Method for creating hybrid sweetgum germplasm by EMS mutagenesis

Abstract

The invention belongs to the technical field of breeding of liquidambar formosana, and particularly relates to a method for creating hybrid liquidambar formosana germplasm through EMS mutagenesis. According to the method, hybridized sweetgum embryogenic callus obtained through callus induction and subculture is used as an in-vitro mutagenesis material, the hybridized sweetgum embryogenic callus is soaked in a phosphate buffer solution containing EMS with the mass concentration being 0.1%-1.0% to be treated for 1-10 h, after somatic embryogenesis culture is conducted, the mutagenized callus is differentiated into a plant, transplanting is conducted after rooting culture, and the hybridized sweetgum embryogenic callus is obtained. Therefore, a large number of hybridized sweetgum mutants are obtained. According to the method, the EMS mutagenic agent is applied to breeding of liquidambar formosana species for the first time, the survival rate of calluses is remarkably increased while high mutagenesis efficiency is guaranteed, the breeding period of a new variety of traditional hybrid liquidambar formosana is shortened, and breeding efficiency is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of liquidambar breeding, and particularly relates to a method for creating hybrid liquidambar germplasm by EMS mutagenesis. BACKGROUND

[0002] Liquidambar formosana Hance is a medicinal plant, and its resin can be used for medicine and has the effects of detoxification, pain relief, hemostasis and skin regeneration. Its roots, leaves and fruits can also be used as medicine and have the effects of dispelling wind and dampness, unblocking collaterals and promoting blood circulation. Its fruits are also known as road road, and the Chinese Pharmacopoeia records that it has the effects of dispelling wind and activating collaterals, promoting water and unblocking channels. Its leaves can detoxify and stop bleeding, and are used to treat acute gastroenteritis, dysentery, postpartum wind and the like. The whole plant of Liquidambar formosana Hance can be used as medicine, but large-scale collection of materials is not conducive to its health and sustainable growth, and it is difficult to meet the market demand.

[0003] Traditional systematic breeding methods have low natural mutation rate, cannot create new genotypes, have limited potential for improvement and enhancement, and are difficult to improve breeding efficiency. Plant mutagenesis technology can accelerate genetic variation of species, so that mutant plants with utilization value can be obtained in a short time, and provide conditions for breeding new varieties, new germplasm and related functional gene research. Radiation breeding is a common new variety breeding method at present, and usually uses Co-γ rays for radiation. Its offspring has high mutation rate and stable mutation traits, and can obtain mutant plants in a short time. 60 In comparison, chemical mutagenesis breeding has the characteristics of low cost, simple operation, high gene recombination rate and short cycle. As a mutagen, ethyl methanesulfonate (EMS) can induce high-density allelic point mutations. Although EMS mutagenesis technology has been widely used in the breeding of herbaceous plants such as crops and flowers, its application in the mutagenesis breeding of woody plants Liquidambar formosana Hance has not been reported and needs further research. SUMMARY

[0004] In order to solve the above problems, the application provides a method for creating hybrid Liquidambar formosana Hance germplasm by EMS mutagenesis. The method provided by the application first applies EMS mutagen to the breeding of Liquidambar species, ensures high mutagenic efficiency, significantly improves the survival rate of callus, shortens the breeding cycle of traditional hybrid Liquidambar formosana Hance new varieties, and improves the breeding efficiency.

[0005] To achieve the above purpose, the specific technical scheme of the application is as follows: The application provides a method for creating hybrid Liquidambar formosana Hance germplasm by EMS mutagenesis, which comprises the following steps: The hybrid maple embryo callus obtained through the induction and subculture is inoculated into an embryo callus propagation culture medium to obtain the mutagenic material; the embryo callus propagation culture medium is based on a modified Blaydes basic culture medium and further includes 1g of hydrolyzed casein, 40g of sucrose, 2.5g-3g of phytagel, 0.5mg-2mg of 2,4-D and 0mg-1mg of 6-BA per liter of the medium; The mutagenic material is immersed in a phosphate buffer containing EMS with a mass concentration of 0.1%-1.0% for 1h-10h, and then subjected to somatic embryogenesis culture to differentiate the mutagenized callus into plants, and the plants are subjected to rooting culture and then transplanted to obtain the mutant of the hybrid maple.

