A method for establishing a carex seed regeneration system and genetic transformation

Abstract

The present application belongs to the field of plant tissue culture and genetic transformation, and particularly relates to a method for establishing a regeneration system of green moss seeds and a genetic transformation method. The subculture medium of the method for establishing the regeneration system of the green moss seeds contains proline and kinetin. The resistant subculture medium of the genetic transformation method of the green moss seeds contains proline and kinetin, and the first and second resistant screening media contain proline and vitamin C. The genetic transformation method of the green moss seeds comprises the steps of cold treatment, vacuum treatment and dry treatment of callus. The subculture medium of the regeneration system of the green moss seeds is optimized, the regeneration efficiency is high, and a large number of regenerated plants can be obtained; the green moss genetic transformation system of the present application can obviously distinguish between the successfully transformed and unsuccessfully transformed experimental materials through twice resistant screening, and the identification accuracy is high.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture and genetic transformation, and specifically relates to a method for establishing a regeneration system for *Carex lucida* seeds, as well as a genetic transformation method. Background Technology

[0002] Carex breviculmis, also known as green sedge, is a perennial herb belonging to the Cyperaceae family. It is characterized by its long green period, low maintenance, and tolerance to shade and drought. As a high-quality native grass species, Carex breviculmis possesses significant landscaping and ecological value. Existing research has extensively explored the physiological response mechanisms of Carex breviculmis under drought, shade, and heavy metal stress. Studies have shown that Carex breviculmis adapts to adversity by regulating photosynthetic characteristics, antioxidant enzyme systems, and osmotic regulators, demonstrating its potential as a stress-resistant breeding material.

[0003] The use of gene editing technologies such as CRISPR / Cas9 for crop trait improvement and functional gene verification has become a research hotspot and a core technology for new germplasm creation both domestically and internationally. However, the application of this technology heavily relies on an efficient genetic transformation system. Currently, its application in major gramineous crops such as rice and wheat, as well as model plants such as Arabidopsis thaliana, is relatively mature. However, in most native grass species and ornamental grasses, including sedges, several technical bottlenecks remain: first, callus induction is difficult, resulting in a low rate of high-quality embryogenic callus; second, differentiation rates are low and vitrified seedlings are high during regeneration, leading to poor plant regeneration stability; third, the plant is less sensitive to infection by transformation-mediated tools such as Agrobacterium, resulting in low transformation efficiency, difficulty in screening positive plants, and poor genetic stability of offspring. These problems severely limit the application of gene editing technology in the genetic improvement of *Carex chinensis*.

[0004] Therefore, current scientific research and experiments require a reliable and efficient method for establishing a regeneration system and genetic transformation of sedge seeds. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a method for establishing a regeneration system and genetic transformation of sedge seeds.

[0006] A subculture medium for establishing a sedge seed regeneration system, wherein the subculture medium contains proline and kinetin.

[0007] A resistance subculture medium, a first resistance selection medium, and a second resistance selection medium for the genetic transformation of sedge seeds, wherein the resistance subculture medium contains proline and kinetin; and the first and second resistance selection media contain proline and vitamin C.

[0008] A method for establishing a regeneration system for sedge seeds includes the following steps:

[0009] Explant treatment steps: *Kleistocene* seeds are used as explants and treated to obtain sterilized seeds; Callus induction culture step: The sterilized seeds are placed in a callus induction medium for callus induction culture to obtain callus fragments; Callus subculture step: The callus fragments are placed in the subculture medium for subculture to obtain callus tissue to be differentiated; Differentiation culture step: The callus tissue to be differentiated is placed in a differentiation medium for differentiation culture to obtain adventitious shoots; Rooting culture step: The adventitious shoots are placed in a rooting medium for rooting culture to obtain small plantlets.

[0010] A method for genetic transformation of sedge seeds, comprising the following steps:

[0011] Preparation steps of bacterial suspension for infection: A bacterial suspension for infection is prepared from Agrobacterium carrying a plasmid vector; Preparation steps of callus for infection: Callus pieces are prepared to obtain callus for infection; Cold treatment step of callus: The callus for infection is placed in a cold treatment solution for cold treatment to obtain cold-treated callus; Vacuum treatment step: The cold-treated callus is immersed in the bacterial suspension for infection and subjected to vacuum treatment and shaking culture to obtain vacuum-treated callus; Drying step: The vacuum-treated callus is dried to obtain dried callus; Co-culture step: The dried callus is placed in a co-culture medium for co-culture to obtain co-cultured callus; Recovery culture step: The co-cultured callus is... The callus tissue is placed in the resistance subculture medium for recovery culture to obtain recovered callus tissue; the first resistance selection culture step: the recovered callus tissue is placed in the first resistance selection medium for first resistance selection culture to obtain first-selected callus tissue; the second resistance selection culture step: the first-selected callus tissue is placed in the second resistance selection medium for second resistance selection culture to obtain second-selected callus tissue; resistant callus differentiation culture: the second-selected callus tissue is placed in the resistance differentiation medium for differentiation culture to obtain resistant adventitious shoots; the rooting culture step: the resistant adventitious shoots are placed in the rooting selection medium for rooting culture to obtain resistant plantlets.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. This invention optimizes the subculture medium of the green sedge seed regeneration system, which has a high regeneration efficiency and can obtain a large number of regenerated plants.

[0014] 2. The invention and establishment of a stable and efficient genetic transformation system for *Carex lucida* is not only an innovative work that breaks through the bottleneck of gene editing technology application in this species, but also provides a reference technical paradigm for molecular breeding of *Carex* and other closely related native grass species, filling the gap in the molecular breeding technology system for native grass species. It has important theoretical value, methodological significance and practical application prospects for promoting the genetic improvement and industrial application of native grass species resources in my country.

[0015] 3. The green sedge genetic transformation system of the present invention undergoes two resistance screenings, which can clearly distinguish between experimental materials that have been successfully transformed and those that have not, with high identification accuracy.

[0016] 4. The various operations and parameters of the regeneration system establishment method and genetic transformation method of the present invention work synergistically and mutually enhance each other, achieving excellent regeneration and genetic transformation effects.

[0017] 5. The regeneration system establishment method and genetic transformation method of the present invention are simple to operate and low in cost, making them suitable for widespread use throughout my country. Attached Figure Description

[0018] Figure 1 To examine different stages of the tissue culture regeneration process of *Carex lucida* in Example 1. A is a photograph of the dedifferentiated *Carex lucida* seeds (i.e., callus fragments) in step S2 of Example 1; B is a photograph of the callus tissue grown from *Carex lucida* before subculture in step S3 of Example 1; C is a photograph of the adventitious buds differentiated in step S4 of Example 1; D is a photograph of the callus tissue obtained from the subculture medium without the addition of Pro and KT in Comparative Example 7; E is a photograph of the callus tissue to be differentiated in step S3 of Example 1 (with the addition of Pro and KT); F is a photograph of the further differentiated adventitious buds grown in step S4 of Example 1; G is a photograph of the small plantlets rooted in step S5 of Example 1; H is a photograph of the regenerated plantlets after transplantation in step S6 of Example 1.

