Method for obtaining barley grass regeneration plant through embryo callus induction

Abstract

The invention discloses a method for obtaining a barley grass regeneration plant through embryo callus induction, and belongs to the technical field of grass industry science and plant tissue culture. According to the method, barley grass seed embryos subjected to endophytic fungus killing, disinfection and cutting treatment serve as explants, callus induction culture, subculture, differentiation culture and rooting culture are conducted through an optimized culture medium, then seedling hardening and transplanting are conducted, and complete regenerated plants are obtained. Experimental results show that the highest callus induction rate of the method for obtaining the barley grass regenerated plants can reach 66.11%, the highest differentiation rate can reach 13.37%, and the obtained callus and regenerated seedlings have high quality. Reliable technical support is provided for genetic engineering breeding and genetic improvement research of the barley grass, and remarkable practical significance and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the fields of grass science and plant tissue culture technology, and in particular to a method for obtaining Buton's barley grass regeneration plants by inducing embryonic callus tissue. Background Art

[0002] Since the first report of successful transformation of tobacco using Agrobacterium infection, genetic engineering has been applied to major food crops to achieve superior phenotypes. With the success of transgenic technology in improving phenotypic traits such as yield and resistance, genetic engineering has been given high hopes, particularly in breeding, where efforts are underway to overcome the limitations of genetic inheritance. Using transgenic technology to cultivate and improve grasses is a convenient and practical method. Furthermore, Agrobacterium transformation technology has matured, offering advantages such as ease of operation, high transformation efficiency, a low number of insert copies, and a relatively simple integration mechanism.

[0003] Hordeum bogdanii, a perennial herb of the genus Hordeum in the family Gramineae, is widely distributed in northern grasslands, showing strong environmental adaptability and strong resistance to drought and salinity. As a forage grass, it has the advantages of high yield per plant, tolerance to mowing, rich protein content and good palatability. However, without long-term domestication, Hordeum bogdanii becomes a natural host for a variety of pathogens (such as rust, leaf spot, powdery mildew). When Hordeum bogdanii is planted in large-scale fields, these diseases will be significantly aggravated starting from the second year, seriously affecting yield and quality. Therefore, cultivating new strains of Hordeum bogdanii with disease resistance is the key to promoting its widespread use as forage.

[0004] Currently, breeding strategies for Buton's barley grass mainly focus on wild species domestication, recurrent selection, and hybrid breeding. However, due to the complexity of the species' genotype, these traditional breeding methods face many challenges and have difficulty achieving breakthrough progress. In this context, it is particularly important to establish a mature genetic transformation system for Buton's barley grass. However, current academic research on the genetic transformation system of Buton's barley grass is scarce, and a mature system has not yet been established. This limits the application of gene editing technologies such as CRISPR / Cas9, hindering the precise modification of its genome and trait improvement.

[0005] Therefore, there is an urgent need to provide a method for callus induction culture and regeneration system of Buton's barley grass, so as to provide a possibility for rapid breeding of Buton's barley grass. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for obtaining regenerated plants of Buton barley grass by inducing embryonic callus, so as to solve the problems existing in the above-mentioned prior art. The present invention explores the effects of different hormone ratios on the steps of callus induction, subculture, differentiation, rooting, etc., completes the screening of callus induction, subculture, differentiation and primary culture medium, and establishes a mature embryo tissue culture system of Buton barley.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for obtaining a regenerated plant of Buton barley grass, comprising the following steps:

[0009] The pretreated embryos of Buton barley grass seeds were used as explants, inoculated into induction medium, and induced to obtain callus tissue.

[0010] Transferring the callus tissue to a subculture medium for subculture, and after subculture twice, transferring it to a differentiation medium for differentiation culture to obtain differentiated seedlings;

[0011] The differentiated seedlings are transferred to a primary culture medium for rooting culture. After the rooting culture is completed, the seedlings are hardened and transplanted to obtain complete regenerated plants.

[0012] Furthermore, the pretreatment includes the following steps: killing endophytic fungi, disinfecting and cutting.

[0013] Furthermore, the killing of endophytic fungi comprises the following steps:

[0014] Vernalized Buton barley grass seeds were taken, dried at 60°C for 7 days, treated at 65°C for 20 days, planted, and mature seeds were harvested for later use.

