Method for culturing resistant callus by momordica grosvenori pulp

Abstract

This invention relates to a method for cultivating resistant callus from monk fruit pulp, belonging to the field of plant biotechnology and genetic transformation technology. Addressing the problems of low genetic transformation efficiency and limited explant sources in existing monk fruit methods, the provided method includes: collecting monk fruit pulp tissue 50 to 60 days after pollination, inoculating it onto a callus induction medium containing atrazine and thiamethoxam to obtain callus tissue; and immersing the callus tissue in Agrobacterium tumefaciens bacterial solution for infection, wherein the OD of the bacterial solution is... 600 The concentration was 0.5 to 0.8, containing 100 to 200 μmol / L acetylsyringone, and the inoculation time was 15 to 25 minutes; after inoculation, the cells were co-cultured for 2 to 4 days; then transferred to a selection medium containing selection and antibacterial agents to screen and obtain resistant callus tissue. The genetic transformation system established in this invention uses monk fruit pulp as explant, which is simple to operate and has high transformation efficiency. It can be used for the verification of functional genes, genetic improvement, and research on the regulation of glycoside metabolism in monk fruit.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and genetic transformation technology, specifically relating to a method for culturing resistant callus tissue from monk fruit pulp. Background Technology

[0002] Monk fruit ( Siraitia grosvenorii Mogroscin, a plant in the Cucurbitaceae family, is both a food and a medicinal herb. Its fruit is rich in mogroscin V, which is characterized by high sweetness and low calories, making it valuable for the development of natural sweeteners. However, mogroscin V accumulates only in the mesocarp tissue of the fruit, and traditional breeding methods are time-consuming and inefficient, making it difficult to meet the need for rapid breeding of new varieties with high mogroscin V content. Therefore, establishing a stable and efficient genetic transformation system is necessary to verify the function and target the regulation of mogroscin V metabolism-related genes through genetic transformation.

[0003] Currently, there are some reports on the genetic transformation of monk fruit. Existing technical literature: Li Wenlan, Gao Yonghua, Li Huaying. Establishment of a genetic transformation system for monk fruit [J]. Molecular Plant Breeding, 2010, 8(05): 997-1002. In the published scheme, monk fruit leaves were used as explants to establish an Agrobacterium-mediated genetic transformation system, and the transformation frequency obtained by PCR detection was 6.86%. Existing technical literature: Li Wenlan, Li Jingjian, Li Huaying. Study on the transformation of monk fruit with the Agrobacterium-mediated acquired resistance regulatory gene (NPR1) [J]. Shanghai Journal of Agricultural Sciences, 2010, 26(04): 15-19. In the published scheme, cotyledons were used as explants, and the transformation efficiency obtained was 1.5%. In addition, existing technical literature: Zeng Lihui, Wu Jinshou, Ke Shishan, et al. Establishment of genetic transformation recipient regeneration system of Luo Han Guo and preliminary study on Agrobacterium rhizogenes transformation [J]. Chinese Agricultural Science Bulletin, 2005, (12): 403-406. The published scheme studied the establishment of genetic transformation recipient regeneration system using test-tube seedling stem segments and leaves as explants. The results showed that the regeneration ability of stem segments was stronger than that of leaves and that they were sensitive to Agrobacterium. And other existing technical literature: Zhou Qiong, Hu Shanshan, Hao Qinglin, et al. Study on transformation of Luo Han Guo with parthenocarpy gene 2A11-iaaM [J]. Guangxi Plants, 2018, 38(12): 1614-1625. The published scheme studied the establishment of genetic transformation system using the leaf disc of female Luo Han Guo as explant and Agrobacterium-mediated transformation. A total of 4 transgenic positive female plants were obtained, but the overall transformation efficiency of this system was not reported.

[0004] Despite the aforementioned research, the following technical deficiencies still exist in the field of Luo Han Guo genetic transformation.

[0005] First, the source of explants is limited to vegetative organs. Reported genetic transformation studies have primarily used explants from vegetative organs such as cotyledons, stem segments, leaves, or leaf discs; a genetic transformation system using mesocarp tissue, which is crucial for the synthesis and accumulation of glycoside V, as explants has not yet been established. Using mesocarp tissue as explants faces technical obstacles in tissue culture, such as easy browning and difficulty in callus induction, leading those skilled in the art to avoid this source for a long time. Therefore, existing systems are difficult to directly apply to the verification of functional genes related to the metabolic regulation of glycoside V.

[0006] Second, the hormone combination for callus induction is singular. Existing technical literature: Mo Haiping, Li Bolin, Su Yuqing, et al. Induction of embryogenic callus and plant regeneration of Siraitia grosvenorii [J]. Guangdong Agricultural Sciences, 2014, 41(12):33-36+2. The published Siraitia grosvenorii tissue culture studies mainly use the combination of 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), with cotyledons as explants. Different ratios of 6-BA to 2,4-D or NAA can induce callus formation. The optimal culture medium is MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L. The obtained callus is pale yellow or yellow, loosely structured, and granular. Other existing technical literature: Zou Qili, Lin Rong. Formation of callus and axillary shoots in the tissue culture of *Siraitia grosvenorii* [J]. *Guangxi Plants*, 1989, (02): 103-104+194. The published scheme indicates that callus can be induced by using *Siraitia grosvenorii* stem segments as explants, but the frequency of redifferentiation to form buds is extremely low. Thiadiazon (TDZ) has been used in *Siraitia grosvenorii* for the adventitious bud differentiation stage, not for the callus induction stage. None of the above existing technical issues involve the application of picrotoxin (PIC) in the callus induction of *Siraitia grosvenorii*. Due to the differences in physiological characteristics between pulp tissue and vegetative organs, it is still unknown whether existing hormone combinations can induce suitable pulp callus for transformation.

