Establishment method of elymus nutans seed callus induction and regeneration system

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for establishing an elymus nutans seed callus induction and regeneration system. The invention provides a tissue culture medium and a tissue culture method for culturing elymus nutans, and in the tissue culture medium provided by the invention, 2, 4-D can induce explants to form calluses and promote cell division and growth. The CH has a good effect on differentiation of embryoid and adventitious buds. ABA can promote the development of seed embryos. The KT can promote the generation of calluses and the growth of adventitious buds. NAA can promote plant growth and promote generation of adventitious roots. Under the combined action of NAA, 2, 4-D, ABA, KT and CH, the callus induction rate of the mature embryo is improved. The invention also provides a regeneration tissue culture method of elymus nutans, the mature embryo is used as an explant for tissue culture, and the callus induction rate of the mature embryo is high.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for establishing a callus induction and regeneration system of Elymus nutans seeds. Background Art

[0002] Elymus nutans ( Elymus nutans ) is also known as hook grass and bent spike grass. It belongs to the genus Elymus, the tribe Triticeae, the subfamily Poaceae, and is a perennial herbaceous sparse clump plant. It is widely distributed in North China, Northeast my country, Northwest China, Southwest China and other places. After a long period of natural selection, Elymus nutans has certain drought resistance, cold resistance, salt and alkali resistance and other stress resistance characteristics. In addition, because Elymus nutans has a well-developed root system and strong invasiveness, it is a high-quality grass species for soil and water conservation. It is also a superior forage grass for artificial grassland construction and natural grassland rejuvenation in high-cold areas, and has high ecological and economic value. In summary, Elymus nutans is a grass species with great development value.

[0003] Tissue culture technology is an asexual reproduction technology based on the theory of plant totipotency. Plant tissue culture uses artificial culture medium to cultivate plant tissues under sterile conditions. It is the basis of bioengineering research and also a basic link in genetic transformation and plant improvement research. Tissue culture technology has the advantages of fast reproduction speed, high production efficiency, maintaining the inherent shape and characteristics of the original variety, and saving reproduction materials. Oryza sativa ),wheat( Triticum aestivum ),corn( Zea mays )、Sorghum( Sorghum bicolor ) etc., the tissue culture technology of Gramineae forage grass started late. In the prior art, young spikelets of Elymus nutans are used as explants for tissue culture, but the callus induction rate is low and the materials are not easy to obtain. Summary of the invention

[0004] The invention provides a method for establishing a callus induction and regeneration system of Elymus nutans seeds. Mature embryos are used as explants, tissue culture is performed using the tissue culture medium and the regeneration tissue culture method of the invention, and the callus induction rate is high.

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions: The present invention provides a tissue culture medium for culturing Elymus nutans, wherein the tissue culture medium comprises an induction medium, a differentiation medium and a rooting medium; The induction medium comprises 4-4.43 g / L MS, 4-6 mg / L 2,4-D, 40-60 mg / L CH, 1-3 mg / LABA, 0.04-0.07 mg / L KT, 28-30 g / L sucrose and 7-9 g / L agar; The differentiation medium comprises 2-2.215 g / L MS, 0.01-0.03 mg / L KT, 0.3-0.5 mg / L NAA, 12.5-15 g / L sucrose and 7-9 g / L agar; The rooting medium comprises 4-4.43 g / L MS, 0.3-0.5 g / L NAA, 28-30 g / L sucrose and 7-9 g / L agar.

[0006] Preferably, the pH value of the induction medium is 5.7-5.9, the pH value of the differentiation medium is 5.7-5.9, and the pH value of the rooting medium is 5.7-5.9.

[0007] The present invention provides a regeneration tissue culture method for Elymus nutans, wherein the regeneration tissue culture method adopts the tissue culture medium described in the above technical solution and comprises the following steps: The mature embryos of Elymus nutans are inoculated into an induction medium for induction culture to obtain callus tissue; Transferring the callus tissue to a differentiation medium for subculture to obtain sterile seedlings; The sterile seedlings are transferred to a rooting medium for rooting culture to obtain rooted seedlings.

[0008] Preferably, the mature embryos are sterilized before inoculation, and the sterilization comprises soaking in a sodium hypochlorite solution with an effective chlorine content of 10% for 28 to 32 minutes, and then rinsing with sterile water for 5 to 7 times.

[0009] Preferably, the temperatures of the induction culture, subculture and rooting culture are 25±2°C, respectively.