[0006] Further, the embryo callus propagation culture medium is based on a modified Blaydes basic culture medium and further includes 1g of hydrolyzed casein, 40g of sucrose, 2.5g-3g of phytagel, 0.5mg-2mg of 2,4-D and 0.25mg-1mg of 6-BA per liter of the medium.

[0007] Further, the somatic embryogenesis culture is to inoculate the EMS mutagenized callus into a somatic embryogenesis culture medium for culture; the somatic embryogenesis culture medium is based on a modified Blaydes basic culture medium and further includes 30g-40g of sucrose and 2g-5g of phytagel per liter of the medium.

[0008] Further, the somatic embryogenesis culture medium is based on a modified Blaydes basic culture medium and further includes 40g of sucrose and 3g of phytagel per liter of the medium.

[0009] Further, the somatic embryogenesis culture is carried out in dark at a temperature of 23℃-27℃.

[0010] Further, the rooting culture is to inoculate the differentiated plants into a modified Blaydes basic culture medium for culture.

[0011] Further, the rooting culture is carried out for 15d-25d.

[0012] Further, the embryo callus propagation culture is carried out in dark at a temperature of 23℃-27℃ for 3 weeks-4 weeks.

[0013] Further, the mutagenesis is to increase the content of phenolic substances in the hybrid maple.

[0014] Further, the hybrid maple is obtained through hybridization of Acer saccharum as the female parent and Acer truncatum as the male parent.

[0015] Compared with the prior art, the present application has the following beneficial effects: The application provides a method for creating hybrid maple germplasm by EMS mutagenesis, and the hybrid maple embryogenic callus obtained through callus induction and subculture is inoculated into embryogenic callus proliferation medium to obtain mutagenized materials; then the mutagenized materials are immersed in a phosphate buffer containing EMS with a mass concentration of 0.1% to 1.0% for 1 to 10 hours, and then subjected to somatic embryogenesis culture to differentiate the mutagenized callus into plants and subjected to rooting culture and transplanting to obtain a large number of hybrid maple mutants. The EMS mutagenesis technology is applied to the hybrid maple embryogenic callus for the first time, and combined with the somatic embryogenesis culture technology, a breakthrough progress in genetic improvement of the hybrid maple is achieved. First, the EMS mutagenesis parameter system of the hybrid maple is established, the EMS treatment concentration and time are systematically screened, and the semi-lethal dose (LD 50 ) is determined, which ensures a high mutagenesis efficiency and significantly improves the survival rate of the callus, thereby providing a stable material basis for subsequent mutant screening and variety breeding. Second, the synergistic application of the somatic embryogenesis culture technology and the EMS mutagenesis technology makes the regeneration and screening process of the mutants more efficient. The high-efficiency regeneration characteristics of the somatic embryogenesis culture combined with the high-density allelic point mutations generated by the EMS mutagenesis not only significantly shorten the breeding period of the new hybrid maple variety, but also improve the breeding efficiency, avoid the cumbersome process of multiple generations of screening in the traditional method, and directly regenerate plants from the mutagenized callus to achieve the goal of rapid screening and creation of new varieties.

[0016] In addition, the application has significant advantages in technical application. On the one hand, the low cost and simple operation of the EMS mutagenizing agent enable the germplasm innovation of the hybrid maple to be carried out on a large scale, greatly reducing the cost of new variety breeding and having a broad application prospect. On the other hand, the mutations generated by the EMS mutagenesis are rich and stable, mainly point mutations, and less chromosomal aberrations, which do not easily cause plant adverse variation and physiological damage, and provide rich genetic variation materials for specific trait improvement of the hybrid maple. At the same time, most of the EMS mutagenesis are dominant mutants, which are easy to screen in early generations, simplify the mutant screening process, and further improve the breeding efficiency. In addition, the high mutagenesis efficiency and wide mutation range of the EMS mutagenesis technology can increase the mutation frequency by 10 to 100 times, can be used for directional improvement of specific traits, and effectively overcomes the problems of low natural mutation rate and inability to create new genotypes, thereby opening up a new path for genetic improvement of the hybrid maple. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without any creative effort.

