Mature embryo callus induction-based achnatherum inebrians genetic transformation system construction method

Abstract

The invention discloses a mature embryo callus induction-based achnatherum inebrians genetic transformation system construction method. According to the method, disinfected seeds are subjected to callus induction and pre-culture, agrobacterium infection and co-culture, recovery culture, screening differentiation and seedling culture as well as seedling hardening and transplanting, so that complete transgenic regenerated plants are obtained. According to the method, by optimizing the endophytic fungus removal and disinfection process, the seed contamination rate is reduced to 5% or below, and the callus rate is increased to 75% or above; a staged antibiotic screening strategy is adopted, so that the false positive rate is effectively reduced; the obtained transgenic regenerated plant is complete, the rooting rate reaches 95% or above, and an efficient technical platform is provided for gene editing and stress-resistant breeding of achnatherum inebrians.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic transformation technology, and in particular relates to a method for constructing a genetic transformation system of *Lysimachia christinae* based on mature embryo callus induction. Background Technology

[0002] *Achnatherum inebrians*, a cool-season perennial grass belonging to the genus *Achnatherum* of the Poaceae family, is widely distributed in arid and semi-arid regions of high altitudes in Northwest my country. This grass species exhibits remarkable resilience, strong self-reproduction ability, and wide adaptability. With its well-developed root system and abundant foliage, it demonstrates exceptional vitality, reproductive capacity, and drought and cold tolerance, making it suitable as a windbreak and soil-conservation plant in non-grazing areas such as desertified and sandy lands. In harsh ecological environments such as arid and semi-arid regions, *Achnatherum inebrians* exhibits a significant growth advantage in grassland communities.

[0003] Crucially, *Drunken Horse Grass* often interacts with endophytic fungi in Gansu (… gansuensis) or endophytic fungi of *Drunken Horse Grass* ( An endophytic relationship is formed between *Inebrians* and other fungi. The presence of these fungi significantly enhances *Inebrians spp.*'s drought resistance, salt tolerance, cold tolerance, heavy metal stress tolerance, as well as its resistance to insects and overfeeding. Based on the excellent resistance characteristics of the *Inebrians spp.* endophytic fungal symbiotic relationship, it can serve as a pioneer plant and ecological improvement species for saline-alkali land, heavy metal polluted areas, and other ecologically fragile regions. It is also a high-quality material for the discovery and utilization of excellent stress-resistance genes.

[0004] Agrobacterium-mediated plant genetic transformation is a highly efficient and stable gene transformation method widely used in plant genetic engineering and molecular breeding. This technology utilizes the natural gene transfer capabilities of Agrobacterium to introduce target genes into plant cells and integrate them into the plant genome, thereby achieving genetic modification.

[0005] The challenges in establishing a genetic transformation system for *Lysimachia foetida* lie in overcoming genotype limitations, plant defense mechanisms, low transformation efficiency, genotype limitations in regenerated tissues, the complexity of technology optimization, post-transformation screening and identification, and balancing issues such as high seed-borne pathogen rates and the rate of healing with contamination. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a method for constructing a genetic transformation system of *Lysimachia christinae* based on mature embryo callus induction.

[0007] This invention is achieved by a method for constructing a genetic transformation system of *Lysimachia christinae* based on mature embryo callus induction, the method comprising the following steps:

[0008] (1) Callus induction and pre-culture: The sterilized seeds were cut in half and inoculated into the induction medium. They were cultured in the dark at 25±1℃ for 2 to 3 weeks until the callus diameter reached 0.5cm. The uncontaminated callus was screened and transferred to the pre-culture medium for 2 days.

[0009] (2) Agrobacterium infection and co-culture: The callus tissue was cut into small pieces of 3±0.5mm, immersed in Agrobacterium suspension, and cultured in a shaker at 28℃ and 100rpm for 15 minutes. Then, it was heat-shocked in a water bath at 42℃ for 5 minutes, and then left to stand for 10 minutes. The callus tissue was then removed, dried, and transferred to co-culture medium. It was co-cultured in the dark at 25℃ for 3 days.

[0010] (3) Recovery culture: After 3 days of co-culture, the callus tissue was transferred to recovery culture medium for one week of recovery culture;

[0011] (4) Screening, differentiation and seedling culture: The resistant callus tissue was transferred to the stem induction medium and cultured at 25℃ under 16 hours of light / 8 hours of darkness for 2 to 3 weeks to induce the differentiation of 2 to 3 cm seedlings. Then, it was transferred to the root induction medium and cultured until the root length reached 5 cm.