[0019] Figure 2 Photographs (one of the culture dishes) showing the browning of callus tissues under different subculture media in Examples 1-3 and Comparative Examples 1-6 in Example 1. Figure 2 Parts 1 to 9 are photographs of callus tissues obtained from the subculture culture media of Comparative Examples 8, 1, 9, 2, 3, 4, 5, 1, and 6, respectively.

[0020] Figure 3These are photographs of various stages in the Agrobacterium-mediated transformation of *Sedgeella asiatica* callus in Example 4. A is a photograph of the prepared callus obtained in step S3; B is a photograph of the callus placed in a cold treatment solution for cold treatment in step S4; C is a photograph of the vacuum treatment in step S5; D is a photograph of the drying treatment in step S6; E and F are photographs of the callus after the second screening in step S11 on the resistance differentiation medium; G is a photograph of the rooted resistant plantlets in step S12; H is a photograph of the *Sedgeella asiatica* resistant regenerated plantlets after transplanting in step S13.

[0021] Figure 4 This is a photograph of the callus tissue after the second screening in Example 4.

[0022] Figure 5 This is a schematic diagram of the identification of the transformed and regenerated plants of *Carex* in Example 4. A is a schematic diagram of the T-DNA structure of the pHDE-35S-RUBY vector; B is an electrophoresis diagram of the PCR amplification detection of the Hyg resistance gene, where columns 1-7 are the plants to be tested, the WT column is the wild type, the positive control column is the plasmid vector pHDE-35S-RUBY, and the negative control column is water.

[0023] Figure 6 Photographs of callus tissue after resistance screening in Comparative Example 10.

[0024] Figure 7 Photographs of callus tissue after resistance screening in Comparative Example 11. Detailed Implementation

[0025] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0026] In a first aspect, the present invention provides a subculture medium for establishing a sedge seed regeneration system, the subculture medium containing proline (Pro) and kinetin (KT).

[0027] In a preferred embodiment, the subculture medium is based on MS basal medium, with the following added: kinetin to a final concentration (unless otherwise specified, all substances in this invention refer to their final concentration in the corresponding medium) of 0.7–1.3 mg / L, preferably 1.0 mg / L; and proline to a final concentration of 1.7–2.3 g / L, preferably 2.0 g / L. The pH of the subculture medium is 5.8–5.9.

[0028] In a further preferred embodiment, the subculture medium is based on MS basal medium, supplemented with: naphthaleneacetic acid (NAA) at a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; 6-benzylaminopurine (6-BA) at a final concentration of 0.1–1.0 mg / L, preferably 0.5 mg / L; 2,4-dichlorophenoxyacetic acid (2,4-D) at a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; kinetin at a final concentration of 0.7–1.3 mg / L, preferably 1.0 mg / L; and proline at a final concentration of 1.7–2.3 g / L, preferably 2.0 g / L.

[0029] The pH value of the above subculture medium is 5.8~5.9.

[0030] In a second aspect, the present invention provides a resistance subculture medium, a first resistance selection medium, and a second resistance selection medium for genetic transformation of sedge seeds; the resistance subculture medium contains proline (Pro) and kinetin (KT), and the first resistance selection medium and the second resistance selection medium contain proline (Pro) and vitamin C (Vc).

[0031] In a preferred embodiment, the above-mentioned resistance subculture medium is based on MS basal medium, with the following added: kinetin at a final concentration of 0.7-1.3 mg / L, preferably 1.0 mg / L; proline at a final concentration of 1.7-2.3 g / L, preferably 2.0 g / L; and termethin at a final concentration of 200-300 mg / L, preferably 250 mg / L.

[0032] In a further preferred embodiment, the above-mentioned resistance subculture medium is based on MS basal medium, supplemented with: naphthaleneacetic acid at a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; 6-benzylaminopurine at a final concentration of 0.1–1.0 mg / L, preferably 0.5 mg / L; 2,4-dichlorophenoxyacetic acid at a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; kinetin at a final concentration of 0.7–1.3 mg / L, preferably 1.0 mg / L; proline at a final concentration of 1.7–2.3 g / L, preferably 2.0 g / L; or proline at a final concentration of 1.7–2.3 g / L, preferably 2.0 g / L; and termethin at a final concentration of 200–300 mg / L, preferably 250 mg / L.

[0033] In a preferred embodiment, the first resistance screening medium is based on MS basal medium, with the following added: proline at a final concentration of 0.5–1.5 g / L, preferably 1.0 g / L; vitamin C at a final concentration of 150–250 mg / L, preferably 200 mg / L; cephalosporin at a final concentration of 150–250 mg / L, preferably 200 mg / L; ampicillin at a final concentration of 2.5–3.5 mmol / L, preferably 3 mmol / L; and hygromycin at a final concentration of 20–40 mg / L, preferably 30 mg / L.

[0034] In a preferred embodiment, the second resistance screening medium is based on MS basal medium, with the following added: proline at a final concentration of 0.5–1.5 g / L, preferably 1.0 g / L; vitamin C at a final concentration of 150–250 mg / L, preferably 200 mg / L; cephalosporin at a final concentration of 150–250 mg / L, preferably 200 mg / L; ampicillin at a final concentration of 2.5–3.5 mmol / L, preferably 3 mmol / L; and hygromycin at a final concentration of 40–60 mg / L, preferably 50 mg / L.

[0035] In a further preferred embodiment, the first resistance screening medium is based on MS basal medium, supplemented with: naphthaleneacetic acid to a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; 6-benzylaminopurine to a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; 2,4-dichlorophenoxyacetic acid to a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; proline to a final concentration of 0.5–1.5 g / L, preferably 1.0 g / L; vitamin C to a final concentration of 150–250 mg / L, preferably 200 mg / L; cephalosporin to a final concentration of 150–250 mg / L, preferably 200 mg / L; ampicillin to a final concentration of 2.5–3.5 mmol / L, preferably 3 mmol / L; and hygromycin to a final concentration of 20–40 mg / L, preferably 30 mg / L.

[0036] In a further preferred embodiment, the second resistance screening medium is based on MS basal medium, supplemented with: naphthaleneacetic acid at a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; 6-benzylaminopurine at a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; 2,4-dichlorophenoxyacetic acid at a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L; proline at a final concentration of 0.5–1.5 g / L, preferably 1.0 g / L; vitamin C at a final concentration of 150–250 mg / L, preferably 200 mg / L; cephalosporin at a final concentration of 150–250 mg / L, preferably 200 mg / L; ampicillin at a final concentration of 2.5–3.5 mmol / L, preferably 3 mmol / L; and hygromycin at a final concentration of 40–60 mg / L, preferably 50 mg / L.

[0037] The pH values ​​of the above-mentioned resistance subculture medium, first resistance screening medium, and second resistance screening medium were all 5.94~5.98.