[0018] Figure 1 The somatic embryo seedlings after greenhouse domestication.

[0019] Figure 2 The statistical diagram of the influence of EMS solutions with different mass concentrations on the survival rate of different test materials.

[0020] Figure 3 The statistical diagram of the influence of EMS solutions with different mass concentrations on the differentiation rate of different test materials.

[0021] Figure 4 The state of somatic embryo maturation of test material A for 50 days under the concentration of 0 w / v% EMS.

[0022] Figure 5 The state of somatic embryo maturation of test material A for 30 days under the concentration of 0.6 w / v% EMS.

[0023] Figure 6 The abnormal somatic embryos generated by callus under different EMS concentrations, and the corresponding EMS solution concentrations from left to right in the diagram are 0.4 w / v%, 0.6 w / v%, 0 w / v%, and 0.8 w / v%, respectively.

[0024] Figure 7 The cotyledon embryo contrast diagram of test material A after being treated with 0.4 w / v% EMS solution for different time, and the corresponding treatment time on the left is 0 h, and the corresponding treatment time on the right is 4 h.

[0025] Figure 8 The total phenol content analysis diagram of the hybrid Feng potential mutation system. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application will be described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort belong to the protection scope of the present application. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] The hybrid sweetgum in the present application is obtained by hybridization of the female parent Liquidambar styraciflua and the male parent sweetgum elite tree. The specific plant information is disclosed in the patent with the publication number CN110896853A. The formula of the improved Blaydes medium in the present application is specifically disclosed in: Merkle SA, Neu KA, Battle PJ, Bailey RL. 1998. Somatic embryogenesis and plantlet regeneration from immature and mature tissues of sweetgum (Liquidambar styraciflua). Plant Science 132: 169-178.

[0028] Each liter of induction medium is based on the improved Blaydes basic medium, and further includes 1 g of hydrolyzed casein, 40 g of sucrose, 3 g of phytagel, 1 mg of 2,4-D and 0.5 mg of 6-BA, with a pH value of 5.8.

[0029] Each liter of embryogenic callus proliferation medium is based on the improved Blaydes basic medium, and further includes 1 g of hydrolyzed casein, 40 g of sucrose, 3 g of phytagel, 1 mg of 2,4-D and 0.5 mg of 6-BA, with a pH value of 5.8.

[0030] Each liter of somatic embryogenesis medium is based on the improved Blaydes basic medium, and further includes 40 g of sucrose and 3 g of phytagel, with a pH value of 5.8.

[0031] Sweetgum is a multifunctional medicinal plant, and its resin, roots, leaves and fruits all have medicinal value, but sustainable collection faces challenges. Traditional breeding methods have limited improvement potential, while plant mutagenesis techniques, especially radiation and chemical mutagenesis, can accelerate genetic variation and improve breeding efficiency. EMS mutagenic agents in chemical mutagenesis are widely used in herbaceous plant breeding, but the mutagenic breeding potential of sweetgum, a woody plant, has not been fully explored, and has important application prospects.

[0032] The application provides a method for creating hybrid maple germplasm by EMS mutagenesis, and the hybrid maple embryogenic callus obtained through callus induction and subculture is used as the in vitro mutagenesis material. The hybrid maple embryogenic callus is immersed in a phosphate buffer containing 0.1% to 1.0% EMS for 1 to 10 hours, then subjected to somatic embryogenesis culture, so that the mutagenized callus is differentiated into plants and roots, and then transplanted, thereby obtaining a large number of hybrid maple mutants. The EMS mutagen is applied to the hybrid maple embryogenic callus for the first time, and the method provided by the application can ensure high mutagenesis efficiency, significantly improve the survival rate of callus, shorten the breeding cycle of hybrid maple new varieties, and improve the breeding efficiency.

