Bromus japonicus tissue culture method

Abstract

This invention discloses a tissue culture method for *Brassica rapa*, comprising the following steps: first, selecting mature *Brassica rapa* seeds as explants and sterilizing them; then, inoculating them into an induction medium and culturing them in the dark to induce callus formation and subculturing; subsequently, transferring the callus to a differentiation medium to differentiate and obtain seedlings; next, transferring them to a rooting medium to induce rooting; and finally, hardening off the seedlings and transplanting them after they have grown into complete plants. This invention establishes for the first time a highly efficient tissue culture regeneration system for *Brassica rapa*, using seeds as explants, which are readily available and low in cost, and a single seed can induce a large number of callus. This method is simple to operate, has good reproducibility, and a high differentiation rate, which can lay a solid technical foundation for genetic transformation and gene editing research of *Brassica rapa*.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture and rapid propagation technology, specifically relating to a tissue culture method for bromegrass. Background Technology

[0002] Bromus japonicus, an annual diploid weed belonging to the genus Bromus in the Poaceae family, has evolved into a dominant weed in wheat fields, severely impacting wheat yields. However, Bromus japonicus also possesses excellent stress resistance traits such as drought tolerance, poor soil tolerance, and low-temperature tolerance, and contains a wealth of high-quality genes. It is an important genetic resource for stress resistance improvement and germplasm innovation in cereal crops, with great potential in molecular breeding and food security. How to efficiently mine and utilize its superior genes while effectively controlling weed damage has become a research hotspot in agricultural biotechnology, and tissue culture is a key technology for achieving this research.

[0003] Currently, biotechnologies such as gene editing and molecular breeding are widely used in crop improvement and weed control, all of which rely on stable and efficient genetic transformation systems. At present, research on *Brassica rapa* (a type of wild oat) focuses primarily on control measures, initial gene exploration, and herbicide resistance identification, lacking a mature and efficient tissue culture regeneration system. This significantly limits research on functional gene discovery, herbicide resistance mechanisms, and gene cloning. Consequently, this results in a series of problems, including low utilization of gene resources, increased herbicide resistance in weeds, and greater difficulty in field control. Therefore, constructing an efficient tissue culture system for *Brassica rapa* is a necessary prerequisite for overcoming existing technological barriers, elucidating the mechanism of herbicide action, and scientifically controlling *Brassica rapa* weeds.

[0004] In summary, tissue culture technology is the core foundation for research on bromegranate gene development and weed control. Currently, the industry still lacks a stable and efficient bromegranate tissue culture system, which seriously hinders its basic research and resource utilization. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by proposing a tissue culture method for bromegranate for the first time, and to develop a bromegranate tissue culture method that can efficiently induce callus tissue and significantly improve the plant regeneration rate.

[0006] The object of this invention is achieved by the following means:

[0007] A tissue culture method for bromelain includes the following steps:

[0008] (1) Disinfection of explants: The seeds of bromegranate were used as explants for disinfection;

[0009] (2) Induction and subculture of callus: Explants were inoculated on induction medium and cultured in the dark to induce callus;

[0010] Induction medium formulation: 4.2-4.4 g / L MS basal salts + 5.8-6.2 mg / L 2,4-D + 1.2-1.3 mg / L CuSO4 + 0.9-1.1 g / L Casein hydrolysate + 0.65-0.7 g / L L-proline + 0.9-1.1 mg / L Thiamine hydrochloride + 28-32 g / L maltose + 4-5 g / L plant gel + 0.3-0.4 g / L inositol, adjust to pH 5.7-5.9;

[0011] (3) Differentiation of callus tissue: The callus tissue was transferred to a differentiation medium to differentiate into shoots;

[0012] Differentiation medium formulation and culture conditions: 4.2-4.4 g / L MS basal salts + 1.2-1.3 mg / L CuSO4 + 0.65-0.7 g / L L-proline + 0.9-1.1 mg / L Thiamine hydrochloride + 0.4-0.6 mg / L IAA + 0.3-0.4 g / L inositol + 28-32 g / L maltose + 4-5 g / L plant gel, adjust pH to 5.7-5.9, and culture under light;

[0013] (4) Rooting: The seedlings are transferred to a rooting medium to differentiate into roots;

[0014] Rooting medium formulation and culture conditions: 2-2.2 g / L MS basal salts + 0.5-0.7 g / L Casein hydrolysate + 28-32 g / L sucrose + 4-5 g / L plant gel, adjust pH to 5.7-5.9, and culture under light;

[0015] (5) After the seedlings have formed a complete plant, harden them off and transplant them.

