Method for creating Rhododendron pulchrum mutant aseptic seedling by combining EMS mutagenesis and tissue culture

Abstract

The invention discloses a method for creating a rhododendron pulchrum mutant aseptic seedling by combining EMS mutagenesis and tissue culture. The method specifically comprises the following steps: (1) determination of EMS semi-lethal dose: preparing EMS solutions with different concentrations, and soaking rhododendron pulchrum seeds in the EMS solutions for mutagenesis to obtain soaked seeds; the soaked seeds are treated, the EMS solution left on the surfaces is removed, and mutagenesis seeds are obtained; placing the mutagenic seeds on wet filter paper in a container, and carrying out dark culture and germination acceleration; the germination rate and the germination potential are counted, and the EMS semi-lethal dose concentration is obtained; and (2) early screening of the rhododendron pulchrum mutant strain: carrying out seed soaking treatment by using the EMS solution with the semi-lethal dose concentration, and screening according to the phenotypic growth index of the rhododendron pulchrum seedling to obtain the rhododendron pulchrum mutant strain. According to the application, the genetic diversity of the rhododendron pulchrum is enriched, the existing development difficulty of the rhododendron pulchrum is broken through, and the wide application of the rhododendron pulchrum in landscape architecture is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of mutagenesis breeding, and specifically to a method for creating axenic seedlings of Rhododendron fortunei mutants by combining EMS mutagenesis and tissue culture. Background Art

[0002] Rhododendron fortunei Lindl. is a wild rare plant with extremely high ornamental value and utilization value. It belongs to the Subg. Hymenanthes of the genus Rhododendron in the family Ericaceae. Rhododendron fortunei is distributed in high-altitude areas with an altitude of 1000 - 2000m. It has the beautiful name of "thousand-flower rhododendron" and is a garden tree species with extremely high ornamental value and great development potential. Currently, with the increasing demand for landscaping and the construction of park cities, the introduction, domestication, development, and utilization of alpine rhododendrons with extremely high ornamental value have become a research hotspot in the genus Rhododendron. As a kind of alpine rhododendron, due to its high distribution altitude, low seed germination rate (18.6%), and difficult cuttage propagation, it is extremely difficult to introduce Rhododendron fortunei, which restricts its development and application in gardens.

[0003] Currently, the development and utilization of Rhododendron fortunei face many challenges, which are mainly reflected in the following aspects: (1) Difficulty in introducing Rhododendron fortunei at low altitudes. Rhododendron fortunei is distributed in high-altitude areas with an altitude of 1000 - 2000m, and it has poor heat resistance and is difficult to be successfully introduced at low altitudes; (2) Difficulty in the reproduction of Rhododendron fortunei. Rhododendron fortunei is difficult to root by cuttage, and the extremely low seed germination rate limits its development and utilization; (3) It is difficult to improve Rhododendron fortunei with current traditional breeding methods. Currently, the breeding of Rhododendron fortunei mainly relies on natural hybridization and selection. Not only is the cycle long, but also the sources of variation are limited, and it is difficult to cultivate new varieties with breakthrough excellent traits in a short time. At the same time, the success rate of natural hybridization is low due to reproductive isolation problems. Secondly, natural hybridization is easily interfered by environmental factors, and the segregation of offspring traits is complex, further increasing the difficulty of screening excellent varieties; (4) Lack of research on the tissue culture of Rhododendron fortunei. Although success has been achieved in the rapid propagation of a few Rhododendron species, there is no report on the successful tissue culture of Rhododendron fortunei.

[0004] In plant variety improvement and new variety breeding, mutagenesis breeding is an efficient means of inducing genetic variation in plants. The chemical mutagen ethyl methanesulfonate (EMS) is widely used due to its advantages such as convenient operation, high mutagenesis efficiency, and the ability to produce a rich variety of mutant types. Existing research has shown that treating Rhododendron aureum with EMS can improve its heat resistance; treating Vaccinium corymbosum L. with EMS can screen out drought-resistant mutants; in patent CN118489557A, EMS and hydroxyproline are used in combination for chemical mutagenesis of Phaseolus vulgaris stress-resistant mutants.

