Elymus plant tissue culture method

Abstract

The invention provides an elymus plant tissue culture method. The elymus plant tissue culture method comprises the following steps: (1) seed treatment; (2) callus induction; (3) carrying out callus subculture; and (4) callus differentiation. By selecting a proper seed treatment method, a callus selection standard and a culture medium used in each step, the method disclosed by the invention effectively improves the induction rate and the differentiation rate of the callus, solves the problems that the callus of elymus dahuricus and siberian wildrye is difficult to obtain and the differentiation rate is low when mature seeds are used as explants, and improves the survival rate of the elymus dahuricus and the siberian wildrye. A high-frequency plant regeneration system of the elymus dahuricus and the siberian wildrye is obtained.

Description

Technical Field

[0001] The present application belongs to the technical field of plant breeding. Specifically, the present application provides a tissue culture method for plants of the genus Elymus. Background Art

[0002] Elymus dahuricus and Elymus sibiricus are perennial herbs of the genus Elymus in the Poaceae family. They are cold-resistant, drought-resistant, alkali-resistant, and wind-sand-resistant, and have good adaptability and ecological plasticity. They are currently widely planted in Northwest my country and the Qinghai-Tibet Plateau. They are an important part of grasslands and meadows, and are also the main forage grass species cultivated and utilized in the region. They have high ecological and feeding value and are important forage germplasm resources.

[0003] In recent years, with the development of biotechnology, the regeneration system of Elymus dahliae and Elymus sibiricus has been preliminarily established, but there are still some problems. For example, the existing regeneration system uses young spikelets as explants, which are limited by season and time. In addition, the hormone content in young spikelets varies greatly, which affects the regeneration rate and genetic stability [Zhao Zhiyan, Pan Junsong, He Yali, et al. Establishment of vegetative organ tissue culture and regeneration system of two tall fescue clones [J]. Acta Prataculturae Sinica, 2009, 18(05):168-175.; Li Junqin, Yun Jinfeng, Yun Lan, et al. Induction of callus tissue of immature embryos of Psathyrostachys ovata [J]. Acta Grasslanda Sinica, 2008, (02):39-42+67.], which seriously limits the mining of excellent stress-resistant genes and germplasm improvement of Elymus dahliae and Elymus sibiricus. Therefore, it is of great significance to establish a stable regeneration system of Elymus dahliae and Elymus sibiricus using mature embryos as explants to break the temporal and spatial limitations of tissue culture regeneration of Elymus sibiricus plants and promote the development of forage industry. Summary of the invention

[0004] The present application provides a plant regeneration method using mature seeds of Elymus salsa and Elymus sibiricus as explants. By regulating the seed treatment method, basic culture medium, exogenous plant hormone ratio, and excellent callus morphology analysis, the problem of difficulty in obtaining callus tissue of Elymus salsa and Elymus sibiricus and low differentiation rate when mature seeds are used as explants is overcome, and a high proportion of granular embryonic callus tissue is obtained, a stable regeneration technology system is established, and a research platform is provided for genetic transformation and trait improvement.

[0005] In one aspect, the present application provides a method for tissue culture of a plant of the genus Elymus, the method comprising:

[0006] (1) Seed treatment;

[0007] (2) callus induction;

[0008] (3) Callus subculture;

[0009] (4) Callus differentiation.

[0010] Furthermore, the Elymus plant is Elymus dahliae or Elymus sibiricus.

[0011] Furthermore, the (1) seed treatment comprises: selecting full seeds, placing them in 3%-5% sodium hypochlorite for disinfection for 20 minutes, and washing them with sterile deionized water for 10 times; peeling off the lemmas outside the seeds; immediately sterilizing the seeds after peeling off the lemmas again with 3%-5% sodium hypochlorite for 20 minutes, and washing them with sterile water for 10 times; and placing the sterilized seeds in a callus induction medium after cutting the embryos and culturing them in the dark.

[0012] Furthermore, (2) callus induction was performed using MS basal medium supplemented with 3 mg L -1 or 4 mg L -1 of 2,4-D and 0.1mg L -1 6-BA.

[0013] Furthermore, (2) during callus induction, callus with yellow color, hard texture and compact structure is selected for subsequent callus subculture.