[0010] Preferably, the induction culture is dark culture; the subculture culture is light-dark alternating culture; and the rooting culture is light culture.

[0011] Preferably, the illumination time of the alternating light and dark is 12~16h / d; the intensity of the illumination is 2200~2500lx; and the darkness time of the alternating light and dark is 8~12h / d.

[0012] Preferably, the light culturing includes white light irradiation.

[0013] Preferably, the induction culture time is 45 to 60 days; the subculture time is 28 to 35 days; and the rooting culture is cultured until the root length is 5 to 7 cm.

[0014] Preferably, after obtaining the rooted seedlings, the method further includes hardening the seedlings and transplanting.

[0015] Beneficial effects of the present invention: The present invention provides a tissue culture medium for culturing Elymus nutans. In the tissue culture medium provided by the present invention, 2,4-D is 2,4-dichlorophenoxy acetic acid; 2,4-D can induce explants to form callus tissue and promote cell division and growth. CH is Hydrolyzed casein, and CH has a good effect on the differentiation of embryoids and adventitious buds. ABA is Abscisic Acid, and ABA can promote the development of seed embryos; KT is Kinetin, and KT can promote the formation of callus tissue and the growth of adventitious buds. NAA can promote plant growth and the formation of adventitious roots. Under the joint action of NAA, 2,4-D, ABA, KT, and CH, the callus induction rate of mature embryos is increased without destroying the endogenous environment of the plant itself.

[0016] The present invention also provides a regeneration tissue culture method for Elymus nutans, which uses mature embryos as explants for tissue culture. Using mature embryos as explants for tissue culture also has the advantage of convenient material collection, and is very suitable for establishing a regeneration system for grasses. The regeneration tissue culture method proposed by the present invention has a high callus induction rate of mature embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the callus obtained by inducing mature embryos of Elymus nutans for 50 days. The scale bar in the figure is 1 cm. Figure 2 These are the green seedlings and adventitious buds obtained from the callus differentiation culture of Elymus nutans for 26 days. The scale bar in the figure is 1 cm. Figure 3 These are seedlings obtained after 18 days of rooting culture of Elymus nutans. The scale bar in the figure is 1 cm. Figure 4 This is the regenerated plant of Elymus nutans 60 days after transplanting. The scale of the figure is 5 cm. Figure 5 The diagrams are callus tissues induced from mature embryos and stems of Elymus nutans in Example 1 and Comparative Example 1, wherein A is a callus tissue diagram of seed (mature embryo) induction culture for 40 days; B is a callus tissue diagram of stem induction culture for 40 days; the scale of Figures A and B is 1 cm; C is a micrograph of callus tissue of seed (mature embryo) induction culture for 40 days; D is a micrograph of callus tissue of stem induction culture for 40 days; the scale of Figures C and D is 100 μm; Figure 6 This is a picture of callus obtained after 40 days of mature embryo induction culture in Comparative Example 2. The scale of the picture is 1 cm; Figure 7 This is a picture of callus obtained after 40 days of mature embryo induction culture in Comparative Example 3. The scale of the picture is 1 cm; Figure 8This is a picture of callus obtained after 40 days of mature embryo induction culture in Comparative Example 4. The scale of the picture is 1 cm; Fig. 9 This is a differentiation diagram of callus tissue in Comparative Example 5 in which mature embryo callus tissue was inoculated into differentiation medium and cultured for 26 days. The scale of the diagram is 1 cm. Fig.10 This is a diagram of the differentiation of callus tissue in Comparative Example 6 after mature embryo callus tissue was inoculated into differentiation medium and cultured for 26 days. The scale of the diagram represents 1 cm; Fig.11 This is a differentiation diagram of callus tissue in comparative example 7, in which mature embryo callus tissue was inoculated into differentiation medium and cultured for 26 days. The scale of the diagram is 1 cm. DETAILED DESCRIPTION

[0018] The invention provides a tissue culture medium for culturing Elymus nutans. The tissue culture medium comprises an induction medium, a differentiation medium and a rooting medium.

[0019] The induction culture medium of the present invention comprises 4-4.43 g / L MS, 4-6 mg / L 2,4-D, 40-60 mg / L CH, 1-3 mg / L ABA, 0.04-0.07 mg / L KT, 28-30 g / L sucrose and 7-9 g / L agar.

