Direct regeneration method of pea tissues

Abstract

The invention belongs to the technical field of pea tissue culture, and particularly relates to a pea tissue direct regeneration method, which comprises the following steps: S1, dedusting pea seeds, fumigating with chlorine, and putting the seeds on a germination culture medium; s2, the seeds germinated and cultured for 24 h are placed on a workbench, complete cotyledonary nodes of the seeds are taken out to serve as explants, and the rinsed explants are inoculated into an induction culture medium; s3, placing the induction culture medium inoculated with the explant in a tissue culture room, and after the explant grows a plurality of regeneration buds, transferring the explant to an elongation culture medium and continuing to culture; s4, transferring the explants into a rooting culture medium for rooting induction to obtain pea seedlings; s5, the pea seedlings are transplanted to the natural environment after being subjected to seedling hardening. According to the method, an in-vitro culture method is adopted, regeneration buds do not need to be generated by inducing calluses, the test period is shortened, only about 73 days are needed from explant preparation to regeneration plant obtaining, the regeneration efficiency is high, and the regeneration plants grow vigorously.

Description

Technical Field

[0001] The invention belongs to the technical field of pea tissue culture, and particularly relates to a direct regeneration method for pea tissue. Background Art

[0002] Pea (Pisum sativum L.) is a globally cultivated and important legume crop. It serves multiple purposes, including food, feed, fertilizer, and medicine. Its nitrogen-fixing, soil-replenishing, and stress-resistance characteristics make it crucial for sustainable agricultural development. As people's health awareness grows, demand for nutritious, green, and healthy foods like peas continues to grow, driving the continuous expansion of pea cultivation.

[0003] In recent years, adverse factors such as pests, diseases, and drought have severely constrained pea yield and quality. As a closed-pollinated crop, pea has experienced significant degradation of varietal characteristics, resulting in low artificial hybridization success rates and difficulty meeting the diverse demands of modern agriculture for pea yield, quality, and resistance. The rapid development of plant molecular biology techniques, coupled with the continuous advancement of crop tissue culture and genetic transformation technologies, has opened up new avenues to address these challenges.

[0004] Currently, pea tissue culture regeneration technology primarily relies on callus induction, whereby callus is formed through dedifferentiation of the explant, followed by redifferentiation to induce adventitious bud and root development. However, callus induction takes 3-4 weeks, and redifferentiation takes 2-3 weeks. The process from explant to regenerated plant typically takes 3-4 months, a lengthy period. Furthermore, long-term culture can easily lead to somatic cell asexuality, compromising the genetic stability of the regenerated plants. Summary of the Invention

[0005] To address the problems existing in the above-mentioned prior art, the present invention provides a method for direct regeneration of pea tissue, which adopts an in vitro culture method and does not require the induction of callus tissue to generate regenerated buds, thereby shortening the experimental period. It only takes about 73 days from preparing the explant to obtaining the regenerated plant, with high regeneration efficiency and strong growth of the regenerated plant.

[0006] The specific technical solution adopted in the present invention is:

[0007] A method for direct regeneration of pea tissue comprises the following steps:

[0008] S1. After removing dust from pea seeds, pea seeds are fumigated with chlorine and then evenly placed on a germination medium.

[0009] S2. Place the seeds after germination and culture for 24 hours on a workbench, remove the seed explants, which are 10-13 cm long and 10-13 mm wide, rinse them in sterile water, and inoculate the rinsed explants into the induction medium;

[0010] S3. Place the induction medium inoculated with the explant in a tissue culture room. After the explant grows multiple regenerated buds, transfer the explant with regenerated buds to an elongation medium and continue culturing in the tissue culture room until the regenerated buds elongate to 6-8 cm.

[0011] S4. Transferring the explants with 6-8 cm regenerated shoots into a rooting medium for rooting induction until a well-developed root system grows to obtain pea seedlings;

[0012] S5. After hardening, the pea seedlings are transplanted into a natural environment.

