The average chromosome doubling success rate was 55.6%. Analyses of agronomic traits showed that the 15 doubled haploid plants reached the standard for awnless triticale, so they are candidate materials for breeding new awnless triticale varieties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anther culture technology of triticale was optimized in this paper, which made it possible to rapidly breed homozygous varieties of awnless triticale.\u003c/p\u003e","manuscriptTitle":"Effects of hormone concentrations on anther cultures and the acquisition of regenerated plants of five awnless triticale genotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-16 14:17:34","doi":"10.21203/rs.3.rs-5176133/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-13T21:28:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-11T18:20:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-06T09:50:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-05T12:34:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-28T22:33:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-27T11:20:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325963480895862390410394728069146808747","date":"2024-10-26T17:00:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13698624207393131170064016768584961411","date":"2024-10-22T10:17:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3286210956432875660640841161330542171","date":"2024-10-21T19:11:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339296911820731835605325733365575540366","date":"2024-10-21T11:23:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6694102910676439407203341289348103011","date":"2024-10-07T08:36:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171574773806034054258600282134070100514","date":"2024-10-06T08:47:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-04T23:31:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-04T01:39:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-04T01:39:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2024-09-29T17:19:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ccf6acf-2036-4244-a1e0-7c6ebdaac3df","owner":[],"postedDate":"December 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T16:05:47+00:00","versionOfRecord":{"articleIdentity":"rs-5176133","link":"https://doi.org/10.1186/s13007-024-01322-z","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2025-01-07 15:57:22","publishedOnDateReadable":"January 7th, 2025"},"versionCreatedAt":"2024-12-16 14:17:34","video":"","vorDoi":"10.1186/s13007-024-01322-z","vorDoiUrl":"https://doi.org/10.1186/s13007-024-01322-z","workflowStages":[]},"version":"v1","identity":"rs-5176133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5176133","identity":"rs-5176133","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}