[0007] Third, the genetic transformation efficiency is low, and there is a lack of research on the problem of transformation efficiency decay during long-term subculture. In existing studies on the genetic transformation of *Siraitia grosvenorii*, the highest transformation efficiency based on PCR detection was 6.86%, and these studies all used callus tissue from the first generation or limited subculture. In plant tissue culture, long-term subculture often leads to loss of embryogenicity and decreased regeneration capacity of callus tissue. However, in the field of *Siraitia grosvenorii*, there is a lack of systematic research on how the number of subcultures affects the genetic transformation efficiency of callus tissue, and no corresponding technical means to maintain transformation activity have been proposed. Summary of the Invention

[0008] One objective of this invention is to address the problem that existing Luo Han Guo genetic transformation systems primarily use explants derived from vegetative organs such as cotyledons, stem segments, and leaves, and have not yet established a genetic transformation method using fruit pulp as explants. This invention provides a genetic transformation method using Luo Han Guo fruit pulp as the starting material.

[0009] To achieve the above objectives, the present invention provides a method for culturing resistant callus tissue from monk fruit pulp, comprising the following steps: Step 1: Take the fruit of the monk fruit 50 to 60 days after pollination and extract the pulp tissue; Step 2: Inoculate the pulp tissue onto a callus induction medium containing thiamethoxam and thiamethoxam, wherein the concentration of thiamethoxam is 1.0 to 4.0 mg / L and the concentration of thiamethoxam is 0.3 to 0.8 mg / L, and culture to obtain callus tissue. Step 3: Immerse the callus tissue from Step 2 in Agrobacterium-mediated bacterial solution. The OD of the Agrobacterium-mediated bacterial solution... 600 The value is 0.5 to 0.8, and the Agrobacterium bacterial suspension contains 100 to 200 μmol / L acetylsylgenone, and the infection time is 15 to 25 minutes; Step 4: Culture the infected callus tissue for 2 to 4 days. Step 5: Transfer the co-cultured callus tissue into a screening medium containing screening agents and antibacterial agents to screen and obtain resistant callus tissue.

[0010] Preferably, in step 2, the concentration of atrazine is 2.0 to 3.0 mg / L and the concentration of thiabendazole is 0.4 to 0.6 mg / L.

[0011] Preferably, in step 3, OD 600 The value was 0.6, the acetylsyl syringone concentration was 150 μmol / L, and the infection time was 20 minutes; the co-culture time in step 4 was 3 days.

[0012] Preferably, the screening agent in step 5 is hygromycin at a concentration of 40 to 60 mg / L; the antibacterial agent is ticarcillin-clavulanate potassium at a concentration of 200 to 400 mg / L.

[0013] Preferably, the concentration of hygromycin is 50 mg / L and the concentration of ticarcillin-clavulanate potassium is 300 mg / L.

[0014] Preferably, the screening in step 5 is carried out in 2 to 6 rounds.

[0015] Preferably, the callus tissue in step 2 is a green, compact type of callus tissue.

[0016] Preferably, in step 2, the concentration of atrazine is 2.5 mg / L and the concentration of thiamethoxam is 0.5 mg / L; in step 3, OD 600The value was 0.6, the concentration of acetylsuccinone was 150 μmol / L, and the infection time was 20 minutes; the co-culture time in step 4 was 3 days; in step 5, the screening agent was hygromycin with a concentration of 50 mg / L, and the antibacterial agent was ticarcillin-clavulanate potassium with a concentration of 300 mg / L.

[0017] Preferably, the subculture process in step 2 further includes the following steps to maintain the high transformation activity of the resistant callus: a) Before each subculture, the callus tissue was subjected to low-temperature treatment at 4℃±1℃ for 24 to 48 hours. Then, the surface part that still retains a bright green color and firm texture was transferred to fresh culture medium, and the yellowish-white, loose or browned central part was discarded. b) After three subcultures, before each subculture, the callus tissue was pre-cultured in MS medium containing 10 to 20 mg / L hygromycin for 5 to 8 days, and then transferred to MS medium without hygromycin and supplemented with 0.1 to 0.3 mg / L abscisic acid, and cultured in the dark at 25±2℃ for 7 to 10 days for recovery culture. c) After the recovery culture is completed, cut the callus particles into 2 to 4 small pieces with a diameter of 0.1 to 0.2 cm, soak them in an Evans blue aqueous solution with a mass-volume ratio of 0.01% to 0.05% for 2 to 5 minutes, pour off the staining solution and rinse with sterile water 3 to 5 times, remove the blue-stained pieces, retain the unstained or only slightly stained pieces and combine them for subsequent subculture or steps 3 to 5.

[0018] The present invention has at least the following beneficial effects: 1. This invention uses the pulp tissue of *Siraitia grosvenorii* fruit 50 to 60 days after pollination as explants. The pulp is the direct site of synthesis and accumulation of mogroitin V. Using it as the starting material, the obtained resistant callus tissue retains the cellular background of mogroitin metabolism and can be directly used for functional verification of genes related to mogroitin V metabolism regulation, eliminating the need for the lengthy process of regenerating plants from vegetative organs and then detecting changes in pulp metabolism. Compared with existing technologies that use cotyledons, leaves, and other vegetative organs as explants, the explant selection in this invention is closer to the actual needs of mogroitin metabolism research.

[0019] 2. This invention uses a combination of picrotoxin (PIC) and thiamethoxam (TDZ) in the callus induction stage, and determines the concentration range of PIC from 1.0 to 4.0 mg / L and TDZ from 0.3 to 0.8 mg / L. Experimental verification shows that under the conditions of PIC 2.5 mg / L + TDZ 0.5 mg / L, the callus induction rate of fruit pulp explants reaches 91.7%, the browning rate is only 5.0%, and the induced callus tissue is green and compact. Compared with the loose, pale yellow callus induced by the 6-BA+NAA combination technique, the compact callus tissue induced by this invention shows higher transformation efficiency and survival rate in subsequent Agrobacterium infection and screening. PIC, as a synthetic auxin, can effectively induce dedifferentiation of fruit pulp cells and maintain embryogenic state, while TDZ, as a highly active cytokinin analog, works synergistically with PIC to regulate the balance of cell division and differentiation, reducing browning.