[0013] Furthermore, the germination medium uses sterile water as a solvent, and each liter of sterile water contains: 2.8-3.2g of B5 medium, 18-22g of sucrose, 5-7g of agar powder, 80-120mg of B5 vitamins, and 0.4-0.6mg of 6-benzylaminopurine, and the pH of the germination medium is 5.6-5.8.

[0014] Furthermore, the induction culture medium uses sterile water as a solvent, and each liter of sterile water contains: 2.8-3.2g of B5 culture medium, 0.65-0.70g of morpholineethanesulfonic acid, 25-35g of sucrose, 6-10g of agar powder, 80-120mg of B5 vitamins, 0.4-0.6mg of 6-benzylaminopurine, 0.4-0.6mg of 6-furfurylaminopurine, 5-7mg of ferrous sulfate, 5-7mg of disodium ethylenediaminetetraacetic acid, 22-26mg of glutamine, and 22-26mg of asparagine, and the pH of the induction culture medium is 5.6-5.8.

[0015] Furthermore, the elongation medium uses sterile water as a solvent, and each liter of sterile water contains: MS medium 3.8-4.3g, morpholineethanesulfonic acid 0.38-0.42g, sucrose 25-35g, agar powder 6-10g, B5 vitamin 80-120mg, indoleacetic acid 0.01-0.02mg, gibberellin 0.4-0.6mg, zeatin 0.4-0.6mg, ferrous sulfate 5-7mg, disodium edetate 5-7mg, glutamine 22-26mg, and asparagine 22-26mg, and the pH of the elongation medium is 5.6-5.8.

[0016] Furthermore, the rooting medium uses sterile water as a solvent, and each liter of sterile water contains: 2.0-2.2g of MS medium, 0.38-0.42g of morpholineethanesulfonic acid, 16-22g of sucrose, 5-7g of agar powder, 80-120mg of B5 vitamins, 0.4-0.6mg of indolebutyric acid, 0.4-0.6mg of naphthaleneacetic acid, 22-26mg of glutamine, and 22-26mg of asparagine. The pH of the rooting medium is 5.6-5.8.

[0017] Furthermore, the seed explant removal described in step S2 specifically includes the following steps: clamping the seed with tweezers, with the hilum of the seed facing upward, fixing it on an operating table, using a scalpel to make a horizontal cut at the radicle of the seed, cutting the seed coat along the hilum direction and removing the seed coat and one cotyledon, leaving the hypocotyl and true leaf on the other cotyledon, using a scalpel to cut off and retain 3-5mm of the hypocotyl, removing the true leaf, and scratching the cotyledon node 2-3 times to obtain the explant, the length and width of the explant are both 10-13mm.

[0018] Furthermore, the environmental conditions of the tissue culture room in step S3 are: room temperature 22-24° C., daily illumination time 14-18 h, and light intensity 2000-3000 lx.

[0019] Furthermore, the environmental conditions for rooting induction in step S4 are: room temperature 18-20°C, daily light duration 10-14 hours, and light intensity 1500-2000 lx.

[0020] Furthermore, the seedling hardening process specifically includes the following steps:

[0021] S501, placing pea seedlings in a culture bottle for culture, injecting sterile water into the culture bottle, first covering half of the opening of the tissue culture bottle, and then culturing for 3-4 days, then completely opening the opening of the tissue culture bottle and continuing to culture for 3-4 days;

[0022] S502, mixing peat, vermiculite, and perlite in proportion to form a transplanting medium, taking out the pea seedlings from the tissue culture bottle, washing the roots with water, and then planting the pea seedlings in the transplanting medium;

[0023] The transplanting matrix includes 2.6-3.4 parts of peat, 1.8-2.3 parts of vermiculite, and 0.8-1.2 parts of perlite by mass;

[0024] S503. Immediately after transplanting, water the pea seedlings and cover them with a transparent plastic cover. After culturing for 7-10 days, complete the seedling hardening process.

