Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale

Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale Jun Ma, Fangyuan Zhao, Xinhui Tian, Wenhua Du This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4608942/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Compared to traditional breeding methods, anther culture method is an effective method for quickly obtaining homozygotes within one generation. The method of cultivating double haploid plants with the anthers of awnless triticale was studied and optimized. Results Young awnless triticale spikes were pretreated at 4°C for 5, 10, 15, 20, or 25 days, and the anthers were cultured on four CHB media with varying hormone concentrations. The callus induction rate (CIR) was highest (28.54%) for A3B3 (anthers pretreated for 15 days and CHB medium containing 1.5 mg/L 2,4-D and 1.5 mg/L KT). The green plantlet differentiation frequency (DFG) was highest (30.20%) for A5B1 (25-days pretreatment and CHB medium containing 0.5 mg/L 2,4-D and 0.5 mg/L KT). The green plantlet production (PPG) was highest (7.98%) for A2B1 (10-days + 0.5 mg/L 2,4-D + 0.5 mg/L KT). The success rate of chromosome doubling for the regenerated green plantlets was 52.8%. Appropriately decreasing the chromosome doubling time may increase the survival rate of the regenerated plants. Ten of the nineteen doubled haploid plants had tip and side awns shorter than 5 mm, implying they may be used for cultivating awnless triticale. Conclusion The anther culture technology of triticale was optimized in this paper, which made it possible to rapidly breed homozygous varieties of awnless triticale, and also accelerated the breeding program of new varieties of awnless triticale. Triticale Anther culture Callus Doubled haploid Flow cytometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Background Triticale (× Triticosecale Wittmack) was produced by the intergeneric hybridization between wheat ( Triticum aestivum ) and rye ( Secale cereale ) [ 1 ]. As a multi-purpose grain-forage species, triticale is an important forage crop [ 2 ]. Triticale is widely cultivated in the alpine pastoral area of northwestern China as a forage crop for feeding livestock [ 3 ]. Most triticale varieties produce awns. If awned varieties are not cut in time, the awns will gradually become waxy and cut the mouth of livestock, thereby increasing the likelihood of pharyngitis, mouth ulcerations, and submandibular edema during feeding [ 4 , 5 ]. Awns can also adversely affect the production of high-quality hay [ 6 ]. Therefore, the breeding of awnless triticale varieties is necessary for improving the palatability of the forage crop and increasing the production of high-quality hay. In addition to traditional breeding methods, anther culture is an indispensable and rapid alternative technique. It has been used to quickly generate haploids and inbred lines of hybrid varieties [ 7 ]. More specifically, it can shorten the time required for breeding new varieties by 3 ~ 5 years, while also decreasing the human labor and financial resources necessary for breeding [ 8 ]. For this technique, the anthers of the F 1 or F 2 heterozygous generation are cultured in vitro . The microspores in the anthers are induced to dedifferentiate to form a callus, which is subsequently induced to redifferentiate to form a haploid plant [ 9 ]. After chromosome doubling, a complete diploid plant is formed, ultimately resulting in the breeding of new or improved varieties [ 10 , 11 ]. The efficiency of the anther culture technique for triticale is affected by many factors, including the temperature during the pretreatment of the anthers as well as the hormone content, plant genotype, pollen grain growth period, and medium type. A pretreatment at high or low temperatures can increase the callus or embryoid body induction rate [ 12 ]. This pretreatment can significantly increase the rice, wheat, and pepper anther callus induction rate [ 13 , 14 ]. A low-temperature pretreatment can cause the developing microspores to deviate from the gametophyte developmental pathway to the sporophyte developmental pathway, leading to the production of haploid callus or embryoid bodies, which subsequently develop into plants [ 15 ]. The signal induced by the exposure to low temperatures initiates the change in microspore development, which is important for embryoid body formation [ 16 ]. In a previous study, the anthers of four Mediterranean japonica rice ( Oryza sativa ) F 2 lines were pretreated for 9 days at 5°C, which increased the anther callus induction rate (16.5%) [ 17 ]. Pretreating the microspore embryos of three sweet pepper genotypes (‘Inspiration F1’, ‘Maratus F1’, and ‘Magno F1’) at 32°C reportedly increases the multinuclear microspore, total embryo, and regenerator production rates [ 18 ]. Although hormones are not major components of the medium, they are indispensable for generating calli or embryoid bodies [ 19 , 20 ]. Chong-Pérez et al. [ 21 ] determined that supplementing the medium with 22.66 µM 2,4-D and 0.54 µM NAA maximizes the number of Vasconcellea pubescens calli (13.9%), whereas Zur et al. [ 22 ] observed that adding NAA and KT to the medium (0.5 and 0.5 mg L − 2 , respectively) could also increase the triticale embryoid body induction rate. Thus, optimizing the hormone content of the medium can increase the callus induction rate. The callus recovery rate varies among genotypes, with the highly responsive genotypes having the highest callus recovery rates [ 23 ]. Lantos et al. [ 24 ] revealed the differences in the responses of 10 winter wheat genotypes to the same anther culture conditions, and 7 of the 10 winter triticale varieties showed significant differences in callus induction rate and differentiation rate, indicating that genotype had a significant effect on the callus induction rate and differentiation rate of anthers. Although the anther culture technique has been successfully applied to develop new triticale varieties, its main limitations include the low callus induction rate and haploid formation rate, genotype dependence, and albinism [ 25 , 26 ]. We hypothesized that modifying the low-temperature pretreatment time and the medium hormone concentration may alter the triticale anther callus induction rate, 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 ). Therefore, the low-temperature pretreatment time and the medium hormone concentration were modified to optimize the triticale anther callus induction rate, green plantlet differentiation rate, and haploid formation rate. Materials and methods Materials The triticale material used in this experiment was awnless triticale line T2020-6337 (hexaploidy, 2n = 6x = 42) derived from a cross between the male parent T17(2013) and female parent J6(2013). After a 30-day vernalization, the awnless triticale seeds were sown in a plastic greenhouse at Gansu Agricultural University (36°03′N, 103°53′E; 1,560 m above sea level), China, in July 2020. A line seeding method was adopted, with a row spacing of 20 cm and a sowing depth of 3 ~ 4 cm. Additionally, 300 kg hm − 2 diammonium phosphate was applied before sowing and 196 kg hm − 2 urea was applied (top dressing) at the seeding and jointing stages. Plants were irrigated as required. 2.2 Induction media Four media were developed by modifying the CHB medium in terms of its hormone contents. Each medium also contained 90 g/L sucrose and 7 g/L agar. Additionally, the pH was adjusted to 5.4. The hormone components of the four media were shown in Table 1 . Table 1 Hormone concentration of induction medium Number Hormone concentration B1 0.5 mg/L 2,4-D + 0.5 mg/L KT B2 1.0 mg/L 2,4-D + 1.0 mg/L KT B3 1.5 mg/L 2,4-D + 1.5 mg/L KT B4 2.0 mg/L 2,4-D + 2.0 mg/L KT Sampling and low-temperature pretreatment of young spikes Samples were collected starting in late October and early November 2020. The young spikes of triticale plants were cut when the pollen grains were in the mid-to-late mononuclear stage (i.e., spikes just reached the middle of the flag leaf and the second leaf from the top). The young spikes were wrapped in aluminum foil, which was labeled with the collection date, and placed in a large beaker containing tap water, and then the beaker was put into a bucket with a lid and several ice packs. They were then transported to the laboratory. Two to four 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 the pollen development stage. The remaining young spikes were divided into five groups and then placed in 500 mL beakers filled with 200 mL tap water for the pretreatment at 4°C for 5 days (A1), 10 days (A2), 15 days (A3), 20 days (A4), and 25 days (A5). Isolation and Culturing of Anthers Upon completion of the low-temperature pretreatment, the flag leaves and the second leaf from the top were removed. The surface of the material was disinfected via a 1-min immersion in 75% ethanol in a biological safety cabinet (Sujingantai Inc., Suzhou, Jiangsu, China). The young spikes were obtained, disinfected for 7 min in a 2% sodium hypochlorite solution, and rinsed with sterile water 4 ~ 5 times. After removing any surface moisture using sterile filter paper (Xinhua Inc., Hangzhou, Zhejiang, China), the young spikes were placed in a sterilized Petri dish (90 mm diameter) (Jingan Inc., Shanghai, China). The anthers were removed from the young spikes using sterile forceps and added to the four induction media (30 ~ 50 anthers per Petri dish). Each treatment was completed using eight Petri dishes, which were placed in the HGZ-250 incubator (Yuejin Inc., Shanghai, China) for a 3-day incubation at 32°C in darkness. The Petri dishes were transferred to another incubator for a 60-day incubation at 28°C in darkness. A few calli were detectable on day 30 of the incubation. On day 60, the number of calli was recorded. Regeneration Calli with a diameter exceeding 1 mm were transferred to the differentiation medium (MS medium supplemented with 1 mg/L IAA, 1 mg/L 6-BA, 10 g/L sorbitol, 30 g/L sucrose, and 7 g/L agar, with a pH of 5.8). The calli were cultured in an incubator set at 27°C with a 14-h light (2,000 ~ 3,000 lx): 10-h dark cycle. The medium was refreshed every 15 days. After 60 days, the number of green plantlets and albino plantlets was recorded. Ploidy Level Analysis of the regeneration green plantlets Flow cytometry was used to determine the ploidy levels of regenerated plants. Briefly, 1 ~ 2 leaves were collected from each plant and then placed in 400 µL extraction buffer (CyStain UV Precise P Kit; Sysmex Co., Hamburg, Germany). The leaves were 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 (CyFlow Cube 6; 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 to determine whether the regenerated plants were haploid plants. Chromosome Doubling, Seedling Training, and Transplanting The green plantlets with more than four leaves were transplanted into plastic pots (Jiesheng Co., Shenzhen, Guangdong, China) containing nutrient soil (Luneng Co., Tianzhu, Gansu, China). The pots were incubated in a climatron set at 25°C with a 14-h light:10-h dark cycle. 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 5.0 h chromosome doubling step. The roots were rinsed by tap water and then the green plantlets were replanted in plastic pots and incubated in the climatron for 3 ~ 5 weeks to resume growth. They were subsequently transferred to a field. Ploidy Level Analysis Flow cytometry was used to determine the ploidy levels of 36 regenerated plants after they grew normally to the jointing stage. The analysis method was the same as ‘Ploidy Level Analysis of the regeneration green plantlets’. Octoploid triticale variety ‘Jinsong No. 49’ (provided by the Hebei Academy of Agriculture and Forestry Science, Shijiazhuang, Hebei, China) was used as a control to determine whether the regenerated plants were DH plants. Agronomic Trait Analysis The following traits of the regenerated plants were examined at the flowering stage: plant height (distance from the ground to the top of the spike, but excluding the awn, which was measured using a ruler), and number of branch per plant. The following spike parameters were examined at the dough stage: tip awn length (length of the longest awn at the top of the spike, which was measured using a vernier caliper), side awn length (length of the longest awn of the middle spikelet on both sides of the spike, which was measured and then the average value was calculated), spike length (length from the basal spikelet to the tip of the spikelet, but excluding the awn), number of spikelets (number of fertile and sterile spikelets), number of grains per spike, and grain weight per spike (determined using an electronic balance). Statistical Analysis Each anther-inoculated Petri dish was used as a replicate, with no fewer than eight replicates per treatment. The number of anthers used for the inoculation, calli, green plantlets, and albino plantlets in each Petri dish was recorded. Additionally, CIR, DFG, DFA, PRG, PRA, and PRR were calculated using the following formulae: 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 number of plantlets/Number of anthers used for the inoculation × 100 The differences in CIR, DFG, DFA, PRG, PRA, and