[0033] Example 1: A method for creating hybrid maple germplasm by EMS mutagenesis 1. Obtaining embryogenic callus After the immature zygotic embryos of the hybrid maple are used as explants and cultured on the induction medium for 3 weeks, the growth rate of the explants begins to slow down and they begin to brown. At this time, the embryogenic callus is selected for subculture, and is transferred to the subculture medium and cultured in the dark at 23-27°C for repeated subculture (subculture every 3 weeks, for 2 times). The subcultured explants (typical embryogenic callus) are transferred to the embryogenic callus proliferation medium and cultured in the dark at 23-27°C. Subculture is performed every 3 weeks to a new embryogenic callus proliferation medium for a total of 2 times.

[0034] The subcultured explants are inoculated on the somatic embryogenesis medium and cultured in the dark at 23-27°C for 10 days and 20 days to obtain embryogenic callus, which is respectively recorded as the 10-day somatic embryogenesis culture material (B) and the 20-day somatic embryogenesis culture material (C). The explants not inoculated on the somatic embryogenesis medium are recorded as the 0-day somatic embryogenesis culture material (A).

[0035] 2. Preparation of EMS solution Under sterile conditions, the EMS stock solution is filtered and sterilized through a 0.22 μm microporous filter using a disposable syringe, and then injected into a high-pressure sterilized 0.01 mol / L, pH 7.0 phosphate buffer to prepare EMS solutions with concentrations of 0.1 w / v%, 0.2 w / v%, 0.4 w / v%, 0.6 w / v%, 0.8 w / v%, 1.0 w / v%, 1.2 w / v%, and 1.4 w / v%.

[0036] 3. Mutagenesis treatment of embryogenic callus Proliferation culture stage: 0.5 g of test materials A, B and C were respectively soaked in different concentrations of EMS solution on a sterile workbench, and the control was treated with phosphate buffer solution without EMS. After 2 h of soaking at each concentration, the test materials were washed with sterile water, pumped with a filter pump, inoculated with filter paper in embryogenic callus proliferation culture medium, and cultured in the dark at a temperature of 23-25°C. Each treatment was repeated 3 times.

[0037] Observation once a week, after 20 d of culture, the relative survival rate of the test materials was calculated to determine the semi-lethal dose (LD 50 ).

[0038] Cell survival rate detection: the test materials treated with different concentrations of EMS were placed in 10 mL test tubes, 2.5 mL of 0.4% TTC and 2.5 mL of 0.1M phosphate buffer solution with a pH of 7.0 were added and mixed, and then incubated in the dark at 22-26°C for 24 h. After 24 h, the supernatant was removed, washed with distilled water for 3 times, and then 10 mL of 95% ethanol was added and incubated in a constant temperature water bath at 65°C for 30 min. After cooling, the supernatant was taken and the absorbance value of the supernatant at 485 nm was measured by Shimadzu UV2550 spectrophotometer, repeated 3 times. The normal subcultured embryogenic tissue was used as a control, and the relative cell survival rate was calculated.

[0039] Relative cell survival rate (%) = TTC value of treated cells / control TTC value x 100%.

[0040] Embryogenic culture stage: the test materials treated by EMS mutagenesis were inoculated on somatic embryogenesis culture medium and cultured in the dark, and the culture temperature was 23-27°C. During the somatic embryogenesis culture of hybrid Altingia, the growth and morphology of somatic embryos were observed, and the somatic embryogenic differentiation rate was calculated.

[0041] Somatic embryogenic differentiation capacity (number / g) = number of differentiated somatic embryos / fresh weight of inoculated embryogenic callus.