[0005] However, there is no relevant research on the treatment of Rhododendron fortunei Lindl. with EMS, nor is there any research on the mutagenesis breeding and rapid cultivation of Rhododendron fortunei Lindl. The research on combining the EMS mutagenesis of Rhododendron fortunei Lindl. with its rapid propagation is still blank. In particular, there is a lack of in-depth research on the key parameters such as the appropriate treatment concentration and treatment time of EMS mutagenesis for Rhododendron fortunei Lindl. seeds, as well as the systematic methods for obtaining tissue culture seedlings from mutagenized seeds through tissue culture technology and improving their propagation coefficient through rapid propagation technology to shorten the breeding cycle and improve the breeding efficiency. This has severely restricted the genetic improvement and new variety selection process of Rhododendron fortunei Lindl., and cannot meet the demand of the flower market for diverse and high-quality Rhododendron fortunei Lindl. varieties.

[0006] Therefore, there is an urgent need to develop a method for mutagenesis breeding of Rhododendron fortunei Lindl. using EMS and obtaining mutant plants by combining tissue culture technology, so as to enrich the genetic diversity of Rhododendron fortunei Lindl., break through the current dilemma of its development and utilization, and realize its wide application in landscape architecture. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a screening method for mutants of Rhododendron fortunei Lindl. based on EMS mutagenesis, which helps to enrich the genetic diversity of Rhododendron fortunei Lindl. and realize its wide application in landscape architecture.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] On the one hand, a method for creating axenic seedlings of mutants of Rhododendron fortunei Lindl. by combining EMS mutagenesis and tissue culture includes the following steps:

[0010] (1) Determination of the EMS semi-lethal dose

[0011] Prepare EMS solutions with different concentrations, soak the seeds of Rhododendron fortunei Lindl. in the EMS solutions with different concentrations for mutagenesis to obtain seeds soaked in different concentrations;

[0012] Remove the residual EMS solution on the surface of the seeds soaked in different concentrations to obtain the mutagenized seeds;

[0013] Place the mutagenized seeds on moist filter paper and incubate them in the dark for germination;

[0014] Count the germination rate and germination potential of the mutagenized seeds, select the concentration of the EMS solution that causes half of the mutagenized seeds to die, obtain the semi-lethal dose concentration of EMS, and determine the optimal conditions for EMS treatment;

[0015] (2) Early screening of mutants of Rhododendron fortunei Lindl.

[0016] Treat the seeds of *Rhododendron fortunei* with the EMS solution at the semi-lethal concentration, then wash and disinfect them, and then use the compound hormone technology and the light-temperature coupling regulation technology for rapid cultivation. Screen according to the phenotypic growth indexes of the *Rhododendron fortunei* seedlings, select the plant individuals with traits different from those of the parents, and obtain the mutant strains of *Rhododendron fortunei*;

[0017] Among them, the rapid cultivation includes initiation culture, proliferation culture, strong seedling culture, root growth promotion culture and seedling hardening and transplanting.

[0018] Preferably, in step (1), the EMS solution uses phosphate buffer as the solvent, and the concentrations are 0, 0.4%, 0.8%, 1.2%, 1.6%, 2.0%. The semi-lethal concentration of the EMS solution is 1.6%. The best effect of the seed soaking treatment is to oscillate in the dark at 23°C for 12 h, but the treatment duration in actual production can be 8-13 h;

[0019] Specifically, the phenotypic growth indexes in step (2) include leaf shape, leaf color, plant type, plant height, and leaf number;

[0020] Specifically, the cleaning and disinfection steps in step (2) are as follows: after the treatment, soak the seeds in 1.5% sodium thiosulfate for 5 minutes, and rinse with running water for 1 hour to remove the residual EMS solution on the seed surface; then wrap the seeds with gauze, soak them in 75% alcohol solution on the sterile operation table for 30 seconds, soak them in sterile water for 30 seconds, and then rinse them with sterile water 3 times. Then soak them in 2% NaClO solution for 6 minutes, soak them in sterile water for 30 seconds and then rinse them with sterile water 3 times. Subsequently, inoculate the seeds on the initiation medium for initiation culture;

[0021] Specifically, the proliferation culture step in step (2) is that when the treated seeds germinate and grow seedlings on the medium, transfer them to the proliferation medium for proliferation culture to grow into cluster buds;

[0022] Specifically, the strong seedling culture step in step (2) is to cut the cluster buds and transfer them to the strong seedling medium for strong seedling culture;

[0023] Specifically, the seedling hardening and transplanting step in step (2) is to cover the plug tray with the culture medium, plant the *Rhododendron fortunei* seedlings after the strong seedlings grow roots in the plug tray, place them in the artificial climate incubator, and water regularly;

