Method for creating malus grandiflora cold-resistant mutant by adopting EMS mutagenic agent

Abstract

The invention belongs to the technical field of agricultural breeding, and particularly relates to a method for creating a malus spectabilis cold-resistant mutant by adopting an EMS mutagenic agent, which comprises the following steps: preparing peripheral calluses of malus spectabilis; performing EMS treatment on the peripheral calluses to obtain inoculated tissues; sequentially inoculating the inoculated tissue to an adventitious bud differentiation culture medium and a rooting culture medium for culturing to obtain a malus speciosa mutant; the method comprises the following steps: inoculating a malus prunifolia mutant to a hydroxyproline rooting seedling culture medium for culture, and then transferring the malus prunifolia mutant to a rooting seedling culture medium without hydroxyproline for recovery culture to obtain a hydroxyproline-resistant variant plant; and culturing into seedlings, and carrying out low-temperature stress and rewarming to obtain the malus speciosus cold-resistant mutant. According to the method, the treatment time and mutagenesis concentration of EMS mutagenesis are set, the half fatality rate and mutagenesis efficiency are guaranteed, the rooting seedlings obtained through EMS mutagenesis of the callus are continuously subjected to HYP directional screening of cold-resistant plants, the most suitable HYP selection pressure is obtained, the mutant screening efficiency is improved, and plants with higher cold resistance are obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural breeding, and in particular relates to a method for creating a cold-resistant mutant of Malus multiflora by using an EMS mutagen. Background Art

[0002] Begoniaⅹbenariensis'BIG' is a perennial herbaceous flower belonging to the Begoniaceae C.Agardh and the Begonia genus BegoniaL. The genus Begonia is a perennial herbaceous plant. It is famous for its diverse leaf shapes, bright leaf colors, rich leaf patterns, gorgeous flowers, and some varieties have fragrance. It is a herbaceous ornamental plant with great development prospects. Some varieties can also be used for medicinal, edible, drinking or as animal feed. As one of the widely used flowers, Begonia plants have high ornamental value and scientific research value.

[0003] Begonia is a perennial herb with a long flowering period and can bloom all year round in a warm environment. However, it has poor cold resistance and can still grow normally at temperatures above 15°C. When the temperature is below 10°C, the plant stops growing and the leaves are easily frostbitten and withered. When the temperature drops to 0°C, a large number of plants will die. The lowest temperature in winter in southern my country is generally around minus 5°C. When Begonia plants are planted outdoors, they usually face the dilemma of being unable to safely overwinter, resulting in the death of a large number of plants. Therefore, it is urgent to cultivate new varieties of Begonia with strong cold resistance. At present, the method of hybrid breeding is used to select Begonia with strong cold resistance, which not only requires a long period of time, but also faces the challenge of accumulating multiple resistance genes in the process of genetic optimization in controlling the quantitative traits of multiple genes and recessive genes; and conventional chemical mutagenesis or radiation mutagenesis is difficult to predict and control because the directionality of mutations is difficult to control, so it is often difficult to screen plants that meet the target traits. Even if cold-resistant plants are screened, their survival rate in low temperature environments is difficult to be satisfactory.

[0004] In summary, there is an urgent need to provide a new method for preparing low-temperature resistant large-flowered Begonia. Summary of the invention

[0005] The invention aims to provide a method for creating a cold-resistant mutant of Malus multiflorus by using EMS mutagen, so as to improve the cold resistance of Malus multiflorus.

[0006] The technical solution adopted by the present invention is: the present invention provides a method for creating a cold-resistant mutant of Malus multiflora by using EMS mutagen. The method comprises the following steps:

[0007] The method comprises the following steps: preparing a four-week callus of Malus melongena; treating the four-week callus with ethyl methanesulfonate in a sterile environment, and removing the damaged part of the four-week callus after the treatment to obtain an inoculated tissue, wherein the treatment condition is to soak the inoculated tissue in ethyl methanesulfonate with a volume fraction of 0.2% to 0.6% in a dark environment for 2 to 4 hours; inoculating the inoculated tissue on an adventitious bud differentiation medium for culture, and then transferring the inoculated tissue to a rooting medium for culture to obtain a Malus melongena mutant; inoculating the Malus melongena mutant on a hydroxyproline rooting seedling medium for culture, and then transferring the inoculated tissue to a rooting seedling medium without hydroxyproline for recovery culture to obtain a hydroxyproline-resistant mutant plant, wherein the formula of the hydroxyproline rooting seedling medium is: : 1mmol-1.5mmol hydroxyproline, 1.0mg-1.2mg naphthaleneacetic acid, 30g-32g sucrose and 7g-8g agar are added to each liter of 1 / 2MS liquid culture medium; the formula of the rooting seedling culture medium without hydroxyproline is: 1mmol-1.5mmol hydroxyproline, 1.0mg-1.2mg naphthaleneacetic acid, 30g-32g sucrose and 7g-8g agar are added to each liter of 1 / 2MS liquid culture medium; the hydroxyproline-resistant mutant plants are cultured until they become seedlings, subjected to low temperature stress, and rewarmed to obtain the large-flowered begonia cold-resistant mutant, and the low temperature stress conditions are: -5°C to -8°C, and the low temperature stress is continuously applied for 12h to 13h.

