A method for tissue culture of Leymus chinensis plants

Abstract

This application provides a method for tissue culture of *Elymus* species, comprising: (1) seed treatment; (2) callus induction; (3) callus subculturing; and (4) callus differentiation. By selecting suitable seed treatment methods, callus selection criteria, and culture media used in each step, the method of this application effectively improves the callus induction rate and differentiation rate, solves the problems of difficulty in obtaining callus and low differentiation rate of *Elymus* and *Elymus sibiricum* when mature seeds are used as explants, and obtains a high-frequency plant regeneration system for *Elymus* and *Elymus sibiricum*.

Description

Technical Field

[0001] This application belongs to the field of plant breeding technology. Specifically, this application provides a method for tissue culture of plants of the genus Leymus. Background Technology

[0002] Elymus dahuricus and Elymus sibiricus are perennial herbaceous plants belonging to the genus Elymus in the family Poaceae. They possess characteristics such as cold resistance, drought resistance, alkali tolerance, and wind and sand resistance, exhibiting good adaptability and ecological plasticity. Currently, they are widely cultivated in Northwest my country and the Qinghai-Tibet Plateau, serving as an important component of grasslands and meadows. They are also the dominant forage grass species cultivated and utilized in these regions, possessing high ecological and feed value and representing important forage germplasm resources.

[0003] In recent years, with the development of biotechnology, regeneration systems for crested wheatgrass and old mangrass have been initially established. However, some problems still exist. For example, existing regeneration systems all use young spikelets as explants, which are limited by season and time. Moreover, the hormone content in young spikelets varies greatly, affecting the regeneration rate and genetic stability [Zhao Zhiyan, Pan Junsong, He Yali, et al. Establishment of tissue culture and regeneration system for vegetative organs 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 from young embryos of new wheatgrass [J]. Chinese Journal of Grassland Science, 2008, (02): 39-42+67.]. This seriously limits the discovery of excellent stress-resistant genes and germplasm improvement of crested wheatgrass and old mangrass. Therefore, establishing a stable regeneration system for crested wheatgrass and old mangrass using mature embryos as explants and breaking the temporal and spatial limitations of crested wheatgrass tissue culture regeneration is of great significance for promoting the development of the forage industry. Summary of the Invention

[0004] This application provides a method for plant regeneration using mature seeds of *Leymus chinensis* and *Triticum aestivum* as explants. By controlling seed treatment, basic culture medium, exogenous plant hormone ratio, and analyzing superior callus morphology, this method overcomes the difficulties in obtaining callus from *Leymus chinensis* and *Triticum aestivum* and the low differentiation rate when using mature seeds as explants. Simultaneously, it obtains a high proportion of granular embryogenic callus, establishes a stable regeneration technology system, and provides a research platform for genetic transformation and trait improvement.

[0005] On the one hand, this application provides a method for tissue culture of *Leymus* species, the method comprising:

[0006] (1) Seed treatment;

[0007] (2) Callus induction;

[0008] (3) Callus subculture;

[0009] (4) Callus differentiation.

[0010] Furthermore, the *Leymus* species mentioned are *Leymus chinensis* or *Leymus chinensis*.

[0011] Further, the seed treatment (1) includes: selecting plump seeds, disinfecting them in 3%-5% sodium hypochlorite for 20 minutes, and washing them 10 times with sterile deionized water; removing the lemma from the seeds; immediately disinfecting the seeds again with 3%-5% sodium hypochlorite for 20 minutes after removing the lemma, and washing them 10 times with sterile water; cutting the embryos of the disinfected seeds and placing them in callus induction medium for light-protected culture.

[0012] Furthermore, (2) MS basal medium was used for callus induction, wherein 3 mg L of MS basal medium was added. -1 Or 4mg L -1 2,4-D and 0.1 mg L -1 6-BA.

[0013] Further, (2) in the callus induction, yellow, hard and dense callus tissues are selected for subsequent callus subculture.

[0014] Further, (2) in the induction of callus tissue, callus tissue that is white, loose in structure, has a high water content and is cotton-like, and contains 1-3 hard nuclei is selected for subsequent callus tissue subculture.