[0012] (5) Hardening off and transplanting: Harden the rooted seedlings in closed bottles for 2 days, then open the bottles after adding tap water for 3 days, wash off the culture medium from the roots, and transplant them into sterilized vermiculite nutrient soil substrate to obtain complete transgenic regenerated plants.

[0013] Preferably, in step (1), the seed disinfection process is as follows: soak the seeds of *Lysimachia foetida* in 50% H2SO4 for 30 minutes, rinse thoroughly with ultrapure water, soak in 75% C2H5OH for 5 minutes, rinse thoroughly with ultrapure water, soak in 5% NaClO for 10 minutes, rinse thoroughly with ultrapure water, rinse 5-8 times with sterile water, and place them on sterile filter paper for later use.

[0014] Preferably, the callus induction medium is: basal medium + 1 mg / L 2,4D + 0.05 mg / L 6BA + 0.6 mg / L CuSO4, pH = 5.8–6.0;

[0015] The pre-culture medium was: callus induction medium + 200 μM acetylsyringone, pH = 5.8–6.0;

[0016] The OD of the Agrobacterium suspension 600 =0.4~0.6, and contains 200μM acetylsyleugenone;

[0017] The recovery culture medium was: callus induction medium + 200 mg / L cephalosporin + 200 mg / L termethin, pH = 5.8–6.0;

[0018] The root induction medium was: 1 / 2 MS basal medium + plasmid-resistant antibiotic, pH=5.8.

[0019] Preferably, the basal culture medium is: 4.33 g / L MS salts + 30 g / L sucrose + 3.5 g / L plant gel, pH = 5.8-6.0, autoclaved at 115°C for 30 minutes.

[0020] Preferably, the preparation process of the Agrobacterium suspension is as follows: Agrobacterium glycerol culture is inoculated into LB medium containing Rif and plasmid-resistant antibiotics, and cultured at 28°C for 36 hours. After that, the Agrobacterium is scraped off and resuspended in an infection medium containing 200 μM acetylsyleugenone, and the OD is adjusted. 600 Up to 0.4 to 0.6.

[0021] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects:

[0022] (1) By optimizing the process of removing and disinfecting endophytic fungi, the seed contamination rate of this invention is reduced to below 5%, and the cure rate is increased to over 75%.

[0023] (2) The present invention adopts a phased antibiotic screening strategy, which effectively reduces the false positive rate;

[0024] (3) The regeneration system of this invention is complete, with a rooting rate of over 95%, providing an efficient technical platform for gene editing and stress-resistant breeding of *Lysimachia foetida*. Attached Figure Description

[0025] Figure 1 Inducing callus tissue in mature embryos of *Lysimachia christinae*;

[0026] Figure 2 To promote the differentiation of green seedlings from callus tissue of *Drunken Horse Grass*;

[0027] Figure 3 For the rooting of the drunken horse grass;

[0028] Figure 4 These are regenerated plants of *Drunken Horse Grass* after transplanting;

[0029] Figure 5 These are the PCR test results for Cas9 transgenic plants. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1: Establishment of a genetic transformation system for *Lysimachia christinae*

[0032] 1. Removal of endophytic fungi and seed disinfection treatment

[0033] (1) Removal of endophytic fungi: Natural seeds of *Lysimachia christinae* collected from Tibet that do not carry endophytic fungi (confirmed by PCR testing to be free of endophytic fungi) were selected. (It belongs to fungal colonization), the grains are plump and free of mold. They are directly packed into kraft paper envelopes, sealed, and labeled with the species name and processing date, saving the need for high-temperature drying and inactivation steps.

[0034] (2) Seed disinfection:

[0035] Take the dried seeds and place them in a 50mL centrifuge tube. Add 30mL of 50% sulfuric acid (H2SO4) and treat with a test tube turntable mixer for 30 minutes to remove the seed coat.

[0036] Pour off the sulfuric acid and rinse vigorously with distilled water 5-8 times until no seed coat debris remains;

[0037] Soak in 75% ethanol (C2H5OH) for 5 minutes, then soak in 5% sodium hypochlorite (NaClO) for 10 minutes.

[0038] Transfer to a clean bench and rinse with sterile water 5-8 times, 1 minute each time;

[0039] Place the seeds on sterile filter paper and let them air dry for later use.