[0020] 3. This invention uses response surface methodology to determine the concentration (OD) of Agrobacterium tumefaciens bacterial culture. 600 The concentration of acetylsuccinone (AS), infection time, and co-culture time were systematically optimized, and the OD was determined. 600 The optimal parameter combination was determined to be 0.6 μmol / L, AS = 150 μmol / L, infection time 20 minutes, and co-culture time 3 days. Under these conditions, the resistant callus acquisition rate reached 59.6%, significantly higher than the 21.3% under the unoptimized conditions. Contour plot analysis revealed that OD... 600 There is a negative interaction between the bacterial culture concentration and the AS concentration. When the bacterial culture concentration is low, a higher AS concentration is required to induce the expression of the virulence gene (Vir gene), while when the bacterial culture concentration is high, a lower AS concentration can achieve a similar transformation efficiency. This discovery allows the transformation parameters to be adjusted according to the actual situation, improving the adaptability of the system.

[0021] 4. This invention determined the screening concentrations of hygromycin (50 mg / L) and timentin (300 mg / L). Experiments showed that 50 mg / L hygromycin significantly inhibited the growth of untransformed fruit pulp callus, while 300 mg / L Timentin effectively inhibited Agrobacterium growth without significant toxicity to callus. Compared with the commonly used antibacterial agent cefotaxime sodium, Timentin showed less inhibitory effect on fruit pulp callus and a higher survival rate of resistant callus. After four rounds of screening, the GFP positivity rate was 83.33%, and the GUS positivity rate was 89.0%, indicating that this screening system can effectively enrich transformed cells and obtain homozygous resistant callus.

[0022] 5. This invention introduces several steps into subculture, including low-temperature pretreatment, low-concentration hygromycin preculture, recovery culture containing abscisic acid, and microdissection combined with Evans blue staining for activity screening. Experimental verification shows that after using these steps, the rate of resistant callus acquisition from *Siraitia grosvenorii* pulp callus remained at 49.8% even after subculture to the 12th generation, while conventional subculture reduced it to 31.2% by the 6th generation and below 10% by the 8th generation. The effective number of generations extended from approximately 5 to over 12. Analysis of the effects of each step showed that: low-temperature pretreatment at 4℃ for 36 hours actively reduced the metabolic activity of low-viability cells, making them more easily eliminated in subsequent hygromycin screening; pre-culture with a low concentration of hygromycin (15 mg / L) for 6 days further inhibited the proliferation of non-embryonic cells; the addition of 0.2 mg / L abscisic acid to the recovery culture induced the expression of cellular stress-resistant proteins, enhancing the callus's tolerance to transformation stress; the cutting treatment broke down the heterogeneity within the callus, allowing Evans blue staining to more accurately identify cells with membrane damage; finally, Evans blue staining screening, using cell membrane integrity as an indicator, objectively removed dead cells, avoiding subjective errors from visual judgment. The synergistic effect of these steps reduced the batch-to-batch coefficient of variation from over 15% in conventional subculture to below 5%.

[0023] Based on the above technical methods, the genetic transformation system for monk fruit pulp callus established in this invention achieves a resistant callus acquisition rate of approximately 59.6%, a GFP positivity rate of approximately 83.33%, and a GUS positivity rate of approximately 89.0%. Compared with the existing technology disclosed in: Li Wenlan, Gao Yonghua, Li Huaying. Establishment of a genetic transformation system for monk fruit [J]. Molecular Plant Breeding, 2010, 8(05):997-1002., this invention achieves a significant improvement in transformation efficiency, providing stable technical support for the verification of monk fruit functional genes, research on glycoside metabolism regulation, and molecular breeding.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 This invention relates to the selection of Luo Han Guo (monk fruit) pulp explants and the process of callus induction. Figure 2 The inhibitory effect of different concentrations of hygromycin on the growth of fruit pulp callus tissue is shown in this invention. Figure 3 The inhibitory effects of different concentrations of ticarcillin-clavulanate potassium on Agrobacterium tumefaciens are shown in this invention. Figure 4 The results of GFP fluorescence detection and GUS staining identification of the transformed positive monk fruit pulp callus tissue of this invention; Figure 5The above are contour plots and 3D surface plots of the genetic transformation parameters optimized by the response surface methodology in Embodiment 5 of the present invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description. It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials described are commercially available unless otherwise specified. It should be understood that terms such as "having," "comprising," and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] Example 1 like Figure 1 As shown, monk fruit was selected 55 days after pollination. The tested monk fruit (Monk Fruit) Siraitia grosvenorii Artificial pollination was conducted on the plants during the peak flowering period, on a sunny morning between 8:00 and 10:00 AM, with the pollination date marked on a tag. Fruits were harvested 55 days after pollination, selecting individuals with intact appearance, no mechanical damage, and free from pests and diseases. The fruit diameter was approximately 5-6 cm transversely and 6-7 cm longitudinally, with the peel changing from green to light yellowish-green. The harvested fruits were placed in a clean bench and the peel surface was wiped twice with 75% (v / v) ethanol. A sterile scalpel was used to make a circumferential incision along the equator of the fruit, breaking open the peel. The outer peel and seeds were removed using sterile forceps, exposing the pulp tissue, which was then cut into appropriately sized slices. The pulp tissue was then removed using a sterile scalpel, discarding the green portion near the peel and the spongy portion near the seeds, extracting a pulp approximately 1-1.5 cm thick. The pulp was cut into small pieces of approximately 0.5 cm × 0.5 cm × 0.5 cm, each weighing approximately 80 to 100 mg, to obtain monk fruit pulp explants. The cut monk fruit pulp explants were immediately placed in sterile covered petri dishes, and surface exudate was blotted away with sterile filter paper. The time from fruit cutting to inoculation should be controlled within 30 minutes to reduce browning.

