A method for obtaining regenerated Button barley grass plants through embryo callus induction

Abstract

This invention discloses a method for obtaining regenerated Button barley grass plants through embryo callus induction, belonging to the fields of grassland science and plant tissue culture technology. This invention uses Button barley grass seed embryos that have undergone endophytic fungus inactivation, disinfection, and wounding treatment as explants. An optimized culture medium is used for callus induction culture, subculture, differentiation culture, and rooting culture, followed by hardening-off and transplanting to obtain complete regenerated plants. Experimental results show that the method of obtaining Button barley grass regenerated plants using this invention achieves a callus induction rate of up to 66.11% and a differentiation rate of up to 13.37%, with high-quality callus and regenerated seedlings. This invention provides reliable technical support for genetic engineering breeding and genetic improvement research of Button barley grass, and has significant practical significance and broad application prospects.

Description

Technical Field

[0001] This invention relates to the fields of grassland science and plant tissue culture technology, and in particular to a method for obtaining regenerated Button barley grass plants through embryo callus induction. Background Technology

[0002] Since the first report of successful tobacco transformation using Agrobacterium-mediated transformation, genetic engineering has been applied to major food crops to obtain superior phenotypes. With the success of transgenic technology in improving yield, resistance, and other phenotypes, genetic engineering is highly anticipated, especially in breeding to overcome the limitations of genes in the laws of heredity. Using transgenic technology to cultivate and improve gramineous forage grasses is a convenient and practical method. Moreover, Agrobacterium-mediated transformation technology is mature, offering advantages such as ease of operation, high transformation efficiency, low copy number of insertions, and a relatively simple integration mechanism.

[0003] Button barley grass (Hordeum bogdanii), a perennial herb belonging to the genus Hordeum in the family Gramineae, is widely distributed in northern grasslands, exhibiting strong environmental adaptability and high tolerance to drought and salinity. As a forage, it boasts advantages such as high yield per plant, resilience to cutting, rich protein content, and good palatability. However, without long-term domestication, Button barley grass becomes a natural host for various pathogens (such as rust, leaf spot, and powdery mildew). When Button barley grass is planted on a large scale, these diseases significantly intensify from the second year onwards, severely impacting yield and quality. Therefore, developing new disease-resistant Button barley grass varieties is crucial for promoting its widespread use as a forage.

[0004] Currently, breeding strategies for Button barley grass mainly focus on wild-type domestication, recurrent selection, and hybridization. However, due to the complexity of the species' genotype, these traditional breeding methods face numerous challenges and are unlikely to achieve breakthroughs. Against this backdrop, establishing a mature genetic transformation system for Button barley grass is particularly important. However, current research on Button barley grass genetic transformation systems is scarce, and a mature system has not yet been established. This limits the application of gene editing technologies such as CRISPR / Cas9, hindering 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 Button barley grass, which would make it possible to quickly realize the breeding of Button barley grass. Summary of the Invention

[0006] The purpose of this invention is to provide a method for obtaining regenerated Button barley grass plants through embryo callus induction, in order to solve the problems existing in the prior art. This invention explores the effects of different hormone ratios on the induction, subculture, differentiation, and rooting of callus tissue, completes the screening of callus induction, subculture, differentiation and primary culture media, and establishes a Button barley mature embryo tissue culture system.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a method for obtaining regenerated Button barley grass plants, comprising the following steps:

[0009] Pretreated Button barley grass seed embryos were used as explants, inoculated into induction medium, and induced to obtain callus tissue.

[0010] The callus tissue was transferred to a subculture medium for subculture, and after two subcultures, it was transferred to a differentiation medium for differentiation culture to obtain differentiated seedlings.

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

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

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

[0014] Take vernalized Button barley grass seeds, dry them at 60℃ for 7 days, treat them at 65℃ for 20 days, plant them, harvest mature seeds, and keep them for later use.

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

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

[0017] The basal culture medium comprises the following components:

[0018] N6 base 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 was conducted at a temperature of 23°C for 25 days under dark conditions.

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

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

[0022] The basal culture medium comprises the following components:

[0023] N6 base 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 photoperiod of the differentiation culture is 16 hours, the dark period is 8 hours; the photoperiod culture temperature is 25°C, the dark period culture temperature is 23°C, and the light intensity is 2000-3000 Lux.

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

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

[0027] The present invention also provides an application of Button barley grass regenerated plants obtained according to the above method in Button barley grass genetic transformation.