[0040] 2. Callus induction and pre-culture

[0041] (1) Embryo treatment and inoculation: The seeds were cut longitudinally with a sterile scalpel to destroy the endosperm and then inoculated into callus induction medium (basal medium + 1 mg / L 2,4D + 0.05 mg / L 6BA + 0.6 mg / L CuSO4, pH 5.8-6.0; the basal medium was: 4.33 g / L MS salts + 30 g / L sucrose + 3.5 g / L plant gel, pH 5.8-6.0, autoclaved at 115℃ for 30 minutes).

[0042] (2) Culture conditions: Culture in the dark at 25±2℃ for 2 to 3 weeks. When the callus diameter reaches 0.5cm, remove the contaminated tissue and cut the callus into small pieces of about 3mm. Transfer to the pre-culture medium (formula is the same as the induction medium + 200μM acetylsyl syringone) and continue to culture for 2 days.

[0043] 3. Agrobacterium infection and co-culture

[0044] (1) Preparation of Agrobacterium suspension:

[0045] Agrobacterium glycerol culture was inoculated into LB solid medium containing Rif (20 μg / mL) and Kan (100 μg / mL) (plasmid resistance) and incubated at 28°C for 36 hours;

[0046] Scrape the bacterial body and resuspend it in the infection medium (containing 200 μM acetylsuccine), and adjust the OD. 600 Up to 0.4 to 0.6.

[0047] (2) Infection procedure:

[0048] Callus tissue pre-cultured for 24 hours was transferred to a suspension containing Agrobacterium (OD600 = 0.4-0.5), and infected by shaking at 100 rpm in a constant temperature shaker at 28°C for 15 minutes, followed by heat shock in a water bath at 42°C for 5 minutes, and then left to stand in a clean bench for 10 minutes to promote full contact between Agrobacterium and plant tissue.

[0049] Remove the callus tissue and transfer it to a clean bench. Place the callus tissue on sterile filter paper, dry the surface moisture, and then transfer it to a co-culture medium (formula same as pre-culture medium) with a layer of sterile filter paper on it. Co-culture at 25°C in the dark for 3 days.

[0050] If Agrobacterium contamination (such as bacterial film or turbid secretions) is observed on the culture medium or callus surface after 24 hours of co-culturing, the uncontaminated callus should be immediately transferred to sterile filter paper without culture medium for further culture.

[0051] 4. Restore culture

[0052] (1) Recovery: The callus tissue was transferred to recovery culture medium (containing 200 mg / L Cef + 200 mg / L Termetine) and cultured for one week.

[0053] 5. Screening, Differentiation, and Seedling Culture

[0054] (1) Stem induction: resistant callus tissue after screening ( Figure 1 The cells were transferred to stem induction medium (basal medium + 3 mg / L 6BA + 0.5 mg / L NAA + 0.6 mg / L CuSO4 + plasmid-resistant antibiotic) and cultured at 25°C under 16 hours of light / 8 hours of darkness for 2 weeks until seedlings of 2-3 cm differentiated. Figure 2 ).

[0055] (2) Root induction: The seedlings were transferred to root induction medium (1 / 2 MS salts + 30 g / L sucrose + plasmid-resistant antibiotics) and cultured until the root length reached 5 cm. Figure 3 ).

[0056] 6. Hardening off seedlings and transplanting

[0057] (1) Hardening treatment: Harden the rooted seedlings in closed bottles for 2 days, then open the bottles after adding tap water for 3 days.

[0058] (2) Transplanting: Wash the roots of the culture medium, transplant them into a sterile substrate (vermiculite: nutrient soil = 1:1), cover with a transparent plastic cover to retain moisture, and remove the cover after the plants have stabilized to obtain transgenic regenerated plants. Figure 4 ).

[0059] 7. Testing and Verification

[0060] (1) PCR detection: DNA was extracted and PCR amplification was performed using GFP and Cas9 gene-specific primers to verify the integration of the target gene.

[0061] (2) Sequencing detection: Sanger sequencing was used to verify the integration of the target gene.

[0062] 7. Results

[0063] (1) Pollution control: After seed disinfection, the pollution rate is reduced to below 5%, and the germination rate is increased to over 75%;

[0064] (2) Conversion efficiency: Shaking culture assisted infection significantly improved the conversion efficiency to 20.3%;

[0065] (3) Regeneration rate: The regeneration rate reaches 21%, the rooting rate reaches over 95%, and the survival rate of transplanted whole plants exceeds 90%;

[0066] (4) Test results: such as Figure 5 As shown in the figure (1# to 8# are some of the identified transgenic seedlings numbered 1 to 8), both PCR and sequencing results confirmed the successful integration of the Cas9 gene.