[0028] Preparation of callus induction medium (CIM): MS basal medium 4.43 g / L, supplemented with 30 g / L sucrose and 7 g / L agar, adjusted pH to 5.8±0.05, autoclaved at 121℃ for 20 minutes. Toxaphene (PIC) stock solution: Weigh 25 mg and dissolve in 1 mL dimethyl sulfoxide (DMSO), bring the volume to 25 mL with sterile water to prepare a 1 mg / mL solution, filter sterilize, and store at -20℃. Thiadiazon (TDZ) stock solution: Weigh 12.5 mg and dissolve in 1 mL 1 M NaOH, bring the volume to 25 mL with sterile water to prepare a 0.5 mg / mL solution, filter sterilize, and store at -20℃. After sterilization, when the culture medium is cooled to 50 to 55°C, add 2.5 mL / L of PIC stock solution to the final concentration of PIC in a laminar flow hood, so that the final concentration of PIC is 2.5 mg / L, and add 1.0 mL / L of TDZ stock solution to the final concentration of TDZ is 0.5 mg / L. Mix thoroughly and pour into sterile disposable culture dishes, about 20 mL per dish.

[0029] In a clean bench, cut explants were picked up with sterile forceps and placed on callus induction medium for tissue culture. The edges of the culture dishes were sealed with Parafilm and incubated in a constant temperature incubator at 25±2℃ in complete darkness. After 7 to 10 days of culture, the explants began to swell; small white callus dots appeared at the edges of the explants at 14 to 18 days; and callus growth was evident at 25 to 30 days. After approximately 30 days of culture, green, compact callus formed at the edges of the explants, distinguishing it from the yellowish original explant tissue. Under a stereomicroscope, the callus cells were small and dense, with large nuclei and thick cytoplasm. The callus induction rate was calculated as (number of explants forming callus / total number of inoculated explants) × 100%. The average induction rate for three independent replicate experiments with ≥30 explants per inoculation was 91.7% ± 2.1%. In a clean bench, sterile forceps and a scalpel were used to separate newly formed callus from the explants, removing any remaining explant tissue and browned portions. The callus was cut into small pieces of approximately 0.3 cm × 0.3 cm and transferred to freshly prepared subculture medium of the same composition. Subculture was carried out at 25 ± 2°C in the dark, with subcultures every 20 to 25 days. After 2 to 3 rounds of subculture, a large amount of uniform, firm, green callus was obtained for subsequent infection and transformation.

[0030] Example 2 To establish a suitable screening system, the sensitivity of callus to hygromycin and the sensitivity of Agrobacterium to ticarcillin-clavulanate potassium were determined.

[0031] Hygromycin B stock solution: Weigh 0.5 g and dissolve in 10 mL of sterile water to prepare a 50 mg / mL solution. Filter sterilize and store at 4°C in the dark. Add the hygromycin stock solution to MS basal medium containing 30 g / L sucrose, 7 g / L agar, and pH 5.8 to achieve final concentrations of 0, 10, 30, and 50 mg / L, respectively. Pour into 60 mm culture dishes, 20 mL per dish. Take healthy, green, firm callus tissue from the second generation of Example 1, cut into uniform small pieces (approximately 0.3 cm × 0.3 cm, fresh weight approximately 15 mg / piece), and inoculate onto the above-mentioned medium containing different concentrations of hygromycin, 10 pieces per dish, with 3 replicates for each concentration. Incubate at 25±2°C in the dark for 14 days and observe the callus growth status as follows. Figure 2 As shown, at 0 mg / L: callus growth was uniform, maintaining a pale yellow-green color, and the volume increased by 2 to 3 times. At 10 mg / L: most callus were slightly larger than the control group, and the color was basically the same as at 0 mg / L, but some callus were smaller. At 30 mg / L: callus were uneven in size, with some being larger and some smaller, and the color was yellowish. At 50 mg / L: the size difference of callus was most obvious, with obvious coexistence of large and small callus, and the color was generally yellowish. Overall growth was severely inhibited, but some cell clusters still survived. Based on the above results, 50 mg / L hygromycin can significantly inhibit the growth of untransformed callus tissue, but a single treatment cannot completely kill all cells. Multiple rounds of screening can be combined to completely remove untransformed cells. Therefore, 50 mg / L was determined as the concentration of hygromycin used in the screening medium.

[0032] Timentin stock solution prepared by mixing ticarcillin disodium and potassium clavulanate in a 15:1 ratio: Weigh 1.0 g of Timentin and dissolve it in 10 mL of sterile water to obtain a ticarcillin concentration of 100 mg / mL. Store at -20℃. Inoculate a single colony of Agrobacterium GV3101 into 5 mL of LB liquid medium containing 50 mg / L rifampin and 50 mg / L kanamycin, and incubate overnight at 28℃ with shaking at 200 rpm until OD. 600 ≈0.8. The overnight bacterial culture was transferred at a ratio of 1:100 (v / v) to Erlenmeyer flasks containing 0, 200, 300, and 400 mg / L Timentin in LB liquid medium. Each flask had a volume of 100 mL, with 20 mL of medium in each flask. Three replicates were prepared for each concentration. After incubation at 28℃ and 200 rpm for 24 hours with shaking, the turbidity and color changes of the culture medium in each flask were observed. Results are as follows: Figure 3As shown: when Timentin is 0 mg / L, the culture medium is turbid and yellow, indicating that Agrobacterium grows in large quantities; when Timentin is 200 mg / L, the culture medium is clearer but still yellow, indicating that the growth of Agrobacterium is partially inhibited; when Timentin is 300 mg / L and 400 mg / L, the culture medium is clear and transparent with a pale yellow color, indicating that the growth of Agrobacterium is significantly inhibited.