[0008] Preferably, the treatment conditions are: immersing in 0.4% by volume ethyl methanesulfonate for 4 hours at a rotation speed of 70 rpm.

[0009] Preferably, the formula of the hydroxyproline rooting seedling culture medium is: 1.5 mmol hydroxyproline, 1.0 mg naphthylacetic acid, 30 g sucrose and 7 g agar are added to each liter of 1 / 2 MS liquid culture medium.

[0010] Preferably, the large-flowered Begonia is a large-flowered Begonia with green leaves and red flowers.

[0011] Preferably, the rewarming condition is: the temperature is uniformly raised to 25° C. within 24 hours.

[0012] Preferably, the steps of preparing the four-week callus tissue are: taking young leaves of Malus macrocarpa, washing them, sterilizing and washing them in a sterile environment, and cutting them into 1 cm 2 ~1.5cm 2 The leaves were inoculated with the leaf surface facing upward into the callus induction medium and pre-cultured for 28 to 29 days to obtain four-week callus.

[0013] Preferably, the process of disinfection and cleaning in a sterile environment is: wash the young leaves of Malus multiflorus with sterile water once, disinfect with 70% to 75% alcohol by volume for 30s to 40s, add 0.1% to 0.15% HgCl2 by mass fraction for disinfection for 8min to 10min, discard HgCl2, wash with sterile water for 3min to 5min each time, and dry the surface moisture of the young leaves of Malus multiflorus.

[0014] Preferably, the formula of the differentiation medium is: 1 mg to 1.2 mg of 6-benzylaminoadenine, 0.2 mg to 0.3 mg of naphthaleneacetic acid, 30 g to 32 g of sucrose, and 7 g to 8 g of agar are added to each liter of MS liquid culture medium; the formula of the rooting medium is: 1 mg to 1.2 mg of naphthaleneacetic acid, 30 g to 32 g of sucrose, and 7 g to 8 g of agar are added to each liter of 1 / 2 MS liquid culture medium.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention provides a method for creating a cold-resistant mutant of Malus melongena by using an EMS mutagen, comprising the following steps: treating a four-week callus with ethyl methanesulfonate in a sterile environment, and after the treatment, cutting off the damaged part of the four-week callus to obtain an inoculated tissue, wherein the treatment condition is to soak the inoculated tissue in ethyl methanesulfonate with a volume fraction of 0.2% to 0.6% in a dark environment for 2 to 4 hours; inoculating the inoculated tissue on an adventitious bud differentiation medium for culture, and then transferring it to a rooting medium for culture to obtain a Malus melongena mutant; inoculating the Malus melongena mutant on a hydroxyproline rooting seedling medium for culture, and then transferring it to a rooting seedling medium without hydroxyproline for recovery culture to obtain a hydroxyproline-resistant variant plant, wherein the hydroxyproline The formula of the ammonia rooting seedling culture medium is as follows: 1mmol-1.5mmol hydroxyproline, 1.0mg-1.2mg naphthaleneacetic acid, 30g-32g sucrose and 7g-8g agar are added to each liter of 1 / 2MS liquid culture medium; the formula of the rooting seedling culture medium without hydroxyproline is as follows: 1mmol-1.5mmol hydroxyproline, 1.0mg-1.2mg naphthaleneacetic acid, 30g-32g sucrose and 7g-8g agar are added to each liter of 1 / 2MS liquid culture medium; the hydroxyproline-resistant mutant plants are cultured to seedlings, subjected to low temperature stress, and rewarmed to obtain the large-flowered begonia cold-resistant mutant, and the low temperature stress conditions are: -5°C to -8°C, and the low temperature stress is continuously applied for 12h to 13h.

[0017] The present invention ensures the median lethality and mutagenesis efficiency by setting the treatment time and mutagenic concentration of EMS mutagenesis, and then adds different concentrations of HYP to the culture medium of the rooted seedlings obtained by EMS mutagenic callus tissue, performs HYP directional screening of cold-resistant plants, obtains the most suitable HYP selection pressure, improves the efficiency of mutant screening, and obtains plants with stronger cold resistance. The present invention further combines low temperature stress and phenotypic identification by adding hydroxyproline-resistant screening variant plants, and can quickly determine cold-resistant variant plants. The large-flowered begonia prepared by the method of the present invention has strong cold resistance and is suitable for large-scale promotion in the Yangtze River Delta region of my country, and also provides new materials for subsequent Begonia plant breeding research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The plants used for the test are Malus macrocarpa.

[0019] Figure 2 The sample is the callus tissue around the leaves of the test Malus multiflora.

[0020] Figure 3 Figure 2 shows the growth status of hydroxyproline-tolerant mutant plants after low temperature stress. The 24 large-flowered Begonia plants shown in A and B are all parallel experimental treatment samples. The white boxes are hydroxyproline-tolerant mutant plants that survived low temperature stress.