[0025] The beneficial effects of the present invention are:

[0026] 1. By establishing a direct regeneration tissue culture technology system for peas, the present invention can achieve the innovation of high-quality germplasm, shorten the breeding cycle, and accelerate the promotion of new varieties. At the same time, it provides technical support for the construction of a genetic transformation platform for genetic engineering, the precise introduction of exogenous excellent functional genes, and the cultivation of new varieties with excellent traits such as multi-resistance and high yield, thereby improving the level of pea breeding.

[0027] 2. Traditional tissue culture relies on the indirect regeneration pathway of callus (explant → dedifferentiation → callus → redifferentiation → regenerated buds). This process requires forced induction of cell dedifferentiation through high concentrations of hormones. However, the dedifferentiation ability of cells in different pea genotypes varies significantly, and the redifferentiation of callus is easily affected by the endogenous hormone balance regulated by the genotype, resulting in unstable regeneration efficiency.

[0028] The present invention directly uses pea cotyledonary nodes as explants, activates the original meristem by scratching the cotyledonary nodes for micro-injury treatment, directly induces latent buds to germinate into seedlings, skips the "dedifferentiation → callus tissue" stage, and avoids the dependence of genotype cells of different plants on dedifferentiation ability.

[0029] 3. Each culture medium in the present invention promotes plant growth through specific component ratios.

[0030] The B5 medium in the germination medium provides basic nutrients; sucrose provides a carbon source for germination; 6-benzylaminopurine inhibits apical dominance and promotes cell activation at the cotyledonary node, laying the foundation for subsequent induction of regenerated buds.

[0031] The 6-benzylaminopurine and 6-furfurylaminopurine in the induction medium work synergistically to stimulate the germination of latent buds at the cotyledonary nodes and increase the number of regenerated buds from a single explant; glutamine and asparagine provide an organic nitrogen source to promote cell division and bud differentiation; ferrous sulfate chelates with disodium ethylenediaminetetraacetic acid to stabilize iron ion absorption and avoid nutrient deficiency; morpholineethanesulfonic acid acts as a buffer to maintain the pH stability of the culture medium and ensure a continuous and efficient induction process.

[0032] Gibberellins in the elongation medium break bud dormancy and promote longitudinal growth of the stem; zeatin cooperates with indoleacetic acid to balance cell division and elongation, avoiding dwarfing or vitrification of the buds; the nitrogen source content in the MS medium is higher, supporting the nutritional needs of the buds during the rapid elongation stage.

[0033] The rooting culture medium uses indolebutyric acid and naphthaleneacetic acid as strong rooting agents, which synergistically promote the formation of adventitious root primordia and root elongation, improve the rooting rate and root robustness, and lay the foundation for transplant survival; at the same time, it reduces the sucrose concentration to avoid excessive sugar leading to poor root development; the MS culture medium is used in a reduced amount to reduce the stimulation of inorganic salts on the root system, which is suitable for the cell differentiation needs in the rooting stage.

[0034] 4. The hardening treatment of the present invention is achieved by gradually opening the bottle mouth, and the open culture is carried out in stages, from half covering the bottle mouth to fully opening the bottle mouth, so that the pea seedlings can gradually adapt to the external humidity and light, reducing transpiration loss and stress damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Place the seeds in germination medium;

[0036] Figure 2 Inoculate the explants into the induction medium;

[0037] Figure 3 The explants were inoculated into induction medium and cultured for 10 days;

[0038] Figure 4 Inoculate the explants into elongation medium;

[0039] Figure 5 Explants were cultured in elongation medium for 15 days;

[0040] Figure 6 Explants were cultured in elongation medium for 30 days;

[0041] Figure 7 Culture the explants in rooting medium;

[0042] Figure 8 Harden the pea seedlings;

[0043] Figure 9 10 days for growing pea seedlings

[0044] Figure 10 30 days for growing pea seedlings

[0045] Figure 11 50 days for pea seedlings to grow DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0047] Example

[0048] A method for direct regeneration of pea tissue comprises the following steps:

[0049] S1. Select plump and healthy pea seeds, place them in a container lined with filter paper, and shake them in a shaker at room temperature for 10 minutes to remove dust from the seed surface. After dust removal, place the pea seeds in a petri dish. The petri dish is opened and placed in a desiccator for chlorine fumigation for 12-14 hours. The chlorine is obtained by slowly adding 10 mL of hydrochloric acid to 100 mL of sodium hypochlorite. Finally, the sterilized seeds are evenly placed on a germination medium.