[0015] Furthermore, the induction medium comprises the following components:

[0016] basal medium, 2 mg / L 2,4-D, and 1 mg / L NAA;

[0017] The basal medium includes the following components:

[0018] N6 basal salt, maltose 30g / L, acid hydrolyzed casein 0.5g / L, proline 0.5g / L, glutamine 0.5g / L, vitamin B1 1mg / L, CuSO4 25mg / L and plant gel 4g / L.

[0019] Furthermore, the induction culture temperature is 23° C., the time is 25 days, and the lighting condition is dark.

[0020] Furthermore, the differentiation medium comprises the following components:

[0021] basal medium, 2.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.05 mg / L KT;

[0022] The basal medium includes the following components:

[0023] N6 basal salt, maltose 30g / L, acid hydrolyzed casein 0.5g / L, proline 0.5g / L, glutamine 0.5g / L, vitamin B1 1mg / L, CuSO4 25mg / L and plant gel 4g / L.

[0024] Furthermore, the differentiation culture has a light cycle of 16 hours and a dark cycle of 8 hours; a light cycle culture temperature of 25° C. and a dark cycle culture temperature of 23° C.; and a light intensity of 2000-3000 Lux.

[0025] Furthermore, the primary culture medium comprises the following components: 1 / 2MS basal salt, 15g / L sucrose and 7g / L agar.

[0026] Furthermore, the photoperiod of the rooting culture is 16 hours, and the dark period is 8 hours; the photoperiod culture temperature is 25° C., and the dark period culture temperature is 23° C.; and the light intensity is 2000-3000 Lux.

[0027] The present invention also provides a use of the regenerated plants of Buton's barley grass obtained according to the above method in the genetic transformation of Buton's barley grass.

[0028] The present invention discloses the following technical effects:

[0029] The present invention uses endophyte-killed, disinfected, and wounded budding barley grass seed embryos as explants, and uses an optimized culture medium for callus induction, subculture, differentiation, and rooting. This is followed by hardening and transplanting to obtain complete regenerated plants. Experimental results show that the present method for obtaining budding barley grass regeneration plants has a callus induction rate of up to 66.11% and a differentiation rate of up to 13.37%. The obtained callus and regenerated seedlings are of high quality. This invention provides reliable technical support for genetic engineering breeding and genetic improvement research of budding barley grass, and has significant practical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1This is a diagram showing the initial effect of killing endophytic fungi in the seeds of Buton barley grass during mature embryo induction. Figure a-1 is a mature embryo contaminated by endophytic fungi during induction; a-2 is the embryo of a-1; b-1 is a mature embryo induced after killing endophytic fungi; b-2 is the embryo of b-1.

[0032] Figure 2 The figures show the results of callus induction and subculture under different auxin ratios. Figure a shows the callus induction under 4 mg / L 2,4-D and 3 mg / L NAA; figure b shows the callus induction under 4 mg / L 2,4-D and 1 mg / L NAA; figure c shows the callus induction under 2 mg / L 2,4-D and 1 mg / L NAA; figure d shows the callus induction under subculture corresponding to figure a; figure e shows the callus induction under subculture corresponding to figure b; figure f shows the callus induction under subculture corresponding to figure c.

[0033] Figure 3 The results of callus regeneration under different auxin and mitogen ratios are shown in Figure 2. Among them, a is the addition of 1.0 mg / L 6-BA; b is the addition of 2.0 mg / L 6-BA; c is the addition of 3.0 mg / L 6-BA; d is the addition of 4.0 mg / L 6-BA; e is the addition of 2 mg / L 6-BA and 0.1 mg / L NAA; f is the addition of 2 mg / L 6-BA and 0.5 mg / L NAA; g is the addition of 2 mg / L 6-BA, 0.5 mg / L NAA and 0.05 mg / L KT; h is the addition of 2 mg / L 6-BA, 0.5 mg / L NAA and 0.1 mg / L KT;

[0034] Figure 4 This is the result of rooting after transfer to primary culture medium. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The Buton barley grass seeds of the present invention were originally collected in Linze, Gansu Province, and the seed material number is E1.

[0041] Example 1

[0042] 1. Kill endophytic fungi

[0043] The Buton barley grass seeds harvested half a year later (after vernalization) were placed in a 60°C oven for 7 days to remove excess moisture generated during seed storage. After the seeds were dried, they were transferred to 65°C for 20 days.

[0044] The heat-killed seeds were planted for recovery, and mature seeds were harvested for subsequent experiments.