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

[0029] This invention uses Button barley grass seed embryos that have undergone endophytic fungus inactivation, disinfection, and wounding treatment as explants. An optimized culture medium is used for callus induction culture, subculture, differentiation culture, and rooting culture. After hardening off and transplanting, complete regenerated plants are obtained. Experimental results show that the method of obtaining Button barley grass regenerated plants using this invention achieves a callus induction rate of up to 66.11% and a differentiation rate of up to 13.37%, with high-quality callus and regenerated seedlings. This invention provides reliable technical support for genetic engineering breeding and genetic improvement research of Button barley grass, and has significant practical value and broad application prospects. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1The images show the initial effects of inducing mature embryos after killing endophytic fungi in Button barley grass seeds. In the images, a-1 is a mature embryo contaminated with endophytic fungi during induction; a-2 is the plumule of a-1; b-1 is a mature embryo induced after killing endophytic fungi; and b-2 is the plumule of b-1.

[0032] Figure 2 Figure 1 shows the results of induction and subculture of well-grown callus under different auxin ratios; where a represents callus lines under 4 mg / L 2,4-D and 3 mg / L NAA; b represents callus lines under 4 mg / L 2,4-D and 1 mg / L NAA; c represents callus lines under 2 mg / L 2,4-D and 1 mg / L NAA; d represents the callus lines after subculture corresponding to a; e represents the callus lines after subculture corresponding to b; and f represents the callus lines after subculture corresponding to c.

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

[0034] Figure 4 The image shows the results of rooting after transfer to the primary culture medium. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0041] Example 1

[0042] 1. Kill endophytic fungi

[0043] Button barley grass seeds, harvested six months prior (after vernalization), were placed in an oven at 60°C for 7 days to remove excess moisture generated during storage. After drying, the seeds were then transferred to a 65°C oven for 20 days.

[0044] The seeds that were killed by the high temperature were replanted and the mature seeds were harvested for subsequent experiments.

[0045] 2. Disinfection and cutting of plant materials

[0046] Select mature Button barley grass seeds of uniform color and size, soak them in a 50% sulfuric acid solution for 30 minutes to remove the outer bran, and rinse them 5 times with running water to obtain clean seeds. Then treat them with 75% alcohol for 60 seconds, rinse them 3 times with sterile water, disinfect them with 10% sodium hypochlorite for 5 minutes, rinse them 5 times with sterile water, place the seeds on sterile filter paper until the seeds are dry, and then use them. Use a sterilized scalpel to cut the seeds longitudinally to create wounds on the seed embryo.

[0047] 3. Culture medium preparation

[0048] Basic culture medium: N6 basic 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: 2,4-D, 6-BA, and NAA were added to the basal medium, and the optimal concentrations were determined. The concentrations of 2,4-D were set at 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, and 4.0 mg / L; the concentrations of 6-BA were set at 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, and 0.5 mg / L; and the concentrations of NAA were set at 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 a subculture medium.

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

[0052] The initial culture medium consisted of 1 / 2 MS basal salt, 15 g / L sucrose, and 7 g / L agar.

[0053] The pH of all the above culture media was adjusted to 5.8-5.9, and then autoclaved at 115℃ for 30 minutes.

[0054] 4. Seed embryo treatment and differentiation induction

[0055] The cultivation conditions for each part are optimized to determine the optimal cultivation results.

[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, and culture them in the dark at 23℃ for 25 days in a constant temperature incubator. The healing rate and growth status are then statistically analyzed.

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

[0058] After two subcultures, the tissues were transferred to differentiation media containing different ratios of mitogens and auxins, and placed in an incubator with a light intensity of 2000-3000 Lux and a light / dark cycle of 16h / 8h. The culture temperature was 25℃ during the light cycle and 23℃ during the dark cycle. The culture medium was changed every 15 days to maintain the hormone concentration. After 30 days, the callus differentiation rate and seedling emergence rate were calculated.

[0059] 5. Rooting and hardening off seedlings

[0060] When the differentiated seedlings grow to 1-2cm, the callus tissue only has clustered buds. The differentiated seedlings are then transferred to the primary culture medium to root. They are cultured under the conditions of 25℃ photoperiod / 23℃ dark period, 2000-3000 Lux light intensity, 16h light / 8h dark, and the rooting rate of the clustered buds is 100%.

[0061] When the roots of the seedlings to be differentiated are about 5cm long, the regenerated seedlings are removed from the artificial climate chamber, the root culture medium residue is washed off, and tap water is added after 2 days to harden the seedlings. After 3 days, the survival rate is measured to be over 95%, and after 5 days, they are transplanted to the greenhouse for cultivation. After washing the roots, they are transferred to high-pressure steam-sterilized soil (vermiculite: nutrient soil = 1:1) for cultivation to obtain complete regenerated plants.

[0062] 6. Experimental Results

[0063] The initial effects of induction of mature embryos are as follows: Figure 1 As shown, after treatment at 60℃ for 7 days, the treatment was transferred to 65℃ for 20 days, and the endophytic fungicide kill rate reached 100%.