[0033] Simultaneously, second-generation callus tissue from Example 1 was cut into 0.3 cm × 0.3 cm pieces and inoculated into MS solid medium containing different concentrations of Timentin (0, 100, 200, 300, 400, and 500 mg / L). The MS solid medium contained 2.5 mg / L PIC and 0.5 mg / L TDZ. The culture dishes were 60 mm in diameter, with 20 mL of medium per dish. Ten pieces were inoculated per dish, and three replicates were set for each concentration. The callus tissue was incubated in the dark at 25 ± 2℃ for 14 days, and the growth status of the callus tissue was observed. The results showed that there was no significant difference in the growth status of the callus tissue within the Timentin concentration range of 0 to 300 mg / L; all callus tissues maintained a bright green color and normal proliferation. Growth was slightly slowed at 400 mg / L. At 500 mg / L, the callus color turned yellow, and growth was inhibited by about 30%.

[0034] Based on the above results, 300 mg / L was selected as the concentration of Timentin, which can completely inhibit Agrobacterium without affecting the normal growth of callus tissue. Therefore, the selection medium (SM) formulation was determined to be: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 50 mg / L hygromycin + 300 mg / L Timentin, supplemented with 30 g / L sucrose and 7 g / L agar, pH 5.8.

[0035] Example 3 The Agrobacterium strain GV3101 carried an expression vector containing the hygromycin phosphotransferase gene *hpt*, the β-glucuronidase gene *gus*, and the green fluorescent protein gene *gfp*, with expression driven by the CaMV 35S promoter. The glycerol-preserved strain was retrieved from -80°C and streaked onto LB agar plates containing 50 mg / L rifampin and 50 mg / L kanamycin, and incubated upside down at 28°C for 48 hours. A single colony was picked and inoculated into 5 mL of LB liquid medium containing the same antibiotics, and cultured overnight at 28°C with shaking at 200 rpm. 2 mL of the overnight culture was transferred to 50 mL of fresh LB liquid medium and cultured with shaking until OD (occurrence limit) was reached. 600 The value is approximately 0.6 to 0.8, and the process takes about 6 to 8 hours.

[0036] Infection solution formulation: 10 mM MgCl2·6H2O (2.03 g / L); 10 mM MES (2.13 g / L morpholinoethanesulfonic acid); adjust pH to 5.6 with 1 M KOH. Add acetylsuccinone (AS) to a final concentration of 150 μmol / L before use. AS stock solution: Weigh 0.196 g and dissolve in 10 mL DMSO to prepare a 100 mmol / L solution. Filter sterilize and store at -20℃. Transfer the Agrobacterium cultured to the logarithmic growth phase to a 50 mL sterile centrifuge tube, centrifuge at 5000×g for 10 minutes at 4℃, discard the supernatant, resuspend in infection solution containing 150 μmol / L LAS, and adjust OD. 600 The bacterial suspension was adjusted to 0.60±0.02, and the resuspended bacterial solution was placed on ice for 1 hour before use.

[0037] Take the green, compact callus tissue from passages 2 to 3 of Example 1, cut it into small pieces of approximately 0.3 cm × 0.3 cm × 0.2 cm, with a fresh weight of 15 to 20 mg per piece, place them on sterile filter paper, and air dry them in a laminar flow hood for 15 to 20 minutes until the surface is slightly dry, with no visible water film but still moist. Immerse 50 to 100 pieces of the pretreated callus tissue in each batch completely in the bacterial solution for 20 minutes, gently shaking them every 5 minutes during this time. After infection, use sterile forceps to remove the callus pieces and place them on multiple layers of sterile filter paper. Gently press with another sheet of filter paper to absorb excess bacterial solution from the surface, avoiding squeezing and damaging the cells, until there is no obvious liquid reflection on the surface.

[0038] Co-culture medium (CCM) composition: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 150 μmol / L AS + 30 g / L sucrose + 7 g / L agar, pH 5.8; AS was added after sterilization and cooling to 55℃. Inoculated callus tissue blocks were evenly inoculated onto the CCM, 8 to 10 blocks per dish, sealed with Parafilm, and co-cultured at 25±2℃ in complete darkness for 3 days (72 hours ± 2 hours), avoiding movement of the culture dishes during this period.

[0039] Example 4 After co-culturing, the callus blocks were removed from the CCM, picked up with sterile forceps, and rinsed twice for 30 seconds each time in sterile water containing 300 mg / L Timentin to remove Agrobacterium bacteria adhering to the surface. They were then transferred to selection medium (SM) with the same formulation as in Example 2, with 8 to 10 blocks inoculated per dish, and cultured under fluorescent light at 25±2°C and 2000 lx, with a light cycle of 16 hours of light followed by 8 hours of darkness. Observations were performed every 7 days. After 10 to 14 days of culture, most callus tissue stopped growing and gradually browned, while a few callus blocks showed new bright green granules at their edges, indicating potential resistant callus. The resistant callus tissue was detached from the browned tissue using a sterile scalpel and transferred to fresh selection medium for a second round of selection. Subculture was performed every 14 days for a total of 4 rounds of selection. Only bright green, firm, and non-browning callus tissue was transferred in each round. After the fourth round of screening, all surviving callus tissues were able to grow stably, with uniform morphology, no browning, and no Agrobacterium contamination.

[0040] Positive identification: Resistant callus tissue blocks obtained from the fourth round of screening were subjected to GFP fluorescence detection and GUS histochemical staining. Results are as follows: Figure 4 As shown, from left to right: ① Bright-field image of resistant callus; ② GFP fluorescence detection image of callus; ③ Combined image of bright-field and GFP fluorescence superimposed to show the correspondence between fluorescence signal and callus location; ④ Bright-field image of another batch of callus used for GUS staining before staining; ⑤ GUS staining result of the corresponding callus. The results show that obvious GFP fluorescence signals can be detected in the callus, and after GUS staining, it appears blue or dark blue or even black, indicating that the obtained resistant callus is a positive transformation material. In the fourth image, the callus is pale yellow-green, which is the natural state of the callus before staining under bright-field; the true GUS positive signal is reflected in the blue staining in the fifth image.