PRR were analyzed using the SPSS 20.0 software. If significant differences were detected, Duncan’s multiple comparison test was performed to compare the differences. RESULTS Microscopic Examination of Anthers The microscopic examination of the collected young spikes of triticale plants showed that most of the microspores were in the late uninucleate stage and the nuclei of individual microspores were located at or near the center, which was ideal for anther cultivation (Fig. 2 a, b). Analysis of Variance The results of the analysis of the differences in CIR, DFR, DFA, PRG, PRA, and PRR are presented in Table 2 . There were extremely significant differences at the 0.01 level in CIR for the low-temperature pretreatment duration, hormone concentrations, and their interactions. There were also extremely significant differences at the 0.01 level in CIR, DFG, DFA, PRG, PRA, and PRR for the interaction between low-temperature pretreatment duration and hormone concentrations (Table 2 ). Table 2 Analysis of the differences in CIR, DFG, DFA, PRG, PRA, and PRR Variation F value CIR (%) DFG (%) DFA (%) PRG (%) PRA (%) PRR (%) Low temperature pretreatment days 138.30 ** – – – – – Hormone concentrations 109.74 ** – – – – – Low temperature pretreatment days × hormone concentration 13.69 ** 34.61 ** 10.62 ** 4.38 ** 54.70 ** 17.14 ** Note: * indicates significant differences at the 0.05 level, whereas ** indicates significant differences at the 0.01 level. CIR: callus induction rate; DFG: green plantlet differentiation frequency; DFA: albino plantlet differentiation frequency; PPG: green plantlet production; PPA: albino plantlet production; PRR: plant regeneration rate. The same as below. Effects of the Low-Temperature Pretreatment Duration, Hormone Concentrations and the interaction between them on the average CIR CIR As the number of low-temperature pretreatment days increased, the average CIR of the triticale anthers initially increased and then decreased. The average CIR for the different hormone concentrations was highest (23.46%) for the 15-day low-temperature pretreatment of the triticale anthers, followed by the 10-day pretreatment and then the 25-day, 20-day, and 5-day pretreatments. The average CIR was similar for the 20-day and 25-day pretreatments, ranging from 14.83–19.23%, which was higher than the average CIR for the 5-day pretreatment (˂10%). Accordingly, the average CIR was highest for the anthers pretreated at a low temperature for 15 days, reflecting the suitability of this pretreatment for the anther culture (Fig. 3 ). Increases in the hormone concentrations were accompanied by a decrease in the average CIR of the triticale anthers pretreated at 4°C for different durations. The average CIR of B1 was significantly higher than that of B2 and B4, but it was not significantly different from that of B3. The average CIR of B4 was significantly lower than that of B1, B2, and B3. Thus, B1 had the highest average CIR, implying it may be best for the anther culture (Fig. 4 ). The analysis of the interaction between the low-temperature pretreatment duration and hormone concentrations indicated that for the same low temperature pretreatment days of A1 and A2, CIR was higher or significantly higher for B1 than for the other media. For A3, CIR was higher or significantly higher for B3 than for the other media. For A4 and A5, CIR was higher or significantly higher for B2 than for the other media. Thus, the hormone concentrations needed for a high CIR varied depending on the duration of the low-temperature pretreatment. In terms of the same hormone concentrations, for both B1 and B3, CIR was highest for A3 and then A2, with significantly lower CIRs for A5, A1, and A4. For B2, CIR was higher or significantly higher for A4 than for the other low-temperature pretreatment durations. For B4, CIR was highest for A2. Hence, the low-temperature pretreatment duration required for a high CIR differed among the tested hormone concentrations. Overall, CIR was significantly higher for A3B3 (28.54%) than for the other treatment combinations, with the exception of A3B1 (Fig. 5 ). Effects of the interaction between the Low-Temperature Pretreatment Duration and Hormone Concentrations on DFG and DFA DFG For A1, DFG was significantly higher for B2 than for the other media (Fig. 6 a), whereas for A2, A4, and A5, DFG was significantly higher for B1 than for the other media. For A3, DFG was significantly higher for B4 than for the other media. Therefore, the hormone concentrations needed for a high DFG varied depending on the low-temperature pretreatment duration. In terms of the same hormone concentrations, for B1, DFG was significantly higher for A5 (47.2%), and A4 than for A1, A2, A3. For B2, DFG was higher or significantly higher for A1 than for A2, A3, A4, and A5. For B3, DFG was highest for A5. For B4, DFG was highest for A3. Thus, the duration of the low-temperature pretreatment required to maximize DFG differed among the hormone concentrations. Overall, DFG was significantly higher for A5B1 than for the other treatment combinations (Fig. 6 a). DFA For the same low temperature pretreatment days of A1 and A2, DFA was higher or significantly higher for B2 than for B3 and the other media (Fig. 6 b). For A3, the DFA of B4 was highest. For A4, the DFA of B3 was highest, and For A5, the DFA of B1 was highest, which were significantly higher for the other media. For the same hormone concentrations, the DFA of A5 was highest in B1, and the DFA of A1 was highest in B2, while For B4, the DFA of A3 was highest, which were significantly higher for the other media. The A1B4, A2B3, A3B1, A4B4, and A5B4 treatment combinations resulted in the lowest DFA (0.00%; not presented in Fig. 6 b), which was suitable for the anther culture. These results reflected the variability in DFA among the hormone concentrations and low-temperature pretreatment durations (Fig. 6 b). Differences in Green Plantlet Production, Albino Plantlet Production and Plant Regeneration Rate there were no significant differences in PRG among B1, B2, and B4. PPG For the same pretreatment day of A2, A3, A4 and A5, PPG was highest for B1. Moreover, there was no significant difference between A2 and A5 in terms of PPG. For A1, the highest PPG (for B2) was significantly higher than the PPG for B1 and B4. For the same hormone concentration treatments, PPG was highest for A2 in B1. For B2, the highest PPG (for A5) was significantly higher than the PPG for A1, A2, A3, A4. For B3, PPG was highest for A3, but it did not differ significantly from that of A5. For B4, the highest PPG (for A3) was significantly higher than the PPG for A1, A2, A4 and A5. These findings indicated that the duration of the low-temperature pretreatment and the hormone concentrations significantly affected PPG (Fig. 7 a). PPA For the same pretreatment day treatment of A1, there were no significant differences in PPA among B1, B2, and B3. However, for A2 and A5, PPA was higher or significantly higher for B1 than for the other media. For A3 and A4, PPA was highest for B2. For the same hormone concentration treatments, PPA was highest for A4 in B2 and B3. Additionally, for B1, PPA was significantly higher for A2 than for A1, A4 and A5. For B4, PPA was significantly higher for A3 than for A2. The A1B4, A2B3, A3B1, A4B4, and A5B4 treatment combinations had the lowest PPA (0.00%; not presented in Fig. 7 b), followed by A1B3, indicative of the significant effects of the duration of the low-temperature pretreatment and the hormone concentrations on PPA (Fig. 7 b). PRR For the same pretreatment day treatment of the A2, A4 and A5, PRR was higher or significantly higher for B1 than for the other media. For A1, the highest PPG (for B2) was significantly higher than the PPG for B1, B3 and B4. For A3, the highest PRR (for B3) was significantly higher than the PRR for B2 and B4. For the same hormone concentration treatments, the analysis of the effects of the different hormone concentrations indicated that PRR was relatively high and consistent for B1 and B2. For B1, PRR was highest (7.98%) for A2, followed by A5 (5.86%) and A4 (4.34%); these values were significantly higher than the corresponding value for A1. For B2 and B3, PRR was highest (4.99% and 3.93%, respectively) for A5. For B4, PRR was significantly higher for A3 (3.50%)than for A1, A2, and A4, and for B4 (Fig. 7 c). Overall, PRR ranged from 0.59–7.98%, with PRR exceeding 5% for two treatments. Specifically, A2B1 resulted in the highest PRR (7.98%), which was followed by A5B1 (5.86%), likely because of the relatively high PRG and PRA associated with these two treatments. The PRR was lowest for A4B4 (0.59%), followed by A1B3 (0.87%), probably because of the relatively low PRG and PRA for these two treatments (Fig. 7 c). Determination of the Ploidy Levels of the Regenerated Plants In the process of anther culture, in addition to pollen microspores, the cells of the anther wall, anther septum, and filament may also dedifferentiate to form callus, and then differentiate into green seedlings. The green seedlings induced by pollen microspores were haploid (triploid in this test), while the green seedlings induced by the cells of the anther wall, anther septum, and filament were diploidized (hexaploid in this test), so the ploidy analysis of the induced green seedlings was carried out by flow cytometry to determine whether the regenerated green seedlings were haploid. The triticale material (T2020-6337) used in this experiment was hexaploid (2n = 6x = 42), and the control triticale ‘Gannon No. 3’ was also hexaploid (2n = 6x = 42). If the fluorescence intensity of the nuclear DNA relative content of the regenerated triticale seedlings detected peaks at 1/2 of the control group, the seedlings are haploid, otherwise they are not haploid. The test results showed that the position of peak of ‘Gannon No. 3’ triticale in the control was between 21000–27000 (Fig. 8 a), while the position of peak of regenerated plants measured was between 8000–15000, and the peak was haploid at 1/2 of the control (Fig. 8 b). A total of 52 haploid plants were obtained by ploidy analysis of the regenerated plants that survived after training (Table 3 ). Table 3 Changes in the number of regenerated plantlet before and after chromosome doubling Name TNGP NHGP NSGPCD SR/% NGPT NSGP DH MOPP SRCD/% T2020-7337 57 52 43 82.7 43 36 21 14 58.3 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; NGPT: the number of green plantlet transplanted in field; NSGP: the number of survival green plantlet; DH: double haploid plant; MOPP: mixoploidy and other ploidy plants; SRCD: the success rate of chromosome doubling. Chromosome doubling and ploidy identification of regenerated seedlings after chromosome doubling After chromosome doubling of haploid plantlets with colchicine solution, 43 plantlets survived, and the survival rate was 82.7%. The 43 plantlets were transplanted to the field, and 36 green plantlets survived and adapted to the growth environment of the field (Table 3 ). The flow cytometry was used to identify the ploidy of 36 green plantlets (before jointing stage) that grew normally after transplanting the field. The control used in this test was octaploid triticale ‘Jinsong 49’. If the tested triticale peaked at 3/4 of the control, the tested plants were hexaploid (double haploid), that is, chromosome doubling was successful. Figure 9 a was the results of flow cytometry analysis of ‘Jinsong 49’ triticale. The peak of ‘Jinsong 49’ triticale ranged from 48000 ~ 60000, while the peak of the tested plants ranged from 32000 ~ 40000, which was at 3/4 of the control (Fig. 9 b). therefore, it is hexaploid (double haploid). Figure 9 c showed that the test plant peaked 16000–28000,42000–48000 and 48000–56000, so it was mixoploid. In the test results, there was a great difference in the high and low peak, which was related to the amount of sample, but not related to plant ploidy. The ploidy analysis of 36 plants that grew normally to the heading stage in the field showed that 21 plants were double haploid (Table 3 , Fig. 9 c), and the rest were mixoploidy or other ploidy plants (Table 3 , Fig. 9 c), and the success rate of chromosome doubling was 58.3% (Table 3 ). Agronomic Trait Analysis The agronomic traits of identified DH plants were examined. The tip and side awn lengths were less than 5 mm for plants 3, 6, 12, 18, 19, 28, 33, 34, 35, and 36 (Table 4 , Fig. 10 ), which satisfies the threshold for awnless triticale. Hence, these 10 plants may be used as preparatory materials for the cultivation of awnless triticale varieties. The remaining plants had tip and side awn lengths of 5–10 mm, making them useful preparatory materials for the cultivation of short-awn triticale varieties. In terms of plant height, the plant height of 19 plants was between 51.77–120.30 cm, and most of the plants were higher than 90 cm, and 6, 7, 18, and 19 plants were between 110-120.30 cm, which was basically close to the plant height of mature triticale varieties. Except the number of branches of 3, 8, 12, 14, 17, 30, 33, 34, 35 and 36 was 3–9, the rest of the plants were more than 10 branches, and the traits were good. The spike length of