[0021] Figure 4 Stomatal closure of plants after low temperature stress. a: Hydroxyproline-tolerant variant plants wilting after low temperature stress; b: cold-resistant variant plants.

[0022] Figure 5 Comparison of the morphology of the control strain of Malus multiflorus and the cold-resistant variant strain after rewarming. a: the control strain cultured at room temperature; b: the common Malus multiflorus after low temperature stress; c: the cold-resistant variant strain.

[0023] Figure 6 Effects of different low temperature stresses on the relative conductivity and MDA content of Malus grandiflora, a: relative conductivity content; b: MDA content.

[0024] Figure 7 Effects of different low temperature stresses on the POD and CAT enzyme activities of Malus grandiflora, a: POD enzyme activity; b: CAT enzyme activity.

[0025] Figure 8 This is the effect of different low temperature stresses on the chlorophyll content of Malus grandiflora. DETAILED DESCRIPTION

[0026] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0027] The inventive concept of the present invention is as follows:

[0028] EMS is a widely used chemical mutagen that causes changes in plant traits by inducing point mutations in alleles. Compared with physical mutagenesis, EMS causes less biological damage and can produce more mutants in a shorter period of time to establish a mutant library. The present invention is different from traditional EMS mutagenesis technology. By adding hydroxyproline, a special chemical substance closely related to a specific metabolic pathway, a specific selection pressure is applied to the material during or after the mutagenesis process to screen out mutants whose physiological function pathways are different from those of the starting material. This method integrates mutagenesis, screening, and rapid propagation into a continuous process, significantly improving the efficiency of mutagenesis.

[0029] During the EMS mutagenesis process, the directionality of the mutation is difficult to predict and control. Therefore, in breeding practice, specific mutation types are usually screened purposefully to cultivate mutants with specific excellent traits. This screening includes conventional screening and special screening. Conventional screening involves observing and evaluating the mutant progeny of crops for many aspects of their traits, including appearance, yield, quality, fertility, and stress and disease resistance, in order to select mutants that exhibit non-original traits or that perform significantly better than the starting materials in certain traits. The special screening method, on the other hand, applies specific selection pressure to the material during or after the mutagenesis process under controlled environmental conditions, often by adding special chemicals that are closely related to specific metabolic pathways to screen out mutants with physiological function pathways that are different from the starting material. As far as the present invention is concerned, hydroxyproline is added to the obtained plants after EMS induction to select cold-resistant materials. Compared with the method of simply using low temperature stress treatment to select cold-resistant plants after EMS induction, the present invention not only carefully optimizes the process of EMS mutagenesis and hydroxyproline stress screening, but also the cold-resistant plants obtained by the method of the present invention have a higher survival rate and lighter cold injury symptoms in a low temperature environment.

[0030] At present, there are no reports on the research on germplasm innovation of Malus grandiflora using EMS mutagenesis technology combined with HYP directional screening, and there is no clear treatment concentration, treatment time and HYP selection pressure. In the EMS mutagenesis method of Malus grandiflora, the present invention solves the following problems: 1. The mutagenesis time and mutagenesis dose of Malus grandiflora are clarified to ensure the half lethality. Mutagenesis time and mutagenesis concentration are the core issues for mutant creation, but there is currently no EMS mutagenesis method for Malus grandiflora as a reference. 2. The most suitable selection pressure for hydroxyproline of EMS-induced Malus grandiflora mutants is provided, ensuring the half lethality of HYP on the induced plants, and obtaining hydroxyproline-resistant mutant plants. 3. A cold-resistant Malus grandiflora is obtained, and the cold-resistant mutant is determined based on the cold damage phenotype of the hydroxyproline-resistant mutant plants after low temperature stress.

[0031] Based on this, the present invention proposes a method for creating a cold-resistant mutant of Malus melongena by using an EMS mutagen, comprising the following steps: treating a four-week callus with ethyl methanesulfonate in a sterile environment, and removing the damaged part of the four-week callus after the treatment to obtain an inoculated tissue, wherein the treatment condition is to soak the inoculated tissue in ethyl methanesulfonate with a volume fraction of 0.2% to 0.6% in a dark environment for 2 hours to 4 hours; inoculating the inoculated tissue on an adventitious bud differentiation medium for culture, and then transferring it to a rooting medium for culture to obtain a mutant of Malus melongena; inoculating the mutant of Malus melongena on a hydroxyproline rooting seedling medium for culture, and then transferring it to a rooting seedling medium without hydroxyproline for recovery culture to obtain a hydroxyproline-resistant mutant plant, wherein the method comprises: treating a four-week callus with ethyl methanesulfonate in a sterile environment, and removing the damaged part of the four-week callus after the treatment to obtain an inoculated tissue, wherein the treatment condition is to soak the inoculated tissue in ethyl methanesulfonate with a volume fraction of 0.2% to 0.6% in a dark environment for 2 hours to 4 hours; inoculating the inoculated tissue on an adventitious bud differentiation medium for culture, and then transferring it to a rooting medium without hydroxyproline for culture to obtain a mutant plant resistant to hydroxyproline. The formula of the hydroxyproline rooting seedling culture medium is as follows: 1 mmol to 1.5 mmol hydroxyproline, 1.0 mg to 1.2 mg naphthaleneacetic acid, 30 g to 32 g sucrose and 7 g to 8 g agar are added to each liter of 1 / 2 MS liquid culture medium; the formula of the rooting seedling culture medium without hydroxyproline is as follows: 1 mmol to 1.5 mmol hydroxyproline, 1.0 mg to 1.2 mg naphthaleneacetic acid, 30 g to 32 g sucrose and 7 g to 8 g agar are added to each liter of 1 / 2 MS liquid culture medium; the hydroxyproline-resistant mutant plants are cultured until they become seedlings, subjected to low temperature stress, and rewarmed to obtain the large-flowered begonia cold-resistant mutant, and the low temperature stress conditions are: -5°C to -8°C, and the low temperature stress is continuously applied for 12h to 13h.