[0045] 2. Disinfection and Cutting of Plant Materials

[0046] Select mature, uniform-sized and color-rich seeds of Buton's barley grass. Soak them in a 50% sulfuric acid solution for 30 minutes to remove the outer bran. Rinse them five times with running water to obtain clean seeds. Then treat them with 75% alcohol for 60 seconds, rinse them three times with sterile water, disinfect them with 10% sodium hypochlorite for 5 minutes, and rinse them five times with sterile water. Place the seeds on sterile filter paper until they are dry and ready for use. Use a sterile scalpel to cut the seeds longitudinally, creating a wound in the embryo.

[0047] 3. Culture Medium Preparation

[0048] Basal culture medium: N6 basal salt, maltose 30g / L, acid hydrolyzed casein 0.5g / L, proline 0.5g / L, glutamine 0.5g / L, vitamin B1 1mg / L, CuSO4 25mg / L, plant gel 4g / L.

[0049] Induction medium: Add 2,4-D, 6-BA, and NAA to the basal medium and determine the optimal concentrations. Set the 2,4-D concentrations to 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, and 4.0 mg / L; the 6-BA concentrations to 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, and 0.5 mg / L; and the NAA concentrations to 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, 1 mg / L, and 2 mg / L (Table 1).

[0050] Subculture medium: Add 2.0 mg / L 2,4-D to the basal medium as subculture medium.

[0051] Differentiation medium: Add the mitogens 6-BA and NAA to the basal medium and determine the optimal concentrations. 6-BA concentrations were set at 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, and 4.0 mg / L; NAA concentrations were set at 0.1 mg / L and 0.5 mg / L; and KT concentrations were set at 0.05 mg / L and 0.1 mg / L (Table 2).

[0052] The primary culture medium is: 1 / 2MS basal salt, 15g / L sucrose, and 7g / L agar.

[0053] The pH of the above culture media was adjusted to 5.8-5.9 and sterilized by high pressure at 115°C for 30 min.

[0054] 4. Treatment of Seed Embryos and Induction of Differentiation

[0055] Optimize the culture conditions of each part and determine under which culture conditions the best culture effect can be achieved.

[0056] The specific culture method is as follows: take the damaged embryos that have been disinfected in step 2, place them in the above-mentioned induction culture medium containing different hormone ratios, culture them in the dark at a constant temperature incubator at 23°C for 25 days, and calculate the healing rate and growth status.

[0057] The callus tissue was transferred to subculture medium and subcultured for 25 days.

[0058] After subculture twice, the cells were transferred to the above-mentioned differentiation medium containing different ratios of mitogen and auxin, placed in an incubator with a light intensity of 2000-3000 Lux, a light / dark cycle of 16h / 8h, and a culture temperature of 25°C for light cycle and 23°C for dark cycle; the culture medium was replaced every 15 days to maintain the hormone concentration, and the callus differentiation rate and seedling emergence rate were counted after 30 days.

[0059] 5. Rooting and hardening

[0060] When the differentiated seedlings grow to 1-2 cm, the callus tissue only has cluster buds. The differentiated seedlings are transferred to the primary culture medium for rooting. They are cultured under the conditions of a light cycle of 25°C / dark cycle of 23°C, a light intensity of 2000-3000 Lux, and a light period of 16 hours / darkness of 8 hours. The rooting rate of the cluster buds is 100%.

[0061] When the roots of the seedlings are approximately 5 cm long, remove the regenerated seedlings from the artificial climate chamber and clean any remaining culture medium from the roots. After 2 days, refill with tap water and harden the seedlings. After 3 days, if the survival rate reaches over 95%, transplant them to the greenhouse after 5 days. After cleaning the roots, transplant them to autoclaved soil (vermiculite:nutrient soil = 1:1) to obtain complete regenerated plants.

[0062] 6. Experimental Results

[0063] The initial effects of mature embryo induction are as follows Figure 1 As shown in the figure, after being treated at 60℃ for 7 days, the temperature was switched to 65℃ for 20 days, and the sterilization rate of endophytic fungi reached 100%.

[0064] Table 1 Effects of different concentrations of 2,4-D, 6-BA and NAA on callus formation of Hordeum vulgare

[0065]

[0066] According to the 2,4-D concentration gradient (Table 1), the induction rate of mature embryos after longitudinal section showed no significant difference between the 2-5 mg / L 2,4-D range, ranging from 44.73% to 47.04%. However, the calli produced at higher concentrations were too dense, while at 2 mg / L, the calli had a grainy texture and rapid callus growth. Therefore, 6-BA was added to 2 mg / L 2,4-D. The callus induction rate decreased with the addition of 6-BA, reaching 34.40% with the addition of 0.05 mg / L 6-BA, lower than the induction rate without 6-BA. When NAA was added to 2 mg / L 2,4-D, the callus induction rate initially increased and then decreased with the addition of NAA. The highest induction rate, 66.11%, was achieved with a hormone combination of 2 mg / L 2,4-D and 1 mg / L NAA.