plants 6, 7, 11, 18, 19, 30 and 34 was higher than 10 cm, while the other plants was less than 10 cm. Except the number of spikelet of 3, 12, 14 and 26 was less than 20, the other plants was greater than 20, ranging from 20.3 to 34.0. Of the 19 DH plants, only two produced more than 30 grains per spike and only two had a grain weight per spike greater than 1.0 g. Accordingly, compared with mature triticale varieties, the DH plants cultured and transplanted in the field in the first year had fewer grains per spike and were less plump (Table 4 ). Table 4 Agronomic traits of nine doubled haploid plants Number Plant height/cm Number of branch Tip awn/mm Side awn/mm Spike length/cm Number of spikelet Number of grains per spike Grain weight per spike/g 3 51.77 ± 3.40g 9 4.01 ± 0.43ef 2.07 ± 0.12ef 5.2 ± 0.7i 13.7 ± 3.5ef 3.0 ± 1.0h 0.06 ± 0.02gh 6 114.17 ± 1.42b 26 4.00 ± 1.97ef 4.16 ± 0.99cd 10.6 ± 0.3cd 26.3 ± 1.5b 38.0 ± 2.1a 1.65 ± 0.11a 7 120.30 ± 0.15a 17 5.11 ± 1.26ef 5.56 ± 0.57c 12.1 ± 0.8bc 33.3 ± 0.7a 41.7 ± 2.7a 1.51 ± 0.08a 8 84.67 ± 0.67d 8 8.54 ± 0.66abc 7.73 ± 0.74a 8.3 ± 0.1fg 22.7 ± 1.2bcd 21.3 ± 1.8cd 0.40 ± 0.05de 9 85.63 ± 0.32d 13 8.48 ± 0.38abc 7.86 ± 0.78a 9.0 ± 0.3defg 22.7 ± 0.7bcd 20.7 ± 1.3cde 0.40 ± 0.03de 10 89.17 ± 1.42d 15 7.89 ± 0.56bcd 7.03 ± 0.26ab 8.6 ± 0.2efg 22.0 ± 0.6bcd 20.0 ± 1.2cde 0.27 ± 0.02efg 11 95.17 ± 1.59c 14 8.55 ± 0.73abc 7.98 ± 0.82a 10.2 ± 0.2de 24.3 ± 0.9bc 24.7 ± 2.7c 0.43 ± 0.07de 12 57.33 ± 0.67fg 5 4.93 ± 0.45def 2.02 ± 0.53fg 5.6 ± 0.1i 12.0 ± 0.6f 0.0 ± 0.0h 0.00 ± 0.00h 14 61.33 ± 1.86f 8 9.32 ± 1.62cd 4.34 ± 0.57cd 6.4 ± 0.3hi 14.3 ± 1.3ef 15.7 ± 2.2ef 0.16 ± 0.02fgh 17 68.00 ± 2.65e 6 9.14 ± 3.18cd 3.61 ± 1.12cde 9.0 ± 0.1defg 20.3 ± 1.5cd 30.3 ± 2.2b 0.76 ± 0.11bc 18 116.50 ± 1.44ab 24 5.48 ± 1.48def 1.45 ± 0.45f 14.0 ± 0.6a 34.0 ± 0.1a 0.0 ± 0.0h 0.00 ± 0.00h 19 114.83 ± 5.63ab 11 3.61 ± 0.98ef 3.96 ± 0.11cde 12.7 ± 1.2ab 32.7 ± 1.8a 9.7 ± 0.9g 0.39 ± 0.04def 26 67.33 ± 1.45e 11 7.38 ± 0.61de 3.32 ± 0.53def 7.7 ± 0.4gh 17.7 ± 0.7de 0.0 ± 0.0h 0.00 ± 0.00h 28 98.40 ± 1.70c 12 1.55 ± 0.45f 4.07 ± 0.57cde 8.4 ± 0.4efg 25.0 ± 3.8bc 0.0 ± 0.0h 0.00 ± 0.00h 30 96.33 ± 0.73c 8 5.10 ± 0.42ef 3.73 ± 0.58cde 12.2 ± 1.5bc 21.3 ± 3.8bcd 15.7 ± 2.3ef 0.32 ± 0.09ef 33 87.07 ± 1.67d 5 2.70 ± 0.35f 3.58 ± 0.13cde 9.7 ± 0.2def 26.7 ± 1.2b 0.0 ± 0.0h 0.00 ± 0.00h 34 94.90 ± 1.16c 3 3.83 ± 0.64ef 2.63 ± 0.53def 10.0 ± 0.1def 24.7 ± 1.2bc 23.0 ± 1.5c 0.79 ± 0.11b 35 97.50 ± 0.79c 3 4.50 ± 0.75ef 2.96 ± 0.37def 9.7 ± 0.2def 25.0 ± 0.6bc 14.7 ± 2.0f 0.57 ± 0.20cd 36 95.73 ± 0.26c 4 3.97 ± 0.21ef 3.87 ± 0.07cde 9.4 ± 0.2defg 22.3 ± 0.9bcd 17.3 ± 1.2def 0.38 ± 0.01def Note: Different lowercase letters in each column indicate significant differences ( P < 0.05). Discussion Effects of a Low-Temperature Pretreatment on CIR and the Differentiation Rate of Triticale Among the many factors affecting the CIR of a triticale anther culture, a low-temperature pretreatment can lead to a significant increase in CIR [ 27 , 28 ]. A low-temperature pretreatment can induce the developing microspores to deviate from the gametophyte developmental pathway and enter the sporophyte developmental pathway, thereby producing haploid calli or embryoid bodies, which will then develop into plants [ 29 , 30 ]. A low-temperature pretreatment can also significantly increase the ABA content in anthers, thereby promoting microspore antioxidant activities in the early stage of the in vitro culture and maintaining microspore viability [ 31 ]. Slusarkiewicz-Jarzina et al. [ 32 ] reported that a pretreatment at 4°C for 14 days can significantly increase the production of green winter triticale plants, while also improving the androgen response of the plants. For wheat anther cultures, researchers increased the number of green plants by including a 14-day low-temperature pretreatment [ 33 ], but other researchers suggested that a 21-day low-temperature pretreatment may be better [ 34 – 36 ]. Many laboratories now consider a low-temperature pretreatment of the young spikes of triticale plants (2 ~ 3 weeks at 4°C) is considered to be optimal for inducing microspore embryogenesis [ 33 ]. In the current study on triticale, young spikes were pretreated at 4°C. The analysis of the effects of the pretreatment duration indicated CIR was highest (28.54%) for the 15-day low-temperature pretreatment. This is consistent with the findings of Slusarkiewicz-Jarzina et al. [ 32 ]. Moreover, it may reflect the ability of the microspores to dedifferentiate during an incubation at low temperatures for 15 days. Increasing the duration of the low-temperature pretreatment beyond 15 days did not have additional positive effects on the anther culture, which may be indicative of cold-induced damage to the anthers and decreased microspore activities. The A3B3 treatment produced the highest CIR (28.54%), which was lower than the CIRs reported by Ślusarkiewicz-Jarzina et al. (39.3%) [ 32 ]. Thus, methods for further increasing the CIR for triticale anthers may need to be developed. In this study, the timing of the initial appearance of calli appeared to influence whether green or albino plantlets were ultimately generated. More specifically, the calli that appeared within the first 30 days continued to grow (diameter greater than 5 mm) and differentiated into green plantlets. In contrast, the calli that appeared after 30 or 60 days grew more poorly (diameter of approximately 1 ~ 2 mm). Additionally, these calli were more likely to develop into brown or albino seedlings during the later induction and differentiation period, possibly because the calli were too small to differentiate into green shoots [ 32 ]. Hence, even though many calli appeared during the latter part of the induction period, these calli produced fewer green seedlings than the calli that appeared earlier. Albinism, which is a common problem associated with plant tissue culture, is due to the inability to produce chloroplasts, resulting in a lack of photosynthesis [ 37 ]. Albinism has been detected in the tissue cultures of monocots, including wheat, barley, and rye. A recent study showed that a prolonged pretreatment often results in a single base pair mutation (C to T), which leads to a missense mutation (Thr to Ile) related albinism [ 38 ]. The cells of albino seedlings produced via tissue culture may contain totipotent nuclei, but their plastids, which cannot differentiate normally, may be affected by a new sporophyte-related process [ 39 ]. For barley anther and microspore cultures, the proportion of albino plantlets reportedly varies widely (1%~99.7%) depending on the genotype, with albinism more common in spring barley than in winter barley [ 40 ]. In the present study, DFA ranged from 0–23.33% among the treatments, with the albino plantlets affecting the plant differentiation efficiency. The production of albino plantlets is mainly related to gene mutations or abnormal gene expression. It is also influenced by culture conditions, including the temperature, light, hormones, and mineral elements. There has been considerable research conducted to decrease DFA. For example, the addition of Cu 2+ or Ag + to the medium reportedly alters the green plantlet-to-albino plantlet ratio by decreasing the number of albino plantlets by 18.7% [ 41 ]. Decreasing the temperature and light intensity may decrease DFA to some extent without affecting normal callus growth and differentiation [ 38 ]. However, genotypes affect the production of albino plantlets considerably more than culture conditions. According to the results of the current study (Fig. 6 a, b), A5B1 may be the ideal treatment for optimizing plantlet differentiation. Specifically, it produced the highest DFG (47.20%), but kept DFA from exceeding 13.90%. Potential alternatives were A1B2 and A4B1, with a DFG of 33.30%, but the DFA for A1B2 (23.33%) was higher than that for A4B1 (16.7%), implying A4B1 may be better than A1B2 for plantlet differentiation. In contrast, A1B3 resulted in the lowest DFG (0.00%) and a high DFA (22.90%), implying this treatment should not be used. The A1B2 treatment had the highest DFA (23.33%), whereas A1B4, A2B3, A3B1, A4B4, and A5B4 had the lowest DFA (0.00%; not presented in Fig. 6 b). Among the 20 treatments, three resulted in a DFA exceeding 20% and 10 resulted in a DFA between 10% and 20%. Hence, measures for decreasing DFA should be assessed in future studies. Effects of Hormones on the Triticale Anther CIR Hormones (type and concentration) have important regulatory effects on cell division, growth, and differentiation. Earlier research on anther cultures indicated that different plant species and different genotypes of the same species vary regarding their hormone requirements [ 42 ]. In the induction medium, different hormone types and concentrations affect callus and plantlet formation [ 43 ]. In the anther cultures of many plants, 2,4-D is often used because of its effects on pollen initiation and division as well as the formation of calli and embryoid bodies [ 44 ]. Moreover, KT is one of the most commonly used cytokinins for increasing the efficiency of winter wheat anther cultures [ 45 , 46 ]. For the triticale anther culture, supplementing the medium with 2 mg/L 2,4-D reportedly increases the average number of calli (13.9%) (compared with the effects of 6-BA) [ 22 ]. In an earlier investigation involving wheat, the relatively few calli generated on medium lacking 2,4-D failed to differentiate into plantlets, but increasing the 2,4-D content in the medium from 0.5 mg/L to 1.0 mg/L increased the number of calli and the plantlet regeneration rate [ 47 ]. However, because prolonging the 2–4 mg/L 2,4-D treatment beyond the initial stage reportedly adversely affects plant regeneration, the 2,4-D concentration in the induction medium should be maintained at 1 ~ 2 mg/L [ 47 ]. In the current study, the pretreatment of triticale anthers at 4°C for 15 days before the inoculation of medium supplemented with 1.5 mg/L 2,4-D and 1.5 mg/L KT resulted in the highest CIR (28.54%). This is in accordance with the findings of a previous study by Zheng and Konzak [ 47 ]. For hybrid rice, the highest CIR (21.66%) is obtained when N6 medium containing 2 mg/L 2,4-D and 0.5 mg/L N6-benzyladenine is used, possibly reflecting the synergistic effects of auxin and cytokinin on calli [ 38 ]. Therefore, the synergistic effects of different auxin and cytokinin concentrations on calli should be explored. Ploidy Level Determination A colchicine treatment can increase the probability of regenerating polyploid plants, but it can also cause seedlings to wither and die [ 48 ]. Chromosome doubling agents inhibit spindle fiber formation, thereby preventing the copied chromosomes from separating and migrating to the poles [ 49 ]. In a previous investigation, the survival rates after the roots and crowns of regenerated oat plants were treated with 0.1% and 0.2% colchicine for 4 h were 97.9% and 93.6%, respectively [ 50 ]. In another study, the survival rate of whole germinated maize seedlings treated with 0.04% colchicine for 5 h was 47.11%, whereas the survival rates following the treatment of maize seedling roots and crowns with 0.1%, 0.4%, and 0.7% colchicine solutions for 5 h were 88.66%, 94.17%, and 89.00%, respectively [ 51 ]. In the present study, the examination of the green triticale plantlets treated with a 0.1% colchicine solution for 5 h revealed that more than half of the plantlets had withered leaf tips after 1 week, while some of the plantlets transferred to plastic pots gradually withered, and the survival rate of green plantlets was 82.7%. When the chromosome doubling time was decreased to 4.5 h, the number of withered plantlets decreased. These findings may reflect the toxicity of colchicine. The detrimental effects of chromosome doubling chemical agents on plant survival have been reported [ 52 ]. Therefore, appropriately decreasing the chromosome doubling time can increase the survival rate of regenerated plants. The utility of colchicine has been assessed in terms of chromosome doubling efficiency and plant survival rates, but the potential toxicity of colchicine to plants and animals (including humans) has often been ignored, so researchers have recently started to screen for less toxic alternatives to colchicine [ 53 ]. Amiprophos-methyl and pronamid, which have been used for the chromosome doubling of maize haploid plants, may be better than other alternatives, with an overall success rate (16.1%) that is close to that (22.1%) of colchicine [ 54 ]. For regenerating cucumber plants, the highest DH regeneration rates for colchicine and trifluralin are 58.33% and 83.33%, respectively, and the chromosome doubling rates for 750 mg/L colchicine and 50 mL/L trifluralin are reportedly 57.41% and 62.50%, respectively, implying trifluralin may be better than colchicine [ 52 ]. Although there are viable alternatives, colchicine is still used in many laboratories for the chromosome doubling of haploid plants. Different colchicine concentrations and treatment times influenced the chromosome doubling rate of the regenerated green seedlings. In our experiment, the roots of the regenerated plants were immersed in a 0.1% colchicine solution for 5 h. Nineteen of the 36 regenerated plants were verified as DH plants, which is lower than the corresponding rates in earlier studies by Slusarkiewicz-Jarzina et al. and Ferrie et al. [ 55 , 50 ]. More specifically, 215 regenerated haploid triticale plantlets were treated with a 4% colchicine solution for 6 h under light at 25°C, which resulted in 128 DH plantlets (i.e., chromosome doubling efficiency of 59.9%) [ 55 ]. The highest haploid transformation rate (80%) for regenerated oat plants was obtained following a 4-h treatment with 0.2% colchicine [ 50 ]. Moreover, soaking the roots of maize seedlings in 0.10% and 0.70% colchicine solutions for 5 h can result in chromosome doubling efficiencies of 31.83% and 27.67%, respectively [ 51 ]. Clearly, colchicine concentrations and treatment times may need to be adjusted to maximize the chromosome doubling efficiency. Conclusion In this study, an improved method for producing DH plants was developed for awnless triticale (Fig. 11 ). This method resulted in significant increases in CIR. A total of 620 calli and 57 regenerated green plants were obtained for genotype T2020-6337. Of the 36 regenerated green plants that were grown in the field, 19 were confirmed as DH plants. The highest CIR was obtained by pretreating anthers for 15 days at 4°C prior to the inoculation of CHB medium containing 1.5 mg/L 2,4-D and 1.5 mg/L KT. In addition, the effects of the duration of the low-temperature pretreatment as well as the hormone types and concentrations on CIR, DFG, DFA, PRG, PRA, and PRR should be considered for triticale anther culture experiments. Furthermore, DFG and PRR must be further improved and methods for decreasing DFA are needed. Future research should also evaluate the utility of this breeding protocol for enhancing other triticale genotypes. 