[0041] (1) GFP fluorescence detection: The resistant callus tissue block was placed under a stereomicroscope and observed first under bright field. The callus tissue block appeared bright white. Figure 4 Bright-field image of resistant callus tissue. Switching to the GFP filter, with excitation wavelength of 470 nm and emission wavelength of 525 nm, under fluorescence field observation, some callus fragments emit bright green fluorescence, indicating expression of GFP protein, while other fragments do not glow, such as... Figure 4 The GFP fluorescence detection image of the callus tissue is shown. Overlaying the bright-field image with the fluorescence image provides a clearer view of the fluorescence signal, and the number of visible luminescent patches is slightly greater after overlay than in a single-channel GFP view, as shown below. Figure 4The combined image of bright-field and GFP fluorescence superimposed is shown, which is due to the image superposition enhancing the display of weak signals. Untransformed wild-type callus was used as a negative control to calculate the GFP positivity rate. Five culture dishes were randomly selected, with 8 to 30 callus pieces per dish. The average GFP positivity rate was 83.33% ± 0.77% in three independent replicate experiments.

[0042] (2) GUS histochemical staining: Take another resistant callus tissue block for GUS staining. The GUS staining solution is prepared as follows: 10 mL of 0.5 M phosphate buffer at pH 7.0, add 0.05 g of 5-bromo-4-chloro-3-indole-β-D-glucuronic acid (X-Gluc), dissolve, then add 10 μL of 0.5 M potassium ferricyanide, 10 μL of 0.5 M potassium ferrocyanide, and 10 μL of 10% Triton X-100, and make up to 20 mL. Store in the dark. Place the resistant callus block in a 1.5 mL centrifuge tube, add 1 mL of GUS staining solution to immerse the tissue, and incubate at 37°C overnight for 12 to 16 hours. Pour off the staining solution, add 70% ethanol to decolorize, and let stand at room temperature for 2 hours, changing the ethanol 2 to 3 times during this period. Under a stereomicroscope, GUS-positive fragments in transformed callus tissue appeared dark blue or even black, while untransformed callus did not exhibit this color. Figure 4 A comparison of the bright-field images of another batch of GUS-stained callus tissue before and after staining revealed that one callus tissue stained a deep blue-black color, while the others remained unstained. A statistical analysis of 50 resistant callus tissues, with three replicates, showed an average GUS positivity rate of 89.0% ± 1.2%. Under these optimized conditions, the resistant callus acquisition rate was approximately 59.6%.

[0043] Example 5 The Box-Behnken design in response surface methodology was used to study the concentration (OD) of Agrobacterium tumefaciens in the culture. 600 The optimization was performed using four factors: Agrobacterium tumefaciens concentration (μmol / L, AS), infection time (minutes, Time), and co-culture time (days). The levels for each factor were set as follows: Agrobacterium tumefaciens concentration: 0.4, 0.6, 0.8 μmol / L; infection time: 100, 150, 200 μmol / L; infection time: 10, 20, 30 minutes; co-culture time: 2, 3, 4 days. The resistant callus acquisition rate was calculated as: resistant callus number / total infected callus number × 100%. Second-generation green, compact callus tissue was prepared according to Example 1. Infection, co-culture, and four rounds of screening were performed according to the methods in Examples 3 and 4. Thirty callus tissues were used for each experimental site, aliquoted into three culture dishes, with ten tissues per dish. All experimental sites were replicated three times. The combinations of factors were executed according to a Box-Behnken design, with a total of 30 experiments, including center point replicates.

[0044] The experimental results were analyzed using Design-Expert 12 software through multiple regression analysis, yielding a quadratic regression equation between the resistant callus acquisition rate (Y, %) and the coded values ​​of each factor: Y=59.57+3.42A+2.85B+1.96C+1.23D-1.85AB-0.95AC-0.68AD-1.12BC-0.45BD-0.32CD-4.21A 2 -3.58B 2 -2.67C 2 -2.01D 2 .

[0045] Analysis of variance results showed that the model's F-value was 18.35, and the P-value was less than 0.0001, indicating that the model was highly significant; the lack-of-fit term had a P-value of 0.082, which was greater than 0.05, and was not significant, indicating that the model fit was good. In the single-factor influence, OD... 600 The linear term coefficients for AS concentrations were the largest and most significant, with p-values ​​all less than 0.005. The negative coefficients of the squared terms indicate the existence of an optimal range. OD 600 The interaction term between AS concentration and AS concentration was significant, with a P-value of 0.031, indicating a negative interaction between the two.

[0046] To visually demonstrate the interactions between the various factors, six sets of contour plots and corresponding 3D surface plots were created, such as... Figure 5 As shown in the figure, the horizontal and vertical axes represent two factors, respectively. The color of the contour lines changes from green to red, indicating that the conversion efficiency increases from low to high. The shape of the contour lines reflects the strength of the interaction, with ellipses indicating a significant interaction and circles indicating a weaker interaction.

[0047] Figure 5 a1 examines the relationship between Agrobacterium concentration and infection duration, where the horizontal axis represents OD. 600 The vertical axis represents the infection time. The results show that when OD 600 When the concentration is below 0.6 and the infection time is less than 20 minutes, the conversion efficiency increases rapidly with the increase of both factors; when the OD... 600 Efficiency decreases when the concentration is above 0.6 and the incubation time exceeds 20 minutes. The optimal combination is OD. 600 The value is approximately 0.6, and the infection time is approximately 20 minutes.

[0048] Figure 5 a2 investigated the interaction between Agrobacterium concentration and co-culture duration, with the x-axis representing OD. 600 The vertical axis represents the number of days of co-culture. Co-culture time shorter than 3 days results in insufficient T-DNA transfer, while co-culture time longer than 3 days leads to Agrobacterium overgrowth and inhibition of callus activity. The optimal match is OD. 600The value is approximately 0.6, and the total culture time is approximately 3 days.