[0016] In step (1), disinfect the seeds with 5% sodium hypochlorite for 1-2 minutes, rinse the seeds thoroughly with sterile water 7-9 times, disinfect with 75% alcohol for 1-2 minutes, rinse the seeds thoroughly with sterile water 7-9 times, and shake them up, down and left and right during the process to ensure thorough sterilization. After disinfection, place the seeds on sterile filter paper and dry the surface moisture of the seeds in a laminar flow hood for 10-20 minutes.

[0017] Step (2) Inoculate the explants onto the induction medium; the preferred induction medium is: 4.3 g / L MS basal salts + 6 mg / L 2,4-D + 1.25 mg / L CuSO4 + 1 g / L Lasein hydrolysate + 0.69 g / L L-proline + 1 mg / L Thiamine hydrochloride + 30 g / L maltose + 4 g / L plant gel + 0.35 g / L inositol, adjusted to pH 5.8.

[0018] Through exploratory experiments, this invention has found that high concentrations of 2,4-D, CuSO4, Casein hydrolysate, and inositol are required during induction culture, along with the addition of L-proline and Thiamine hydrochloride. Maltose should also be used instead of sucrose. The synergistic effect of these factors has resulted in a high induction rate and good growth status.

[0019] Step (2) Incubate in the dark at 20℃-25℃.

[0020] Step (2) Place 15-20 seeds in a 9 cm petri dish; preferably 15 seeds.

[0021] Step (2) Subculture the induced callus. The subculture medium contains 3 mg / L of 2,4-D and the other components are the same as the induction medium. The culture conditions are the same as the induction treatment. The medium needs to be changed every 20-22 days until sufficient callus is obtained. The callus should not be subcultured more than 4 times.

[0022] Callus formation begins after 14 days of dark culture at 25°C. The callus induction rate is approximately 80% at 21 days and reaches as high as 94% at 28 days. It is recommended that the callus of *Brassica oleracea* be subcultured for no more than 4 generations to maintain good differentiation ability.

[0023] The preferred differentiation medium for step (3) is: 4.3 g / L MS basal salts + 1.25 mg / L CuSO4 + 0.69 g / L L-proline + 1 mg / L Thiamine hydrochloride + 0.5 mg / L IAA + 0.35 g / L inositol + 30 g / L maltose + 4 g / L plant gel, adjusted to pH 5.8.

[0024] Through exploratory experiments, this invention has found that differentiation culture requires the use of high concentrations of CuSO4 and inositol, as well as the addition of L-proline and thiamine hydrochloride, and the use of maltose instead of sucrose. The synergistic effect of these factors has achieved a high differentiation rate and good growth status.

[0025] The differentiation medium in step (3) begins to differentiate buds 7 days after transfer, and the seedlings of bromegrass can be seen after 14 days, with a differentiation rate of over 96%.

[0026] Step (4) Optimal rooting medium: 2.15 g / L MS basal salts + 0.6 g / L Casein hydrolysate + 30 g / L sucrose + 4 g / L plant gel, adjusted to pH 5.8.

[0027] Steps (3) and (4) are cultured under light at 20℃-25℃ with a light intensity of 8000-20000 Lx for 12-14 h per day.

[0028] Step (4) When the bud grows to 1.0-2.0 cm in height, transfer it to the rooting medium to differentiate roots and form a complete plant.

[0029] Step (5): After hardening off the seedlings by opening the lid for 1-2 days, transplant them into nutrient soil, paying attention to moisture retention. The transplant survival rate reaches 100%.

[0030] In the field of genetic resource discovery and conservation, traditional methods of preserving germplasm through field planting are highly susceptible to natural disasters and environmental fluctuations, resulting in high preservation risks and difficulty in achieving long-term stable preservation. Constructing germplasm banks of *Brassica oleracea* cell lines, callus tissues, and regenerated plants using tissue culture technology enables the long-term safe in vitro preservation of wild genetic resources, providing a reliable material guarantee for subsequent genetic resource development and utilization. This invention successfully establishes a highly efficient tissue culture regeneration system for *Brassica oleracea* for the first time. Mature seeds are selected as explants, making material acquisition convenient and cost-effective; a single seed can induce a large amount of callus tissue. The entire culture process is simple to operate, exhibits good stability and reproducibility, and produces high plant differentiation rates, providing stable and efficient technical support for subsequent genetic transformation and gene editing research. Attached Figure Description