[0032] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0033] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art. The abbreviations described in the present invention are shown in Table 1.

[0034] Table 1 Abbreviations

[0035] abbreviation English full name Chinese LH GreenleafsafflowertypeofBegoniabenariensis Green leaves and red flowers of Begonia EMS Ethylmethanesulfonate Ethyl methanesulfonate MDA Malondialdehyde Malondialdehyde POD Peroxidase Peroxidase 6-BA N-(Phenylmethyl)-9H-purin-6-amine 6-Benzylaminoadenine 2，4-D 2,4-Dichlorophenoxyaceticacid 2,4-Dichlorophenoxyacetic acid HYP Hydroxyproline Hydroxyproline CAT Catalase Catalase ％ percentage percentage d Days Number of days h Hour Hour

[0036] 1. Experimental Materials: The present invention selects the green-leafed and red-flowered large-flowered Begonia of the "Bige" series as experimental materials. Figure 1 shown.

[0037] 2. Experimental instruments: The main instruments used in the present invention are shown in Table 2.

[0038] Table 2 Main instruments

[0039] instrument Company Clean bench China Instrument Technology Co., Ltd. pH Meter Shanghai INESA Scientific Instrument Co., Ltd. Electronic balance Shanghai Youke Instrument Co., Ltd. High Pressure Steam Sterilizer Japan SANYO Group Electric heating blast drying oven Thermo Corporation Pipette Eppendorf, Germany Elix water purifier Millipore Corporation Inoculation sterilizer Jinan Aite Instrument Equipment Co., Ltd. UV-Vis Spectrophotometer ABI Corporation Centrifuge Thermo Corporation Light incubator Shanghai Sanfa Scientific Instrument Co., Ltd.

[0040] 3. Experimental reagents are as follows:

[0041] (1) Preparation of EMS solution:

[0042] EMS is an organic substance with the molecular formula C3H8O3S. It is a colorless, transparent, oily liquid, slightly soluble in water, and needs to be diluted with a phosphate buffer with a pH of 7.0. It is toxic, so please pay attention to protection during operation.

[0043] Take 31.21g of NaH2PO4 and dissolve it in 1L of distilled water and stir until it is completely dissolved, which is solution 1; take 71.64g of Na2HPO4 and dissolve it in 1L of distilled water and stir until it is completely dissolved, which is solution 2. Take 61.0mL of solution 1 and 39.0mL of solution 2 and make the volume to 200mL, make up to the volume with distilled water, prepare a phosphate buffer with a pH of 7.0, filter and sterilize it for use.

[0044] Use the prepared phosphate buffer to prepare EMS solutions with volume fractions of 0.2% to 1% as mutagenic solutions of different concentrations. Sterilize the 100% EMS mother solution by filtering it with a 1mL syringe with a 0.22μm filter membrane under a sterile environment, draw 1mL of the mother solution and dilute it to 5mL with sterile water to prepare a 0.2% EMS treatment solution. According to this method, prepare EMS solutions of other concentrations in turn. All the above operations are carried out in a clean bench.

[0045] (2) The preparation method of 6-BA and 2,4-D solution is as follows:

[0046] Take 50 μg of 6-BA powder, dissolve it in 1N NaOH solution, and then make up to 100 mL with distilled water. Mix well and set aside for use. Prepare a 0.5 mg / L solution and store it at 4°C. Prepare it before use to avoid crystallization and affect its use.

[0047] Take 10 mL of 10 mg / L 2,4-D solution and dilute to 100 mL with distilled water to make a 1 mg / L solution. Store at 4°C and mix well for later use.

[0048] 4. Data statistics, the method is as follows:

[0049] Microsoft Excel 2015 was used for graphing and SPSS 19.0 software was used for data and statistical analysis. The measured data were expressed as the mean plus or minus the standard deviation.

[0050] HYP screening survival rate = number of surviving EMS-induced seedlings after growing on HYP medium for 20 days / number of EMS-induced seedlings tested in HYP × 100%.

[0051] Example 1

[0052] A method for creating a cold-resistant mutant of Malus multiflora using an EMS mutagen comprises the following steps:

[0053] S1: Prepare the callus tissue of Malus dasyphylla as follows:

[0054] The present invention selects callus tissue around the leaves of the green-leaf and red-flower type "Bigo" as an induction material, and the induction material is pre-cultured by the following method.