[0024] Preferably, the ratio of nutrient soil: perlite: vermiculite in the culture medium for seedling hardening and transplanting is 1:1:1;

[0025] On the other hand, the rapid propagation method of the mutant strains of *Rhododendron fortunei* includes the compound hormone technology and the light-temperature coupling regulation technology;

[0026] The compound hormone technology includes:

[0027] Starting medium: basic medium WPM, 0.5 - 1.0 mg / L naphthaleneacetic acid, 0.5 - 1.0 mg / L zeatin, 30 g / L sucrose, 7 g / L agar, pH 5.6 - 5.8;

[0028] Proliferation medium: basic medium WPM, 1.0 - 2.0 mg / L naphthaleneacetic acid, 1.0 - 2.0 mg / L zeatin, 30 g / L sucrose, 7 g / L agar, pH 5.4 - 5.6;

[0029] Seedling strengthening medium: basic medium WPM, 1.0 - 2.0 mg / L naphthaleneacetic acid, 1.0 - 2.0 mg / L indoleacetic acid, 30 g / L sucrose, 7 g / L agar, pH 5.4 - 5.6;

[0030] Rooting medium: basic medium WPM, 2.0 mg / L indolebutyric acid, 30 g / L sucrose, 6.5 g / L agar, pH 5.4 - 5.6.

[0031] Specifically, the light - temperature coupling regulation technology includes:

[0032] During the starting culture, proliferation culture, seedling strengthening culture, and rooting culture of the Rhododendron fortunei, the culture light intensity is 1500 - 2000 Lx, the light time is 12 - 14 h / d, the temperature is 20 - 23 °C, and the humidity is 30% - 45%;

[0033] During the acclimatization and transplanting of the Rhododendron fortunei, the culture light intensity is 1500 - 1800 Lx, the light time is 12 - 14 h / d, the temperature is 20 - 24 °C, and the humidity is 40% - 45%.

[0034] On the one hand, the Rhododendron fortunei mutant screened and obtained in this application; the application of the obtained Rhododendron fortunei mutant plant in the breeding and variety improvement of new Rhododendron varieties.

[0035] The beneficial effects of the present invention are:

[0036] (1) Based on the EMS mutagenesis of Rhododendron fortunei seeds and combined with tissue culture technology, this application helps to enrich the genetic diversity of Rhododendron fortunei. This technology may have caused chromosomal mutations. Through the chemical mutagenesis technology of this application, the number of new bud proliferations has been increased, the rooting rate and acclimatization survival rate of plants have been effectively improved, and morphological variations such as leaf color, leaf shape, and stem of the seedlings have occurred.

[0037] (2) This application helps to improve the growth rate of Rhododendron fortunei, shorten the breeding cycle, and improve the breeding efficiency. Description of the Drawings

[0038] Figure 1Effects of EMS mutagenesis treatments of different qualities on seed germination of Rhododendron yunjinensis in Example 1;

[0039] Figure 2 Comparison of the growth status of the mutant plants and the non-mutated plants in Example 1;

[0040] Figure 3 Effects of EMS mutagenesis treatments of different qualities on seed germination of Rhododendron yunjinensis in Example 2;

[0041] Figure 4 Comparison of the growth status of the mutant plants and the non-mutated plants in Example 2;

[0042] Figure 5 Partial pictures of the ESM half-lethal concentration screening process (A is 12 hours of incubation at an ESM concentration of 0; B is 24 hours of incubation at an ESM concentration of 0);

[0043] Figure 6 This is a diagram of the seed germination process on the starting medium;

[0044] Figure 7 This is a diagram of the plant state after germination and before transfer to the proliferation medium;

[0045] Figure 8 is a diagram of the state of the plant in the proliferation medium;

[0046] Figure 9 This is a diagram of the plant status in the seedling culture medium;

[0047] Figure 10 rooting culture;

[0048] Figure 11 Seedlings out of the bottle;

[0049] Figure 12 Plug tray seedling hardening;

[0050] Figure 13 one of the primary mutants and the control group;

[0051] Figure 14 First-generation mutation - growing from red stem to red leaf (A first-generation mutation red stem; B first-generation mutation red leaf);

[0052] Figure 15 First generation mutation - branching;

[0053] Figure 16 Primary mutation - deformed curl;

[0054] Figure 17 First generation mutation - Red Leaf;

[0055] Figure 18 The first generation mutation had one light yellow leaf;

[0056] Figure 19 Primary mutation - leaf overlap;

[0057] Figure 20 The yellow leaf trait is stable and used as a control. Specific implementation manners

[0058] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following description.