[0020] As an optional embodiment, the concentration of MS in the induction medium provided by the present invention is 4-4.43 g / L, or 4-4.2 g / L. In a specific embodiment of the present invention, the concentration of MS in the induction medium may be 4.0, 4.1, 4.2 or 4.43 g / L. The concentration of 2,4-D in the induction medium provided by the present invention is 4-6 mg / L; in a specific embodiment of the present invention, the concentration of 2,4-D in the induction medium may be 4.0, 4.3, 4.5, 5.0, 5.5, 5.7 or 6.0 mg / L. 2,4-D can induce explants to form callus tissue and promote cell division and growth. The concentration of CH in the induction medium provided by the present invention is 40-60 mg / L; in a specific embodiment of the present invention, the concentration of CH in the induction medium may be 40, 45, 50, 55 or 60 mg / L. CH has a good effect on the differentiation of embryoids and adventitious buds. The concentration of ABA in the induction medium provided by the present invention is 1-3 mg / L. In a specific embodiment of the present invention, the concentration of ABA in the induction medium may be 1, 1.5, 2 or 3.0 mg / L. ABA can promote the development of seed embryos. The concentration of KT in the induction medium provided by the present invention is 0.04-0.07 mg / L, and may also be 0.05-0.65 mg / L. In a specific embodiment of the present invention, the concentration of KT in the induction medium may be 0.04, 0.05, 0.06 or 0.07 mg / L. KT can promote the formation of callus. The concentration of sucrose in the induction medium provided by the present invention is 28-30 g / L. In a specific embodiment of the present invention, the concentration of sucrose in the induction medium may be 28, 29 or 30 g / L. The concentration of agar in the induction medium provided by the present invention is 7-9 g / L. In a specific embodiment of the present invention, the concentration of agar in the induction medium may be 7, 8 or 9 g / L. In the present invention, the pH value of the induction medium may be 5.7-5.9, or 5.80.

[0021] The differentiation medium of the present invention comprises 2-2.215 g / L MS, 0.01-0.03 mg / L KT, 0.3-0.5 mg / L NAA, 12.5-15 g / L sucrose and 7-9 g / L agar.

[0022] As an optional embodiment, the concentration of MS in the differentiation medium provided by the present invention is 2~2.215g / L. In a specific embodiment of the present invention, the concentration of MS in the differentiation medium may be 2.0, 2.1, 2.2 or 2.215g / L. The concentration of KT in the differentiation medium provided by the present invention is 0.01~0.03mg / L, or 0.15~0.25mg / L; in a specific embodiment of the present invention, the concentration of KT in the differentiation medium is 0.01, 0.015, 0.02 or 0.03mg / L. The concentration of NAA in the differentiation medium provided by the present invention is 0.3~0.5mg / L; in a specific embodiment of the present invention, the concentration of NAA in the differentiation medium is 0.3, 0.35, 0.4, 0.45 or 0.5mg / L. NAA and KT jointly promote the formation of adventitious buds from callus tissue at an appropriate concentration ratio. The concentration of sucrose in the differentiation medium provided by the present invention is 12.5-15 g / L; in a specific embodiment of the present invention, the concentration of sucrose in the differentiation medium is 12.5, 13, 13.5, 14 or 15 g / L. The concentration of agar in the differentiation medium provided by the present invention is 7-9 g / L. In a specific embodiment of the present invention, the concentration of agar in the differentiation medium can be 7, 8 or 9 g / L. In the present invention, the pH value of the differentiation medium can be 5.7-5.9, or 5.80.

[0023] The rooting culture medium of the present invention comprises 4-4.43 g / L MS, 0.3-0.5 g / L NAA, 28-30 g / L sucrose and 7-9 g / L agar.

[0024] As an optional embodiment, the concentration of MS in the rooting medium provided by the present invention is 4~4.43g / L, or 4~4.2g / L. In a specific embodiment of the present invention, the concentration of MS in the rooting medium may be 4.0, 4.1, 4.2 or 4.43g / L. The concentration of NAA in the rooting medium provided by the present invention is 0.3~0.5g / L, or 0.35~0.45g / L. In a specific embodiment of the present invention, the concentration of NAA in the rooting medium may be 0.3, 0.35, 0.4, 0.45 or 0.5g / L. NAA can promote the rooting of adventitious buds of Elymus davidianus. The concentration of sucrose in the rooting medium provided by the present invention is 28~30g / L. In a specific embodiment of the present invention, the concentration of sucrose in the rooting medium may be 28, 29 or 30g / L. The concentration of agar in the rooting medium provided by the present invention is 7~9g / L. In a specific embodiment of the present invention, the concentration of agar in the rooting medium can be 7, 8 or 9 g / L. In the present invention, the pH value of the rooting medium can be 5.7-5.9, or 5.80.