[0031] Figure 1 Images of callus induced by bromegranate seeds in the optimal callus induction medium of this invention, at 0 d (A), 14 d (B), and 21 d (C);

[0032] Figure 2Images of the optimal callus subculture conditions of the present invention at 7 days (A) and the optimal differentiation medium for callus differentiation at 14 days (B);

[0033] Figure 3 Images show the callus differentiation seedlings of this invention after being transplanted into the optimal rooting medium for 3 days (A), 7 days (B), and transplanted into pots (C). Detailed Implementation

[0034] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1:

[0036] Bromosia japonicus seeds were disinfected with 5% sodium hypochlorite for 1-2 minutes, then thoroughly rinsed with sterile water (8 times), disinfected with 75% alcohol for 1-2 minutes, and thoroughly rinsed with sterile water (8 times). During this process, the seeds were shaken up and down and left and right to ensure thorough sterilization. After disinfection, the seeds were placed on sterile filter paper and dried on the surface of the seeds in a laminar flow hood (10-20 minutes).

[0037] Example 2: Callus induction and subculture

[0038] The sterilized explants were inoculated into the induction medium (see Table 1) and cultured in the dark at 25°C; approximately 15 seeds were placed in a 9 cm culture dish.

[0039] The subculture medium contained 3 mg / L of 2,4-D, and the remaining components were the same as those in the induction medium. The culture conditions were the same as those for induction, and the medium needed to be changed after about 21 days until sufficient callus was obtained.

[0040] Callus formation begins after 14 days of dark culture at 25°C. The callus induction rate is approximately 80% at 21 days and reaches as high as 94% at 28 days. It is recommended that the callus subculture of Broccoli not exceed 4 generations to maintain good differentiation ability.

[0041] The results are shown in Table 2.

[0042] Table 1 Formulations of different induction culture media

[0043]

[0044] Table 2. Induction of callus in different culture media for *Brassica oleracea*

[0045]

[0046] Note: Data are expressed as mean ± standard deviation (n = 3 independent replicates, 15 explants per replicate). Different lowercase letters indicate significant differences in callus induction rates among different culture media (P < 0.05). Specifically, 'a' indicates a significant difference in callus induction rates between YD-1 medium and YD-2 and YD-3 media labeled 'b' and 'c', respectively; 'b' indicates a significant difference in callus induction rates between YD-2 medium and YD-3 medium labeled 'c'. No letter is used to represent callus browning rate, indicating no significant difference in browning rates among the three culture media.

[0047] The results above show that YD-3 medium is significantly better than the other mediums.

[0048] Example 3: Differentiation Culture

[0049] The induced bromeliad callus was placed on differentiation medium (see Table 3) and cultured at 25°C under light at an intensity of 12000 Lx for 12-14 h per day. The differentiation results are shown in Table 4.

[0050] Table 3 Formulations of different differentiation culture media

[0051]

[0052] Table 4. Effects of different differentiation media on callus differentiation

[0053]

[0054] Note: Data are expressed as mean ± standard deviation (n = 3 independent replicates, each replicate inoculated with 10 mature calluses). Different lowercase letters indicate significant differences in callus differentiation rates of *Brassica napus* in different culture media (P < 0.05). Specifically, 'a' indicates no significant difference in callus differentiation rates between *Brassica napus* in FH-1, FH-2, and FH-4 media, but a significant difference compared to the callus differentiation rates in FH-3 and FH-5 media (labeled b and c). 'b' indicates a significant difference in callus differentiation rates between FH-3 and FH-5 media (labeled c).

[0055] The results above show that FH-1 and FH-2 are significantly better than FH-3 culture medium, and FH-1 is the optimal choice.

[0056] Example 4:

[0057] The differentiated seedlings were placed on a rooting medium: 2.15 g / L MS basal salts + 0.6 g / L caseinhydrolysate + 30 g / L sucrose + 4 g / L plant gel, adjusted to pH 5.8, and cultured at 25°C under light at an intensity of 12000 Lx for 14 h per day. Rooting rate was 100%, and complete plants formed in 15 days.

[0058] Example 5: Transplanting

[0059] After hardening off the seedlings by removing the covers for 1-2 days, transplant them into nutrient soil (black soil: rice substrate: vermiculite = 1:1:1), and keep the soil moist (60% humidity, 25℃). The transplant survival rate will reach 100%.