[0059] The present invention will be described in detail below in conjunction with specific embodiments.

[0060] 1. Main reagents and drugs

[0061] The plant growth regulators naphthylacetic acid (NAA), zeatin (ZT), and indolebutyric acid (IBA) are all purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The disinfection reagents and all reagents, sucrose, agar powder, etc. in the basic culture medium formula are all domestic analytical pure.

[0062] 2. Culture environment

[0063] The inoculated Rhododendron fortunei seeds and seedlings need to be cultured and observed in a tissue culture room under specified light and temperature conditions. The culture outside the tissue culture bottle needs to be assisted by an artificial climate chamber.

[0064] Example 1

[0065] (1) Seed pretreatment

[0066] Select plump and healthy Rhododendron fortunei seeds (from Longchi Subalpine Botanical Garden), wrap them with gauze and soak them in normal temperature water, soak them in the dark for 12 hours to make them absorb water and germinate. Take out the gauze and hang it until it stops dripping, then soak it in 0.5% sodium hypochlorite solution for 8 minutes for disinfection, and then soak and rinse it with sterile water 3 times for preliminary disinfection treatment.

[0067] (2) Screening of the semi-lethal concentration and treatment time of EMS

[0068] Using 0.1 mol / L phosphate buffer solution with pH 7.0 as the solvent, prepare EMS solutions with concentrations of 0, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0%; soak the pretreated Rhododendron fortunei seeds in the EMS solution, seal it, and perform dark oscillation treatment at 24 °C for 12, 24, and 36 hours; after the mutagenesis treatment, soak the seeds in 1.5% sodium thiosulfate for 5 minutes and rinse them with running water for 1 hour to remove the residual EMS solution on the seed surface; inoculate the treated seeds on a culture dish lined with wet filter paper - the seeds of each concentration are evenly inoculated in 5 culture dishes, with 40 seeds in each culture dish. After labeling each culture dish with the treatment concentration, time, and group number, place it in a 24 °C culture room for dark culture and germination promotion. The blank control group uses phosphate buffer solution to soak the seeds for 12, 24, and 36 hours respectively, and then performs sowing using the same treatment method as above. Observe every day, and statistically calculate the germination rate and germination potential. Finally, obtain the semi-lethal dose and determine the optimal conditions for EMS treatment (EMS treatment concentration 1.6%, induction time 12 h). The specific results are as Figure 1 shown.

[0069] (3) Mutagenesis treatment of a large number of Rhododendron fortunei

[0070] In a fume hood, using 0.1 mol / L phosphate buffer solution with pH 7.0 as the solvent, prepare a 1.6% EMS solution; wrap the pretreated Rhododendron fortunei seeds with gauze, fully soak them in the EMS solution, and place them in the fume hood. Perform dark oscillation treatment at 24 °C at 80 r / min for 12 hours.

[0071] (4) Cleaning and disinfection

[0072] After the mutagenesis treatment, place the soaked seeds in a beaker containing 1.5% sodium thiosulfate solution and soak for 5 minutes to remove the residual EMS solution on the seed surface, and rinse with running water for more than 1 hour; then replace the gauze wrapping the mutagenized seeds, put them into a pre-sterilized sterile operating table, soak them in 75% alcohol solution for 30 seconds, soak them in sterile water for 30 seconds, then rinse with sterile water 3 times. After that, try to drain the water from the gauze wrapped with seeds, soak them in 2% NaClO solution for 6 minutes, soak them in sterile water for 30 seconds, and then rinse with sterile water 3 times. On the ultra-clean workbench, inoculate the disinfected seeds on the sterilized initiation medium. Inoculate 25 seeds in each tissue culture bottle. The initiation medium is WPM basic medium added with 0.5 mg / L naphthaleneacetic acid (NAA) and 0.5 mg / L zeatin (ZT). The pH of the medium is 5.8. Place the tissue culture bottles inoculated with seeds in a 25 °C tissue culture room and perform shading treatment to initiate the germination of the seeds. When the seeds germinate, transfer them to light culture. Under these conditions, the germination rate of Rhododendron fortunei seeds is 53.8%.