[0067] Example 2: Optimization and Validation of Different Disinfection Combinations

[0068] 1. The steps in this embodiment are the same as those in Embodiment 1, except that the seed disinfection step is adjusted as follows:

[0069] Disinfection combination: 30 minutes 50% H2SO4 + 5 minutes 75% C2H5OH + 12 minutes 5% NaClO.

[0070] 2. Results: The contamination rate decreased to 2%–5%, and the initial healing rate stabilized at 75%–85%.

[0071] Because *Lysimachia christinae* is symbiotic with endophytic fungi, it is easily contaminated by fungi. The results of this embodiment show that the seed disinfection step provided by the present invention can enable callus tissue to achieve a high initial healing rate with a low contamination rate.

[0072] Example 3: Phased antibiotic screening validation

[0073] This embodiment follows the same steps as Example 1, except that the steps for screening differentiation and seedling culture are adjusted as follows: gradient antibiotic concentrations are used (3 mg / L gradually reduced to 2 mg / L). The results show that the false positive rate is reduced to below 0.11%, and stable transformed callus tissue is effectively screened.

[0074] This example demonstrates that higher antibiotic concentrations result in slower growth rates, but can accelerate the growth of transgenic seedlings when positive seedlings are effectively screened.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a genetic transformation system of Achnatherum inebrium based on mature embryo callus induction, characterized in that, The method comprises the following steps: (1) callus induction and pre-culture: the sterilized seeds are cut in half, inoculated on callus induction medium, and cultured at 25±1°C in dark conditions for 2-3 weeks until the callus diameter reaches 0.5 cm, and the uncontaminated callus is transferred to pre-culture medium for 2 days of culture; (2) Agrobacterium infection and co-culture: the callus is cut into small pieces of 3±0.5 mm, immersed in an Agrobacterium suspension, and subjected to 15 minutes of shaking culture at 28°C and 100 rpm, followed by 5 minutes of 42°C water bath heat shock, 10 minutes of standing, and then the callus is taken out, dried, and transferred to co-culture medium for 3 days of co-culture at 25°C in dark; (3) recovery culture: after 3 days of co-culture, the callus is transferred to recovery culture medium for one week of recovery culture; (4) selection and differentiation and seedling culture: the resistant callus is transferred to stem induction medium, and cultured at 25°C under 16 hours of light / 8 hours of dark for 2-3 weeks to promote the differentiation of 2-3 cm seedlings, and then transferred to root induction medium for culture until the root length reaches 5 cm; (5) hardening and transplanting: the rooted seedlings are hardening in a closed bottle for 2 days, hardening in an open bottle for 3 days after injection of tap water, washed with root medium, and transplanted into a sterile vermiculite nutrient soil matrix to obtain complete transgenic regenerated plants.

2. The method of claim 1, wherein, In step (1), the seed sterilization process of the seeds is as follows: the seeds are soaked in 50% H2SO4 for 30 minutes, washed with ultrapure water, soaked in 75% C2H5OH for 5 minutes, washed with ultrapure water, soaked in 5% NaClO for 10 minutes, washed with ultrapure water, washed with sterile water for 5-8 times, and placed on sterile filter paper for standby.

3. The method of claim 1, wherein, The callus induction medium is: basic medium + 1 mg / L 2,4D + 0.05 mg / L 6BA + 0.6 mg / L CuSO4, pH = 5.8-6.0; The pre-culture medium is: callus induction medium + 200 μM acetosyringone, pH = 5.8-6.0; OD of the Agrobacterium suspension 600 = 0.4-0.6 and contains 200 μM acetosyringone; The recovery culture medium is: callus induction medium + 200 mg / L cefotaxime + 200 mg / L trimethoprim, pH = 5.8-6.0; The root induction medium is: 1 / 2MS basic medium + plasmid resistance antibiotics, pH = 5.

8.

4. The method of claim 3, wherein, The basic medium is: 4.33 g / L MS salts + 30 g / L sucrose + 3.5 g / L plant gel, pH = 5.8-6.0, 115°C high-pressure sterilization for 30 minutes.

5. The method of claim 1, wherein, The preparation process of the Agrobacterium suspension is as follows: Agrobacterium glycerol bacteria liquid is inoculated into LB medium containing Rif and plasmid resistance antibiotic, and cultured at 28°C for 36 hours. Then, the Agrobacterium is scraped and resuspended in an infection medium containing 200 μM acetosyringone, and the OD is adjusted to 0.4-0.

6. 600 0.4-0.6.

Images