[0015] Furthermore, (3) MS basal medium was used for callus subculturing, wherein 5 mg L of MS basal medium was added. -1 2,4-D and 0.1 mg L -1 6-BA.

[0016] Furthermore, (4) MS basal medium was used for callus differentiation, wherein 2 mg L of MS basal medium was added. -1 2,4-D and 0.1 mg L -1 6-BA.

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

[0018] On the other hand, this application provides the application of the above-mentioned methods in genetic manipulation, breeding, or germplasm resource conservation of Leymus plants. Attached Figure Description

[0019] Figure 1 This section shows the induction, subculturing, and transformation of *Leymus chinensis* callus; Part A: *Leymus chinensis* callus induction; Part B: callus induction for 6 weeks; Part C: 2 mg / L... -12,4-D, 0.1 mg / L -1 Callus tissue cultured in 6-BA subculture medium for 4 weeks; Part D: 5 mg / L -1 2,4-D, 0.1 mg / L -1 Callus tissue after 4 weeks of subculture in 6-B medium; Part E: Rooting culture; Parts F and G: Different types of callus tissue; Part H: Callus tissue selected under a stereomicroscope; Part I: Adventitious shoots differentiated under a stereomicroscope.

[0020] Figure 2 This shows the different callus types of Leymus chinensis.

[0021] Figure 3 This study demonstrates the induction, subculturing, and transformation of *Ophiopogon japonicus* callus tissue; Part A: *Ophiopogon japonicus* callus induction; Part B: callus induction for 6 weeks; Part C: 2 mg / L... -1 2,4-D, 0.1 mg / L -1 Callus tissue cultured in 6-BA subculture medium for 4 weeks; Part D: 5 mg / L -1 2,4-D, 0.1 mg / L -1 Callus tissue after 4 weeks of subculture in 6-B medium; Part E: Rooting culture; Parts F and G: Different types of callus tissue; Part H: Callus tissue No. 4 under a stereomicroscope; Part I: Adventitious shoots differentiated under a stereomicroscope.

[0022] Figure 4 This shows the different callus types of old wheat. Detailed Implementation

[0023] Example 1: Leymus chinensis tissue culture

[0024] Materials and methods

[0025] Test materials:

[0026] The seeds of *Leymus chinensis* used in the experiment were sourced from Beijing Best Grass Industry Co., Ltd., and were newly produced seeds of *Leymus chinensis* that year.

[0027] Test method:

[0028] Seed disinfection and peeling:

[0029] The following three methods are used for seed treatment:

[0030] Treatment 1: Select plump seeds and disinfect them in 3%-5% sodium hypochlorite solution for 20 minutes. Wash them 10 times with sterile deionized water and remove the lemma and palea. Immediately after removing the lemma and palea, disinfect the seeds again with 3%-5% sodium hypochlorite solution for 20 minutes and wash them 10 times with sterile water. After the disinfected seeds have had their embryos injured, place them in callus induction medium and culture them in the dark.

[0031] Treatment 2: Select plump seeds and disinfect them in 3%-5% sodium hypochlorite solution for 20 minutes. Wash them 10 times with sterile deionized water and remove the lemma and palea. Air-dry the seeds at room temperature for half a month, then disinfect them again with 3%-5% sodium hypochlorite solution for 20 minutes and wash them 10 times with sterile water. After disinfection, injure the embryos of the seeds and place them in callus induction medium for culture in the dark.

[0032] Treatment 3: Select plump seeds and disinfect them in 3%-5% sodium hypochlorite solution for 20 minutes, then wash them 10 times with sterile deionized water. Without removing the lemma, cut the embryo directly and disinfect it again in 3%-5% sodium hypochlorite solution for 20 minutes, then wash it 10 times with sterile water. After the disinfected seeds have been cut to injure the embryo, place them in callus induction medium for culture in the dark.

[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 medium, with 30 g / L added. -1 Maltose, 1g L -1 Proline, 1g L -1 Acid-hydrolyzed casein, 6-BA 0.1 mg / L -1 2,4-D 0-9 mg / L -1 and 4g L -1 Gel, pH 5.8, was used as a callus induction medium (Table 1).