[0042] 4. Rooting and transplanting of regenerated seedlings The differentiated plants were transferred to modified Blaydes basic medium without plant growth regulators for rooting culture, and then transplanted to flowerpots containing sandy soil after 15 d to obtain a large number of hybrid Altingia mutants (M Figure 1 ).

[0043] Example 2: Screening of mutagenesis conditions and identification of mutants 1. Screening of EMS mutagenesis concentration and test material combination In this study, three different maturity levels of hybrid sweetgum embryogenic calli (A: no culture; B: 10 days of culture; C: 20 days of culture) were compared for their response to different concentrations of EMS treatment. As shown in Table 1, Figure 2 and Figure 3 With the increase of EMS concentration, the relative survival rate and somatic embryo differentiation rate of all tested materials showed a downward trend.

[0044] Table 1 Relative survival rate and somatic embryo differentiation rate of different tested materials under different EMS concentration treatments As can be seen from Table 1, Figure 2 and Figure 3 Material A is the most sensitive to EMS treatment, with a relative survival rate of 54.3% ± 1.0% at 0.4% EMS concentration, close to the semi-lethal dose. In contrast, the survival rates of materials B and C under the same concentration were 76.4% ± 0.1% and 73.3% ± 1.0%, respectively, showing strong tolerance.

[0045] Figure 4 Figure 1 shows the state of somatic embryo maturation of tested material A at 0w / v% EMS concentration after 50 days, Figure 5 Figure 2 shows the state of somatic embryo maturation of tested material A at 0.6w / v% EMS concentration after 30 days, Figure 6 Figure 3 shows the abnormal somatic embryos produced by calli under different EMS concentration treatments.

[0046] As can be seen from Figure 4 and Figure 5 After treatment with 0.6w / v% EMS concentration, the embryogenic calli were severely browned, but some white spherical embryos were also produced. As can be seen from Figure 6 The somatic embryos treated with EMS grew faster and had higher resistance, but were prone to produce abnormal embryos.

[0047] The somatic embryo differentiation ability was also significantly affected by EMS concentration. Under 0.4% EMS treatment, the differentiation rates of tested materials A, B, and C were 456.4 / g ± 12.3 / g, 507.6 / g ± 25.3 / g, and 632.2 / g ± 23.9 / g, respectively. It is worth noting that although tested material C maintained a high survival rate at higher EMS concentration, its differentiation ability decreased sharply with increasing concentration.

[0048] According to the principle of semi-lethal dose, the most suitable EMS concentration is 0.4w / v% combined with tested material A as the optimal mutagenesis condition, which ensures a sufficient mutation rate while maintaining good regeneration ability.

[0049] 2. Screening of mutagenesis time 2.1, total phenol content determination method The embryogenic callus after different treatment times was washed with sterile water, and after filtration with a filtration pump, the test material was inoculated with filter paper in embryogenic callus proliferation medium, and dark culture was carried out for 40 d, with subculture once every 20 d, and especially at the time of subculture, well-growing embryogenic callus was selected, and the culture temperature was 23-25°C. Well-conditioned embryogenic callus was selected for total phenol content determination.

[0050] Preparation of gallic acid standard solution: 0.1 g of gallic acid was accurately weighed, dissolved with ultrapure water, and diluted to 100 mL to obtain a gallic acid standard solution with a mass concentration of 1 mg / mL. 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the gallic acid standard solution were taken in a 10 mL volumetric flask, and ultrapure water was added to make up the volume, obtaining a series of standard solutions with mass concentrations of 0 mg / L, 20 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L.

[0051] Extraction of total phenol from hybrid maple embryogenic callus: 0.500 g of embryogenic callus was accurately weighed in a mortar and ground, and the ground sample was placed in a 10 mL centrifuge tube. 5 mL of methanol containing 0.1% hydrochloric acid was added, and ultrasonic extraction (300 W) was carried out at room temperature for 1 h. The extract was placed in the dark at 4°C for 24 h, and the clean supernatant was poured into a 20 mL collection bottle and stored in the dark at 4°C. 5 mL of methanol containing 0.1% hydrochloric acid was added again, and ultrasonic extraction was repeated three times. The combined extracts were evaporated to dryness by rotary evaporation at 40°C under reduced pressure, and then transferred to a 10 mL volumetric flask and diluted to the mark with ultrapure water.