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 NAA Naphthalene acetic acid BA Benzyladenine CIR Callus induction rate DFG Green plantlet differentiation frequency DFA Albino plantlet differentiation frequency PPG Green plantlet production PPA Albino plantlet production PRR Plant regeneration rate Declarations Author contributions WD and JM designed the experiment. JM and FZ 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. 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) 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 Evtushenko EV, Lipikhina YA, Stepochkin PI, Vershinin AV. Cytogenetic and molecular characteristics of rye genome in octoploid triticale (× Triticosecale Wittmack). Comp Cytogenet. 2019;13:423–34. https://doi.org/10.3897/CompCytogen.v13i4.39576 . 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Seifert F, Bössow S, Kumlehn J, Gnad H, Scholten S. Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar Svilena. Bmc Plant Biol. 2016;16:97–113. https://doi.org/10.1186/s12870-016-0782-8 . Maheshwari P, Laurie JD. Triticale Isolated Microspore Culture for Doubled Haploid Production. Methods Mol Biol. 2021;2287:295–312. https://doi.org/10.1007/978-1-0716-1315-3-16 . Migneault A, Sandhu H, McCord P, Zhao D, Erickson J. Albinism in sugarcane: significance, research gaps, and potential future research developments. Sugar Tech. 2018;21:536–41. https://doi.org/10.1007/s12355-018-0668-1 . Pattnaik SS, Dash B, Bhuyan SS, Katara JL, Parameswaran C, Verma R, Ramesh N, Samantaray S. Anther culture efficiency in quality hybrid rice: a comparison between hybrid rice and its ratooned plants. Plants (Basel). 2020;9:1306. https://doi.org/10.3390/plants9101306 . Ohnoutkova L, Vlcko T, Ayalew M. Barley Anther Culture. Methods Mol. Biol. 2019;1900,37–52. https://doi.org/10.1007/978-1-4939-8944-7_4 . Makowska K, Oleszczuk S. Albinism in barley androgenesis. Plant Cell Rep. 2014. https://doi.org/10.1007/s00299-013-1543-x . 33,385 – 92. Warcho M, Katarzyna J, Dziurka K, Czyczyo-Mysza I, Skrzypek E. The Effect of Zinc, Copper, and Silver Ions on Oat ( Avena sativa L). Androgenesis Plants. 2021;10248. https://doi.org/10.3390/plants10020248 . Lordan J, Fazio G, Francescatto P, Robinson T. Effects of apple (Malus × domestica) rootstocks on scion performance and hormone concentration. Sci Horticulturae. 2017;225:96–105. https://doi.org/10.1016/j.scienta.2017.06.050 . Juzoń-Sikora K, Nowicka A, Plaková L, Doleal K, Zur I. Hormonal homeostasis associated with effective induction of triticale microspore embryogenesis. Plant Cell Tiss Organ Cult. 2022;152:583–604. https://doi.org/10.1007/s11240-022-02433-y . Dissanayake L, Perera P, Attanayaka T, Heberle E, Jayawardhana M. Early Development of Direct Embryos in the Cultured Anthers of Manihot esculenta Crantz. Plants (Basel). 2020. https://doi.org/10.3390/plants9101315 . 9,1315. Wang J, Gao J, Fan W, Dong J, Tang F, Shi F. Construction of Tissue Culture System of Onobrychis viciaefolia Scop ‘Mengnong’ Anthers. Legume Res. 2023;46:855–61. https://doi.org/10.18805/lrf-729 . Kruppa J, Kanbar OZ, Tóth-Lencsés KA, Kiss E, Bóna L, Lantos C, Pauk J. Induction of triticale ( ×Triticosecale Wittmack) in vitro androgenesis in anther cultures of F 1 hybrid combinations, varieties and homogeneity testing of offspring generation. Life. 2023;131709. https://doi.org/10.3390/life13101970 . Zheng MY, Konzak CF. Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat ( Triticum aestivum L). Plant Cell Rep. 1999;19:69–73. https://doi.org/10.1007/s002990050712 . Huy NP, Tam DTT, Luan VQ, Tung HT, Hien VH, Ngan HTM, Duy PN, Nhut DT. In vitro polyploid induction of Paphiopedilum villosum using colchicine. Sci Hort. 2019;252:283–90. https://doi.org/10.1016/j.scienta.2019.03.063 . Broughton S, Castello M, Liu L, Killen J, Hepworth A, O'Leary R. The effect of caffeine and trifluralin on chromosome doubling in wheat anther culture. Plants (Basel). 2020. https://doi.org/10.3390/plants9010105 . 9,105. Ferrie AMR, Irmen KI, Beattie AD, Rossnagel BG. Isolated microspore culture of oat ( Avena sativa L.) for the production of doubled haploids: effect of pre-culture and post-culture conditions. Plant Cell Tiss Org. 2014;116:89–96. https://doi.org/10.1007/s11240-013-0385-0 . 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. https://doi.org/10.3390/plants9040459 . 9,459. Ebrahimzadeh H, Soltanloo H, Shariatpanahi ME, Eskandari A, Ramezanpour SS. Improved chromosome doubling of parthenogenetic haploid plants of cucumber ( Cucumis sativus L.) using colchicine, trifluralin, and oryzalin. Planr Cell Tissue Organ Cult. 2018;135:407–17. https://doi.org/10.1007/s11240-018-1473-y . Touchell DH, Palmer IE, Ranney TG. In vitro Ploidy Manipulation for Crop Improvement. Front. Plant Sci. 2020;11:722. https://doi.org/10.3389/fpls.2020.00722 . Melchinger AE, Molenaar WS, Mirdita V, Schipprack W. Colchicine alternatives for chromosome doubling in maize haploids for doubled-haploid production. Crop Sci. 2015;56:559–69. https://doi.org/10.2135/cropsci2015.06.0383 . Slusarkiewiczjarzina A, Ponitka A. Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal Res Commun. 2003;31:289–96. https://doi.org/10.1079/9780851996530.0447 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4608942","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317755257,"identity":"1e8393b3-9604-4725-915f-b937c1053b5b","order_by":0,"name":"Jun Ma","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ma","suffix":""},{"id":317755258,"identity":"41a02808-f8e2-44f3-b9fe-0cdfb81bf9b3","order_by":1,"name":"Fangyuan Zhao","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fangyuan","middleName":"","lastName":"Zhao","suffix":""},{"id":317755259,"identity":"a8020d60-6c44-4fe3-bab9-04972eba36d0","order_by":2,"name":"Xinhui Tian","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinhui","middleName":"","lastName":"Tian","suffix":""},{"id":317755260,"identity":"e037303c-e6f1-4b48-9b6f-224ac17cb13c","order_by":3,"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":"2024-06-20 03:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4608942/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4608942/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60054339,"identity":"f0f5e5f5-2a0c-4b72-8690-60f5b6443337","added_by":"auto","created_at":"2024-07-11 07:01:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3617442,"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-4608942/v1/1fe9930da00c31666e7c2a2c.png"},{"id":60055363,"identity":"2110173a-cc32-4a99-8956-c13b653214b5","added_by":"auto","created_at":"2024-07-11 07:17:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3306990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eMicroscopic examination of the microspore mononuclear border stage; \u003cstrong\u003eb\u003c/strong\u003e: the magnified microspore mononuclear border stage\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/c346abe2b55346c58be84491.png"},{"id":60053487,"identity":"4569f12e-3dec-47c6-bae7-9a0bbd2128d6","added_by":"auto","created_at":"2024-07-11 06:53:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59410,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the duration of the low-temperature pretreatment on the average CIR for different hormone concentrations. Different letters above the columns indicate significant differences at the 0.05 level. CIR: callus induction rate.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/a446d9deffe49bfdb083d4c9.png"},{"id":60055362,"identity":"119c32ba-75be-4248-b421-3b7c6bdbdac6","added_by":"auto","created_at":"2024-07-11 07:17:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58169,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of hormone concentrations on the average CIR of triticale for different low-temperature pretreatment durations. CIR: callus induction rate.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/257cb67dc3b344f06458efd5.png"},{"id":60054742,"identity":"c5c464c5-4a01-4c34-b4ac-47326ac4436a","added_by":"auto","created_at":"2024-07-11 07:09:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99307,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the interaction between the low-temperature pretreatment duration and hormone concentration on CIR. CIR: callus induction rate.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/901ef54fb8b88e8609a3bc39.png"},{"id":60053477,"identity":"5602dbf3-fab3-4e95-b5e5-f8a204748900","added_by":"auto","created_at":"2024-07-11 06:53:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":571496,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the interaction between the low-temperature pretreatment duration and hormone concentration on (a) green plantlet differentiation frequency (DFG)and (b) albino plantlet differentiation frequency (DFA).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/70d892266c81899427f02f67.png"},{"id":60054743,"identity":"8d9d4894-3320-42ba-b256-4d493b460615","added_by":"auto","created_at":"2024-07-11 07:09:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":601396,"visible":true,"origin":"","legend":"\u003cp\u003eThe difference of green plantlet production(PPG) (a), albino plantlet production(PPA)(b) and plant regeneration rate(PRR) (c) in treatments\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/e45c3ca115dc290d59bda353.png"},{"id":60053478,"identity":"5b332c41-b60e-4979-8e8a-dc0649710f3e","added_by":"auto","created_at":"2024-07-11 06:53:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":574530,"visible":true,"origin":"","legend":"\u003cp\u003eResults of DNA content in regenerated seedlings of triticale determined by flow cytom\u003c/p\u003e\n\u003cp\u003eNote: \u003cstrong\u003e(a)\u003c/strong\u003e: ‘Gannong No. 3’ triticale (CK); \u003cstrong\u003e(b)\u003c/strong\u003e: haploid regenerated seedlings\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/f6a7de39871e4db28870df8d.png"},{"id":60053484,"identity":"2fe97ca1-7fa4-4ef6-a6be-76a790f7dcc3","added_by":"auto","created_at":"2024-07-11 06:53:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":669659,"visible":true,"origin":"","legend":"\u003cp\u003eResults of DNA content in regenerated seedlings of triticale determined by flow cytom\u003c/p\u003e\n\u003cp\u003eNote: \u003cstrong\u003e(a)\u003c/strong\u003e: Octoploid triticale variety ‘Jinsong No. 49’ (CK); \u003cstrong\u003e(b)\u003c/strong\u003e: double haploid regenerated seedlings; \u003cstrong\u003e(c)\u003c/strong\u003e: mixoploid regenerated seedlings\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/b8b52764fe50214de53b9439.png"},{"id":60053480,"identity":"839c8e94-2527-4d1b-bd8a-683bdaa7883f","added_by":"auto","created_at":"2024-07-11 06:53:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3678053,"visible":true,"origin":"","legend":"\u003cp\u003eSpike of triticale plant 36\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/0335c0c5b1db476252943c11.png"},{"id":60054346,"identity":"e200e592-9d84-4176-a9bd-cda27435b044","added_by":"auto","created_at":"2024-07-11 07:01:17","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2278289,"visible":true,"origin":"","legend":"\u003cp\u003eProduction of doubled haploid triticale plants.\u003cstrong\u003e (a) \u003c/strong\u003eCollected triticale young spikes.\u003cstrong\u003e (b) \u003c/strong\u003eInduction medium inoculated with anthers.