[0050] S201, the seeds after germination and culture for 24h were placed on a clean bench, and tweezers and a scalpel were autoclaved. First, pea seeds were clamped with tweezers, with the hilum facing up, and fixed on the clean bench. A sharp scalpel was used to cut a knife horizontally at the radicle of the seed. The seed coat was then cut open along the hilum, taking care to avoid damaging the embryo inside. Under a dissecting microscope, the seed coat was removed using tweezers and a scalpel. Two cotyledons were separated, and one cotyledon had a hypocotyl and true leaves. A scalpel was used to cut and retain a 3-5mm hypocotyl, and the true leaves were excised. Three cuts were made at the cotyledon node. The cotyledon node is the area where the cotyledons are connected to the hypocotyl. During the excision process, the size of the cotyledon node explant was ensured to be moderate and complete.

[0051] S202, removing the intact cotyledonary node of the seed as an explant, placing the explant in a culture dish filled with sterile water and rinsing for 3-5 minutes to remove any disinfectant and other impurities that may remain on the surface of the explant, and inoculating the rinsed explant into an induction medium;

[0052] S3. The induction medium inoculated with the explants was placed in a tissue culture room at 22°C, 16 h light / 8 h dark, and a light intensity of 2500 lx. The culture was carried out for 10 days. After multiple regenerated buds grew at the cotyledonary nodes, the explants with regenerated buds were transferred to elongation medium and cultured in the tissue culture room for another 14-20 days. After replacing the elongation medium, the culture was continued for another 14-20 days until the regenerated buds elongated to 6-8 cm.

[0053] S4. The explants with 6-8 cm regenerated shoots were transferred to a rooting medium for root induction. The induction environment conditions were room temperature 20°C, 12 h light / 12 h dark, and a light intensity of 1800 lx. A well-developed root system grew 15-20 days after inoculation to obtain pea seedlings.

[0054] S501, culturing pea seedlings in a culture flask, injecting 20 mL of sterile water containing 1 mg / L amphotericin B into the culture flask, first covering half of the opening of the tissue culture flask, culturing for 3 days, then fully opening the opening of the tissue culture flask, and continuing to culture for 3 days;

[0055] S502, mixing peat, vermiculite, and perlite in a ratio of 3:2:1 to form a transplanting medium, taking out the pea seedlings from the tissue culture bottle and rinsing the roots with water to remove the culture medium attached to the roots, then planting the pea seedlings in the transplanting medium, covering the roots with the transplanting medium and gently pressing;

[0056] S503. After transplanting, water thoroughly immediately and cover with a transparent plastic cover for 7-10 days, and then transplant the pea seedlings to a natural environment.

[0057] In step S1, chlorine fumigation is used for disinfection. Chlorine fumigation can act more evenly on the surface of seeds, reduce the impact of chemical residues on subsequent germination, and avoid the problem of excessive water absorption of seeds or penetration of disinfectants into the seed coat caused by immersion disinfection.

[0058] In step S2, the pea cotyledonary node is used as an explant, and the cotyledonary node is scratched to cause micro-injury to activate the original meristem, thereby directly inducing the latent buds to germinate into seedlings.

[0059] In steps S3 and S4, 22°C and 16 h of light were used in the induction and elongation stages, and 20°C and 12 h of light were used in the rooting stage, with targeted designs for the physiological needs of pea at different developmental stages.

[0060] In step S5, the seedling hardening treatment is achieved by gradually opening the bottle mouth, and the open culture is carried out in stages, from half covering the bottle mouth to fully opening the bottle mouth, so that the pea seedlings can gradually adapt to the external humidity and light, reduce transpiration loss and stress damage, and thus improve the transplant survival rate.