[0052] Determination of total phenol in hybrid maple embryogenic callus: 0.5 mL of the gallic acid standard solution was accurately taken according to the gradient increase in a 10 mL cuvette, 2.0 mL of diluted Folin phenol (10% v / v) reagent was added, and after 5 min, 2.5 mL of 7.5% sodium carbonate solution was added. After standing for 1 h, the absorbance was measured at a wavelength of 765 nm, and a standard curve was drawn. The mixture of 0 mg / L gallic acid standard solution and reagent was set as blank. The determination method of the total phenol extract of hybrid maple was consistent with that of the gallic acid standard solution. The measured absorbance was substituted into the standard curve to obtain the concentration of total phenol in the embryogenic callus of hybrid maple, and the total phenol content was calculated using the following formula:

[0053] In the formula, C represents the measured total phenol concentration (mg / L); V T represents the total volume of the extract (mL); N represents the dilution factor; M represents the sample mass (g); 2.2, Screening of mutagenesis time Based on the optimal EMS concentration of 0.4 w / v%, the tested material A was used as the experimental material, and the effects of different EMS mutagenesis times on mutagenesis results were determined according to the above steps of embryogenic callus mutagenesis treatment.

[0054] Figure 7 Figure 2 is a comparison chart of cotyledon embryos of the tested material A after being treated in 0.4 w / v% EMS solution for different times. The left side corresponds to a treatment time of 0 h, and the right side corresponds to a treatment time of 4 h.

[0055] From Figure 7 It can be seen that after being treated in 0.4 w / v% EMS solution for 4 h, the hypocotyls of the cotyledon embryos of the control group are significantly elongated, indicating that 0.4 w / v% EMS solution treatment for 4 h can promote the formation of total phenols, further improve the stress resistance of somatic embryos, and promote the maturation of somatic embryos.

[0056] Table 2 Effects of different EMS solution treatment times on induction results The statistical results of the effects of different EMS solution treatment times on induction results are shown in Table 2. It can be found that the total phenol content of the callus after 4 hours of treatment is higher, so more mutants can be obtained relative to 2 h of treatment. The survival rate and differentiation rate of the tested material gradually decrease with the extension of the EMS treatment time: after 4 hours of treatment, the survival rate decreases to 44.0% ± 3.7%, and the differentiation rate decreases to 384.0 / g ± 24.0 / g. After 8 hours of treatment, these two indicators further decrease to 15.5% ± 2.5% and 75.0 / g ± 3.0 / g.

[0057] From Figure 8 It can be seen that the total phenol content changes in a trend of first increasing and then decreasing: when treated for 4 hours, the total phenol content reaches a peak of 38.0 mg / g ± 2.0 mg / g, which is significantly higher than that of the control (18.3 mg / g ± 2.0 mg / g) and other treatment groups (P < 0.01). This result indicates that 4 hours of treatment can effectively induce callus to produce a stress response and promote the accumulation of phenolic substances, thereby possibly increasing the mutation frequency. P <0.01). This result indicates that 4 hours of treatment can effectively induce callus to produce a stress response and promote the accumulation of phenolic substances, thereby possibly increasing the mutation frequency.

[0058] Considering the mutation efficiency and material survival rate, 4 hours is the optimal EMS treatment time, which can obtain the highest mutation frequency while maintaining a certain regeneration ability.

[0059] 3, Mutant screening Through the above optimal conditions (EMS concentration is 0.4 w / v %, the tested material A, treatment time is 4h), 20 potential mutant systems are obtained, which are respectively marked (T1-T20) and inoculated into the induction medium to re-induce the embryonic callus, and then 0.5g of the T1-T20 embryonic callus is taken to determine the total phenol content. As shown in Table 3 and Figure 3 The total phenol content of the mutant system is significantly different.