[0073] (5) Proliferation culture

[0074] When the treated seeds germinate and grow seedlings higher than 0.5 cm on the culture medium, cut them from the base and transfer them to a proliferation medium supplemented with 1.0 mg / L naphthaleneacetic acid (NAA), 2.0 mg / L zeatin (ZT) and a pH of 5.6 for proliferation culture. Place 5-10 seedlings in each bottle and allow them to grow into clustered buds.

[0075] (6) Seedling cultivation

[0076] After the seedlings sprout from the base and grow into dense clusters with the plants higher than 1 cm, cut each seedling and inoculate it into a seedling medium with 1.0 mg / L naphthaleneacetic acid (NAA) and 2.0 mg / L indolebutyric acid (IBA) and a pH of 5.5. Place no more than 5 seedlings in each bottle. After the plant grows to more than 2 cm as a whole and the base begins to turn brown and lignified.

[0077] (7) Rooting culture

[0078] (6) Plants were cut short at the base and transferred to a rooting medium containing 2.0 mg / L indolebutyric acid (IBA) and a pH of 5.5 for rooting. Care was taken not to insert the seedlings too deeply into the medium. The culture conditions were a light intensity of 1500 Lx, a photoperiod of 12 h / d, a temperature of 23°C, and a humidity of 45%. When the plants had developed a large number of fibrous roots, they could be hardened.

[0079] (8) Hardening and transplanting

[0080] Use a culture medium mixed with nutrient soil, perlite and vermiculite in a ratio of 1:1:1, cover the hole tray with the mixed medium, plant the Yunjin Rhododendron seedlings after they have grown strong roots in the hole tray, place them in an artificial climate incubator, and water them regularly. The culture conditions are a light intensity of 1500Lx, a light time of 10h / d, a temperature of 24℃, and a humidity of 40%.

[0081] (9) Mutant screening

[0082] During the cultivation process, the phenotypic growth indicators of the seedlings, such as plant height, number of leaves, and leaf shape, are observed, and individual plants with traits different from those of the parents are selected for cultivation, so that individual plants that can be stably inherited by offspring are obtained and proliferated.

[0083] Under the above conditions, a comparison found that after replacing the proliferation medium, the number of new shoots proliferated by a young bud increased by 53.85% to 71.43% compared to the non-mutated plants; when transferred to the seedling medium, the survival rate increased by 1.55%; the rooting rate of the rooting medium increased by 36.93%, the survival rate of the seedlings increased by 37.40%, and the morphological variation rate of leaf color, leaf shape, stem, etc. in the seedlings reached 7.2%. Therefore, the method of this application may have caused chromosome mutations. Specific results are shown in Figure 2 .

[0084] Example 2

[0085] (1) Seed pretreatment

[0086] Healthy, plump Rhododendron yunjinensis seeds from the Longchi Subalpine Botanical Garden were placed in a non-woven bag and soaked in room temperature water, protected from light, for at least 12 hours to encourage germination. The bag was then removed and drained, then soaked in a 1.2% NaClO solution for 5 minutes for disinfection. The seeds were then quickly rinsed with sterile water at least five times for initial disinfection.

[0087] (2) Screening of EMS half-lethal concentration and treatment time

[0088] Using 0.1 mol / L, pH 7.0 phosphate buffer as solvent, prepare EMS solutions with concentrations of 0, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0%; soak the (1) pretreated Rhododendron yunjinensis seeds in EMS solution and shake them in a fume hood at 26°C in the dark for 12, 24, and 36 hours; after the mutagenesis treatment, soak the seeds in 1.5% sodium thiosulfate for 8 minutes and rinse with running water for 1 hour to remove the EMS solution remaining on the seed surface; inoculate the treated seeds in a culture dish covered with moist filter paper, with 40 seeds in each culture dish, and seeds of each concentration were inoculated in an average of 5 culture dishes. Each culture dish was marked with the treatment concentration, time, and group number, and then placed in a 24°C culture room for dark culture and germination. The blank control group was soaked in phosphate buffer for 12, 24, and 36 hours, and then sown in the same treatment method as above. Observation was performed every day, and the germination rate and germination potential were calculated. Finally, the half-lethal dose was obtained and the optimal conditions for EMS treatment were determined (EMS treatment concentration 1.6%, induction time 12h). The specific results are shown in Figure 3 .