[0061] The germination medium uses sterile water as a solvent. Each liter of sterile water contains: 3.1 g of B5 medium, 20 g of sucrose, 6 g of agar powder, 100 mg of B5 vitamins, and 0.5 mg of 6-benzylaminopurine. The pH of the germination medium is 5.8.

[0062] The B5 medium in the germination medium provides basic nutrients; sucrose provides a carbon source for germination; 6-benzylaminopurine inhibits apical dominance and promotes cell activation at the cotyledonary node, laying the foundation for subsequent induction of regenerated buds.

[0063] The induction culture medium uses sterile water as a solvent, and each liter of sterile water contains: 3.1g B5 culture medium, 0.68g morpholineethanesulfonic acid, 30g sucrose, 8g agar powder, 100mg B5 vitamins, 0.5mg 6-benzylaminopurine, 0.5mg 6-furfurylaminopurine, 6mg ferrous sulfate, 6mg disodium ethylenediaminetetraacetic acid, 25mg glutamine, and 25mg asparagine. The pH of the induction culture medium is 5.7.

[0064] The 6-benzylaminopurine and 6-furfurylaminopurine in the induction medium work synergistically to stimulate the germination of latent buds at the cotyledonary nodes and increase the number of regenerated buds from a single explant; glutamine and asparagine provide an organic nitrogen source to promote cell division and bud differentiation; ferrous sulfate chelates with disodium ethylenediaminetetraacetic acid to stabilize iron ion absorption and avoid nutrient deficiency; morpholineethanesulfonic acid acts as a buffer to maintain the pH stability of the culture medium and ensure a continuous and efficient induction process.

[0065] The elongation medium uses sterile water as a solvent, and each liter of sterile water contains: 4.0 g of MS medium, 0.41 g of morpholineethanesulfonic acid, 30 g of sucrose, 8 g of agar powder, 100 mg of B5 vitamins, 0.02 mg of indoleacetic acid, 0.5 mg of gibberellin, 0.5 mg of zeatin, 6 mg of ferrous sulfate, 6 mg of disodium ethylenediaminetetraacetic acid, 25 mg of glutamine, and 25 mg of asparagine. The pH of the elongation medium is 5.6.

[0066] Gibberellins in the elongation medium break bud dormancy and promote longitudinal growth of the stem; zeatin cooperates with indoleacetic acid to balance cell division and elongation, avoiding dwarfing or vitrification of the buds; the nitrogen source content in the MS medium is higher, supporting the nutritional needs of the buds during the rapid elongation stage.

[0067] The rooting medium uses sterile water as a solvent, and each liter of sterile water contains: 2.15g of MS medium, 0.41g of morpholineethanesulfonic acid, 20g of sucrose, 6g of agar powder, 100mg of B5 vitamins, 0.5mg of indolebutyric acid, 0.5mg of naphthaleneacetic acid, 25mg of glutamine, and 25mg of asparagine. The pH of the rooting medium is 5.7.

[0068] The rooting culture medium uses indolebutyric acid and naphthaleneacetic acid as strong rooting agents, which synergistically promote the formation of adventitious root primordia and root elongation, improve the rooting rate and root robustness, and lay the foundation for transplant survival; at the same time, it reduces the sucrose concentration to avoid excessive sugar leading to poor root development; the MS culture medium is used in a reduced amount to reduce the stimulation of inorganic salts on the root system, which is suitable for the cell differentiation needs in the rooting stage.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and the embodiment is only that the disinfection method in step S1 is different, and the remaining steps are the same as those in the embodiment. The disinfection method in Comparative Example 1 is:

[0071] The seeds were soaked in 75% ethanol for 3 minutes, and then the soaked seeds were placed in a sodium hypochlorite solution containing 1.8% available chlorine, shaken and soaked for 30 minutes. Finally, the seeds were taken out and rinsed with sterile water.