[0035] Inoculation: Sterilized seeds were placed under a dissecting microscope. Seeds with intact embryos were selected, and the embryo portion was cut with a scalpel. The seeds were then placed in callus induction medium, 10 dishes of each type per inoculation, with 16 seeds per dish. The inoculation was carried out at 25°C in the dark. After 4 weeks, the callus induction rate was calculated as (number of explants with callus formation ÷ total number of explants × 100%). After 6 weeks, callus selection and subculture were performed.

[0036] Subculture: Six weeks after callus induction, superior callus tissues were selected and inoculated onto two subculture media, with MS (M519) as the basal medium supplemented with 30g L. -1 Maltose, 1g L -1 Proline, 1g L -1Acid-hydrolyzed casein, 6-BA 0.1 mg / L -1 2,4-D 2 / 5mg L -1 and 4g L -1 Gel, pH 5.8, was used as a subculture medium.

[0037] Healing selection:

[0038] Six weeks after callus induction, superior callus tissue was selected. Callus tissue induced from individual explants was placed under a dissecting microscope, classified, and the proportion of each callus type was calculated. Callus tissues containing embryoids and possessing proliferation and differentiation potential (superior callus tissues) were selected. The number of superior callus tissues relative to the total number of callus tissues in different culture media was counted, and the selection rate was calculated as (number of superior callus tissues ÷ total number of callus tissues × 100%).

[0039] Based on the understanding of the structure of the induced callus, the callus tissue that appears during the induction process is divided into six categories, numbered 0-5. Figure 2 Callus #0 is unique, as it differentiates into adventitious buds during the induction process. Callus #1 and #2 are white, loosely structured, have high water content, resemble cotton, and grow rapidly. Callus #1 lacks a hard core, while callus #2 contains 1-3 hard cores. Callus #3 and #4 are yellow, grow vigorously, and easily differentiate into seedlings. Callus #3 has a dense structure, while callus #4 has a loose and brittle structure. Callus #5 has weak regeneration ability and is prone to browning and death.

[0040] Healing and Succession:

[0041] The callus tissue was divided into sections with a volume of 0.5 cm. 3 Small pieces were inoculated onto different subculture media. Subculture medium: MS 4.43 g / L -1 30g L -1 Maltose, 1g L -1 Proline, 1g L -1 Acid-hydrolyzed casein, 6-BA 0.1 mg / L -1 2,4-D 5mg / L -1 Or 2mg L -1 and 4g L -1 Gel, pH 5.8.

[0042] Callus differentiation experiment:

[0043] The callus tissue was divided into 0.5cm sections. 3 Small pieces were inoculated onto two different differentiation media (MS 4.43 g L). -1 30g L -1 Maltose, 6-BA 0.1 mg / L -1 2,4-D 2mg / L -1and 4g L -1 Gel, pH 5.8; MS 4.43 g / L -1 30g L -1 Maltose, 6-BA 0.2 mg / L -1 and 4g L -1 The callus tissue (pH 5.8) was used for differentiation culture to induce the generation of regenerated shoots. After 4 weeks of differentiation culture, dark green regenerated shoots appeared on the pale yellow callus tissue. These shoots were then transferred to rooting medium to induce further development of clustered shoots.

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

[0045] Table 1. Culture media used in Example 1

[0046]

[0047]

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

[0049] As shown in Table 2, under the same induction medium, the treatment method of seeds has a significant impact on the callus induction rate. Treatment 3 (cutting the embryo directly without removing the lemma) had the lowest callus induction rate. The induction rate of seeds in Treatment 2 (removing the lemma and then placing the embryo in the treatment) was significantly lower than that of Treatment 1.

[0050] Different basal culture media and hormone ratios were designed to induce callus formation in seeds. Seeds were cultured on callus induction media with different formulations, and the callus rate was calculated after 30 days (Table 2). The induction results showed that, under the same treatment and exogenous hormone addition, the induction rate of MS basal medium was higher than that of N6 basal medium. Using MS as the basal medium, changing the exogenous hormone ratio under the same treatment significantly altered the induction rate of *Leymus chinensis* seeds, with a 2,4-D concentration of 4 mg / L... -1 The concentration of 6-BA was 0.1 mg / L. -1 At this time, the callus emergence rate was higher, and the callus selection rate (number of excellent callus tissue ÷ total number of callus × 100%) was the highest. In summary, MS basal medium and 4 mg L... -1 2,4-D, 0.1 mg / L -1 For 6-BA, the optimal induction method is to induce callus tissue immediately after the lemma is removed from the seed.