[0038] Thirdly, the present invention provides a method for establishing a regeneration system for sedge seeds, the method comprising the following steps:

[0039] Explant preparation steps:

[0040] Using sedge seeds as explants, the seeds were washed, soaked, and stirred with sterile water to remove the bran; then disinfected with ethanol solution; after rinsing with sterile water, they were soaked in sodium hypochlorite solution while stirring; after rinsing with sterile water, the seeds were placed on sterile paper to absorb the moisture, thus obtaining sterilized seeds.

[0041] All of the above operations are performed in a clean bench.

[0042] Callus induction and culture steps:

[0043] The sterilized seeds were placed in a callus induction medium for callus induction culture, and small callus pieces were obtained after selection.

[0044] The callus induction medium described above is based on MS basal medium, with the following added: naphthaleneacetic acid to a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; 6-benzylaminopurine to a final concentration of 0.1–1.0 mg / L, preferably 0.5 mg / L; and 2,4-dichlorophenoxyacetic acid to a final concentration of 1.5–2.5 mg / L, preferably 2.0 mg / L.

[0045] The culture conditions for the above-mentioned callus induction culture include: temperature 22-28℃, preferably 25℃, and culture in the dark.

[0046] The induction and culture time for the above-mentioned callus tissue is 6 to 10 weeks, preferably 8 weeks.

[0047] The diameter of the callus pieces mentioned above is 1~3 mm; the selection method includes: peeling off the outer layer of the callus mass from a single seed, and picking out the callus pieces with a dark color and dense texture in the core part.

[0048] Callus subculture steps:

[0049] The callus fragments were placed in the subculture medium provided in the first aspect of the present invention and subcultured to obtain callus tissue to be differentiated.

[0050] The culture conditions for the above-mentioned subculture include: temperature 22-28℃, preferably 25℃, and culture in the dark.

[0051] The above-mentioned subculture includes: first, culturing the callus fragments in the dark for 2-4 weeks (preferably 3 weeks), then selecting callus tissue and culturing it in fresh subculture medium for 3-5 weeks (preferably 4 weeks) to obtain callus tissue to be differentiated; preferably, the callus tissue to be differentiated is then subcultured in the above-mentioned subculture medium 2-4 times (preferably 3 times), with each subculture spaced 3-5 weeks apart (preferably 4 weeks) to obtain more callus tissue to be differentiated. The callus tissue used for each subculture is: well-grown, brightly colored, firm in texture, and with a large number of embryoids visible on the surface under a dissecting microscope.

[0052] The addition of proline to the above-mentioned subculture medium can alleviate osmotic stress, scavenge reactive oxygen species, and reduce oxidative damage and browning of callus tissue during culture. The addition of kinetin to the subculture medium can delay cell senescence and maintain cell metabolic activity by regulating endogenous hormone balance. Therefore, the combined use of proline and kinetin in the subculture medium can work synergistically to reduce the browning rate of callus tissue and prolong the subculture cycle.

[0053] Differentiation culture steps:

[0054] The callus tissue to be differentiated was placed in a differentiation medium and cultured for differentiation to obtain adventitious shoots.

[0055] The differentiation medium described above is based on MS basal medium, with the following added: naphthaleneacetic acid to a final concentration of 1.0–2.0 mg / L, preferably 1.5 mg / L; and 6-benzylaminopurine to a final concentration of 0.5–1.5 mg / L, preferably 1.0 mg / L.

[0056] The culture conditions for the above differentiation culture include: a temperature of 22–28℃, preferably 25℃; a photoperiod of 16 h light / 8 h dark; and a light intensity of 150–250 μmol·m⁻¹. -2 ·s -1Preferably 200 μmol·m -2 ·s -1 .

[0057] The differentiation culture time is 3 to 5 weeks, preferably 4 weeks.

[0058] Rooting culture steps:

[0059] The adventitious buds were placed in a rooting medium for rooting culture to obtain small plantlets.

[0060] The above-mentioned rooting medium is a 1 / 2 MS basal medium.

[0061] The above-mentioned rooting culture conditions include: temperature 23~25℃, photoperiod of 16 h light / 8 h dark, and light intensity of 150~250 μmol·m⁻¹. -2 ·s -1 Preferably 200 μmol·m -2 ·s -1 .

[0062] The rooting culture time is 3 to 5 weeks, preferably 4 weeks.

[0063] Afterwards, the small plant was hardened off and transplanted to obtain a regenerated green sedge plant.

[0064] The pH value of the culture medium used in the third aspect above is 5.8~5.9.

[0065] Fourthly, the present invention provides a method for genetic transformation of sedge seeds, the method comprising the following steps:

[0066] Preparation steps of bacterial suspension for infection:

[0067] Agrobacterium carrying the plasmid vector was inoculated into YEP resistant liquid medium and cultured with shaking to obtain an expanded culture broth. The expanded culture broth was then centrifuged, and the precipitate was retained. The precipitate was then resuspended in LB liquid medium to obtain a suspension. Acetyleugenol (AS) was added to the suspension, and after standing, the bacterial suspension for infection was obtained.

[0068] The above-mentioned YEP resistance liquid culture medium is based on YEP basic culture medium, with the following added: kanamycin to a final concentration of 10~80 mg / L, preferably 50 mg / L; and rifampin to a final concentration of 10~30 mg / L, preferably 20 mg / L.

[0069] The temperature for the above-mentioned shaking culture is 25~30℃, preferably 28℃.

[0070] The OD600 values ​​of the above expanded culture solutions were 0.8~1.0.

[0071] The centrifugation speed is 2000~400 rpm, preferably 3000 rpm, and the time is 5~15 min, preferably 10 min.

[0072] The OD600 values ​​of the above suspensions are 0.8 to 1.0.

[0073] The final concentration of acetylsuccinone in the bacterial solution used for infection is 100~300μmol / L, preferably 200μmol / L.

[0074] The temperature for the above-mentioned standing is 25~30℃, preferably 28℃, and the time is 20~40min, preferably 30min.

[0075] Steps for preparing callus for infection:

[0076] The callus tissue to be differentiated obtained by the method for establishing the regeneration system of sedge seeds according to the third aspect of the present invention is transferred to a pre-culture medium and pre-cultured for 2 to 4 days (preferably 3 days) before being cut into callus tissue for infection.

[0077] Preferably, the method for preparing the above-mentioned callus to be differentiated includes: culturing the callus pieces described in the third aspect under dark conditions for 2 to 4 weeks (preferably 3 weeks), and then placing the obtained callus on the above-mentioned subculture medium for 3 to 5 weeks (preferably 4 weeks) to obtain the above-mentioned callus to be differentiated.

[0078] The above-mentioned pre-culture medium is based on MS basal medium, with the following added: 6-BA to a final concentration of 0.05-0.15 mg / L, preferably 0.1 mg / L; 2,4-D to a final concentration of 2.5-3.5 mg / L, preferably 3.0 mg / L; and KT to a final concentration of 0.5-1.5 mg / L, preferably 1.0 mg / L.