[0072] Comparative Example 2

[0073] The difference between Comparative Example 2 and the embodiment is only step S501, and the remaining steps are the same as those of the embodiment. Step S501 of Comparative Example 2 is specifically as follows:

[0074] S501. Place pea seedlings in a culture bottle for culture, inject sterile water into the culture bottle, completely open the opening of the tissue culture bottle, and culture for 6 days.

[0075] Comparative Example 3

[0076] S1 Germination culture: After disinfection and soaking, pea seeds are inoculated with the hilum downwards onto germination medium and cultured for 4-6 days to germinate sterile seedlings;

[0077] S2 bud induction sterile seedlings were removed from the cotyledons, epicotyls and radicles, leaving 2-4 cm of the hypocotyls, and a small wound was made at the junction of the two cotyledons. The seedlings were then transferred to an adventitious bud induction medium containing B5 medium + 1.0 mg / L benzylaminoadenine + 1.0 mg / L kinetin + 0.1 mg / L indolebutyric acid and induced to sprout for 10-15 days.

[0078] S3: Cultivation and elongation: The explant obtained in step S2 is transferred to an adventitious bud elongation medium and cultured for 20 to 25 days until the adventitious buds elongate to 3 to 4 cm;

[0079] S4 rooting and transplanting: After the seedlings have grown to about 3-4 cm, they are transferred to a rooting medium for 10-15 days. After more than 3 roots, each about 3 cm long, are formed, they are transplanted to obtain regenerated plants; the rooting medium is 1 / 2 MS medium + 1 mg / L IBA.

[0080] 2. Test and Inspection

[0081] Pea tissue culture was performed according to the methods in Example 1 and Comparative Examples 1-2, the culture period and growth conditions were recorded, and the survival rate of pea seeds was calculated. The results are shown in Table 1.

[0082] Table 1

[0083]

[0084] As can be seen from Table 1, compared with Comparative Example 1, the disinfection method of the embodiment has a very strong seed surface disinfection effect, is easy to operate, and can disinfect a large number of different varieties of mung bean seeds at one time. The survival rate of peas after disinfection in the embodiment reaches 100%, while the survival rate of peas after disinfection in Comparative Example 1 is only 78.60%.

[0085] During the seedling hardening period, compared to Comparative Example 2, the Example injected 20 mL of sterile water containing 1 mg / L amphotericin B into the culture flask, effectively preventing environmental bacterial contamination. Furthermore, the hardening process in the Example was divided into two stages: first, the opening of the tissue culture flask was partially covered for 3 days, and then fully opened for another 3 days. This gradual opening helped the seedlings gradually adapt to the external environment, thereby improving the transplant survival rate of the regenerated pea seedlings.

[0086] Comparative Example 3 and the Example both used Zhongwan No. 6 as the test variety, with 100 seeds in each test. In Comparative Example 3, Zhongwan No. 6 obtained 37 regenerated plants, with 1 to 3 adventitious buds elongated per explant and an average regeneration frequency of 2.1 per explant. In the Example, 86 regenerated plants were obtained from Zhongwan No. 6, with 5 to 7 adventitious buds elongated per explant and an average regeneration frequency of 5.6 per explant. Compared with the regeneration system of Comparative Example 3, the Example has strong repeatability, a short test process cycle (<80 days), produces many adventitious buds (5 to 7 per explant), a high regeneration frequency (5.6 per explant), and the regenerated plants grow robustly.

Claims

1. A method for direct regeneration of pea tissue, characterized in that: The following steps are involved: S1. After removing dust from pea seeds, pea seeds are fumigated with chlorine and then evenly placed on a germination medium. S2. Place the seeds after germination and culture for 24 hours on a workbench, remove the seed explants, rinse the explants in sterile water, and inoculate the rinsed explants into the induction medium; S3. Place the induction medium inoculated with the explant in a tissue culture room. After the explant grows multiple regenerated buds, transfer the explant with regenerated buds to an elongation medium and continue culturing in the tissue culture room until the regenerated buds elongate to 6-8 cm. S4. Transferring the explants with 6-8 cm regenerated shoots into a rooting medium for rooting induction until a well-developed root system grows to obtain pea seedlings; S5. After hardening, the pea seedlings are transplanted into a natural environment.

2. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The germination culture medium uses sterile water as a solvent. Each liter of sterile water contains: 2.8-3.2g of B5 culture medium, 18-22g of sucrose, 5-7g of agar powder, 80-120mg of B5 vitamins, and 0.4-0.6mg of 6-benzylaminopurine. The pH of the germination culture medium is 5.6-5.

8.

3. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The induction culture medium uses sterile water as a solvent, and each liter of sterile water contains: 2.8-3.2g of B5 culture medium, 0.65-0.70g of morpholineethanesulfonic acid, 25-35g of sucrose, 6-10g of agar powder, 80-120mg of B5 vitamins, 0.4-0.6mg of 6-benzylaminopurine, 0.4-0.6mg of 6-furfurylaminopurine, 5-7mg of ferrous sulfate, 5-7mg of disodium ethylenediaminetetraacetic acid, 22-26mg of glutamine, and 22-26mg of asparagine. The pH of the induction culture medium is 5.6-5.

8.

4. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The elongation medium uses sterile water as a solvent, and each liter of sterile water contains: 3.8-4.3g of MS medium, 0.38-0.42g of morpholineethanesulfonic acid, 25-35g of sucrose, 6-10g of agar powder, 80-120mg of B5 vitamins, 0.01-0.02mg of indoleacetic acid, 0.4-0.6mg of gibberellin, 0.4-0.6mg of zeatin, 5-7mg of ferrous sulfate, 5-7mg of disodium ethylenediaminetetraacetic acid, 22-26mg of glutamine, and 22-26mg of asparagine. The pH of the elongation medium is 5.6-5.

8.

5. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The rooting medium uses sterile water as a solvent. Each liter of sterile water contains: 2.0-2.2g of MS medium, 0.38-0.42g of morpholineethanesulfonic acid, 16-22g of sucrose, 5-7g of agar powder, 80-120mg of B5 vitamins, 0.4-0.6mg of indolebutyric acid, 0.4-0.6mg of naphthaleneacetic acid, 22-26mg of glutamine, and 22-26mg of asparagine. The pH of the rooting medium is 5.6-5.

8.

6. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The step S2 of removing the seed explant specifically includes the following steps: Use tweezers to hold the seeds with the hilum facing up and fix them on the operating table. Use a scalpel to make a horizontal cut at the radicle of the seeds. Cut the seed coat along the hilum and remove the seed coat and one cotyledon. Leave the hypocotyl and true leaves on the other cotyledon. Use a scalpel to cut off and retain 3-5mm of the hypocotyl, remove the true leaves, and make 2-3 cuts at the cotyledon node to obtain the explant. The length and width of the explant are both 10-13mm.

7. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The environmental conditions of the tissue culture room in step S3 are: room temperature 22-24° C., daily light duration 14-18 h, and light intensity 2000-3000 lx.

8. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The environmental conditions for rooting induction in step S4 are: room temperature 18-20°C, daily light duration 10-14 hours, and light intensity 1500-2000 lx.

9. The method for direct regeneration of pea tissue according to claim 1, characterized in that: The seedling hardening process specifically comprises the following steps: S501, placing pea seedlings in a culture bottle for cultivation, injecting sterile water into the culture bottle, first covering half of the opening of the tissue culture bottle, culturing for 3-4 days, then completely opening the opening of the tissue culture bottle, and continuing to culture for 3-4 days; S502, mixing peat, vermiculite, and perlite in proportion to form a transplanting medium, taking out the pea seedlings from the tissue culture bottle, washing the roots with water, and then planting the pea seedlings in the transplanting medium; The transplanting matrix includes 2.6-3.4 parts of peat, 1.8-2.3 parts of vermiculite, and 0.8-1.2 parts of perlite by mass; S503. Immediately after transplanting, water the pea seedlings and cover them with a transparent plastic cover. After culturing for 7-10 days, complete the seedling hardening process.

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