[0051] Table 2. Callus induction rate of *Leymus chinensis* under different treatments and different callus induction culture medium components.

[0052]

[0053]

[0054] like Figure 1 As shown, after 6 weeks of callus induction, different colors and states of callus tissue were observed: Callus No. 0 had differentiated into adventitious buds; Callus No. 1 was white or slightly yellow, with high water content, soft structure, and no dense nucleate callus; this type of callus could not differentiate and was extremely difficult to subculture to produce callus tissue suitable for transformation. Callus No. 2 was white or slightly yellow, with high water content, soft structure, and few dense nucleate callus; this type of callus could produce callus tissue suitable for transformation after subculture and selection. Callus Nos. 3 and 4 were yellow, hard in texture, dense in structure, and had high regeneration efficiency. Callus No. 5 had weak regeneration ability, was prone to browning, and was prone to death. Callus No. 0 easily differentiated into seedlings; Callus Nos. 3 and 4 had high embryogenicity, were easy to form seedlings, and could maintain embryogenicity through multiple subcultures; Callus No. 2 might grow into Callus No. 3 after multiple subcultures.

[0055] Four weeks after passage, the hormone ratio was found to be 0.1 mg / L 6-BA. -1 2,4-D 5mg / L -1 On the subculture medium, callus types 3 and 4 grew normally, and callus type 2 was subcultured into callus type 3. The hormone ratio was 0.1 mg / L 6-BA. -1 2,4-D 2mg / L -1 Callus types 3 and 4 on the subculture medium showed abnormal growth. The newly formed callus was yellowish-brown, loosely structured, spongy with high water content, lacked a hard core, and degenerated into callus type 1 with severe browning. In summary, MS basal medium and 5 mg / L... -1 2,4-D, 0.1 mg / L -1 6-BA is the optimal culture medium formulation for subculture.

[0056] The callus tissue was divided into sections with a volume of 0.5 cm. 3 Small pieces of callus were inoculated onto different differentiation media for differentiation culture to induce the formation of shoot clusters. The differentiation rate was calculated after 3 weeks (Table 3). The differentiation results showed that callus No. 3 was more likely to differentiate into shoots, and the ratio of exogenous hormones in the culture medium could change the differentiation rate of the callus.

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

[0058]

[0059]

[0060] Example 2: Tissue Culture of Old Mango

[0061] Materials and Methods:

[0062] Test materials:

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

[0064] Test method:

[0065] Seed disinfection and peeling

[0066] Seed treatments were the same as those for crested wheatgrass, namely treatment 1 and treatment 2.

[0067] Callus induction, subculture and differentiation experiments

[0068] Same treatment as for crested wheatgrass.

[0069] Results and Analysis:

[0070] Different combinations of basal culture media and hormone ratios were designed to induce callus formation in seeds. Seeds were cultured on different callus induction media, and the callus rate was calculated after 30 days (Table 4). Similar to the results of *Leymus chinensis* induction, under the same induction medium, the seed treatment method had a significant impact on the callus induction rate. Seeds treated by removing the lemma and placing them in the medium all showed a significant decrease in induction success rate. As shown in Table 4, changing the ratio of exogenous hormones can alter the callus induction rate of *Leymus chinensis* seeds to some extent. When the 2,4-D concentration reached 3 mg / L... -1 At or above, the concentration of 6-BA is 0.1 mg / L. -1 At that time, the callus formation rate of *Ophiopogon japonicus* did not increase with increasing 2,4-D concentration, but the ratio of exogenous hormones could alter the quality of induced callus tissue. When the 2,4-D concentration was 3 mg / L... -1 At this time, the callus selection rate (potential callus count ÷ total number of callus × 100%) was the highest, and the callus emergence rate was also the highest. In summary, MS basal medium and 3 mg / L... -1 2,4-D, 0.1 mg / L -1 The optimal induction method for callus induction is to immediately perform 6-BA after disinfection and removal of the lemma, which can achieve a callus formation rate of 94%.