[0055] Select the green-leafed and red-flowered "Bigo" plants planted in the intelligent greenhouse of Hefei Hualu Seedling Co., Ltd. When the plants grow to 50 cm in height, collect the plants with good horticultural traits as the mother plant. Take the tender leaves of the mother plant that are basically the same size, wash them with detergent, then clean them with distilled water, and place them on a clean workbench for disinfection. The specific disinfection steps are: first wash them with sterile water, wash them with 75% alcohol by volume for 30 seconds, then add 0.1% HgCl2 by mass to disinfect for 10 minutes, pour out the HgCl2 and wash them repeatedly with sterile water for 5 times, each washing time is 3 minutes, obtain sterile leaves, and then place them in a 7cm culture dish covered with sterile filter paper to absorb the moisture on the surface of the leaves, use a sterile scalpel to cut the leaves into about 1cm×1cm along the main vein direction, and inoculate them on the callus induction medium with the leaves facing up. Pre-culture for 28 days to prepare the callus tissue around the leaves of Malus multiflora. Figure 2 shown.

[0056] The formula of the callus induction medium is: add 1 mg of 6-benzylaminoadenine, 0.1 mg of naphthaleneacetic acid, 0.1 mg of 2,4-dichlorophenoxyacetic acid, 30 g of sucrose and 7 g of agar per liter of MS liquid basic medium, and the pH value is 6.0. The culture conditions are: light intensity of 2000 Lx, light time of 12 h, and temperature of 25°C.

[0057] S2: Under a sterile environment, the four-week callus tissue is treated with ethyl methanesulfonate, and the damaged part of the four-week callus tissue is removed after treatment to obtain the inoculated tissue, as follows:

[0058] 90 callus tissues around the leaves of green-leafed and red-flowered Begonia were selected as in vitro mutagenesis materials. In a clean bench, the in vitro mutagenesis materials were soaked in a glass bottle containing sterilized EMS, with a volume fraction of 0.4%, and shaken for 4 hours. Shaking conditions: avoid light, temperature 20°C, shaker speed 70rpm. After soaking, rinse with sterile water 3 times, dry the surface moisture on sterile paper, and remove the damaged parts.

[0059] S3: The inoculated tissue is inoculated on an adventitious bud differentiation medium and then transferred to a rooting medium to obtain a Malus multiflora mutant, as follows:

[0060] After the damaged part was removed, the remaining part was inoculated on the adventitious bud differentiation medium, and the plants were subcultured every 50 days. After the plants grew to about 2 cm, they were transferred to the rooting medium to obtain the Malus multiflora mutant. The callus tissue around the leaves could differentiate into adventitious buds in 9d±1d. The differentiation of adventitious buds was observed 30d after inoculation, and the callus survival rate and adventitious bud induction rate were calculated, as shown in Table 3.

[0061] Differentiation medium formula: add 1 mg of 6-benzylaminoadenine, 0.2 mg of naphthaleneacetic acid, 30 g of sucrose, and 7 g of agar per liter of MS liquid medium, pH 6.0. Culture conditions: 25°C, light-dark cycle 16h / 8h, light intensity 2000Lx, relative air humidity 80% in the culture room.

[0062] Rooting medium formula: add 1.0 mg naphthaleneacetic acid, 30 g sucrose and 7 g agar per liter of 1 / 2MS liquid medium, pH value 6.0. Culture conditions: 25°C, light-dark cycle 16h / 8h, light intensity 2000Lx, relative air humidity 80% in the culture room.

[0063] S4: The Malus multiflora mutant is inoculated into a hydroxyproline rooting seedling medium for culture, and then transferred to a rooting seedling medium without hydroxyproline for recovery culture to obtain a hydroxyproline-tolerant mutant plant, the method is as follows:

[0064] The mutant of Malus multiflorus was used as HYP directional screening material after mutagenesis and rooting. The mutant of Malus multiflorus was collected and inoculated into a rooting seedling medium containing 1mmol / L HYP. After 20 days of culture, the number of surviving rooting seedlings after HYP screening was recorded. The surviving rooting seedlings were then inoculated into a rooting seedling medium without HYP for recovery culture, and the rooting seedlings that survived were counted, which were hydroxyproline-resistant mutant plants. The number of surviving rooting seedlings was counted, and the screening survival rate was calculated, as shown in Table 3.

[0065] HYP rooting seedling medium formula: add 1mmol hydroxyproline, 1.0mg naphthaleneacetic acid, 30g sucrose and 7g agar per liter of 1 / 2MS liquid medium, and adjust the pH to 6.0. HYP-free rooting seedling medium formula: add 1.0mg naphthaleneacetic acid, 30g sucrose and 7g agar per liter of 1 / 2MS liquid medium, and adjust the pH to 6.0. Culture conditions: 25℃, light-dark cycle 16h / 8h, light intensity 2000Lx, relative air humidity in the culture room 80%.