[0049] Figure 5 a3 examines the pairing of Agrobacterium concentration and AS concentration, with the x-axis representing OD. 600 The vertical axis represents AS concentration. The contour lines are distinctly elliptical and distorted along the diagonal, indicating a significant negative interaction between the two. When OD 600 At a lower concentration (0.4), a higher AS concentration (150 to 200 μmol / L) is required to achieve higher efficiency; when OD... 600 For a higher concentration (0.8), a lower AS concentration (100-120 μmol / L) is acceptable. The optimal combination is OD. 600 It equals 0.6, and the AS concentration equals 150 μmol / L.

[0050] Figure 5 The a4 plot examines the synergistic effect of infection time and co-culture time, with the x-axis representing infection time and the y-axis representing the number of days of co-culture. Insufficient infection time leads to incomplete adsorption of Agrobacterium, which cannot be compensated for even with extended co-culture; conversely, too short a co-culture time prevents T-DNA transfer. The optimal combination is 20 minutes of infection and 3 days of co-culture.

[0051] Figure 5 The a5 plot examines the interaction between infection duration and AS concentration, with the x-axis representing infection time and the y-axis representing AS concentration. The contour lines show a distinct ellipse, indicating a significant interaction. When the infection time is short (10 minutes), a higher AS concentration (greater than 150 μmol / L) is required to compensate; when the infection time is moderate (20 minutes), an AS concentration of 150 μmol / L achieves optimal efficiency. The optimal combination is 20 minutes of infection and an AS concentration of 150 μmol / L.

[0052] Figure 5 The a6 plot investigated the interaction between co-culture duration and AS concentration, with the x-axis representing the number of co-culture days and the y-axis representing the AS concentration. AS-induced activation of Agrobacterium virulence genes requires sufficient co-culture time to complete T-DNA transfer. The optimal combination was 3 days of co-culture and an AS concentration of 150 μmol / L.

[0053] According to the regression model prediction, the optimal condition is: OD 600 =0.62, AS=152 μmol / L, infection time=20.5 minutes, co-culture time=3.1 days. For ease of practical operation, rounded to the nearest whole number: OD 600 =0.6, AS=150 μmol / L, infection time=20 minutes, co-culture time=3 days. Under these conditions, the model predicted a resistant callus acquisition rate of 59.57%.

[0054] Three independent validation experiments were conducted under the above rounding conditions, each using 30 callus tissues. The actual resistant callus acquisition rates were 58.3%, 60.0%, and 59.3%, respectively, with an average of 59.2% ± 0.9%, which highly matched the model predictions. For comparison, at OD... 600 Under unoptimized conditions of 0.4 μmol / L, AS = 100 μmol / L, infection time 10 minutes, and co-culture time 4 days, the average resistant callus acquisition rate was only 21.3% ± 2.5% after 3 transformations using the same method.

[0055] Therefore, the optimal genetic transformation parameter was determined to be: Agrobacterium tumefaciens bacterial concentration OD 600 =0.6, acetylsyringone concentration 150 μmol / L, infection time 20 minutes, co-culture time 3 days. Under these optimized conditions, the resistant callus acquisition rate of monk fruit pulp callus was approximately 59.6%, the GFP positivity rate was approximately 83.33%, and the GUS positivity rate was approximately 89%.

[0056] Comparative Example 1 Comparative Example 1 was set up according to the conventional tissue culture method using cotyledons of sterile seedlings as explants. Cotyledons of sterile *Siraitia grosvenorii* seedlings were used as explants. The callus induction medium was MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L. The *Agrobacterium* strain was LBA4404. The infection solution OD... 600 =0.5, AS concentration 100 μmol / L, infection time 25 minutes, co-culture for 4 days, screening medium was MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L + hygromycin 20 mg / L + cefotaxime sodium 300 mg / L, positive plants were obtained by PCR detection. The average transformation efficiency of 3 independent replicate experiments was 6.86%, which is much lower than 59.57% in Example 4 of this invention.

[0057] Comparative Example 2 Comparative Example 2 used *Siraitia grosvenorii* pulp 55 days after pollination as explants, but callus induction employed the hormone combination commonly used in *Siraitia grosvenorii* research: MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L. The remaining steps, including *Agrobacterium* infection, co-culture, and screening, were the same as in Examples 3 and 4. The results showed that the callus induction rate was only 45% ± 3.5%, and the induced callus tissue was mostly water-soaked, loose, and prone to browning, with a browning rate of approximately 25%, failing to form green, compact callus. Subsequent *Agrobacterium* infection resulted in a resistant callus acquisition rate of less than 5%, which could not meet the requirements for genetic transformation.

[0058] Comparative Example 3 Comparative Example 3 used a PIC+TDZ combination, but the concentration ratio was based on the optimized ratio for *Lilium spp.*: MS+PIC 2.0 mg / L+TDZ 0.1 mg / L. The explants were still fruit pulp 55 days after pollination, and the remaining steps were the same as in Examples 3 and 4. Results: The callus induction rate was approximately 60%±4.2%, but the callus tissue grew slowly, was mostly yellowish-white and loose, with some browning, and the browning rate was approximately 15%. After *Agrobacterium* infection, the resistant callus acquisition rate was only approximately 12%±2.1%. This indicates that too low a TDZ concentration cannot induce high-quality, firm callus suitable for transformation, and the transformation efficiency is significantly lower than the 59.57% of the present invention.

[0059] Comparative Example 4 Comparative Example 4 used the optimal callus induction scheme of Example 1: PIC 2.5 mg / L + TDZ 0.5 mg / L, and obtained green and firm fruit pulp callus, but the Agrobacterium transformation parameters used conventional, unoptimized values: OD 600 =0.4, AS 100 μmol / L, infection time 10 minutes, co-culture for 4 days, the rest were the same as in Examples 3 and 4. Results: The average resistant callus acquisition rate was 21.3% ± 2.5%, significantly lower than 59.6% in Example 4. This indicates that even with high-quality callus recipient material, efficient transformation cannot be achieved if the transformation parameters are not re-optimized for the characteristics of fruit pulp callus.