\u003cstrong\u003e (c) \u003c/strong\u003eCalli. \u003cstrong\u003e(d) \u003c/strong\u003eDifferentiated green plantlet and albino plantlet.\u003cstrong\u003e (e) \u003c/strong\u003eGrowing green plantlet.\u003cstrong\u003e (f) \u003c/strong\u003eGreen plantlets before chromosome doubling.\u003cstrong\u003e (g) \u003c/strong\u003eGreen plantlets after chromosome doubling in a climatron.\u003cstrong\u003e (h) \u003c/strong\u003eGreen plantlets in the field.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/5d064ebb996b272e2bfc9d4b.png"},{"id":62168297,"identity":"0838a2a8-2e7f-4e9f-ab61-a133e66af633","added_by":"auto","created_at":"2024-08-10 04:01:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23079366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4608942/v1/3924076a-6042-46ca-8f2e-3a68b959d36f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale","fulltext":[{"header":"Background","content":"\u003cp\u003eTriticale (\u0026times;\u003cem\u003eTriticosecale\u003c/em\u003e Wittmack) was produced by the intergeneric hybridization between wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) and rye (\u003cem\u003eSecale cereale\u003c/em\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As a multi-purpose grain-forage species, triticale is an important forage crop [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Triticale is widely cultivated in the alpine pastoral area of northwestern China as a forage crop for feeding livestock [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Most triticale varieties produce awns. If awned varieties are not cut in time, the awns will gradually become waxy and cut the mouth of livestock, thereby increasing the likelihood of pharyngitis, mouth ulcerations, and submandibular edema during feeding [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Awns can also adversely affect the production of high-quality hay [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the breeding of awnless triticale varieties is necessary for improving the palatability of the forage crop and increasing the production of high-quality hay. In addition to traditional breeding methods, anther culture is an indispensable and rapid alternative technique. It has been used to quickly generate haploids and inbred lines of hybrid varieties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. More specifically, it can shorten the time required for breeding new varieties by 3\u0026thinsp;~\u0026thinsp;5 years, while also decreasing the human labor and financial resources necessary for breeding [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For this technique, the anthers of the F\u003csub\u003e1\u003c/sub\u003e or F\u003csub\u003e2\u003c/sub\u003e heterozygous generation are cultured \u003cem\u003ein vitro\u003c/em\u003e. The microspores in the anthers are induced to dedifferentiate to form a callus, which is subsequently induced to redifferentiate to form a haploid plant [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. After chromosome doubling, a complete diploid plant is formed, ultimately resulting in the breeding of new or improved varieties [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe efficiency of the anther culture technique for triticale is affected by many factors, including the temperature during the pretreatment of the anthers as well as the hormone content, plant genotype, pollen grain growth period, and medium type. A pretreatment at high or low temperatures can increase the callus or embryoid body induction rate [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This pretreatment can significantly increase the rice, wheat, and pepper anther callus induction rate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A low-temperature pretreatment can cause the developing microspores to deviate from the gametophyte developmental pathway to the sporophyte developmental pathway, leading to the production of haploid callus or embryoid bodies, which subsequently develop into plants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The signal induced by the exposure to low temperatures initiates the change in microspore development, which is important for embryoid body formation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In a previous study, the anthers of four Mediterranean \u003cem\u003ejaponica\u003c/em\u003e rice (\u003cem\u003eOryza sativa\u003c/em\u003e) F\u003csub\u003e2\u003c/sub\u003e lines were pretreated for 9 days at 5\u0026deg;C, which increased the anther callus induction rate (16.5%) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Pretreating the microspore embryos of three sweet pepper genotypes (\u0026lsquo;Inspiration F1\u0026rsquo;, \u0026lsquo;Maratus F1\u0026rsquo;, and \u0026lsquo;Magno F1\u0026rsquo;) at 32\u0026deg;C reportedly increases the multinuclear microspore, total embryo, and regenerator production rates [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although hormones are not major components of the medium, they are indispensable for generating calli or embryoid bodies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Chong-P\u0026eacute;rez et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e] determined that supplementing the medium with 22.66 \u0026micro;M 2,4-D and 0.54 \u0026micro;M NAA maximizes the number of \u003cem\u003eVasconcellea pubescens\u003c/em\u003e calli (13.9%), whereas Zur et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e] observed that adding NAA and KT to the medium (0.5 and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, respectively) could also increase the triticale embryoid body induction rate. Thus, optimizing the hormone content of the medium can increase the callus induction rate. The callus recovery rate varies among genotypes, with the highly responsive genotypes having the highest callus recovery rates [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Lantos et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e] revealed the differences in the responses of 10 winter wheat genotypes to the same anther culture conditions, and 7 of the 10 winter triticale varieties showed significant differences in callus induction rate and differentiation rate, indicating that genotype had a significant effect on the callus induction rate and differentiation rate of anthers. Although the anther culture technique has been successfully applied to develop new triticale varieties, its main limitations include the low callus induction rate and haploid formation rate, genotype dependence, and albinism [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We hypothesized that modifying the low-temperature pretreatment time and the medium hormone concentration may alter the triticale anther callus induction rate, 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). Therefore, the low-temperature pretreatment time and the medium hormone concentration were modified to optimize the triticale anther callus induction rate, green plantlet differentiation rate, and haploid formation rate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe triticale material used in this experiment was awnless triticale line T2020-6337 (hexaploidy, 2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42) derived from a cross between the male parent T17(2013) and female parent J6(2013). After a 30-day vernalization, the awnless triticale seeds were sown in a plastic greenhouse at Gansu Agricultural University (36\u0026deg;03\u0026prime;N, 103\u0026deg;53\u0026prime;E; 1,560 m above sea level), China, in July 2020. A line seeding method was adopted, with a row spacing of 20 cm and a sowing depth of 3\u0026thinsp;~\u0026thinsp;4 cm. Additionally, 300 kg hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e diammonium phosphate was applied before sowing and 196 kg hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e urea was applied (top dressing) at the seeding and jointing stages. Plants were irrigated as required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Induction media\u003c/h2\u003e \u003cp\u003eFour media were developed by modifying the CHB medium in terms of its hormone contents. Each medium also contained 90 g/L sucrose and 7 g/L agar. Additionally, the pH was adjusted to 5.4. The hormone components of the four media were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\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\u003eB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;0.5 mg/L KT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 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\u003eB3\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\u003eB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;2.0 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=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSampling and low-temperature pretreatment of young spikes\u003c/h2\u003e \u003cp\u003eSamples were collected starting in late October and early November 2020. The young spikes of triticale plants were cut when the pollen grains were in the mid-to-late mononuclear stage (i.e., spikes just reached the middle of the flag leaf and the second leaf from the top). The young spikes were wrapped in aluminum foil, which was labeled with the collection date, and placed in a large beaker containing tap water, and then the beaker was put into a bucket with a lid and several ice packs. They were then transported to the laboratory.\u003c/p\u003e \u003cp\u003eTwo to four 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 the pollen development stage. The remaining young spikes were divided into five groups and then placed in 500 mL beakers filled with 200 mL tap water for the pretreatment at 4\u0026deg;C for 5 days (A1), 10 days (A2), 15 days (A3), 20 days (A4), and 25 days (A5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and Culturing of Anthers\u003c/h2\u003e \u003cp\u003eUpon completion of the low-temperature pretreatment, the flag leaves and the second leaf from the top were removed. The surface of the material was disinfected via a 1-min immersion in 75% ethanol in a biological safety cabinet (Sujingantai Inc., Suzhou, Jiangsu, China). The young spikes were obtained, disinfected for 7 min in a 2% sodium hypochlorite solution, and rinsed with sterile water 4\u0026thinsp;~\u0026thinsp;5 times. After removing any surface moisture using sterile filter paper (Xinhua Inc., Hangzhou, Zhejiang, China), the young spikes were placed in a sterilized Petri dish (90 mm diameter) (Jingan Inc., Shanghai, China). The anthers were removed from the young spikes using sterile forceps and added to the four induction media (30\u0026thinsp;~\u0026thinsp;50 anthers per Petri dish). Each treatment was completed using eight Petri dishes, which were placed in the HGZ-250 incubator (Yuejin Inc., Shanghai, China) for a 3-day incubation at 32\u0026deg;C in darkness. The Petri dishes were transferred to another incubator for a 60-day incubation at 28\u0026deg;C in darkness. A few calli were detectable on day 30 of the incubation. On day 60, the number of calli was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRegeneration\u003c/h2\u003e \u003cp\u003eCalli with a diameter exceeding 1 mm were transferred to the differentiation medium (MS medium supplemented with 1 mg/L IAA, 1 mg/L 6-BA, 10 g/L sorbitol, 30 g/L sucrose, and 7 g/L agar, with a pH of 5.8). The calli were cultured in an incubator set at 27\u0026deg;C with a 14-h light (2,000\u0026thinsp;~\u0026thinsp;3,000 lx): 10-h dark cycle. The medium was refreshed every 15 days. After 60 days, the number of green plantlets and albino plantlets was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePloidy Level Analysis of the regeneration green plantlets\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to determine the ploidy levels of regenerated plants. Briefly, 1\u0026thinsp;~\u0026thinsp;2 leaves were collected from each plant and then placed in 400 \u0026micro;L extraction buffer (CyStain UV Precise P Kit; Sysmex Co., Hamburg, Germany). The leaves were 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 (CyFlow Cube 6; 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 to determine whether the regenerated plants were haploid plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eChromosome Doubling, Seedling Training, and Transplanting\u003c/h2\u003e \u003cp\u003eThe green plantlets with more than four leaves were transplanted into plastic pots (Jiesheng Co., Shenzhen, Guangdong, China) containing nutrient soil (Luneng Co., Tianzhu, Gansu, China). The pots were incubated in a climatron set at 25\u0026deg;C with a 14-h light:10-h dark cycle. 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 5.0 h chromosome doubling step. The roots were rinsed by tap water and then the green plantlets were replanted in plastic pots and incubated in the climatron for 3\u0026thinsp;~\u0026thinsp;5 weeks to resume growth. They were subsequently transferred to a field.