[0066] S5: Cultivate the hydroxyproline-tolerant mutant plants until they are mature, subject them to low temperature stress, and rewarm them to obtain the cold-tolerant mutant of Malus multiflora, as follows:

[0067] By observing the changes in the appearance of plants under low temperature stress, we can intuitively judge the strength of their cold resistance. The hydroxyproline-tolerant variant plants were cultured to the seedling state, placed at -5°C for 12 hours, and then slowly rewarmed. The temperature rose uniformly within 24 hours to 25°C. Figure 3 As shown in the figure, the three plants in the white frame still grew normally after 12 hours of low temperature stress at -5℃, and the area of ​​leaf dehydration and wilting was small. The three surviving plants were recorded as M4-1, M4-2, and M4-3 from left to right and from top to bottom. They quickly returned to normal after rewarming and began to grow new leaves on the 10th day. However, the other hydroxyproline-tolerant plants wilted due to severe water loss after 12 hours of low temperature stress at -5℃. After slow rewarming, the leaves thawed and lost a lot of water. In the process of rewarming and resuming growth, new leaves could not sprout and they quickly died of dehydration.

[0068] After low temperature stress, the hydroxyproline-resistant variant plants that survived the rewarming culture were the cold-resistant variants of Malus multiflora.

[0069] At the same time, EMS treatment control 1 to EMS treatment control 11 were set, and the experimental method was referred to S1 and S2 of Example 1, wherein the specific EMS treatment time and volume fraction in S2 were shown in Table 3, and the other conditions were exactly the same as in Example 1. As can be seen from Table 3, the survival rate after EMS treatment and the adventitious bud induction rate after EMS treatment in EMS treatment control 1 to EMS treatment control 11 were far from reaching the median lethality of 50%, so EMS treatment control 1 to EMS treatment control 11 were no longer subjected to hydroxyproline treatment.

[0070] Embodiment 2 to Embodiment 8

[0071] A method for creating a cold-resistant mutant of Malus multiflora using EMS mutagen, the method is as follows:

[0072] In Examples 2 to 8, callus tissues around leaves of the green-leaf, red-flowered variety "Bige" were treated with EMS to prepare large-flowered begonia mutants; hydroxyproline-tolerant mutant plants were subsequently screened, and then subjected to low temperature stress to create large-flowered begonia cold-resistant mutants; the specific EMS treatment time and volume fraction and HYP screening concentration are shown in Table 3. The survival rate after EMS induction treatment, the adventitious bud induction rate, and the HYP screening survival rate are shown in Table 3, and the other conditions are exactly the same as in Example 1.

[0073] In order to prove the effect of the present invention, the present invention has made comparative examples 1 to 8.

[0074] Comparative Example 1 to Comparative Example 8

[0075] A method for creating a cold-resistant mutant of Malus multiflora using EMS mutagen, the method is as follows:

[0076] Comparative Examples 1 to 8 are to treat the callus tissue around the leaves of the green-leaf and red-flower type "Bige" by EMS induction treatment to prepare the large-flowered begonia mutant; then screen the hydroxyproline-resistant mutant plants, and then subject them to low temperature stress to create the large-flowered begonia cold-resistant mutant; wherein the specific EMS treatment time and volume fraction and HYP screening concentration are shown in Table 3. The survival rate after EMS induction treatment, the adventitious bud induction rate and the HYP screening survival rate are shown in Table 3, and the other conditions are exactly the same as those in Example 1.

[0077] Table 3 EMS and HYP treatment conditions and survival rates

[0078]

[0079]

[0080] Note: “-” means there is no such item.

[0081] As shown in Table 3, the callus tissue around the leaves of the green-leafed and red-flowered "Bige" was treated with 0.4% EMS for 4 hours, and the survival rate and adventitious bud induction rate were 47.78% and 44.44%, respectively, which was closest to the median lethality of 50%. Therefore, it was determined that the more suitable mutagenesis conditions for the callus tissue around the leaves were 0.4% EMS concentration and 4 hours of treatment time; under the 1.5mmol / L HYP concentration culture, the survival rate was 49.67%, which was close to 50%, and 10 plants survived after the recovery culture. Therefore, the concentration of 1.5mmol / L was determined to be the most suitable selection pressure for selecting cold-resistant mutants.

[0082] The low temperature damage phenotype of the cold-resistant mutant of Malus multiflora obtained in Example 1 was identified as follows:

[0083] Low temperature stress has a great impact on plant phenotype. Under low temperature conditions, the chloroplast structure of plants is easily damaged, the activity of chloroplast pigment synthase is reduced, and the chlorophyll content is affected, causing the plants to show symptoms of chlorosis and yellowing. By observing the changes in the appearance of plants under low temperature stress, its cold resistance can be intuitively judged.

[0084] After the hydroxyproline-tolerant mutant plants were exposed to low temperature stress at -5°C for 12 hours, the growth conditions of the plants were as follows: Figure 3 As shown in the figure, the three plants in the white frame still grew normally after being subjected to low temperature stress at -5℃ for 12 hours, and the area of ​​leaf dehydration and wilting was small; the stomata could adaptively close in the face of adversity, reducing water loss and maintaining the normal state of the leaves, such as Figure 4 shown; Figure 3After other plants were subjected to low temperature stress at -5℃ for 12 hours, the low temperature caused the root system's ability to absorb water and transport it to the branches to decrease rapidly. At the same time, when the plants sensed water deficiency, the ability of stomatal closure decreased, causing the plants to wilt due to severe water loss.