[0025] The induction medium, differentiation medium and rooting medium provided by the present invention all contain sucrose, which can be a carbon source required for plant growth, promote the generation and proliferation of callus, the growth and rooting of buds. The tissue culture medium provided by the present invention has good application effect, and can improve the callus induction rate of mature embryos of Elymus nutans. The present invention does not specifically limit the sources of each component in the induction medium, differentiation medium and rooting medium, and conventional commercially available products can be used. The innovation of the present invention is to use the mature embryos of Elymus nutans as explants, obtain high-quality and dense embryonic callus by regulating exogenous hormones and culture medium formulas, establish a technical system for plant regeneration, and provide a research platform for genetic transformation and trait improvement.

[0026] The present invention provides a regeneration tissue culture method for Elymus nutans, wherein the regeneration tissue culture method adopts the tissue culture medium described in the above technical solution and comprises the following steps: The mature embryos of Elymus nutans are inoculated into an induction medium for induction culture to obtain callus tissue; Transferring the callus tissue to a differentiation medium for subculture to obtain sterile seedlings; The stem segments of the sterile seedlings are transferred to a rooting medium for rooting culture to obtain rooted seedlings.

[0027] The present invention inoculates the mature embryos of Elymus nutans into an induction medium for induction culture to obtain callus tissue. As an embodiment, the preparation method of the mature embryos of Elymus nutans described in the present invention may include: removing the glume of Elymus nutans seeds with full grains to obtain mature embryos. Taking mature embryos as explants also has the advantage of convenient material collection. The present invention obtains mature embryos, and also includes disinfection before inoculation, and the disinfection may also include disinfection with a sodium hypochlorite solution with an effective chlorine content of 10% for 28 to 32 minutes, and rinsing with sterile water for 5 to 7 times. The present invention can use a sodium hypochlorite solution with an effective chlorine content of 10% for disinfection for 28 to 32 minutes, and can also disinfect for 30 minutes. After the present invention uses the sodium hypochlorite solution for disinfection, it can also use sterile water for cleaning, and the number of times of cleaning can be 5 to 7 times, or it can be 6 times. The disinfection method of the present invention is simple and effective.

[0028] After sterilizing the mature embryos of the present invention, the mature embryos of Elymus nutans can be dried with sterile filter paper and then inoculated into an induction medium for induction culture to obtain callus. As an embodiment, the inoculation method of the present invention is preferably placed on an induction medium.

[0029] As an embodiment, the temperature of the induction culture can be 25±2°C, or 25°C; the time of the induction culture can be 45-60 days, or 45-50 days, or even 45 days. The induction culture is dark culture. The time of the induction culture of the present invention is calculated from the time when the mature embryos are inoculated on the induction medium until a white dense callus the size of a soybean grain is obtained. Dark culture helps promote the formation of callus in mature embryos.

[0030] After obtaining the callus tissue, the present invention transfers the callus tissue to a differentiation medium for subculture to obtain sterile seedlings. As an embodiment, the temperature of the subculture can be 25±2°C, or 25°C; the time for the seedling growth culture can be 25~35d, or 27~32d, or even 28d. The subculture can be carried out under alternating light and dark conditions, and the illumination time of the alternating light and dark can be 12~16h / d, or 14~16h / d; the dark time of the alternating light and dark can be 8~12h / d, or 8~10h / d; the intensity of the illumination can be 2200~2500lx, or 2300~2500lx. The subculture of the present invention can be carried out until the height of the sterile seedlings is 2~3cm.

[0031] After obtaining the sterile seedlings, the present invention can divide the sterile seedlings into individual plants, transfer them to a rooting medium for rooting culture, and obtain rooted seedlings.

[0032] As an embodiment, the temperature of the rooting culture of the present invention can be 25±2°C, or 25°C; the rooting culture is carried out until the root length of the rooted seedling is 5-7cm. The rooting culture of the present invention is light culture, and the rooting culture can be carried out under light conditions, and the light can be white light irradiation; the white light irradiation can be carried out using ordinary fluorescent lamps; the light intensity of the white light irradiation can be 2200-2500lx, or 2300-2500lx. Rooting culture enables the sterile seedlings to grow strong roots.