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePloidy Level Analysis\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to determine the ploidy levels of 36 regenerated plants after they grew normally to the jointing stage. The analysis method was the same as \u0026lsquo;Ploidy Level Analysis of the regeneration green plantlets\u0026rsquo;. Octoploid triticale variety \u0026lsquo;Jinsong No. 49\u0026rsquo; (provided by the Hebei Academy of Agriculture and Forestry Science, Shijiazhuang, Hebei, China) was used as a control to determine whether the regenerated plants were DH plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAgronomic Trait Analysis\u003c/h2\u003e \u003cp\u003eThe following traits of the regenerated plants were examined at the flowering stage: plant height (distance from the ground to the top of the spike, but excluding the awn, which was measured using a ruler), and number of branch per plant. The following spike parameters were examined at the dough stage: tip awn length (length of the longest awn at the top of the spike, which was measured using a vernier caliper), side awn length (length of the longest awn of the middle spikelet on both sides of the spike, which was measured and then the average value was calculated), spike length (length from the basal spikelet to the tip of the spikelet, but excluding the awn), number of spikelets (number of fertile and sterile spikelets), number of grains per spike, and grain weight per spike (determined using an electronic balance).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEach anther-inoculated Petri dish was used as a replicate, with no fewer than eight replicates per treatment. The number of anthers used for the inoculation, calli, green plantlets, and albino plantlets in each Petri dish was recorded. Additionally, CIR, DFG, DFA, PRG, PRA, and PRR were calculated using the following formulae:\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 number of plantlets/Number of anthers used for the inoculation \u0026times; 100\u003c/p\u003e \u003cp\u003eThe differences in CIR, DFG, DFA, PRG, PRA, and PRR were analyzed using the SPSS 20.0 software. If significant differences were detected, Duncan\u0026rsquo;s multiple comparison test was performed to compare the differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic Examination of Anthers\u003c/h2\u003e \u003cp\u003eThe microscopic examination of the collected young spikes of triticale plants showed that most of the microspores were in the late uninucleate stage and the nuclei of individual microspores were located at or near the center, which was ideal for anther cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Variance\u003c/h2\u003e \u003cp\u003eThe results of the analysis of the differences in CIR, DFR, DFA, PRG, PRA, and PRR are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There were extremely significant differences at the 0.01 level in CIR for the low-temperature pretreatment duration, hormone concentrations, and their interactions. There were also extremely significant differences at the 0.01 level in CIR, DFG, DFA, PRG, PRA, and PRR for the interaction between low-temperature pretreatment duration and hormone concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eAnalysis of the differences in CIR, DFG, DFA, PRG, PRA, and PRR\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=\"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 \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\u003eLow temperature pretreatment days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e138.30\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormone concentrations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e109.74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow temperature pretreatment days \u0026times; hormone concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.69\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.61\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.62\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.38\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.70\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.14\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: * indicates significant differences at the 0.05 level, whereas ** indicates significant differences at the 0.01 level. CIR: callus induction rate; DFG: green plantlet differentiation frequency; DFA: albino plantlet differentiation frequency; PPG: green plantlet production; PPA: albino plantlet production; PRR: plant regeneration rate. The same as below.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the Low-Temperature Pretreatment Duration, Hormone Concentrations and the interaction between them on the average CIR\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCIR\u003c/strong\u003e \u003cp\u003eAs the number of low-temperature pretreatment days increased, the average CIR of the triticale anthers initially increased and then decreased. The average CIR for the different hormone concentrations was highest (23.46%) for the 15-day low-temperature pretreatment of the triticale anthers, followed by the 10-day pretreatment and then the 25-day, 20-day, and 5-day pretreatments. The average CIR was similar for the 20-day and 25-day pretreatments, ranging from 14.83\u0026ndash;19.23%, which was higher than the average CIR for the 5-day pretreatment (˂10%). Accordingly, the average CIR was highest for the anthers pretreated at a low temperature for 15 days, reflecting the suitability of this pretreatment for the anther culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIncreases in the hormone concentrations were accompanied by a decrease in the average CIR of the triticale anthers pretreated at 4\u0026deg;C for different durations. The average CIR of B1 was significantly higher than that of B2 and B4, but it was not significantly different from that of B3. The average CIR of B4 was significantly lower than that of B1, B2, and B3. Thus, B1 had the highest average CIR, implying it may be best for the anther culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis of the interaction between the low-temperature pretreatment duration and hormone concentrations indicated that for the same low temperature pretreatment days of A1 and A2, CIR was higher or significantly higher for B1 than for the other media. For A3, CIR was higher or significantly higher for B3 than for the other media. For A4 and A5, CIR was higher or significantly higher for B2 than for the other media. Thus, the hormone concentrations needed for a high CIR varied depending on the duration of the low-temperature pretreatment. In terms of the same hormone concentrations, for both B1 and B3, CIR was highest for A3 and then A2, with significantly lower CIRs for A5, A1, and A4. For B2, CIR was higher or significantly higher for A4 than for the other low-temperature pretreatment durations. For B4, CIR was highest for A2. Hence, the low-temperature pretreatment duration required for a high CIR differed among the tested hormone concentrations. Overall, CIR was significantly higher for A3B3 (28.54%) than for the other treatment combinations, with the exception of A3B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the interaction between the Low-Temperature Pretreatment Duration and Hormone Concentrations on DFG and DFA\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDFG\u003c/strong\u003e \u003cp\u003eFor A1, DFG was significantly higher for B2 than for the other media (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), whereas for A2, A4, and A5, DFG was significantly higher for B1 than for the other media. For A3, DFG was significantly higher for B4 than for the other media. Therefore, the hormone concentrations needed for a high DFG varied depending on the low-temperature pretreatment duration. In terms of the same hormone concentrations, for B1, DFG was significantly higher for A5 (47.2%), and A4 than for A1, A2, A3. For B2, DFG was higher or significantly higher for A1 than for A2, A3, A4, and A5. For B3, DFG was highest for A5. For B4, DFG was highest for A3. Thus, the duration of the low-temperature pretreatment required to maximize DFG differed among the hormone concentrations. Overall, DFG was significantly higher for A5B1 than for the other treatment combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDFA\u003c/strong\u003e \u003cp\u003eFor the same low temperature pretreatment days of A1 and A2, DFA was higher or significantly higher for B2 than for B3 and the other media (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). For A3, the DFA of B4 was highest. For A4, the DFA of B3 was highest, and For A5, the DFA of B1 was highest, which were significantly higher for the other media. For the same hormone concentrations, the DFA of A5 was highest in B1, and the DFA of A1 was highest in B2, while For B4, the DFA of A3 was highest, which were significantly higher for the other media. The A1B4, A2B3, A3B1, A4B4, and A5B4 treatment combinations resulted in the lowest DFA (0.00%; not presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), which was suitable for the anther culture. These results reflected the variability in DFA among the hormone concentrations and low-temperature pretreatment durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferences in Green Plantlet Production, Albino Plantlet Production and Plant Regeneration Rate\u003c/b\u003e there were no significant differences in PRG among B1, B2, and B4.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePPG\u003c/strong\u003e \u003cp\u003eFor the same pretreatment day of A2, A3, A4 and A5, PPG was highest for B1. Moreover, there was no significant difference between A2 and A5 in terms of PPG. For A1, the highest PPG (for B2) was significantly higher than the PPG for B1 and B4. For the same hormone concentration treatments, PPG was highest for A2 in B1. For B2, the highest PPG (for A5) was significantly higher than the PPG for A1, A2, A3, A4. For B3, PPG was highest for A3, but it did not differ significantly from that of A5. For B4, the highest PPG (for A3) was significantly higher than the PPG for A1, A2, A4 and A5. These findings indicated that the duration of the low-temperature pretreatment and the hormone concentrations significantly affected PPG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePPA\u003c/strong\u003e \u003cp\u003eFor the same pretreatment day treatment of A1, there were no significant differences in PPA among B1, B2, and B3. However, for A2 and A5, PPA was higher or significantly higher for B1 than for the other media. For A3 and A4, PPA was highest for B2. For the same hormone concentration treatments, PPA was highest for A4 in B2 and B3. Additionally, for B1, PPA was significantly higher for A2 than for A1, A4 and A5. For B4, PPA was significantly higher for A3 than for A2. The A1B4, A2B3, A3B1, A4B4, and A5B4 treatment combinations had the lowest PPA (0.00%; not presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), followed by A1B3, indicative of the significant effects of the duration of the low-temperature pretreatment and the hormone concentrations on PPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePRR\u003c/strong\u003e \u003cp\u003eFor the same pretreatment day treatment of the A2, A4 and A5, PRR was higher or significantly higher for B1 than for the other media. For A1, the highest PPG (for B2) was significantly higher than the PPG for B1, B3 and B4. For A3, the highest PRR (for B3) was significantly higher than the PRR for B2 and B4. For the same hormone concentration treatments, the analysis of the effects of the different hormone concentrations indicated that PRR was relatively high and consistent for B1 and B2. For B1, PRR was highest (7.98%) for A2, followed by A5 (5.86%) and A4 (4.34%); these values were significantly higher than the corresponding value for A1. For B2 and B3, PRR was highest (4.99% and 3.93%, respectively) for A5. For B4, PRR was significantly higher for A3 (3.50%)than for A1, A2, and A4, and for B4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, PRR ranged from 0.59\u0026ndash;7.98%, with PRR exceeding 5% for two treatments. Specifically, A2B1 resulted in the highest PRR (7.98%), which was followed by A5B1 (5.86%), likely because of the relatively high PRG and PRA associated with these two treatments. The PRR was lowest for A4B4 (0.59%), followed by A1B3 (0.87%), probably because of the relatively low PRG and PRA for these two treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the Ploidy Levels of the Regenerated Plants\u003c/h2\u003e \u003cp\u003eIn the process of anther culture, in addition to pollen microspores, the cells of the anther wall, anther septum, and filament may also dedifferentiate to form callus, and then differentiate into green seedlings. The green seedlings induced by pollen microspores were haploid (triploid in this test), while the green seedlings induced by the cells of the anther wall, anther septum, and filament were diploidized (hexaploid in this test), so the ploidy analysis of the induced green seedlings was carried out by flow cytometry to determine whether the regenerated green seedlings were haploid. The triticale material (T2020-6337) used in this experiment was hexaploid (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42), and the control triticale \u0026lsquo;Gannon No. 3\u0026rsquo; was also hexaploid (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42). If the fluorescence intensity of the nuclear DNA relative content of the regenerated triticale seedlings detected peaks at 1/2 of the control group, the seedlings are haploid, otherwise they are not haploid. The test results showed that the position of peak of \u0026lsquo;Gannon No. 3\u0026rsquo; triticale in the control was between 21000\u0026ndash;27000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea), while the position of peak of regenerated plants measured was between 8000\u0026ndash;15000, and the peak was haploid at 1/2 of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). A total of 52 haploid plants were obtained by ploidy analysis of the regenerated plants that survived after training (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\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\u003eChanges in the number of regenerated plantlet before and after chromosome doubling\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\u003eName\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 \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNGPT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNSGP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMOPP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\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\u003eT2020-7337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e58.