[0085] Depend on Figure 5 It can be seen that the leaves of the control group of large-flowered begonia plants grown at an appropriate temperature are tender green, brightly colored, and spread outward with good morphology; after low temperature treatment, the leaves all showed varying degrees of yellowing and wilting. After 12 hours of low temperature stress at -5℃, most of the leaves of the control group of large-flowered begonia showed intercellular ice. After slow rewarming, the leaves thawed and lost a lot of water. In the process of rewarming and resuming growth, new leaves could not sprout and they soon died of dehydration. After 12 hours of low temperature stress at -5℃, the cold-resistant variant grew basically normally, with a small area of ​​leaf dehydration and wilting, and maintained a good leaf morphology. After rewarming and resuming growth, the leaves of the cold-resistant variant quickly returned to normal, and new leaves began to grow in 5d to 10d.

[0086] The physiological responses of the cold-tolerant variant of Malus macrocarpa to low temperature stress are as follows:

[0087] (1) Effects of low temperature stress on cell membrane of Malus grandiflora.

[0088] Under low temperature conditions, the first part of the plant to be affected is its biofilm structure. By measuring the relative conductivity and malondialdehyde content of the plant, the degree of damage to the cell membrane can be assessed. Figure 6 As shown in the figure, the relative conductivity of the control strain CK and the three cold-resistant variants M4-1, M4-2, and M4-3 of Malus multiflora showed an increasing trend as the temperature continued to decrease. At 20℃, the relative conductivity of the control strain and the cold-resistant variant strain was relatively similar. After the temperature dropped to 5℃, the relative conductivity of the two groups differed and slowly increased. When the temperature dropped to -5℃, the relative conductivity of the control plant was 82.59%, which was seriously damaged; the relative conductivity of the cold-resistant variant strain was in the range of 54.54% to 65.67%, which was much lower than that of the control plant. Its cell frostbite was mild and its cold resistance was strong. The MDA content of the control strain and the cold-resistant variant strain of Malus multiflora gradually increased with the decrease of stress temperature. Under different low temperature stresses, the MDA content of the cold-resistant variant strain was always lower than that of the control group. The MDA content was the highest at -5℃, the control strain was 3.64nmol / g, and the MDA content of the cold-resistant mutant strain was in the range of 1.57nmol / g to 2.13nmol / g. The MDA content of the cold-resistant mutant strain was lower than that of the control strain. It can be seen that the cell membrane damage of the cold-resistant mutant plants induced by EMS was less than that of the control strain. The degree of cell membrane damage caused by low temperature stress on M4-1, M4-2, and M4-3 was similar.

[0089] (2) Effect of low temperature stress on the activities of POD and CAT enzymes in Malus large-flowered.

[0090] Low temperature stress causes the balance between the production and removal of free radicals in plant cells to be broken, which in turn leads to the accumulation of a large amount of reactive oxygen, which damages the structure and function of the cell membrane. POD and CAT enzymes have the function of protecting cells from damage by hydrogen peroxide and delaying aging in plants.

[0091] Depend on Figure 7 It can be seen that the POD enzyme activity of the control strain and the cold-resistant variant strain of Malus multiflora first decreased and then increased with the decrease in temperature. When the treatment temperature was 20℃ and 5℃, the POD enzyme activity of the cold-resistant variant strain and the control strain was close. When the temperature was as low as 0℃, the POD enzyme activity of the cold-resistant variant strain showed a slow upward trend and reached a maximum value of 361.67U / (min·g) at -5℃; while the POD enzyme activity of the control strain continued to decrease and began to increase at -5℃, reaching a maximum value of only 209.33U / (min·g). It can be seen that under low temperature adverse environment, the POD enzyme activity of the cold-resistant variant strain is higher than that of the control plant.

[0092] Depend on Figure 7 It can be seen that the CAT enzyme activity of the control strain and the cold-resistant variant strain gradually increased with the decrease in temperature. When the treatment temperature was 20℃ and 5℃, the CAT enzyme activity of the control strain and the cold-resistant variant strain increased slowly. When the temperature continued to drop, the POD enzyme activity of the control strain increased slowly, while that of the cold-resistant variant strain increased rapidly. At -5℃, the CAT enzyme activity of the control strain was 14.06U / g, and the CAT enzyme activity of the cold-resistant variant strain was in the range of 23.35U / g to 30.4U / g. The CAT enzyme activity of M4-3 was 2.16 times that of the control strain.

[0093] In summary, the EMS mutant plants screened by HYP showed a stronger ability to remove reactive oxygen than the control group. The results further confirmed that the cold-resistant mutant plants induced by EMS have a stronger ability to resist low temperatures. M4-1, M4-2, and M4-3 have similar abilities to remove reactive oxygen under low temperature stress.