[0033] The rooted seedlings are obtained, and the present invention may further include hardening and transplanting the rooted seedlings to obtain complete regenerated plants. The hardening time of the present invention may be 3 days, and the substrate used for transplanting may include peat soil and vermiculite, and the volume ratio of the peat soil to the vermiculite may be 1:1. The present invention does not specifically limit the temperature of the hardening and the management method of transplanting, and the conventional management method of Elymus nutans can be adopted.

[0034] In the prior art, tissue culture of Elymus nutans usually uses young spikelets as explants, and the callus induction rate is low. The present invention confirms that compared with callus induced by mature embryos, the callus induction rate induced by stems as explants is only 26%, and the induced callus is spongy and severely vitrified. Figure 5 B and D in the figure. The innovation of the present invention is to use the mature embryos of Elymus nutans as explants, provide a method for establishing a callus induction and regeneration system of mature embryos of Elymus nutans, and regulate the ratio of exogenous hormones in the callus induction medium to improve the induction rate of callus of Elymus nutans. At present, there are few reports on the research of plant tissue culture using mature embryos of Elymus nutans as explants. Therefore, it is very necessary to establish a tissue culture regeneration system using mature embryos of Elymus nutans as explants. The method for establishing a callus induction and regeneration system of mature embryos of Elymus nutans established by the present invention has the characteristics of fast reproduction speed, high production efficiency, and maintaining the inherent shape and characteristics of the original variety. It provides a research platform for the smooth development of gene editing, genetic transformation, and trait improvement memory planting creation, and also lays a foundation for future gene editing and genetic transformation research.

[0035] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] The seeds of Elymus nutans used in the following examples and comparative examples were provided by the State Key Laboratory of Three Rivers Source Ecology and Plateau Agriculture and Animal Husbandry jointly established by the Qinghai University and the Ministry of Agriculture. The seeds of Elymus nutans with full grains were selected for de-glume cultivation to obtain mature embryos; the de-glume treatment was to soak the seeds in water for 8 to 12 hours for artificial de-hulling to obtain mature embryos of Elymus nutans, and the seeds could not be polished, because the seed coat would be worn off after polishing, and the embryo would be damaged. The seeds of Elymus nutans were from the same batch of seeds.

[0037] Example 1 Establishment of callus induction and regeneration system of Elymus nutans 1. Disinfection of Explants Take mature embryos of Elymus nutans and soak them in 40% sodium hypochlorite solution for disinfection for 30 min. The effective chlorine concentration of 40% sodium hypochlorite solution is 10%. After disinfection, place them on a clean bench and rinse them with sterile water for 5 to 7 times under sterile conditions. Then wipe the surface of the mature embryo dry with sterilized filter paper.

[0038] 2. Inducing callus formation The mature embryos of Elymus nutans were placed in the callus induction medium (i.e., induction medium) for induction culture. The callus induction medium was: 4g / L MS, 4mg / L 2, 4-D, 50mg / L CH, 2mg / L ABA, 0.04mg / L KT, 30g / L sucrose and 8g / L agar. The induction culture was dark culture at 25±2℃. After 45 days of culture, white callus was induced until the white dense callus of the size of soybean grains appeared. After 50 days of induction culture, Figure 1 .

[0039] 3. Subculture in Differentiation Medium The induced callus tissue was inoculated into the differentiation medium for subculture. The composition of the differentiation medium was: 2g / L MS, 0.02 mg / L KT, 0.4mg / L NAA, 15g / L sucrose and 8g / L agar. The light intensity of the subculture was 2500 lx, the light time was 16 h / d, the dark time was 8 h / d, the culture temperature was 25±2℃, and the culture time was 28 days. The subculture was continued until the seedlings were 2~3cm long.

[0040] Adventitious buds obtained from callus differentiation culture of Elymus nutans for 26 days Figure 2 .

[0041] 4. Rooting culture The differentiated seedlings were transferred to the rooting medium for rooting culture. The rooting medium consisted of 4g / L MS, 0.5 g / L NAA, 30g / L sucrose and 8g / L agar. The rooting culture was placed under a normal fluorescent light source with a light intensity of 2300lx and a temperature of 25±2℃. The roots were cultured until they were strong and 5~7cm long. Figure 3 .