[0014] Furthermore, (2) during callus induction, callus that is white, loose in structure, cotton-like in appearance, and contains 1-3 hard cores is selected for subsequent callus subculture.

[0015] Furthermore, (3) the callus tissue was subcultured using MS basal medium supplemented with 5 mg L -1 of 2,4-D and 0.1mg L -1 6-BA.

[0016] Furthermore, (4) callus differentiation was performed using MS basal medium supplemented with 2 mg L -1 of 2,4-D and 0.1mg L -1 6-BA.

[0017] Furthermore, the method also includes a rooting culture step.

[0018] On the other hand, the present application provides the application of the above method in genetic manipulation, breeding or germplasm resource protection of Elymus plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows the induction, subculture and transformation of Elymus dahliae callus; Part A: Induction of Elymus dahliae callus; Part B: Callus induction for 6 weeks; Part C: 2 mg L -12,4-D, 0.1mg L -1 Callus tissue cultured in 6-BA subculture medium for 4 weeks; Part D: 5 mg L -1 2,4-D, 0.1mg L -1 6-B Callus tissue after 4 weeks of culture on subculture medium; Part E: rooting culture; Part F and Part G: different types of callus tissue; Part H: callus tissue selected under a stereomicroscope; Part I: adventitious buds differentiated under a stereomicroscope.

[0020] Figure 2 Showing the different callus types of Elymus salsa.

[0021] Figure 3 Shows the induction, subculture and transformation of callus tissue of Elymus sibiricus; Part A: Induction of callus tissue of Elymus sibiricus; Part B: Callus induction for 6 weeks; Part C: 2 mg L -1 2,4-D, 0.1mg L -1 Callus tissue cultured in 6-BA subculture medium for 4 weeks; Part D: 5 mg L -1 2,4-D, 0.1mg L -1 6-B callus after 4 weeks of culture on subculture medium; Part E: rooting culture; Part F and Part G: different types of callus; Part H: No. 4 callus under a stereomicroscope; Part I: adventitious buds produced by differentiation under a stereomicroscope.

[0022] Figure 4 Showing different callus types of Elymus sibiricus. DETAILED DESCRIPTION

[0023] Example 1 Elymus dahliae tissue culture

[0024] Materials and methods

[0025] Test materials:

[0026] The seeds of Elymus dahliae used in the experiment came from Beijing Best Grass Industry Co., Ltd. and were new seeds of Elymus dahliae produced that year.

[0027] Test method:

[0028] Seed disinfection and peeling:

[0029] There are three methods for seed treatment:

[0030] Treatment 1: Select full seeds, place them in 3%-5% sodium hypochlorite for disinfection for 20 minutes, wash them with sterile deionized water 10 times, and peel off the lemma outside the seeds. Immediately sterilize the seeds with 3%-5% sodium hypochlorite for 20 minutes, and wash them with sterile water 10 times. After the sterilized seeds are cut, the embryos are placed in the callus induction medium and cultured in the dark.

[0031] Treatment 2: Select full seeds, place them in 3%-5% sodium hypochlorite for disinfection for 20 minutes, wash them with sterile deionized water 10 times, and peel off the lemma. The seeds with the lemma peeled off were dried in the shade at room temperature and stored for half a month, and then disinfected with 3%-5% sodium hypochlorite for 20 minutes, and washed with sterile water 10 times. After the disinfected seeds were cut into embryos, they were placed in callus induction medium and cultured in the dark.

[0032] Treatment 3: Select full seeds, place them in 3%-5% sodium hypochlorite for disinfection for 20 minutes, and wash them with sterile deionized water 10 times. Cut the embryos directly without removing the lemma and place them in 3%-5% sodium hypochlorite for disinfection again for 20 minutes, wash them with sterile water 10 times, and place the embryos of the disinfected seeds in callus induction medium for dark culture.

[0033] Callus induction and subculture:

[0034] Culture medium: Two basic culture media were used for callus induction: MS (M519) and N6 (C416). MS or N6 was used as the basic culture medium, and 30 g L -1 Maltose, 1g L -1 Proline, 1 g L -1 Acid hydrolyzed casein, 6-BA 0.1mg L -1 、2,4-D0~9mg L -1 and 4g L -1 gel, pH 5.8, was used as callus induction medium (Table 1).