[0079] The pre-culture conditions mentioned above include: a temperature of 22–28°C, preferably 25°C, and incubation in the dark.

[0080] The specifications of the callus tissue used for infection are (1.5~2.5) mm × (1.5~2.5) mm, preferably 2 mm × 2 mm.

[0081] All of the above callus tissues grew well and showed a large number of embryoids.

[0082] Cold treatment steps for callus tissue:

[0083] The above-mentioned infected callus was placed in a cold treatment solution for cold treatment to obtain cold-treated callus.

[0084] The above-mentioned cold treatment solution includes: glutamine (Gln) with a final concentration of 200~400μmol / L, preferably 300μmol / L; and maltose with a final concentration of 1~5%, preferably 3%.

[0085] The above-mentioned cold treatment operation includes: immersing the infected callus tissue in a cold treatment solution, ice bath for 10-30 minutes, preferably 20 minutes, and then discarding the cold treatment solution.

[0086] Vacuum treatment steps:

[0087] The callus tissue after cold treatment is immersed in the above-mentioned bacterial solution for infection, and then glutamine is added to the bacterial solution for infection until the final concentration is 200~400μmol / L, preferably 300μmol / L, to obtain the material to be vacuumed; the material to be vacuumed is subjected to vacuum treatment to obtain the vacuum-treated material; the vacuum-treated material is then subjected to shaking culture to obtain the vacuum-treated callus tissue.

[0088] The vacuum treatment pressure is -1.0 to -0.5 MPa, preferably -0.8 MPa, and the time is 5 to 15 minutes, preferably 10 minutes. During this vacuum treatment, oscillation is performed at a speed of 60 to 100 rpm, preferably 80 rpm.

[0089] The temperature for the above-mentioned shaking culture is 25~30℃, preferably 28℃, the rotation speed is 60~100rpm, preferably 80rpm, and the time is 10~30min, preferably 20min.

[0090] Drying process steps:

[0091] The callus tissue after vacuum treatment was dried to obtain dried callus tissue.

[0092] The above drying process must be carried out in a sterile environment at room temperature for 1.5 to 2.5 hours, preferably 2 hours. For example, the vacuum-treated callus tissue can be placed on three layers of sterile filter paper and air-dried in a laminar flow hood.

[0093] The three-step combined infection method (CVD) of cold treatment, vacuum treatment, and drying treatment for callus infection achieves the highest conversion efficiency compared to single vacuum drying (VD) or permeation cold treatment (PVD). Because different plant callus tissues have significantly different tolerances to cold, vacuum, and drying, while vacuum or drying treatments have some applications, the idea of ​​combining them with a cold treatment system has not been tested in practice. In the cold treatment step, excessively high temperatures or prolonged times can cause cold damage. In the vacuum step, excessively high vacuum or prolonged time can lead to cell collapse, while excessively low vacuum or short time results in poor permeation. After vacuum treatment, the material is shaken and cultured to further evenly distribute the bacterial solution. In the drying step, the vacuum-treated callus tissue needs to be air-dried in a sterile environment to avoid secondary contamination; insufficient drying time results in weak bacterial adhesion, while excessive drying time leads to cell dehydration and death.

[0094] Co-cultivation steps:

[0095] The dried callus tissue was placed in a co-culture medium for co-culture to obtain co-cultured callus tissue.

[0096] The above co-culture medium is based on MS basal medium, with the following added: 6-BA to a final concentration of 0.05-0.15 mg / L, preferably 0.1 mg / L; and AS to a final concentration of 50-150 μM, preferably 100 μM.

[0097] The above co-culture conditions include: a temperature of 23–30°C, preferably 26°C, and incubation in the dark.

[0098] The co-cultivation time is 2-4 days, preferably 3 days.

[0099] The above co-culture is carried out by placing the dried callus tissue on filter paper soaked in liquid co-culture medium.

[0100] Recovery culture steps:

[0101] The co-cultured callus was placed in the resistant subculture medium provided in the second aspect of the present invention for recovery culture to obtain the recovered callus.

[0102] The recovery culture time is 4 to 10 days, preferably 7 days.

[0103] The above-mentioned recovery culture conditions include: a temperature of 22-28℃, preferably 25℃, and culture in the dark.

[0104] First resistance screening culture steps:

[0105] The recovered callus was placed in the first resistance screening medium provided in the second aspect of the present invention for the first resistance screening culture to obtain the first screened callus.

[0106] The culture conditions for the first resistance screening culture mentioned above include: a temperature of 22-28℃, preferably 25℃, and culture in the dark.

[0107] The culture time for the first resistance screening is 1 to 3 weeks, preferably 2 weeks.

[0108] Second resistance screening culture steps:

[0109] The callus tissue after the first screening was placed in the second resistance screening medium provided in the second aspect of the present invention for a second resistance screening culture to obtain the callus tissue after the second screening.

[0110] The culture conditions for the second resistance screening culture mentioned above include: a temperature of 22-28℃, preferably 25℃, and culture in the dark.

[0111] The second resistance screening culture period is 1 to 3 weeks, preferably 2 weeks.

[0112] Culture of resistant callus differentiation:

[0113] The callus tissue after the second screening was placed in a resistance differentiation medium for resistance differentiation culture to obtain resistant adventitious shoots.

[0114] The above-mentioned resistance differentiation medium is based on MS basal medium, supplemented with: NAA at a final concentration of 0.5-1.5 mg / L, preferably 1 mg / L; 6-BA at a final concentration of 2.5-3.5 mg / L, preferably 3.0 mg / L; Vc at a final concentration of 150-250 mg / L, preferably 200 mg / L; cephalosporin at a final concentration of 150-250 mg / L, preferably 200 mg / L; ampicillin at a final concentration of 2.5-3.5 mmol / L, preferably 3 mmol / L; and hygromycin at a final concentration of 150-250 mg / L, preferably 200 mg / L.

[0115] The culture conditions for the above-mentioned resistance differentiation culture include: a temperature of 22–28℃, preferably 25℃; a photoperiod of 16 h light / 8 h dark; and a light intensity of 150–250 μmol·m⁻¹. -2 ·s -1 Preferably 200 μmol·m -2 ·s -1 .

[0116] The culture time for the above-mentioned resistance differentiation is 3 to 5 weeks, preferably 4 weeks.

[0117] The first and second resistance selection media, supplemented with both vitamin C and protein (Pro), synergistically alleviated callus browning and enhanced callus viability. The core cause of browning is the oxidation of phenolic substances. Vitamin C, as a strong reducing agent, reduces quinones, preventing their polymerization into melanin; protein (Pro) maintains cell integrity by ensuring cellular water balance and energy supply; and vitamin C removes leaked phenolic oxides. This dual approach, working synergistically, significantly reduces the browning rate of callus.

[0118] Rooting culture steps:

[0119] The resistant adventitious buds were placed in a rooting selection medium for rooting culture to obtain resistant plantlets.