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\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; NGPT: the number of green plantlet transplanted in field; NSGP: the number of survival green plantlet; DH: double haploid plant; MOPP: mixoploidy and other ploidy plants; SRCD: the 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=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChromosome doubling and ploidy identification of regenerated seedlings after chromosome doubling\u003c/h2\u003e \u003cp\u003eAfter chromosome doubling of haploid plantlets with colchicine solution, 43 plantlets survived, and the survival rate was 82.7%. The 43 plantlets were transplanted to the field, and 36 green plantlets survived and adapted to the growth environment of the field (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The flow cytometry was used to identify the ploidy of 36 green plantlets (before jointing stage) that grew normally after transplanting the field. The control used in this test was octaploid triticale \u0026lsquo;Jinsong 49\u0026rsquo;. If the tested triticale peaked at 3/4 of the control, the tested plants were hexaploid (double haploid), that is, chromosome doubling was successful. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea was the results of flow cytometry analysis of \u0026lsquo;Jinsong 49\u0026rsquo; triticale. The peak of \u0026lsquo;Jinsong 49\u0026rsquo; triticale ranged from 48000\u0026thinsp;~\u0026thinsp;60000, while the peak of the tested plants ranged from 32000\u0026thinsp;~\u0026thinsp;40000, which was at 3/4 of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). therefore, it is hexaploid (double haploid). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec showed that the test plant peaked 16000\u0026ndash;28000,42000\u0026ndash;48000 and 48000\u0026ndash;56000, so it was mixoploid. In the test results, there was a great difference in the high and low peak, which was related to the amount of sample, but not related to plant ploidy.\u003c/p\u003e \u003cp\u003eThe ploidy analysis of 36 plants that grew normally to the heading stage in the field showed that 21 plants were double haploid (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), and the rest were mixoploidy or other ploidy plants (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), and the success rate of chromosome doubling was 58.3% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAgronomic Trait Analysis\u003c/h2\u003e \u003cp\u003eThe agronomic traits of identified DH plants were examined. The tip and side awn lengths were less than 5 mm for plants 3, 6, 12, 18, 19, 28, 33, 34, 35, and 36 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), which satisfies the threshold for awnless triticale. Hence, these 10 plants may be used as preparatory materials for the cultivation of awnless triticale varieties. The remaining plants had tip and side awn lengths of 5\u0026ndash;10 mm, making them useful preparatory materials for the cultivation of short-awn triticale varieties. In terms of plant height, the plant height of 19 plants was between 51.77\u0026ndash;120.30 cm, and most of the plants were higher than 90 cm, and 6, 7, 18, and 19 plants were between 110-120.30 cm, which was basically close to the plant height of mature triticale varieties. Except the number of branches of 3, 8, 12, 14, 17, 30, 33, 34, 35 and 36 was 3\u0026ndash;9, the rest of the plants were more than 10 branches, and the traits were good. The spike length of plants 6, 7, 11, 18, 19, 30 and 34 was higher than 10 cm, while the other plants was less than 10 cm. Except the number of spikelet of 3, 12, 14 and 26 was less than 20, the other plants was greater than 20, ranging from 20.3 to 34.0. Of the 19 DH plants, only two produced more than 30 grains per spike and only two had a grain weight per spike greater than 1.0 g. Accordingly, compared with mature triticale varieties, the DH plants cultured and transplanted in the field in the first year had fewer grains per spike and were less plump (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\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\u003eAgronomic traits of nine doubled haploid plants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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=\"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 \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\u003eNumber of branch\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTip awn/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSide awn/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpike length/cm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber of spikelet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNumber of grains per spike\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\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\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.77\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02gh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e114.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1fg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3defg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2efg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02efg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67fg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53fg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3hi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02fgh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1defg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e116.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e114.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.63ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04def\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4gh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4efg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09ef\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20cd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07cde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2defg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2def\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01def\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eNote: Different lowercase letters in each column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffects of a Low-Temperature Pretreatment on CIR and the Differentiation Rate of Triticale\u003c/h2\u003e \u003cp\u003eAmong the many factors affecting the CIR of a triticale anther culture, a low-temperature pretreatment can lead to a significant increase in CIR [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A low-temperature pretreatment can induce the developing microspores to deviate from the gametophyte developmental pathway and enter the sporophyte developmental pathway, thereby producing haploid calli or embryoid bodies, which will then develop into plants [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A low-temperature pretreatment can also significantly increase the ABA content in anthers, thereby promoting microspore antioxidant activities in the early stage of the \u003cem\u003ein vitro\u003c/em\u003e culture and maintaining microspore viability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Slusarkiewicz-Jarzina et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e] reported that a pretreatment at 4\u0026deg;C for 14 days can significantly increase the production of green winter triticale plants, while also improving the androgen response of the plants. For wheat anther cultures, researchers increased the number of green plants by including a 14-day low-temperature pretreatment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e], but other researchers suggested that a 21-day low-temperature pretreatment may be better [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany laboratories now consider a low-temperature pretreatment of the young spikes of triticale plants (2\u0026thinsp;~\u0026thinsp;3 weeks at 4\u0026deg;C) is considered to be optimal for inducing microspore embryogenesis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the current study on triticale, young spikes were pretreated at 4\u0026deg;C. The analysis of the effects of the pretreatment duration indicated CIR was highest (28.54%) for the 15-day low-temperature pretreatment. This is consistent with the findings of Slusarkiewicz-Jarzina et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, it may reflect the ability of the microspores to dedifferentiate during an incubation at low temperatures for 15 days. Increasing the duration of the low-temperature pretreatment beyond 15 days did not have additional positive effects on the anther culture, which may be indicative of cold-induced damage to the anthers and decreased microspore activities. The A3B3 treatment produced the highest CIR (28.54%), which was lower than the CIRs reported by Ślusarkiewicz-Jarzina et al. (39.3%) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, methods for further increasing the CIR for triticale anthers may need to be developed.\u003c/p\u003e \u003cp\u003eIn this study, the timing of the initial appearance of calli appeared to influence whether green or albino plantlets were ultimately generated. More specifically, the calli that appeared within the first 30 days continued to grow (diameter greater than 5 mm) and differentiated into green plantlets. In contrast, the calli that appeared after 30 or 60 days grew more poorly (diameter of approximately 1\u0026thinsp;~\u0026thinsp;2 mm). Additionally, these calli were more likely to develop into brown or albino seedlings during the later induction and differentiation period, possibly because the calli were too small to differentiate into green shoots [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Hence, even though many calli appeared during the latter part of the induction period, these calli produced fewer green seedlings than the calli that appeared earlier.\u003c/p\u003e \u003cp\u003eAlbinism, which is a common problem associated with plant tissue culture, is due to the inability to produce chloroplasts, resulting in a lack of photosynthesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Albinism has been detected in the tissue cultures of monocots, including wheat, barley, and rye. A recent study showed that a prolonged pretreatment often results in a single base pair mutation (C to T), which leads to a missense mutation (Thr to Ile) related albinism [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The cells of albino seedlings produced via tissue culture may contain totipotent nuclei, but their plastids, which cannot differentiate normally, may be affected by a new sporophyte-related process [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For barley anther and microspore cultures, the proportion of albino plantlets reportedly varies widely (1%~99.7%) depending on the genotype, with albinism more common in spring barley than in winter barley [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the present study, DFA ranged from 0\u0026ndash;23.33% among the treatments, with the albino plantlets affecting the plant differentiation efficiency. The production of albino plantlets is mainly related to gene mutations or abnormal gene expression. It is also influenced by culture conditions, including the temperature, light, hormones, and mineral elements. There has been considerable research conducted to decrease DFA. For example, the addition of Cu\u003csup\u003e2+\u003c/sup\u003e or Ag\u003csup\u003e+\u003c/sup\u003e to the medium reportedly alters the green plantlet-to-albino plantlet ratio by decreasing the number of albino plantlets by 18.7% [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Decreasing the temperature and light intensity may decrease DFA to some extent without affecting normal callus growth and differentiation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, genotypes affect the production of albino plantlets considerably more than culture conditions.