[0035] Inoculation: Place the sterilized seeds under a dissecting microscope, select seeds with intact embryos, cut the embryo parts with a scalpel, and place them in callus induction medium, 10 dishes of each type each time, 16 seeds per dish. Culture at 25℃ in the dark. After 4 weeks, calculate the callus induction rate (number of explants with callus formation ÷ total number of explants × 100%), and select callus and subculture after 6 weeks.

[0036] Subculture: Six weeks after callus induction, select excellent callus tissue and inoculate it on two subculture media, using MS (M519) as the basic medium and adding 30g L -1 Maltose, 1g L -1 Proline, 1 g L -1Acid hydrolyzed casein, 6-BA 0.1mgL -1 , 2,4-D 2 / 5mg L -1 and 4g L -1 gel, pH 5.8, was used as the secondary culture medium.

[0037] Healing selection:

[0038] After 6 weeks of callus induction, excellent callus tissue was selected. The callus tissue induced by a single explant was placed under a dissecting microscope, the callus tissue was classified, the proportion of various callus types was counted, and callus with embryoids and potential for proliferation and differentiation (excellent callus tissue) was selected. We counted the number of excellent callus to the total number of callus in different culture media and calculated the selection rate (number of excellent callus tissues ÷ total number of callus × 100%).

[0039] Based on the understanding of the structure of the induced callus, the callus that appeared during the induction process was divided into six categories (0-5). Figure 2 ). Callus No. 0 is quite special, and adventitious buds have been differentiated during the induction process. Callus No. 1 and No. 2 are white, loose in structure, cotton-like in water content, and grow fast. Callus No. 1 has no hard core, and callus No. 2 contains 1-3 hard cores. Callus No. 3 and No. 4 are yellow, grow vigorously, and are easy to differentiate into seedlings. Callus No. 3 has a dense structure, and callus No. 4 has a brittle structure. Callus No. 5 has weak regeneration ability and is prone to browning and death.

[0040] Healing subculture:

[0041] The callus tissue was divided into 0.5 cm 3 Small pieces were inoculated into different subculture medium. Subculture medium MS4.43g L -1 , 30g L -1 Maltose, 1g L -1 Proline, 1 g L -1 Acid hydrolyzed casein, 6-BA 0.1mg L -1 、2,4-D 5mgL -1 or 2 mg L -1 and 4g L -1 Gel, pH 5.8.

[0042] Callus differentiation experiment:

[0043] The callus tissue was cut into 0.5 cm 3 Small pieces were inoculated into two different differentiation media (MS4.43 g L -1 , 30g L -1 Maltose, 6-BA 0.1mg L -1 , 2,4-D 2mg L -1and 4g L -1 Gel, pH 5.8; MS 4.43 gL -1 , 30g L -1 Maltose, 6-BA 0.2mg L -1 and 4g L -1 After 4 weeks of differentiation culture, dark green regenerated buds appeared on the pale yellow callus, which were transferred to rooting medium to induce further development of clustered buds.

[0044] The composition of the culture medium used in the method is shown in Table 1:

[0045] Table 1 Culture medium used in Example 1

[0046]

[0047]

[0048] Note: 200×VB stock (1L): 2g thiamine hydrochloride (VB1), 1.9g pyridoxine zincate (VB6), 0.9g nylon acid. Results and analysis:

[0049] As shown in Table 2, under the same induction medium, the seed treatment method has a great influence on the callus induction rate. The seed callus induction rate of treatment 3 (directly cutting the embryo without stripping the lemma) is the lowest; the seed callus induction rate of treatment 2 (stripping the lemma and then placing it in a place) is significantly lower than that of treatment 1.

[0050] Different basal culture media and hormone ratios were designed to induce callus in seeds. The seeds were cultured on callus induction medium with different formulas, and the callus rate was calculated after 30 days (Table 2). The induction results showed that under the same treatment method and the same exogenous hormone addition, the induction rate of MS basal culture medium was higher than that of N6 basal culture medium. Using MS as the basal culture medium, changing the ratio of exogenous hormones under the same treatment can significantly change the induction rate of Elymus dahliae seeds. When the concentration of 2,4-D was 4 mg L -1 , 6-BA concentration was 0.1 mg L -1 When , the callus rate is higher, and the callus selection rate (number of high-quality callus tissues ÷ total number of calli × 100%) is the highest. In summary, MS basal medium, 4 mg L -1 2,4-D, 0.1mg L -1 For 6-BA, the best induction method is to induce callus tissue immediately after removing the lemma from the seeds.