[0120] The above-mentioned rooting selection medium is based on 1 / 2 MS basal medium, with the following added: NAA to a final concentration of 0.1-1.0 mg / L, preferably 0.5 mg / L; and indolebutyric acid (IBA) to a final concentration of 0.1-1.0 mg / L, preferably 0.5 mg / L.

[0121] The above-mentioned rooting culture conditions include: temperature 23~25℃, photoperiod of 16 h light / 8 h dark, and light intensity of 150~250 μmol·m⁻¹. -2 ·s -1 Preferably 200 μmol·m -2 ·s -1 .

[0122] Afterwards, the resistant plantlets were hardened off and transplanted to obtain sedge-transformed regenerated plants.

[0123] In the co-culture steps described above, if the co-culture time is too short, it will affect the transformation efficiency; if the co-culture time is too long, it will cause Agrobacterium to overgrow and become difficult to inhibit, harming plant cells and increasing false positives.

[0124] In the aforementioned first and second resistance selection cultures, as well as the resistant callus differentiation culture, the concentrations of hygromycin and ampicillin are crucial. Hygromycin kills non-transformed cells lacking the resistance gene, allowing only transformed cells to grow. If the hygromycin concentration is too low, it cannot effectively inhibit the growth of non-transformed cells; if the concentration is too high, it is toxic to all cells (including transformed cells), inhibiting callus growth and differentiation, and even leading to complete cell death. Ampicillin kills residual Agrobacterium, preventing its excessive growth from interfering with callus growth. If the Ampicillin concentration is too low, it cannot completely eliminate Agrobacterium, leading to subsequent culture contamination, callus browning, and death; if the Ampicillin concentration is too high, it may cause unnecessary physiological stress or toxicity to plant cells.

[0125] The pH value of the culture medium used in the fourth aspect above is 5.94~5.98.

[0126] The MS basic culture medium used in this invention includes: MS medium dry powder, sucrose, agar, and deionized water; the final concentration of MS powder is 4.43 g / L, the final concentration of sucrose is 30 g / L, and the final concentration of agar is 6 g / L.

[0127] The 1 / 2MS basic culture medium used in this invention includes: MS medium dry powder, sucrose, agar, and deionized water; the final concentration of MS medium dry powder is 2.215 g / L, the final concentration of sucrose is 15 g / L, and the final concentration of agar is 4 g / L.

[0128] The MS culture medium powder mentioned above can be purchased commercially. For example, MS culture medium powder purchased from XMJ Scientific, brand name Phytotech, catalog number M524 (Murashige & Skoog (MS) Basal Salt Mixture).

[0129] The *Carex spp.* used in the following examples, test cases, and comparative examples is the cultivar "Siji" *Carex spp.*, bred by the Beijing Academy of Agricultural and Forestry Sciences. It has been approved by the National Forestry and Grassland Administration (National S-SV-CL-006-2010). The inventor can be contacted at 010-81127836 to obtain it. "Siji" *Carex spp.* is preserved at the National Ornamental Grass Germplasm Resource Center, with the number BJCY-Cb-0002. For the purpose of implementing the technical solution of this invention, anyone can contact the inventor to obtain the above-mentioned plant cultivar within the patent term from the date of application of this invention.

[0130] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.

[0131] Example 1

[0132] This embodiment illustrates the method for establishing a regeneration system for green sedge provided by the present invention, which includes the following steps.

[0133] S1. Explant treatment:

[0134] Select plump seeds of "Four Seasons" green sedge (national S-SV-CL-006-2010). In a clean bench, wash the seeds three times with sterile water; then soak and stir in sterile water for 30 minutes to remove the bran; then disinfect with 75% ethanol for 30 seconds; after rinsing with sterile water, soak in 0.1% sodium hypochlorite and stir in a magnetic stirrer for 30 minutes; then rinse with sterile water 3-5 times, place on sterile paper, absorb the moisture, and inoculate into the culture medium to obtain sterilized seeds.

[0135] S2. Callus induction and culture steps:

[0136] The sterilized seeds were placed in callus induction medium and cultured for 8 weeks. After selection, callus pieces were obtained.

[0137] The callus induction medium described above was based on MS basal medium, with the following additions: NAA to a final concentration of 1.5 mg / L, 6-BA to a final concentration of 0.5 mg / L, and 2,4-D to a final concentration of 2.0 mg / L.

[0138] The culture conditions for the above callus induction culture were: temperature 25℃, culture in the dark.

[0139] The above selection was performed under a dissecting microscope to screen for embryogenic callus. The outer layer of the callus mass derived from a single seed was removed, and small callus pieces with a diameter of about 1-3 mm with a dark color and dense texture were selected from the core area.

[0140] S3. Callus subculture procedure:

[0141] The callus fragments were placed on a subculture medium and subcultured to obtain callus tissue to be differentiated.

[0142] The above subculture medium was based on MS basal medium, supplemented with: NAA to a final concentration of 1.5 mg / L, 6-BA to a final concentration of 0.5 mg / L, 2,4-D to a final concentration of 2.0 mg / L, Pro to a final concentration of 2.0 g / L, and KT to a final concentration of 1.0 mg / L.

[0143] The culture conditions for the above subculture were: temperature 25℃, culture in the dark.

[0144] The above subculture process includes: first, culturing the callus pieces in the dark for 3 weeks, then selecting the callus tissue and placing it in fresh subculture medium, subculturing 3 times, with an interval of 4 weeks between each subculture, and finally obtaining the callus tissue to be differentiated.

[0145] The callus tissue selected for each subculture was characterized by good growth, bright color, firm texture, and the presence of numerous embryoids on the surface visible under a dissecting microscope.

[0146] S4. Differentiation culture:

[0147] The callus tissue to be differentiated was placed in differentiation medium and cultured for 4 weeks to obtain adventitious shoots.

[0148] The differentiation medium described above was based on MS basal medium, with the following additions: NAA to a final concentration of 1.5 mg / L and 6-BA to a final concentration of 1.0 mg / L.

[0149] The culture conditions for the above differentiation culture included: a temperature of 25℃, a photoperiod of 16 h light / 8 h dark, and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 .

[0150] S5. Rooting Culture:

[0151] The adventitious buds were placed on a rooting medium and rooted for 4 weeks to obtain small plantlets.

[0152] The above-mentioned rooting medium is a 1 / 2 MS basal medium.

[0153] The above-mentioned rooting culture conditions include: a temperature of 23–25℃, a photoperiod of 16 h light / 8 h dark, and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 .

[0154] S6. Seedling hardening and transplanting:

[0155] Remove the cap from the culture bottle, harden the small plantlets in the culture room for 1-2 days, then transplant them into flowerpots filled with nutrient soil, place them in a light incubator for 2 weeks, and then transplant them into a greenhouse with natural light to grow, thus obtaining regenerated plantlets.

[0156] In this embodiment, 30 seeds were used in step S1, and 21 regenerated plants were obtained in step S6.