[0042] 5. Hardening and transplanting After the test tube seedlings of Elymus nutans were opened and hardened for 3 days, the test tube seedlings were taken out and the residual culture medium on the roots was washed off. The complete regenerated plants were transplanted. The regenerated plants of Elymus nutans 60 days after transplantation showed Figure 4 .

[0043] Comparative Example 1 The same as Example 1, the only difference is that the young stems of suitable size are cut from the seed culture seedlings of Elymus nutans as explants, and the specific method of obtaining the young stems is: take sterile Elymus nutans seedlings, and cut the seedling stems into stem segments of suitable size of about 1 cm with a sterilized blade or scissors as explants. The sterile seedlings do not need to be disinfected.

[0044] The callus initiation rate and differentiation rate of the callus tissues of Example 1 and Comparative Example 1 were statistically analyzed, and the specific method was as follows: The induction time and induction rate of callus induced by different explants in Example 1 and Comparative Example 1 were statistically analyzed. There were 50 explants in each plate and 5 repeated experiments were set.

[0045] After the callus tissue was transferred to the differentiation medium for 2 weeks, the differentiation rate was counted. Twenty callus tissues were transferred to each plate and five replicate experiments were set up.

[0046] Induction rate (%) = (number of healed explants / number of infected explants) × 100 Differentiation rate (%) = (number of sprouted callus / number of infected callus) × 100 The callus tissue induced from the seeds (mature embryos) and stems of Elymus nutans in Example 1 and Comparative Example 1 is shown in Figure 5 It can be seen that the callus induced by the seeds of Elymus nutans is light yellow and has a dense structure; the callus induced by the stems of Elymus nutans is spongy, vitrified, and has a loose structure; the callus induction rate of different explants is significantly different. The callus induction rate of mature embryo structure of Elymus nutans seeds is 55%~65%, and the callus induction rate of stems is only 14%~26%. Although both mature embryo-induced callus and stem-induced callus can differentiate and sprout, there are also obvious differences in differentiation rates. The differentiation rate of mature embryo-induced callus on differentiation medium is 40%~46%, and the differentiation rate of stem-induced callus is lower, with only 1 differentiated seedling obtained from 100 callus.

[0047] Comparative Example 2 The same as Example 1, only 1 and 2 in Example 1 were performed, the only difference being that the composition of the induction medium was changed, and the composition of the induction medium was 4 g / L MS, 3 mg / L 2,4-D, 30 mg / L CH, 0.02 mg / L KT, 30 g / L sucrose and 8 g / L agar.

[0048] Compared with Example 1, the culture medium of the induction culture medium of Comparative Example 2 can culture callus tissue, but the callus induction rate is greatly reduced as observed by the ratio of the number of callus tissues to the total number of mature embryos in one culture dish, and the density of the callus tissue is lower than that of Example 1. Figure 6 .

[0049] Comparative Example 3 The same as Example 1, only 1 and 2 in Example 1 were performed, the only difference being that the composition of the induction medium was changed, and the composition of the induction medium was 4 g / L MS, 4 mg / L 2,4-D, 40 mg / L CH, 0.04 mg / L KT, 1 mg / LABA, 1 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0050] Compared with Example 1 and Comparative Example 2, the induction medium of Comparative Example 3 can culture callus. Although the callus initiation rate of Comparative Example 3 is higher than that of Comparative Example 2, the callus formed by induction has rooting and more seedlings, resulting in a low density of callus. Figure 7 .

[0051] Comparative Example 4 Same as Example 1, only 1 and 2 in Example 1 were performed, the only difference being that the composition of the induction medium was changed to 4 g / L MS, 5 mg / L 2,4-D, 50 mg / L CH, 0.06 mg / L KT, 2 mg / LABA, 2 mg / L NAA, 30 g / L sucrose and 8 g / L agar; Compared with Example 1, the callus induced by mature embryos was of suitable size and compact structure when NAA hormone was added to the induction medium, but the number of plants was large and the root system was seriously developed. Figure 8 .

[0052] Comparative Example 5 The same as Example 1, only 1 to 3 in Example 1 were performed, the only difference being that the composition of the differentiation medium was changed, and the composition of the differentiation medium was: 2 g / L MS, 0.02 mg / L KT, 0.2 mg / L NAA, 15 g / L sucrose and 8 g / L agar.