[0051] Table 2 Callus induction rate of Elymus dahliae under different treatments and different callus induction medium components

[0052]

[0053]

[0054] like Figure 1 As shown in the figure, after 6 weeks of callus induction, callus tissues were observed to be in different colors and states: callus tissue No. 0 had differentiated into adventitious buds; callus tissue No. 1 was white or slightly yellow, with high water content, loose structure, and no dense nuclear callus. Such callus tissues could not differentiate and were extremely difficult to subculture into callus tissues that could be used for transformation. Callus tissue No. 2 was white or slightly yellow, with high water content, loose structure, and few dense nuclear callus. Such callus tissues could produce callus tissues that could be used for transformation after subculture selection. Callus tissues No. 3 and No. 4 were yellow, hard in texture, dense in structure, and had high regeneration efficiency. Callus No. 5 had weak regeneration ability, was easy to brown, and was easy to die. Callus No. 0 was easy to differentiate into seedlings, and callus tissues No. 3 and No. 4 were highly embryonic, easy to form seedlings, and could be subcultured multiple times to maintain embryonic characteristics; callus No. 2 could grow into callus No. 3 after multiple subcultures.

[0055] After four weeks of subculture, the hormone ratio was found to be 6-BA 0.1mg L -1 , 2,4-D 5mg L -1 On the subculture medium, callus No. 3 and No. 4 grew normally, and callus No. 2 subcultured to callus No. 3. The hormone ratio was 6-BA 0.1mgL -1 , 2,4-D 2mg L -1 The calli No. 3 and No. 4 on the subculture medium grew abnormally. The new calli were yellow-brown, loose in structure, spongy with high water content, without hard core, and degenerated into callus No. 1 with severe browning. In summary, MS basal medium, 5 mg L -1 2,4-D, 0.1mg L -1 6-BA is the best formula of subculture medium.

[0056] The callus tissue was divided into 0.5 cm 3 The small pieces were inoculated into different differentiation mediums for differentiation culture to induce the production of clustered buds. The differentiation rate was calculated after 3 weeks (Table 3). The differentiation results showed that callus No. 3 was more likely to differentiate into seedlings, and the ratio of exogenous hormones in the culture medium could change the differentiation rate of callus.

[0057] Table 1 Differentiation rates of different callus types in different differentiation media

[0058]

[0059]

[0060] Example 2 Tissue culture of Elymus sibiricus

[0061] Materials and Methods

[0062] Test materials:

[0063] The old sibiricum seeds used in the experiment came from Inner Mongolia Agricultural University and were new old sibiricum seeds produced that year.

[0064] Test method:

[0065] Seed disinfection and peeling

[0066] The seed treatments were the same as those for Elymus salsa, Treatment 1 and Treatment 2.

[0067] Experiments on callus induction, subculture and differentiation

[0068] Treat with Elymus salsa.

[0069] Results and Analysis:

[0070] Different basal culture media and hormone ratios were designed to induce callus in seeds. The seeds were cultured on different callus induction media, and the callus induction rate was calculated after 30 days (Table 4). Similar to the results of Elymus salsa induction, under the same induction medium, the seed treatment method had a great influence on the callus induction rate. The induction success rate of seeds placed after the lemma was removed was greatly reduced. As shown in Table 4, changing the ratio of exogenous hormones can change the callus induction rate of Elymus sibiricus seeds to a certain extent. When the 2,4-D concentration reached 3 mg L -1 and above, 6-BA concentration is 0.1 mg L -1 The callus rate of Elymus sibiricus did not increase with the increase of 2,4-D concentration, but the ratio of exogenous hormones could change the quality of induced callus. -1 When , the callus selection rate (number of potential callus tissues ÷ total number of calli × 100%) was the highest, and the callus emergence rate was the highest. In summary, MS basal medium, 3 mg L -1 2,4-D, 0.1mg L -1 6-BA, the best induction method is to induce callus tissue immediately after disinfection and peeling of the lemma, and the callus rate can reach 94%.