[0157] Examples 2-5

[0158] The methods for establishing the regeneration system of *Carex lucida* in Examples 2-5 are the same as those in Example 1, except that the final concentrations of Pro and KT in the subculture medium in step S3 of Example 1 are replaced with the following:

[0159] Example 2: The final concentration of Pro in the subculture medium used in step S3 was 2.3 g / L, and the final concentration of KT was 0.7 mg / L.

[0160] Example 3: The final concentration of Pro in the subculture medium used in step S3 was 1.7 g / L, and the final concentration of KT was 1.3 mg / L.

[0161] Example 4: The final concentration of Pro in the subculture medium used in step S3 was 0.7 g / L, and the final concentration of KT was 1.7 mg / L.

[0162] Example 5: The final concentration of Pro in the subculture medium used in step S3 was 2.3 g / L, and the final concentration of KT was 1.3 mg / L.

[0163] Comparative Examples 1-9:

[0164] Comparative Examples 1-9 are used to illustrate the effects of subculture media with different component ratios on callus tissue.

[0165] The methods for establishing the regeneration systems of *Carex lucida* in Comparative Examples 1-9 are the same as in Example 1, except that the final concentrations of Pro and KT in the subculture medium in step S3 of Example 1 are replaced with the following:

[0166] Comparative Example 1: The final concentration of Pro in the subculture medium used in step S3 was 0.5 g / L, and the final concentration of KT was 1.0 mg / L.

[0167] Comparative Example 2: The final concentration of Pro in the subculture medium used in step S3 was 1.0 g / L, and the final concentration of KT was 0.5 mg / L.

[0168] Comparative Example 3: The final concentration of Pro in the subculture medium used in step S3 was 1.0 g / L, and the final concentration of KT was 1.0 mg / L.

[0169] Comparative Example 4: The final concentration of Pro in the subculture medium used in step S3 was 1.0 g / L, and the final concentration of KT was 1.5 mg / L.

[0170] Comparative Example 5: The final concentration of Pro in the subculture medium used in step S3 was 2.0 g / L, and the final concentration of KT was 0.5 mg / L.

[0171] Comparative Example 6: The final concentration of Pro in the subculture medium used in step S3 was 2.0 g / L, and the final concentration of KT was 1.5 mg / L.

[0172] Comparative Example 7: The final concentration of Pro in the subculture medium used in step S3 was 0 g / L, and the final concentration of KT was 0 mg / L.

[0173] Comparative Example 8: The final concentration of Pro in the subculture medium used in step S3 was 0.5 g / L, and the final concentration of KT was 0.5 mg / L.

[0174] Comparative Example 9: The final concentration of Pro in the subculture medium used in step S3 was 0.5 g / L, and the final concentration of KT was 1.5 mg / L.

[0175] Detection Example 1

[0176] This test example is used to illustrate the browning of the subcultured callus obtained by step S3 of the tissue culture method of *Carex lucida* in Examples 1-5 and Comparative Examples 1-9.

[0177] Ensuring uniform callus size and growth, the callus fragments obtained in step S2 of Example 1 were placed in 10 different subculture media with varying Pro and kt concentrations, as shown in Table 1. Nine fragments were inoculated into each subculture medium, with four replicates. Callus tissue blocks were obtained by culturing at 24±1℃ under 16 h light / 8 h dark conditions. After 4 weeks, the browning rate of the callus tissue blocks in each treatment was calculated. Browning was defined as callus tissue blocks with a surface area greater than or equal to 50% turning brown. Browning rate = (Number of browned callus tissue blocks / Total number of inoculated callus tissue blocks) × 100%.

[0178] As shown in Table 1, the browning rate and growth status of callus blocks obtained from 14 different subculture media in Examples 1-5 and Comparative Examples 1-9 were compared to evaluate the inhibitory effect of different subculture media formulations on browning. Among them, the subculture media formulation in Example 1 with 2.0 g / L proline + 1.0 mg / L kinetin showed the best effect in inhibiting the browning rate of *Carex lucida*, and the subculture media in Examples 2-5 also significantly improved the inhibition of browning rate.

[0179] Table 1: Statistical table of browning rate for different subculture medium formulations

[0180]

[0181] like Figure 1 As shown, obtaining high-quality embryogenic callus is a crucial step in establishing the *Carex lucida* regeneration system. Both the culture medium supplemented with Pro and KT showed normal differentiation into seedlings, and the browning rate decreased during the same period. Figure 1 E).

[0182] Figure 2 The boundary of "browning" is not absolutely clear; the brown and dark areas represent tissues with different degrees of browning, while the light yellow areas represent healthy, unbrowned callus tissue. It can be seen that the callus tissue of Example 1 on medium 8 has the lowest degree of browning, while the callus tissue of Comparative Example 5 on medium 7 has a severe degree of browning.

[0183] Example 4

[0184] This embodiment illustrates the genetic transformation method of *Carex lucida* provided by the present invention, which includes the following steps.

[0185] S1. Preparation of Agrobacterium carrying plasmid vector pHDE-35S-RUBY:

[0186] To enable effective visualization and monitoring during the genetic transformation process, the RUBY reporter system was used as a selection marker, and the selected plasmid vector was pHDE-35S-RUBY. This plasmid vector is described in Sun H, Wang S, Yang K, et al. Development of dual‐visible reporter assays to determine the DNA–protein interaction[J]. The Plant Journal, 2023, 113(5): 1095-1101. The structural diagram of this plasmid vector is shown below. Figure 5 As shown in A. For the purpose of implementing the technical solution of this invention, anyone can contact the inventor to obtain the above-mentioned plasmid vector within the patent term from the date of application of this invention.

[0187] The vector pHDE-35S-RUBY carrying the 35S:RUBY plasmid was introduced into Agrobacterium strain GV3101 via a freeze-thaw method to obtain Agrobacterium carrying the plasmid vector. The specific procedures included: taking competent Agrobacterium cells stored at -80℃, partially thawing them to an ice-water mixture at room temperature and then placing them on ice; subsequently, adding 1 μg of the plasmid vector pHDE-35S-RUBY to 100 μl of competent cells and gently mixing. Then, the following steps were performed sequentially: ice bath for 5 minutes, liquid nitrogen treatment for 5 minutes, heat shock at 37℃ for 5 minutes, and ice bath for 5 minutes. Next, 700 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 28℃ with shaking for 2–3 hours. Afterwards, the cells were collected by centrifugation at 6000 rpm for 1 minute, discarding most of the supernatant and retaining approximately 100 μL of supernatant. The bacterial cells were gently resuspended and spread on LB agar plates containing 50 μg / ml kanamycin and 20 μg / ml rifampin. The plates were incubated upside down at 28°C for 72 hours to obtain Agrobacterium carrying the plasmid vector.

[0188] The above LP plates are based on LB solid medium, wherein 1L of LB solid medium contains: 5g yeast extract, 10g tryptone, 10g NaCl, and 8g agar powder.