[0067] Therefore, the optimal induction medium for mature embryos of Hordeum vulgare is: basal medium + 2 mg / L 2,4-D + 1 mg / L NAA.

[0068] After two subcultures, three callus lines with different states were obtained, such as Figure 2 As shown, callus induction was observed ( Figure 2 ac) and secondary culture ( Figure 2 In the case of df), select Figure 2 The compact, pale yellow calli shown in c and f were used for subsequent experiments.

[0069] Table 2 Effects of different concentrations of 6-BA and NAA on the differentiation ability of wild barley callus

[0070]

[0071] Since the regeneration of Buton barley grass is slow, the regeneration status was counted at 30 days, and the differentiation medium was replaced once on the 15th day. Figure 3 As shown in Table 2, when the 6-BA concentration gradually increased, the differentiation rate showed a trend of first increasing and then decreasing. At 2.0 mg / L 6-BA, the differentiation rate was 6.22%. However, the root hair growth of the callus was severe during the regeneration process, which affected the production of differentiated buds. Adding 0.5 mg / L NAA to 2.0 mg / L 6-BA inhibited root production. Adding 0.05 mg / L KT increased the differentiation rate of the callus. Therefore, 2.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.05 mg / L KT were used as the optimal hormone ratio for the Buton barley grass callus differentiation medium.

[0072] Rooting results Figure 4 As shown, the rooting rate of differentiated seedlings was 100%.

[0073] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for obtaining regenerated plants of Buton barley grass, characterized in that: The following steps are involved: The pretreated embryos of Buton barley grass seeds were used as explants, inoculated into induction medium, and induced to obtain callus tissue. Transferring the callus tissue to a subculture medium for subculture, and after subculture twice, transferring it to a differentiation medium for differentiation culture to obtain differentiated seedlings; The differentiated seedlings are transferred to a primary culture medium for rooting culture. After the rooting culture is completed, the seedlings are hardened and transplanted to obtain complete regenerated plants.

2. The method according to claim 1, wherein The pretreatment comprises the following steps: killing endophytic fungi, disinfecting and cutting.

3. The method according to claim 2, wherein The killing of endophytic fungi comprises the following steps: Vernalized Buton barley grass seeds were taken, dried at 60°C for 7 days, treated at 65°C for 20 days, planted, and mature seeds were harvested for later use.

4. The method according to claim 1, wherein The induction medium includes the following components: basal medium, 2 mg / L 2,4-D, and 1 mg / L NAA; The basal medium includes the following components: N6 basal salt, maltose 30g / L, acid hydrolyzed casein 0.5g / L, proline 0.5g / L, glutamine 0.5g / L, vitamin B1 1mg / L, CuSO4 25mg / L and plant gel 4g / L.

5. The method according to claim 1, wherein The induction culture was carried out at a temperature of 23° C., for 25 days, and under dark conditions.

6. The method according to claim 1, wherein The differentiation medium comprises the following components: basal medium, 2.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.05 mg / L KT; The basal medium includes the following components: N6 basal salt, maltose 30g / L, acid hydrolyzed casein 0.5g / L, proline 0.5g / L, glutamine 0.5g / L, vitamin B1 1mg / L, CuSO4 25mg / L and plant gel 4g / L.

7. The method according to claim 1, wherein The differentiation culture has a light cycle of 16 hours and a dark cycle of 8 hours; a light cycle culture temperature of 25° C. and a dark cycle culture temperature of 23° C.; and a light intensity of 2000-3000 Lux.

8. The method according to claim 1, wherein The primary culture medium includes the following components: 1 / 2MS basal salt, 15g / L sucrose and 7g / L agar.

9. The method according to claim 1, wherein The photoperiod of the rooting culture is 16 hours, and the dark period is 8 hours; the culture temperature during the photoperiod is 25° C., and the culture temperature during the dark period is 23° C.; and the light intensity is 2000-3000 Lux.

10. Use of the regenerated plant of Bouton's barley grass obtained by the method according to any one of claims 1 to 9 in the genetic transformation of Bouton's barley grass.

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