[0064] Table 1. Effects of different concentrations of 2,4-D, 6-BA, and NAA on callus formation in *Barley grass* (Butterton's barley grass).

[0065]

[0066] Based on the 2,4-D concentration gradient (Table 1), the induction rate of longitudinally sectioned mature embryos showed no significant difference in the 2-5 mg / L 2,4-D range, with an induction rate of 44.73-47.04%. However, higher concentrations resulted in overly dense callus tissue; at a concentration of 2 mg / L, the callus tissue had a granular texture and grew rapidly. Therefore, 6-BA was added to the 2 mg / L 2,4-D concentration. The callus induction rate decreased after adding 6-BA, reaching 34.40% with 0.05 mg / L 6-BA, lower than the rate without 6-BA. Adding NAA to the 2 mg / L 2,4-D concentration resulted in a callus induction rate that first increased and then decreased with increasing NAA concentration. The highest induction rate of 66.11% was achieved when the hormone ratio was 2 mg / L 2,4-D and 1 mg / L NAA.

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

[0068] After two generations, it obtained three different states of the Healing type, such as Figure 2 As shown, callus induction can be observed. Figure 2 (middle ac) and successor ( Figure 2 In the case of df), choose as follows Figure 2 The compact, pale yellow callus tissue shown in C and F was used for subsequent experiments.

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

[0070]

[0071] Because Button barley grass regeneration is slow, regeneration progress was assessed at 30 days, with the differentiation medium changed on day 15. Callus regeneration status is as follows: Figure 3 As shown in Table 2, the differentiation rate initially increased and then decreased with increasing 6-BA concentration. At 2.0 mg / L 6-BA, the differentiation rate was 6.22%, but severe root hair growth occurred during callus regeneration, hindering the formation of differentiated shoots. Adding 0.5 mg / L NAA to the 2.0 mg / L 6-BA concentration inhibited root formation. Adding 0.05 mg / L KT increased the callus differentiation rate. Therefore, 2.0 mg / L 6-BA, 0.5 mg / L NAA, and 0.05 mg / L KT were considered the optimal hormone ratio for Button's barley grass callus differentiation culture medium.

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

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

Claims

1. A method for obtaining regenerated Button barley grass plants, characterized in that, Includes the following steps: Pretreated Button barley grass seed embryos were used as explants, inoculated into induction medium, and induced to obtain callus tissue. The callus tissue was transferred to a subculture medium for subculture, and after two subcultures, it was transferred to a differentiation medium for differentiation culture to obtain differentiated seedlings. The differentiated seedlings were transferred into the primary culture medium for rooting culture. After the rooting culture was completed, the seedlings were hardened off and transplanted to obtain complete regenerated plants. The induction medium consisted of: N6 basal salt, maltose 30 g / L, acid-hydrolyzed casein 0.5 g / L, proline 0.5 g / L, glutamine 0.5 g / L, vitamin B1 1 mg / L, CuSO4 25 mg / L, plant gel 4 g / L, 2 mg / L 2,4-D and 1 mg / L NAA; The differentiation medium consisted of: N6 basal salt, maltose 30 g / L, acid-hydrolyzed casein 0.5 g / L, proline 0.5 g / L, glutamine 0.5 g / L, vitamin B1 1 mg / L, CuSO4 25 mg / L, plant gel 4 g / L, 6-BA 2.0 mg / L, NAA 0.5 mg / L, and KT 0.05 mg / L. The primary culture medium consisted of 1 / 2 MS basal salt, 15 g / L sucrose, and 7 g / L agar.

2. The method as described in claim 1, characterized in that, The pretreatment includes the following steps: killing endophytic fungi, disinfection, and cutting.

3. The method as described in claim 2, characterized in that, The process of killing endophytic fungi includes the following steps: Take vernalized Button barley grass seeds, dry them at 60℃ for 7 days, treat them at 65℃ for 20 days, plant them, harvest mature seeds, and keep them for later use.

4. The method as described in claim 1, characterized in that, The induction culture was conducted at a temperature of 23°C for 25 days under dark conditions.

5. The method as described in claim 1, characterized in that, The differentiation culture had a photoperiod of 16 h and a darkperiod of 8 h; the photoperiod culture temperature was 25℃ and the darkperiod culture temperature was 23℃; the light intensity was 2000-3000 Lux.

6. The method as described in claim 1, characterized in that, The photoperiod for the rooting culture was 16 h, and the dark period was 8 h; the photoperiod culture temperature was 25℃, and the dark period culture temperature was 23℃; the light intensity was 2000-3000 Lux.

7. The application of Button barley grass regenerated plants obtained by the method according to any one of claims 1-6 in Button barley grass genetic transformation.

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