[0071] like Figure 3 As shown in the figure, after screening the subculture medium, it was found that the hormone ratio after four weeks of subculture was 6-BA 0.1mg L -1 , 2,4-D 2mg L -1 The excellent callus on the subculture medium grew abnormally and degenerated into callus No. 1 without embryoids. The hormone ratio was 6-BA 0.1mg L-1, 2,4-D 5mg L -1In summary, MS basal medium, 5 mg L -1 2,4-D, 0.1mg L -1 6-BA is the best formula of subculture medium.

[0072] The callus tissue was cut into 0.5 cm 3 The small pieces were inoculated into different differentiation mediums for differentiation culture to induce the formation of clustered buds. The differentiation rate was calculated after 3 weeks (Table 5). -1 2,4-D,0.1mg L -1 The differentiation medium with 6-BA formula makes it easier for Elymus sibiricus callus to differentiate, and its No. 2 callus is easier to differentiate into seedlings.

[0073] Table 4 Callus induction medium components and callus induction rates of each treatment group of Elymus sibiricus

[0074]

[0075] Table 5 Differentiation rates of different callus types of Elymus sibiricus in different differentiation media

[0076]

[0077]

[0078] The implementation of the present invention helps to accelerate the biotechnology breeding process of monocotyledonous plants, and has considerable social and economic benefits. The method of the present invention uses mature seeds of Elymus dahliae and Elymus sibiricus as explants, and obtains clustered buds through induction, proliferation, and differentiation of callus tissue, thereby overcoming the difficulties of obtaining explants and inconsistent explant development during the regeneration of Elymus dahliae and Elymus sibiricus. At the same time, it also lays a technical foundation for the molecular biological operation of Elymus dahliae and Elymus sibiricus.

[0079] In the embodiments of the present invention, callus tissues produced by mature embryos of Elymus dahliae and Elymus sibiricus are divided into 6 types, and the morphological characteristics of each type of callus tissue are clearly pointed out. Excellent callus tissues can be easily obtained by two selections with the help of a microscope. The method is applicable to Elymus dahliae and Elymus sibiricus, is easy to operate, and only takes 14 weeks to obtain excellent cell lines produced by a single seed, thus overcoming the difficulty of obtaining explants during the regeneration of Elymus dahliae and Elymus sibiricus.

Claims

1. A method for tissue culture of Elymus spp., characterized in that: The method comprises: (1) Seed treatment; (2) callus induction; (3) Callus subculture; (4) Callus differentiation. 2 . The method according to claim 1 , wherein the plant of the genus Elymus is Elymus dahliae or Elymus sibiricus.

3. The method according to claim 1 or 2, wherein (1) seed treatment comprises: Select full seeds, place them in 3%-5% sodium hypochlorite for disinfection for 20 minutes, and wash them with sterile deionized water for 10 times; peel off the lemmas outside the seeds; immediately disinfect the seeds with the lemmas peeled off again with 3%-5% sodium hypochlorite for 20 minutes, and wash them with sterile water for 10 times; after cutting the embryos of the disinfected seeds, place them in a callus induction medium and culture them in the dark.

4. The method according to any one of claims 1 to 3, wherein (2) the callus induction is carried out using MS basal medium supplemented with 3 mg L -1 or 4 mg L -1 of 2,4-D and 0.1mg L -1 of 6-BA.

5. The method according to any one of claims 1 to 4, wherein (2) during callus induction, callus that is yellow in color, hard in texture, and compact in structure is selected for subsequent callus subculture.

6. The method according to any one of claims 1 to 5, wherein (2) during callus induction, a callus that is white, loose in structure, has a high water content and is cotton-like, and contains 1 to 3 hard cores is selected for subsequent callus subculture.

7. The method according to any one of claims 1 to 6, wherein (3) the callus subculture is carried out using MS basal medium supplemented with 5 mg L -1 of 2,4-D and 0.1mg L -1 of 6-BA.

8. The method according to any one of claims 1 to 7, wherein (4) MS basal medium is used for callus differentiation, wherein the MS basal medium is supplemented with 2 mg L -1 of 2,4-D and 0.1mg L -1 of 6-BA.

9. The method according to any one of claims 1 to 8, further comprising a rooting culture step.

10. Use of the method according to any one of claims 1 to 9 in genetic manipulation, breeding or germplasm resource protection of plants of the genus Elymus.

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