Claims

1. A tissue culture method of Bromus japonicus, characterized by comprising the steps of, Includes the following steps: (1) Disinfection of explants: The seeds of bromegranate were used as explants for disinfection; (2) Induction and subculture of callus: Explants were inoculated on induction medium and cultured in the dark to induce callus; Induction medium formulation: 4.2-4.4 g / L MS basal salts + 5.8-6.2 mg / L 2,4-D + 1.2-1.3 mg / L CuSO4 + 0.9-1.1 g / L Casein hydrolysate + 0.65-0.7 g / L L-proline + 0.9-1.1 mg / L Thiaminehydrochloride + 28-32 g / L maltose + 4-5 g / L plant gel + 0.3-0.4 g / L inositol, adjust to pH 5.7-5.9; (3) Differentiation of callus tissue: The callus tissue was transferred to a differentiation medium to differentiate into shoots; Differentiation medium formulation and culture conditions: 4.2-4.4 g / L MS basal salts + 1.2-1.3 mg / L CuSO4 + 0.65-0.7 g / L L-proline + 0.9-1.1 mg / L Thiamine hydrochloride + 0.4-0.6 mg / L IAA + 0.3-0.4 g / L inositol + 28-32 g / L maltose + 4-5 g / L plant gel, adjust pH to 5.7-5.9, and culture under light; (4) Rooting: The seedlings are transferred to a rooting medium to differentiate into roots; Rooting medium formulation and culture conditions: 2-2.2 g / L MS basal salts + 0.5-0.7 g / L Casein hydrolysate + 28-32 g / L sucrose + 4-5 g / L plant gel, adjust pH to 5.7-5.9, and culture under light; (5) After the seedlings have formed a complete plant, harden them off and transplant them.

2. The method according to claim 1, characterized in that, In step (1), disinfect the seeds with 5% sodium hypochlorite for 1-2 minutes, rinse the seeds thoroughly with sterile water 7-9 times, disinfect with 75% alcohol for 1-2 minutes, rinse the seeds thoroughly with sterile water 7-9 times, and shake them up, down and left and right during the process to ensure thorough sterilization. After disinfection, place the seeds on sterile filter paper and dry the surface moisture of the seeds in a laminar flow hood for 10-20 minutes.

3. The method according to claim 1, characterized in that, Step (2) Inoculate the explants onto the induction medium; Induction medium: 4.3 g / L MS basal salts + 6 mg / L 2,4-D + 1.25 mg / L CuSO4 + 1 g / L Caseinhydrolysate + 0.69 g / L L-proline + 1 mg / L Thiamine hydrochloride + 30 g / L maltose + 4 g / L plant gel + 0.35 g / L inositol, adjusted to pH 5.

8.

4. The method of claim 1, wherein, Step (2) Incubate in the dark at 20℃-25℃.

5. The method according to claim 1 or 3 or 4, characterized in that, Step (2): Place 15-20 seeds in a 9 cm petri dish.

6. The method according to claim 1, 3, or 4, characterized in that, Step (2) Subculture the induced callus. The subculture medium contains 3 mg / L of 2,4-D and the other components are the same as the induction medium. The culture conditions are the same as the induction treatment. The medium needs to be changed every 20-22 days until sufficient callus is obtained. The callus should not be subcultured more than 4 times.

7. The method according to claim 1, characterized in that, Differentiation medium for step (3): 4.3 g / L MS basal salts + 1.25 mg / L CuSO4 + 0.69 g / L L-proline + 1 mg / L Thiamine hydrochloride + 0.5 mg / L IAA + 0.35 g / L Inositol + 30 g / L Maltose + 4 g / L Plant gel, adjusted to pH 5.8; Step (4) Rooting medium: 2.15 g / L MS basal salts + 0.6 g / L Casein hydrolysate + 30 g / L sucrose + 4 g / L plant gel, adjusted to pH 5.

8.

8. The method according to claim 1, characterized in that, Steps (3) and (4) are cultured under light at 20℃-25℃ with a light intensity of 8000-20000 Lx for 12-14 h per day.

9. The method according to claim 1 or 8, characterized in that, Step (4) When the bud grows to 1.0-2.0 cm in height, transfer it to the rooting medium to differentiate roots and form a complete plant.

10. The method according to claim 1, characterized in that, Step (5) After opening the lid and hardening off the seedlings for 1-2 days, transplant them into the nutrient soil and keep them moist.

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