[0089] (3) Mutagenesis treatment of large quantities of Rhododendron yunjinensis

[0090] A 1.6% EMS solution was prepared in a fume hood using 0.1 mol / L, pH 7.0 phosphate buffer as a solvent. Rhododendron fortunei seeds pretreated with non-woven bags were fully immersed in the EMS solution and placed in a fume hood under a shaking condition of 80 rpm at 26°C for 12 hours in the dark.

[0091] (4) Cleaning and disinfection

[0092] After the mutagenesis treatment, the seeds treated by soaking were placed in a beaker containing 1.5% sodium thiosulfate solution and soaked for 5 minutes to remove the residual EMS solution on the seed surface, and then rinsed with running water more than 5 times; the seeds were put into a new non-woven bag, placed in a pre-prepared sterilized sterile operating table, soaked in 75% alcohol solution for 30 seconds, then soaked and rinsed with sterile water for 15 seconds, and rinsed 3 times with three bottles of sterile water. After that, the water on the gauze wrapped with seeds was drained as much as possible, soaked in 2% NaClO solution for 2 minutes, and then soaked in sterile water for 15 seconds for 3 times. On the ultra-clean workbench, the disinfected seeds were inoculated onto the sterilized starting medium. 25 seeds were inoculated in each tissue culture bottle. The starting medium was WPM basal medium added with 0.5 mg / L naphthaleneacetic acid (NAA) and 1.0 mg / L zeatin (ZT), and the pH of the medium was 5.6. The tissue culture bottles inoculated with seeds were placed in a tissue culture room at 24 °C and shaded to initiate seed germination. Under these conditions, the germination rate of Rhododendron fortunei seeds was 50.4%.

[0093] (5) Proliferation culture

[0094] After the seeds germinated, light culture was carried out. When the seedlings grown from the treated seeds on the medium were higher than 0.5 cm, they were cut off from the base and transferred to a proliferation medium added with 2.0 mg / L naphthaleneacetic acid (NAA) and 2.0 mg / L zeatin (ZT) and with a pH of 5.5 for proliferation culture. 5 - 10 seedlings were placed in each bottle to proliferate from the base into cluster buds.

[0095] (6) Strong seedling culture

[0096] After the seedlings germinated from the base and grew to a dense cluster and most of the plants were more than 1 cm, each seedling was cut and inoculated into a strong seedling medium added with 2.0 mg / L naphthaleneacetic acid (NAA) and 1.0 mg / L indolebutyric acid (IBA) and with a pH of 5.5. 3 - 5 seedlings were placed in each bottle and cultured until the whole plant grew to more than 2 cm and lignification began to appear at the base.

[0097] (7) Rooting culture

[0098] The plants in (6) were cut short from the base and transferred to a rooting medium added with 2.0 mg / L indolebutyric acid (IBA) and with a pH of 5.5 for rooting culture; note that the seedlings should not be inserted too deep into the medium. The culture conditions were light intensity of 1600 Lx, light time of 10 h / d, temperature of 24 °C, and humidity of 40%. When the plants grew more fibrous roots and the base was more completely lignified, the acclimatization treatment could be carried out.

[0099] (8) Hardening off and transplanting

[0100] Line the plug tray with a culture medium made by mixing nutrient soil and vermiculite in a ratio of 1:1. Plant the Rhododendron fortunei seedlings with well-developed roots into the plug tray, place them in an artificial climate incubator, and water regularly. The cultivation conditions are as follows: light intensity is 1600 Lx, light duration is 10 h / d, temperature is 24 °C, and humidity is 45%.

[0101] (9) Mutant screening

[0102] During the cultivation process, observe the phenotypic growth indicators such as plant height, number of leaves, and leaf shape of the seedlings. Select the plant individuals with traits different from those of the parent plants for cultivation, obtain the plant individuals with stable inheritance in the offspring, and proliferate them.

[0103] Under the above conditions, it is found by comparison that after changing the proliferation medium, the number of proliferated new buds of a single young bud is increased by 33.33% - 100.00% compared with that of the non-mutated plants; when transferred to the strong seedling medium, the survival rate is increased by 6.86%; the rooting rate of the rooting medium is increased by 38.10%, the survival rate of hardening off is increased by 48.21%, and the morphological variation rate of the seedlings in terms of leaf color, leaf shape, and stem reaches 8.9%. Therefore, the method of this application may cause chromosomal mutations. For specific details, see Figure 4 .