[0094] (3) Effect of low temperature stress on the chlorophyll content of Malus grandiflora.

[0095] When plants are subjected to low temperature stress, the outer membrane structure of the chloroplasts will break or even disintegrate due to severe damage, which causes the leaves of the plants to fade. The chloroplast content is one of the indicators of plant damage caused by low temperature. Figure 8It can be seen that as the temperature continued to drop, the chloroplast content of the control strain and the three cold-resistant variants M4-1, M4-2, and M4-3 all decreased. Among them, the chlorophyll content of the cold-resistant variants decreased more slowly than that of the control group. At -5°C, the content was in the range of 0.87mg / g to 1.21mg / g, and the chloroplast disintegration was less. The chlorophyll content of the control strain decreased rapidly as the temperature dropped, and it was as low as 0.22mg / g at -5°C. This significant difference directly led to a large-scale chlorosis of the leaves in the control group at -5°C.

[0096] Based on the above analysis, it can be concluded that the cold-resistant mutant plants induced by EMS are less vulnerable to low temperature stress. The stable maintenance of their chloroplast content enables the cold-resistant mutant plants to better maintain normal growth in a low temperature environment.

[0097] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several changes and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for creating a cold-resistant mutant of Malus multiflora using EMS mutagen, characterized in that: The following steps are involved: Preparing callus tissue of Malus melongena; Treating the callus tissue around the periphery with ethyl methanesulfonate in a sterile environment, and removing the damaged part of the callus tissue around the periphery after the treatment to obtain the inoculated tissue, wherein the treatment condition is to immerse the tissue in ethyl methanesulfonate with a volume fraction of 0.2% to 1% in the dark for 2 hours to 4 hours; The inoculated tissue was inoculated on an adventitious bud differentiation medium and then transferred to a rooting medium to obtain a Malus melongena mutant. The mutant of Malus multiflora is inoculated into a hydroxyproline rooting seedling medium for culture, and then transferred to a rooting seedling medium without hydroxyproline for recovery culture, so as to obtain a hydroxyproline-resistant mutant plant, wherein the formula of the hydroxyproline rooting seedling medium is as follows: 1 mmol to 1.5 mmol hydroxyproline, 1.0 mg to 1.2 mg naphthaleneacetic acid, 30 g to 32 g sucrose and 7 g to 8 g agar are added to each liter of 1 / 2 MS liquid medium; the formula of the rooting seedling medium without hydroxyproline is as follows: 1 mmol to 1.5 mmol hydroxyproline, 1.0 mg to 1.2 mg naphthaleneacetic acid, 30 g to 32 g sucrose and 7 g to 8 g agar are added to each liter of 1 / 2 MS liquid medium; The hydroxyproline-tolerant mutant plants were cultured until they became seedlings, subjected to low temperature stress, and rewarmed to obtain a cold-resistant mutant of Malus multiflora. The low temperature stress conditions were: -5°C to -8°C, and the low temperature stress was continued for 12h to 13h.

2. The method according to claim 1, characterized in that The treatment conditions are: immersion in ethyl methanesulfonate with a volume fraction of 0.4%, immersion for 4 hours, and a rotation speed of 70 rpm.

3. The method according to claim 1, characterized in that The formula of the hydroxyproline rooting seedling culture medium is as follows: 1.5 mmol hydroxyproline, 1.0 mg naphthaleneacetic acid, 30 g sucrose and 7 g agar are added into each liter of 1 / 2 MS liquid culture medium.

4. The method according to claim 1, characterized in that: The large-flowered begonia is a large-flowered begonia with green leaves and red flowers.

5. The method according to claim 1, characterized in that The rewarming conditions are: The temperature was raised uniformly to 25°C over 24 hours.

6. The method according to claim 1, characterized in that The steps of preparing the four-week callus are: Take the young leaves of the large-flowered crabapple, wash them, disinfect and wash them in a sterile environment, and cut them into 1cm 2 ~1.5cm 2 The leaves were inoculated with the leaf surface facing upward into the callus induction medium and pre-cultured for 28 to 29 days to obtain four-week callus.

7. The method according to claim 6, characterized in that The process of disinfection and cleaning in a sterile environment is as follows: Wash the young leaves of Begonia grandiflora with sterile water, disinfect with 70% to 75% alcohol by volume for 30s to 40s, add 0.1% to 0.15% HgCl2 by mass and disinfect for 8min to 10min, discard HgCl2, wash with sterile water 3 to 5 times, each washing time is 3min to 5min, and dry the surface moisture of the young leaves of Begonia grandiflora.

8. The method according to claim 1, characterized in that The formula of the differentiation medium is: Add 1 mg to 1.2 mg of 6-benzylaminoadenine, 0.2 mg to 0.3 mg of naphthaleneacetic acid, 30 g to 32 g of sucrose, and 7 g to 8 g of agar per liter of MS liquid medium; The formula of the rooting culture medium is as follows: 1 mg to 1.2 mg of naphthaleneacetic acid, 30 g to 32 g of sucrose and 7 g to 8 g of agar are added to each liter of 1 / 2 MS liquid culture medium.

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