[0053] Compared with Example 1, the callus tissue of Comparative Example 5 was in a differentiated state, and no obvious differentiation phenomenon occurred. This was due to the low level of NAA hormone. Fig. 9 .

[0054] Comparative Example 6 The same as Example 1, only 1 to 3 in Example 1 were performed, the only difference being that the composition of the differentiation medium was changed, and the composition of the differentiation medium was: 2 g / L MS, 0.02 mg / L KT, 0.6 mg / L NAA, 15 g / L sucrose and 8 g / L agar.

[0055] Compared with Example 1, when excessive NAA was added to the differentiation medium, the callus did not differentiate into regenerated shoots, but took root severely, which resulted in the original callus losing its differentiation ability after taking root. Fig.10 .

[0056] Comparative Example 7 The same as Example 1, only 1 to 3 in Example 1 were performed, the only difference being that the composition of the differentiation medium was changed, and the composition of the differentiation medium was: 2 g / L MS, 0.04 mg / L KT, 0.4 mg / L NAA, 15 g / L sucrose and 8 g / L agar.

[0057] Compared with Example 1, when the KT content in the differentiation medium was too high, the germination rate was low and the seedlings turned yellow after germination, and the growth of the seedlings was poor. Fig.11 .

[0058] In summary, the induction rate of the densely structured callus of the present invention reaches 65%, and in the subsequent differentiation culture process, 46% of the callus differentiates into adventitious buds and produces regenerated plants. The Agrobacterium genetic transformation system based on mature embryos of Elymus nutans established by the present invention provides certain technical guidance for exploring the gene function of Elymus nutans, and also lays a foundation for the directed molecular design to improve the varieties of Elymus nutans.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture medium for culturing Elymus nutans, characterized in that: The tissue culture medium includes an induction medium, a differentiation medium and a rooting medium; The induction medium includes 4-4.43 g / L MS, 4-6 mg / L 2,4-D, 40-60 mg / L CH, 1-3 mg / L ABA, 0.04-0.07 mg / L KT, 28-30 g / L sucrose and 7-9 g / L agar; The differentiation medium comprises 2-2.215 g / L MS, 0.01-0.03 mg / L KT, 0.3-0.5 mg / L NAA, 12.5-15 g / L sucrose and 7-9 g / L agar; The rooting medium comprises 4-4.43 g / L MS, 0.3-0.5 g / L NAA, 28-30 g / L sucrose and 7-9 g / L agar.

2. The tissue culture medium according to claim 1, characterized in that The pH value of the induction medium is 5.7-5.9, the pH value of the differentiation medium is 5.7-5.9, and the pH value of the rooting medium is 5.7-5.

9.

3. A regeneration tissue culture method for Elymus nutans, characterized in that: The regeneration tissue culture method adopts the tissue culture medium according to claim 1 or 2, comprising the following steps: The mature embryos of Elymus nutans are inoculated into an induction medium for induction culture to obtain callus tissue; Transferring the callus tissue to a differentiation medium for subculture to obtain sterile seedlings; The sterile seedlings are transferred to a rooting medium for rooting culture to obtain rooted seedlings.

4. The regeneration tissue culture method according to claim 3, characterized in that: The mature embryos are sterilized before inoculation, wherein the sterilization comprises soaking in a sodium hypochlorite solution with an effective chlorine content of 10% for 28 to 32 minutes, and then washing with sterile water for 5 to 7 times.

5. The regeneration tissue culture method according to claim 3, characterized in that: The temperatures of the induction culture, subculture and rooting culture are 25±2°C respectively.

6. The regeneration tissue culture method according to claim 3, characterized in that: The induction culture is dark culture; the subculture culture is light-dark alternating culture; and the rooting culture is light culture.

7. The regeneration tissue culture method according to claim 6, characterized in that: The illumination time of the light-dark alternation is 12-16 h / d; the illumination intensity is 2200-2500 lx; ​​the darkness time of the light-dark alternation is 8-12 h / d.

8. The regeneration tissue culture method according to claim 6, characterized in that: The light culturing includes white light irradiation.

9. The regeneration tissue culture method according to claim 3, characterized in that: The induction culture time is 45 to 60 days; the subculture time is 28 to 35 days; and the rooting culture is cultured until the root length is 5 to 7 cm.

10. The regeneration tissue culture method according to claim 3, characterized in that: After obtaining the rooted seedlings, the process also includes seedling hardening and transplanting.

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