[0060] Table 3 Determination results of the total phenol content of the mutant system Through the determination of the total phenol content of the 20 lines, the content of 6 lines is more than 1 times of the control, so 6 mutants are screened.

[0061] The mutant screening takes 2 times (36.6 mg / g) of the total phenol content of the control (CK) as the threshold, and 6 high-phenol mutants are identified, including T3 (44.8 mg / g± 0.6 mg / g), T5 (42.2 mg / g± 0.5 mg / g), T10 (40.2 mg / g± 0.5 mg / g), T11 (40.5 mg / g± 0.5 mg / g), T15 (38.5 mg / g± 0.4 mg / g) and T19 (34.3 mg / g± 0.5 mg / g). The total phenol content of these mutants is significantly higher than that of the control (P<0.01), indicating that the EMS treatment successfully induces gene mutation and affects the phenolic metabolic pathway. P <0.01), indicating that the EMS treatment successfully induces gene mutation and affects the phenolic metabolic pathway.

[0062] The mutation rate analysis shows that the frequency of the beneficial mutants obtained by the method provided in the application is 30% (6 / 20), which proves that the mutagenesis system has high efficiency. These high-phenol mutants provide valuable material basis for further studying the secondary metabolic regulation mechanism of hybrid maple and breeding excellent new varieties.

[0063] It should be noted that when the numerical range is involved in the application, it should be understood that each numerical range of the two endpoints and any number between the two endpoints can be selected. Since the same steps and examples are adopted, in order to prevent repetition, the preferred embodiments are described in the application. Although the preferred embodiments of the application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the application.

[0064] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A method for creating a hybrid red maple germplasm using EMS mutagenesis, characterized in that, The method comprises the following steps: The hybrid maple embryogenic callus obtained through the induction of callus and subculture is inoculated on an embryogenic callus proliferation culture medium to obtain mutagenized materials; the embryogenic callus proliferation culture medium is based on a modified Blaydes basic culture medium and further comprises 1 g of hydrolyzed casein, 40 g of sucrose, 2.5 g to 3 g of plant gel, 0.5 mg to 2 mg of 2,4-D and 0 mg to 1 mg of 6-BA per liter of the medium; The mutagenized materials are immersed in a phosphate buffer containing EMS with a mass concentration of 0.1% to 1.0% for 1 h to 10 h, then subjected to somatic embryogenesis culture to differentiate the mutagenized callus into plants, and subjected to rooting culture and then transplanting to obtain the mutants of the hybrid maple.

2. The method of claim 1, wherein, The somatic embryogenesis culture is to inoculate the EMS mutagenized embryogenic callus on a somatic embryogenesis culture medium; the somatic embryogenesis culture medium is based on a modified Blaydes basic culture medium and further comprises 30 g to 40 g of sucrose and 2 g to 5 g of plant gel per liter of the medium.

3. The method of claim 2, wherein, The somatic embryogenesis culture medium is based on a modified Blaydes basic culture medium and further comprises 40 g of sucrose and 3 g of plant gel per liter of the medium.

4. The method of claim 2, wherein, The somatic embryogenesis culture is performed in darkness at a temperature of 23 ℃ to 27 ℃.

5. The method of claim 1, wherein, The rooting culture is to inoculate the differentiated plants on a modified Blaydes basic culture medium.

6. The method of claim 5, wherein, The rooting culture is performed for 15 d to 25 d.

7. The method of claim 1, wherein, The embryogenic callus proliferation culture is performed in darkness at a temperature of 23 ℃ to 27 ℃ for 3 weeks to 4 weeks.

8. The method of claim 1, wherein, The mutagenesis is to increase the content of phenolic substances in the hybrid maple.

9. The method of claim 1, wherein, The hybrid maple is obtained through hybridization of Acer saccharum as the female parent and Acer truncatum as the male parent.

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