[0104] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be changed within the scope of the concept described herein through the above teachings or the techniques or knowledge in related fields. Any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for creating aseptic seedlings of mutant Rhododendron fortunei by combining EMS mutagenesis and tissue culture, characterized in that: It includes the following steps: (1) Determination of the semi-lethal dose of EMS Prepare EMS solutions with different concentrations, soak the seeds of Rhododendron fortunei Lindl. in the EMS solutions with different concentrations for mutagenesis to obtain seeds soaked in different concentrations; Remove the residual EMS solution on the surface of the seeds soaked in different concentrations to obtain the mutagenized seeds; Place the mutagenized seeds on moist filter paper and conduct dark culture for germination acceleration; Count the germination rate and germination potential of the mutagenized seeds, select the concentration of the EMS solution that causes half of the mutagenized seeds to die, obtain the semi-lethal dose concentration of EMS, and determine the optimal conditions for EMS treatment; (2) Early screening of Rhododendron fortunei Lindl. mutant strains Use the EMS solution with the semi-lethal dose concentration to soak the seeds of Rhododendron fortunei Lindl., then conduct cleaning and disinfection, and then use the compound hormone technology and light-temperature coupling regulation technology for rapid cultivation. Screen according to the phenotypic growth indexes of the Rhododendron fortunei Lindl. seedlings, and select plant individuals with traits different from those of the parents to obtain the Rhododendron fortunei Lindl. mutant strains; Among them, the rapid cultivation includes initiation culture, proliferation culture, strong seedling culture, root growth promotion culture, and acclimatization and transplantation.

2. The method according to claim 1, wherein: In step (1), the EMS solution uses phosphate buffer as the solvent, and the concentrations are 0, 0.4%, 0.8%, 1.2%, 1.6%, 2.0%. The semi-lethal dose concentration of the EMS solution is 1.6%. The seed soaking treatment is carried out under dark oscillation at 23°C for 8 - 13 h.

3. The method according to claim 1, characterized in that: In step (2), the phenotypic growth indexes include leaf shape, leaf color, plant type, plant height, and number of leaves.

4. The method according to claim 1, wherein: The components of the culture medium used for the acclimatization and transplantation include, by weight: 1 part of nutrient soil; 1 part of perlite; 1 part of vermiculite.

5. The method according to claim 1, characterized in that: The culture medium used in the compound hormone technology includes initiation medium, proliferation medium, strong seedling medium, and rooting medium.

6. The method according to claim 5, wherein: The initiation medium includes basic medium WPM, 0.5 - 1.0 mg / L of naphthylacetic acid, 0.5 - 1.0 mg / L of zeatin, 30 g / L of sucrose, 7 g / L of agar, pH 5.6 - 5.8; And / or, the proliferation medium: basic medium WPM, 1.0 - 2.0 mg / L of naphthylacetic acid, 1.0 - 2.0 mg / L of zeatin, 30 g / L of sucrose, 7 g / L of agar, pH 5.4 - 5.6; And / or, the strong seedling medium: basic medium WPM, 1.0 - 2.0 mg / L of naphthylacetic acid, 1.0 - 2.0 mg / L of indoleacetic acid, 30 g / L of sucrose, 7 g / L of agar, pH 5.4 - 5.6; And / or, the rooting medium: basic medium WPM, 2.0 mg / L of indolebutyric acid, 30 g / L of sucrose, 6.5 g / L of agar, pH 5.4 - 5.

6.

7. The method according to claim 1, characterized in that: The light-temperature coupling regulation technology includes: During the initiation culture, proliferation culture, strong seedling medium culture, and rooting culture of Rhododendron fortunei Lindl., the culture light intensity is 1500 - 2000 Lx, the light time is 12 - 14 h / d, the temperature is 20 - 23°C, and the humidity is 30% - 45%; The light intensity for cultivating the Rhododendron fortunei Lindl. during the seedling hardening and transplanting is 1500 - 1800 Lx, the light duration is 12 - 14 h / d, the temperature is 20 - 24 °C, and the humidity is 40% - 45%.

8. The Rhododendron fortunei Lindl. mutant obtained by screening according to the method described in any one of claims 1 - 7.

9. The application of the Rhododendron fortunei Lindl. mutant obtained by screening according to the method described in any one of claims 1 - 7 or the Rhododendron fortunei Lindl. mutant described in claim 8 in the breeding and variety improvement of new rhododendron varieties.

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