[0071] like Figure 3 As shown, after screening the subculture medium, it was found that, similar to the subculture of *Leymus chinensis* callus, the hormone ratio after four weeks of subculture was 6-BA 0.1 mg / L. -1 2,4-D 2mg / L -1 On the subculture medium, the excellent callus growth was abnormal, degenerating into callus number 1 without embryoids, while the hormone ratio was 0.1 mg L⁻¹ 6-BA and 5 mg L⁻¹ 2,4-D. -1Callus growth was normal on the subculture medium. In summary, MS basal medium and 5 mg / L... -1 2,4-D, 0.1 mg / L -1 6-BA is the optimal culture medium formulation for subculture.

[0072] The callus tissue was divided into 0.5cm sections. 3 Small pieces were inoculated onto different differentiation media for differentiation culture to induce the formation of shoot clusters. The differentiation rate was calculated after 3 weeks (Table 5). The differentiation results showed that 2 mg L... -1 2,4-D, 0.1 mg / L -1 The differentiation medium formulated with 6-BA made it easier for the callus tissue of *Eriocheir sinensis* to differentiate, and its No. 2 callus tissue was more likely to differentiate into seedlings.

[0073] Table 4. Composition of callus induction culture medium and callus induction rate for each treatment group of *Mallotus spp.*

[0074]

[0075] Table 5 Differentiation rates of different callus types from *Mallotus spp.* on different differentiation media.

[0076]

[0077]

[0078] The implementation of this invention helps accelerate the biotechnology breeding process of monocotyledonous plants, yielding considerable social and economic benefits. The method described in this invention uses mature seeds of *Leymus chinensis* and *Leymus chinensis* as explants, and obtains clustered shoots through callus induction, proliferation, and differentiation, overcoming the difficulties of obtaining explants and inconsistent explant development during the regeneration of *Leymus chinensis* and *Leymus chinensis*. It also lays the technical foundation for realizing molecular biological manipulation of *Leymus chinensis* and *Leymus chinensis*.

[0079] In the embodiments of this invention, callus tissues produced from mature embryos of *Elymus sibiricum* and *Triticum aestivum* are classified into six types, and the morphological characteristics of each type of callus tissue are clearly indicated. High-quality callus tissues are easily obtained through two selection processes using a microscope. This method is applicable to *Elymus sibiricum* and *Triticum aestivum*, is easy to operate, and can obtain excellent cell lines from single seeds in just 14 weeks, overcoming the difficulty of obtaining explants during the regeneration of *Elymus sibiricum* and *Triticum aestivum*.

Claims

1. A method for tissue culture of *Leymus* species, characterized in that, The method includes: (1) Seed treatment: Select plump seeds, disinfect them in 3%-5% sodium hypochlorite for 20 min, and wash them 10 times with sterile deionized water; peel off the lemma and chrysanthemum on the outside of the seeds; immediately disinfect the seeds again with 3%-5% sodium hypochlorite for 20 min after peeling off the lemma and chrysanthemum, and wash them 10 times with sterile water; cut the embryo of the disinfected seeds and place them in callus induction medium for culture in the dark. (2) Callus induction: Incubate at 25°C in the dark for 6 weeks on callus induction medium; (3) Callus subculture: Select yellow, hard, and dense callus or white, loose, cotton-like callus with high water content and 1-3 hard nuclei for callus subculture; place them on subculture medium for culture. (4) Callus differentiation: The callus obtained in step (3) is divided into 0.5 cm³ sections. 3 Small pieces were inoculated onto differentiation medium and cultured. The species mentioned is *Leymus* spp.; The callus induction culture medium was supplemented with 3 mg·L⁻¹ -1 or 4 mg·L -1 2,4-D and 0.1 mg·L -1 MS basal medium containing 6-BA; The subculture medium was supplemented with 5 mg·L⁻¹ -1 2,4-D and 0.1 mg·L -1 MS basal medium containing 6-BA; The differentiation medium was supplemented with 2 mg·L⁻¹ -1 2,4-D and 0.1 mg·L -1 MS basal medium containing 6-BA.

2. The method according to claim 1, further comprising a rooting culture step.

3. The application of the method according to claim 1 or 2 in genetic manipulation, breeding or germplasm resource protection of Leymus plants.

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