[0189] S2. Preparation of bacterial suspension for infection:

[0190] Agrobacterium carrying the plasmid vector was inoculated into YEP resistant liquid medium and cultured in a shaker at 28°C to obtain an expanded culture with an OD600 value of 0.8-1.0. The expanded culture was then centrifuged (3000 rpm, 10 min) and the precipitate was retained. The precipitate was then resuspended in LB liquid medium to obtain a suspension. Acetyleugenol (AS) was added to the suspension to a final concentration of 200 μmol / L, and the suspension was allowed to stand at 28°C for 30 min to obtain the bacterial suspension for infection.

[0191] The above-mentioned YEP resistance liquid culture medium is based on YEP basal medium, with the addition of: kanamycin at a final concentration of 50 mg / L and rifampin at a final concentration of 20 mg / L.

[0192] S3. Preparation of callus tissue for infection:

[0193] Following the method described in Example 1, callus fragments were obtained through explant treatment and callus induction culture. These fragments were then cultured in the dark for 3 weeks. The resulting callus was then placed in the subculture medium described in Example 1 for 4 weeks to obtain ready-to-differentiate callus (i.e., callus to be differentiated). This ready-to-differentiate callus was then transferred to a pre-culture medium and pre-cultured at 25°C in the dark for 3 days, before being cut into callus for infection (approximately 2mm × 2mm in size).

[0194] All of the above callus tissues grew well and showed a large number of embryoids.

[0195] The above pre-medium was based on MS basal medium, with the following added: 6-BA to a final concentration of 0.1 mg / L, 2,4-D to a final concentration of 3.0 mg / L, and KT to a final concentration of 1.0 mg / L.

[0196] S4. Cold treatment of callus tissue:

[0197] The above-mentioned callus tissue for infection was placed in a cold treatment solution for cold treatment: the above-mentioned callus tissue for infection was immersed in the cold treatment solution in an ice bath for 20 minutes, and then the cold treatment solution was discarded to obtain the cold-treated callus tissue.

[0198] The aforementioned cold treatment solution includes: glutamine (Gln) at a final concentration of 300 μmol / L and maltose at a final concentration of 3% by mass.

[0199] S5, Vacuum treatment:

[0200] The callus tissue after cold treatment was immersed in the bacterial solution for infection obtained in step S2, and then glutamine was added to the bacterial solution for infection to a final concentration of 300 μmol / L to obtain the material to be vacuumed; the material to be vacuumed was subjected to vacuum treatment at -0.8 MPa for 10 min, and oscillated at 80 rpm during the vacuum treatment to obtain the material after vacuum treatment; the material after vacuum treatment was then cultured at 28°C and 80 rpm for 20 min to obtain the callus tissue after vacuum treatment.

[0201] S6. Drying treatment:

[0202] The vacuum-treated callus was transferred onto three layers of sterile filter paper and air-dried in a clean bench for 2 hours to obtain dried callus.

[0203] S7, Co-cultivation:

[0204] The dried callus tissue was placed on two layers of sterile filter paper soaked in 500 μL of co-culture medium (liquid) and co-cultured at 26°C in the dark for 3 days to obtain the co-cultured callus tissue.

[0205] The above co-culture medium was based on MS basal medium (without agar), with the following added: 6-BA to a final concentration of 0.1 mg / L and AS to a final concentration of 100 μM.

[0206] S8, Recovery Culture:

[0207] The co-cultured callus was placed in a resistant subculture medium and cultured in the dark at 25°C for 7 days to obtain the recovered callus.

[0208] The above-mentioned resistance subculture medium was based on MS basal medium, supplemented with: NAA to a final concentration of 1.5 mg / L, 6-BA to a final concentration of 0.5 mg / L, 2,4-D to a final concentration of 2.0 mg / L, kinetin to a final concentration of 1.0 mg / L, proline to a final concentration of 2.0 g / L, and termethin to a final concentration of 250 mg / L.

[0209] S9, First resistance selection culture:

[0210] The recovered callus was placed in the first resistance selection medium and cultured in the dark at 25°C for 2 weeks to obtain the first-selection callus.

[0211] The first resistance screening medium was based on MS basal medium, supplemented with: NAA to a final concentration of 1.5 mg / L, 6-BA to a final concentration of 2.0 mg / L, 2,4-D to a final concentration of 2.0 mg / L, proline to a final concentration of 1.0 g / L, vitamin C to a final concentration of 200 mg / L, cephalosporin to a final concentration of 200 mg / L, ampicillin to a final concentration of 3 mmol / L, and hygromycin to a final concentration of 30 mg / L.

[0212] S10, Second Resistance Selection Culture:

[0213] The callus tissue obtained after the first screening was placed in the second resistance screening medium and cultured in the dark at 25°C for 2 weeks to obtain the callus tissue after the second screening.

[0214] The second resistance screening medium was based on MS basal medium, supplemented with: NAA to a final concentration of 1.5 mg / L, 6-BA to a final concentration of 2.0 mg / L, 2,4-D to a final concentration of 2.0 mg / L, proline to a final concentration of 1.0 g / L, vitamin C to a final concentration of 200 mg / L, cephalosporin to a final concentration of 200 mg / L, ampicillin to a final concentration of 3 mmol / L, and hygromycin to a final concentration of 50 mg / L.

[0215] S11, resistant callus differentiation culture:

[0216] The callus tissues selected after the second screening were placed in a resistant differentiation medium and incubated at 25°C with a photoperiod of 16 h light / 8 h dark and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 After 4 weeks of differentiation culture, adventitious buds that show a RUBY red color are selected to obtain resistant adventitious buds.

[0217] The above-mentioned resistance differentiation medium was based on MS basal medium, supplemented with: NAA to a final concentration of 1 mg / L, 6-BA to a final concentration of 3.0 mg / L, Vc to a final concentration of 200 mg / L, cephalosporin to a final concentration of 200 mg / L, ampicillin to a final concentration of 3 mmol / L, and hygromycin to a final concentration of 200 mg / L.

[0218] S12. Rooting Culture Steps:

[0219] The resistant adventitious buds were placed in a rooting medium for rooting culture to obtain resistant plantlets.

[0220] The above resistance selection medium was based on 1 / 2 MS basal medium, with the addition of NAA to a final concentration of 0.5 mg / L and IBA to a final concentration of 0.5 mg / L.

[0221] The conditions for rooting culture mentioned above include: a temperature of 24±1℃, a light exposure of 16 h / dark period, and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 .

[0222] S13: Hardening off and transplanting:

[0223] Remove the cap from the culture bottle, harden the small plantlets in the culture room for 1-2 days, then transplant the small plantlets into flower pots filled with nutrient soil, place them in a light incubator for 2 weeks, and then transplant them into a greenhouse with natural light to grow, thus obtaining sedge regeneration plants.

[0224] The experimental results and analysis of the genetic transformation method of *Carex lucida* in this embodiment are as follows.