\u003c/p\u003e \u003cp\u003eAccording to the results of the current study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b), A5B1 may be the ideal treatment for optimizing plantlet differentiation. Specifically, it produced the highest DFG (47.20%), but kept DFA from exceeding 13.90%. Potential alternatives were A1B2 and A4B1, with a DFG of 33.30%, but the DFA for A1B2 (23.33%) was higher than that for A4B1 (16.7%), implying A4B1 may be better than A1B2 for plantlet differentiation. In contrast, A1B3 resulted in the lowest DFG (0.00%) and a high DFA (22.90%), implying this treatment should not be used. The A1B2 treatment had the highest DFA (23.33%), whereas A1B4, A2B3, A3B1, A4B4, and A5B4 had the lowest DFA (0.00%; not presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Among the 20 treatments, three resulted in a DFA exceeding 20% and 10 resulted in a DFA between 10% and 20%. Hence, measures for decreasing DFA should be assessed in future studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Hormones on the Triticale Anther CIR\u003c/h2\u003e \u003cp\u003eHormones (type and concentration) have important regulatory effects on cell division, growth, and differentiation. Earlier research on anther cultures indicated that different plant species and different genotypes of the same species vary regarding their hormone requirements [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the induction medium, different hormone types and concentrations affect callus and plantlet formation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In the anther cultures of many plants, 2,4-D is often used because of its effects on pollen initiation and division as well as the formation of calli and embryoid bodies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, KT is one of the most commonly used cytokinins for increasing the efficiency of winter wheat anther cultures [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. For the triticale anther culture, supplementing the medium with 2 mg/L 2,4-D reportedly increases the average number of calli (13.9%) (compared with the effects of 6-BA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In an earlier investigation involving wheat, the relatively few calli generated on medium lacking 2,4-D failed to differentiate into plantlets, but increasing the 2,4-D content in the medium from 0.5 mg/L to 1.0 mg/L increased the number of calli and the plantlet regeneration rate [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, because prolonging the 2\u0026ndash;4 mg/L 2,4-D treatment beyond the initial stage reportedly adversely affects plant regeneration, the 2,4-D concentration in the induction medium should be maintained at 1\u0026thinsp;~\u0026thinsp;2 mg/L [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the current study, the pretreatment of triticale anthers at 4\u0026deg;C for 15 days before the inoculation of medium supplemented with 1.5 mg/L 2,4-D and 1.5 mg/L KT resulted in the highest CIR (28.54%). This is in accordance with the findings of a previous study by Zheng and Konzak [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For hybrid rice, the highest CIR (21.66%) is obtained when N6 medium containing 2 mg/L 2,4-D and 0.5 mg/L N6-benzyladenine is used, possibly reflecting the synergistic effects of auxin and cytokinin on calli [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, the synergistic effects of different auxin and cytokinin concentrations on calli should be explored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePloidy Level Determination\u003c/h2\u003e \u003cp\u003eA colchicine treatment can increase the probability of regenerating polyploid plants, but it can also cause seedlings to wither and die [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Chromosome doubling agents inhibit spindle fiber formation, thereby preventing the copied chromosomes from separating and migrating to the poles [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In a previous investigation, the survival rates after the roots and crowns of regenerated oat plants were treated with 0.1% and 0.2% colchicine for 4 h were 97.9% and 93.6%, respectively [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In another study, the survival rate of whole germinated maize seedlings treated with 0.04% colchicine for 5 h was 47.11%, whereas the survival rates following the treatment of maize seedling roots and crowns with 0.1%, 0.4%, and 0.7% colchicine solutions for 5 h were 88.66%, 94.17%, and 89.00%, respectively [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In the present study, the examination of the green triticale plantlets treated with a 0.1% colchicine solution for 5 h revealed that more than half of the plantlets had withered leaf tips after 1 week, while some of the plantlets transferred to plastic pots gradually withered, and the survival rate of green plantlets was 82.7%. When the chromosome doubling time was decreased to 4.5 h, the number of withered plantlets decreased. These findings may reflect the toxicity of colchicine. The detrimental effects of chromosome doubling chemical agents on plant survival have been reported [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, appropriately decreasing the chromosome doubling time can increase the survival rate of regenerated plants. The utility of colchicine has been assessed in terms of chromosome doubling efficiency and plant survival rates, but the potential toxicity of colchicine to plants and animals (including humans) has often been ignored, so researchers have recently started to screen for less toxic alternatives to colchicine [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Amiprophos-methyl and pronamid, which have been used for the chromosome doubling of maize haploid plants, may be better than other alternatives, with an overall success rate (16.1%) that is close to that (22.1%) of colchicine [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. For regenerating cucumber plants, the highest DH regeneration rates for colchicine and trifluralin are 58.33% and 83.33%, respectively, and the chromosome doubling rates for 750 mg/L colchicine and 50 mL/L trifluralin are reportedly 57.41% and 62.50%, respectively, implying trifluralin may be better than colchicine [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Although there are viable alternatives, colchicine is still used in many laboratories for the chromosome doubling of haploid plants.\u003c/p\u003e \u003cp\u003eDifferent colchicine concentrations and treatment times influenced the chromosome doubling rate of the regenerated green seedlings. In our experiment, the roots of the regenerated plants were immersed in a 0.1% colchicine solution for 5 h. Nineteen of the 36 regenerated plants were verified as DH plants, which is lower than the corresponding rates in earlier studies by Slusarkiewicz-Jarzina et al. and Ferrie et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. More specifically, 215 regenerated haploid triticale plantlets were treated with a 4% colchicine solution for 6 h under light at 25\u0026deg;C, which resulted in 128 DH plantlets (i.e., chromosome doubling efficiency of 59.9%) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The highest haploid transformation rate (80%) for regenerated oat plants was obtained following a 4-h treatment with 0.2% colchicine [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Moreover, soaking the roots of maize seedlings in 0.10% and 0.70% colchicine solutions for 5 h can result in chromosome doubling efficiencies of 31.83% and 27.67%, respectively [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Clearly, colchicine concentrations and treatment times may need to be adjusted to maximize the chromosome doubling efficiency.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, an improved method for producing DH plants was developed for awnless triticale (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). This method resulted in significant increases in CIR. A total of 620 calli and 57 regenerated green plants were obtained for genotype T2020-6337. Of the 36 regenerated green plants that were grown in the field, 19 were confirmed as DH plants. The highest CIR was obtained by pretreating anthers for 15 days at 4\u0026deg;C prior to the inoculation of CHB medium containing 1.5 mg/L 2,4-D and 1.5 mg/L KT. In addition, the effects of the duration of the low-temperature pretreatment as well as the hormone types and concentrations on CIR, DFG, DFA, PRG, PRA, and PRR should be considered for triticale anther culture experiments. Furthermore, DFG and PRR must be further improved and methods for decreasing DFA are needed. Future research should also evaluate the utility of this breeding protocol for enhancing other triticale genotypes.\u003c/p\u003e \u003cp\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\u0026prime;,6-Diamidino-2-phenylindole dihydrochloride\u003c/p\u003e\n\u003cp\u003eABA \u0026nbsp; \u0026nbsp;Abscisic acid\u003c/p\u003e\n\u003cp\u003eIAA \u0026nbsp; \u0026nbsp; Indoleacetic acid\u003c/p\u003e\n\u003cp\u003e6-BA \u0026nbsp; \u0026nbsp;N6-benzyladenine\u003c/p\u003e\n\u003cp\u003eNAA \u0026nbsp; \u0026nbsp;Naphthalene acetic acid\u003c/p\u003e\n\u003cp\u003eBA \u0026nbsp; \u0026nbsp; \u0026nbsp;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\u0026nbsp;plantlet\u0026nbsp;differentiation frequency\u003c/p\u003e\n\u003cp\u003ePPG \u0026nbsp; \u0026nbsp;Green\u0026nbsp;plantlet production\u003c/p\u003e\n\u003cp\u003ePPA \u0026nbsp; \u0026nbsp;Albino\u0026nbsp;plantlet production\u003c/p\u003e\n\u003cp\u003ePRR \u0026nbsp; \u0026nbsp;Plant regeneration rate\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 and FZ 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. 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\u003c/p\u003e\n\u003cp\u003eWe 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) 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\u003cli\u003e\u003cspan\u003eEvtushenko EV, Lipikhina YA, Stepochkin PI, Vershinin AV. 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Cereal Res Commun. 2003;31:289\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/9780851996530.0447\u003c/span\u003e\u003cspan address=\"10.1079/9780851996530.0447\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triticale, Anther culture, Callus, Doubled haploid, Flow cytometry","lastPublishedDoi":"10.21203/rs.3.rs-4608942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4608942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCompared to traditional breeding methods, anther culture method is an effective method for quickly obtaining homozygotes within one generation. The method of cultivating double haploid plants with the anthers of awnless triticale was studied and optimized.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e Young awnless triticale spikes were pretreated at 4\u0026deg;C for 5, 10, 15, 20, or 25 days, and the anthers were cultured on four CHB media with varying hormone concentrations. The callus induction rate (CIR) was highest (28.54%) for A3B3 (anthers pretreated for 15 days and CHB medium containing 1.5 mg/L 2,4-D and 1.5 mg/L KT). The green plantlet differentiation frequency (DFG) was highest (30.20%) for A5B1 (25-days pretreatment and CHB medium containing 0.5 mg/L 2,4-D and 0.5 mg/L KT). The green plantlet production (PPG) was highest (7.98%) for A2B1 (10-days\u0026thinsp;+\u0026thinsp;0.5 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;0.5 mg/L KT). The success rate of chromosome doubling for the regenerated green plantlets was 52.8%. Appropriately decreasing the chromosome doubling time may increase the survival rate of the regenerated plants. Ten of the nineteen doubled haploid plants had tip and side awns shorter than 5 mm, implying they may be used for cultivating awnless triticale.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e The anther culture technology of triticale was optimized in this paper, which made it possible to rapidly breed homozygous varieties of awnless triticale, and also accelerated the breeding program of new varieties of awnless triticale.\u003c/p\u003e","manuscriptTitle":"Effects of the duration of a low-temperature pretreatment and hormone concentrations on anther cultures and the regeneration of awnless triticale","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-11 06:53:12","doi":"10.21203/rs.3.rs-4608942/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ccf6acf-2036-4244-a1e0-7c6ebdaac3df","owner":[],"postedDate":"July 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-04T15:36:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-11 06:53:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4608942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4608942","identity":"rs-4608942","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}