[0225] (1) Based on the optimized high-efficiency regeneration system and the visualized RUBY reporter system, a high-efficiency genetic transformation system was developed, which enabled Agrobacterium to successfully infect the explants. The explant incision site was clearly observed to show the red color of the RUBY reporter system. Figure 3 E, F); approximately 24 days after infection, embryogenic callus tissue begins to differentiate at the explant wound; finally, the resistant adventitious shoots obtained in step S11, which exhibit a ruby-red color, are rooted ( Figure 3 G) and seedling transplanting ( Figure 3 F).

[0226] (2) such as Figure 4 As shown, after the second resistance selection culture of S10, the callus tissue obtained after the second selection grew well and had a certain degree of distinguishability. Some of the untransformed tissues were screened to death, while the other part of the successfully transformed tissues containing the resistance gene survived.

[0227] (3) In this embodiment, a total of 90 green sedge seedlings (i.e., sedge transformation and regeneration plants) were obtained. These 90 plants were subjected to PCR amplification, and 7 of them amplified the target band of 257bp (e.g., Figure 5 (As shown in B) These are the true positive seedlings, while the target band was not amplified in the wild-type WT; this indicates that these 7 plants have hygromycin (Hyg) resistance, and the plasmid vector was successfully transformed into sedge, with a genetic transformation rate of 7.78%.

[0228] Comparative Examples 10-11

[0229] The genetic transformation method of 10-11 Comparative proportions of *Carex lucida* was used to illustrate the effect of a single resistance selection culture on callus tissue.

[0230] In Comparative Example 10, the recovered callus obtained in step S8 of Example 4 was placed in the first resistance selection medium and cultured in the dark at 25°C for 4 weeks to obtain the selected callus. Figure 6 As shown, all the screened callus tissues grew well, and it was impossible to distinguish between successfully transformed and unsuccessfully transformed callus tissues.

[0231] In Comparative Example 11, the recovered callus obtained in step S8 of Example 4 was placed in a second resistance selection medium and cultured in the dark at 25°C for 4 weeks to obtain the selected callus. Figure 7 As shown, most of the callus tissue after the above screening turned black and could not continue to survive.

[0232] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A subculture medium for establishing a sedge seed regeneration system, characterized in that: The subculture medium contains proline and kinetin.

2. The culture medium established according to the sedge seed regeneration system of claim 1, characterized in that: The culture medium is a subculture medium, which is based on MS basal medium and supplemented with: The final concentration of kinetin is 0.7–1.3 mg / L, preferably 1.0 mg / L; the final concentration of proline is 1.7–2.3 g / L, preferably 2.0 g / L.

3. A resistance subculture medium, a first resistance selection medium, and a second resistance selection medium for genetic transformation of sedge seeds, characterized in that: The resistance subculture medium contains proline and kinetin; the first resistance selection medium and the second resistance selection medium contain proline and vitamin C.

4. The resistant subculture medium according to claim 3, characterized in that: The resistance subculture medium was based on MS basal medium, with the following added: The final concentration of kinetin is 0.7–1.3 mg / L, preferably 1.0 mg / L; the final concentration of proline is 1.7–2.3 g / L, preferably 2.0 g / L; and the final concentration of termethin is 200–300 mg / L, preferably 250 mg / L.

5. The first resistance screening medium according to claim 3, characterized in that: The first resistance screening medium was based on MS basal medium, with the following added: The final concentration of proline is 0.5–1.5 g / L, preferably 1.0 g / L; the final concentration of vitamin C is 150–250 mg / L, preferably 200 mg / L; the final concentration of cephalosporin is 150–250 mg / L, preferably 200 mg / L; the final concentration of ampicillin is 2.5–3.5 mmol / L, preferably 3 mmol / L; and the final concentration of hygromycin is 20–40 mg / L, preferably 30 mg / L.

6. The second resistance screening medium according to claim 3, characterized in that: The culture medium is a second-stage resistance screening medium, which is based on MS basal medium and supplemented with: The final concentration of proline is 0.5–1.5 g / L, preferably 1.0 g / L; the final concentration of vitamin C is 150–250 mg / L, preferably 200 mg / L; the final concentration of cephalosporin is 150–250 mg / L, preferably 200 mg / L; the final concentration of ampicillin is 2.5–3.5 mmol / L, preferably 3 mmol / L; and the final concentration of hygromycin is 20–40 mg / L, preferably 30 mg / L.

7. A method for establishing a regeneration system for sedge seeds, the method comprising the following steps: Explant treatment steps: Carex seeds were used as explants and treated to obtain sterilized seeds; Callus induction and culture steps: The sterilized seeds were placed in a callus induction medium to induce callus culture and obtain callus fragments. Callus subculture steps: The callus fragments are placed in the subculture medium described in claim 1 or 2 and subcultured to obtain callus tissue to be differentiated. Differentiation culture steps: The callus tissue to be differentiated was placed in a differentiation culture medium for differentiation culture to obtain adventitious shoots; Rooting culture steps: The adventitious buds were placed in a rooting medium for rooting culture to obtain small plantlets.

8. A method for genetic transformation of sedge seeds, the method comprising the following steps: Preparation steps of bacterial suspension for infection: Agrobacterium carrying a plasmid vector was used to prepare a bacterial solution for infection. Steps for preparing callus for infection: The callus fragments described in claim 7 are used to prepare callus tissue for infection. Cold treatment steps for callus tissue: The infected callus was placed in a cold treatment solution for cold treatment to obtain cold-treated callus. Vacuum treatment steps: The cold-treated callus was immersed in the infection solution for vacuum treatment and shake culture to obtain vacuum-treated callus. Drying process steps: The vacuum-treated callus tissue was dried to obtain dried callus tissue. Co-cultivation steps: The dried callus tissue was placed in a co-culture medium and co-cultured to obtain co-cultured callus tissue. Recovery culture steps: The co-cultured callus is placed in the resistant subculture medium described in claim 3 or 4 for recovery culture to obtain the recovered callus. First resistance screening culture steps: The recovered callus is placed in the first resistance screening medium as described in claim 3 or 5 for the first resistance screening culture to obtain the first-screened callus. Second resistance selection culture steps: The callus tissue after the first screening was placed in the second resistance screening medium as described in claim 3 or 6 for the second resistance screening culture to obtain the callus tissue after the second screening. Culture of resistant callus differentiation: The callus tissue after the second screening was placed in a resistance differentiation medium for differentiation culture to obtain resistant adventitious shoots; Rooting culture steps: The resistant adventitious buds were placed on a rooting selection medium for rooting culture to obtain resistant plantlets.

9. The genetic transformation method for sedge seeds according to claim 8, characterized in that: In the vacuum treatment step, the pressure of the vacuum treatment is -1.0 to -0.5 MPa, preferably -0.8 MPa; the time is 5 to 15 minutes, preferably 10 minutes.

10. The genetic transformation method for sedge seeds according to claim 8, characterized in that: In the drying process, the drying time is 1.5 to 2.5 hours, preferably 2 hours.

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