[0060] To determine the optimal concentration of atrazine (PIC) for callus induction, *Siraitia grosvenorii* pulp 55 days post-pollination was used as explants. Different concentrations of PIC (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 mg / L) were added to MS medium, with a fixed TDZ concentration of 0.5 mg / L. Thirty explants were treated with each concentration, with three replicates. Callus induction rate and browning rate were recorded after 30 days of culture. The results are shown in Table 1.

[0061] Table 1. Effects of different PIC concentrations on callus induction in fruit pulp. As can be seen, the highest callus induction rate (91.7%) and the lowest browning rate (5.0%) were observed at a PIC concentration of 2.5 mg / L, with the ideal green and compact callus morphology. Concentrations ≤1.0 mg / L resulted in low induction rates and loose callus, while concentrations ≥4.0 mg / L led to callus hardening and accelerated browning. Therefore, the preferred range for PIC is 2.0 to 3.0 mg / L, with 2.5 mg / L being optimal.

[0062] With a fixed PIC concentration of 2.5 mg / L, different concentrations of TDZ (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1.0 mg / L) were added to MS medium, with other conditions remaining the same. The effect of TDZ concentration on callus induction and subsequent growth was investigated, and the results are shown in Table 2.

[0063] Table 2. Effects of different TDZ concentrations on callus induction in fruit pulp. As can be seen, the highest callus induction rate (91.7%) was observed at a TDZ concentration of 0.5 mg / L, with the callus morphology being green and compact, exhibiting vigorous subculture growth. Concentrations that were too low (0.2 mg / L) resulted in loose callus and slow proliferation; concentrations that were too high (≥0.8 mg / L) easily led to vitrification or water-soaked appearance. Therefore, the preferred range for TDZ is 0.4 to 0.6 mg / L, with 0.5 mg / L being optimal.

[0064] Under the optimal callus induction conditions of fixed PIC 2.5 mg / L + TDZ 0.5 mg / L, single-factor experiments were conducted to investigate the effects of Agrobacterium infection parameters on transformation efficiency. The test ranges and optimal values ​​of each parameter are shown in Table 3.

[0065] Table 3 Results of single-factor optimization of Agrobacterium infection parameters As can be seen, OD 600 The highest conversion efficiency (59.6%) was observed at a concentration of 0.6 μmol / L, followed by 58.9% at an AS concentration of 150 μmol / L, 57.8% at an infection time of 20 minutes, and 60.1% after 3 days of co-cultivation. Further response surface methodology analysis revealed that OD... 600 There is a significant negative interaction between OD and AS concentration; the optimal combination is OD. 600 With a concentration of 0.6 μmol / L, an AS concentration of 150 μmol / L, an infection time of 20 minutes, and a co-culture time of 3 days, the conversion efficiency reached approximately 59.57%.

[0066] To determine the optimal screening concentration of hygromycin, untransformed fruit pulp callus from the second generation of Example 1 was inoculated onto MS medium containing different concentrations of hygromycin (0, 10, 30, 50, 60, and 80 mg / L). The MS medium also contained 2.5 mg / L PIC and 0.5 mg / L TDZ. 30 callus pieces were inoculated at each concentration, with three replicates, and the callus was incubated in the dark at 25±2℃ for 14 days. The results showed that when the hygromycin concentration was ≤30 mg / L, all callus tissues exhibited varying degrees of growth. At 50 mg / L, the callus size variation was most pronounced, and the color was generally yellowish, indicating severely inhibited overall growth, although some cell clusters still survived. These clusters could be gradually eliminated through multiple rounds of screening. At 60 and 80 mg / L, the callus rapidly browned and died within 7 to 10 days. Therefore, 50 mg / L was determined as the optimal concentration of hygromycin in the screening medium, and multiple rounds of screening were conducted to obtain homozygous resistant callus.

[0067] To determine the optimal inhibitory concentration of Timentin, a single colony of Agrobacterium GV3101 was inoculated into 5 mL of LB liquid medium containing 50 mg / L rifampin and 50 mg / L kanamycin, and cultured overnight at 28°C with shaking at 200 rpm until OD500. 600≈0.8. The overnight bacterial culture was transferred at a ratio of 1:100 (v / v) to Erlenmeyer flasks containing different concentrations of Timentin (0, 200, 300, and 400 mg / L). Each flask had a volume of 100 mL, with 20 mL of culture medium per flask. Three replicates were prepared for each concentration. The flasks were incubated at 28°C and 200 rpm for 24 hours with shaking. The turbidity and color changes of the culture medium in each flask were then observed. Results: At 0 mg / L Timentin, the culture medium was turbid and yellow, indicating abundant Agrobacterium growth; at 200 mg / L, the culture medium was relatively clear but still yellow, indicating partial inhibition of Agrobacterium growth; at 300 and 400 mg / L, the culture medium was clear and transparent with a pale yellow color, indicating complete inhibition of Agrobacterium growth. Simultaneously, second-generation callus tissue from Example 1 was cut into 0.3 cm × 0.3 cm pieces and inoculated into MS solid medium containing different concentrations of Timentin (0, 100, 200, 300, 400, and 500 mg / L). The MS solid medium contained 2.5 mg / L PIC and 0.5 mg / L TDZ. The culture dishes were 60 mm in diameter, each containing 20 mL, with 10 pieces inoculated per dish. Three replicates were set for each concentration. The culture was incubated in the dark at 25 ± 2℃ for 14 days, and the callus growth status was observed. The results showed that within the Timentin concentration range of 0 to 300 mg / L, there was no significant difference in the growth status of the callus tissue, which remained bright green and proliferated normally. At 400 mg / L, growth was slightly slowed down; at 500 mg / L, the callus color turned yellow, and growth was inhibited by about 30%. Based on the above results, 300 mg / L, which could completely inhibit Agrobacterium without affecting the normal growth of callus tissue, was selected as the Timentin concentration.

[0068] Example 6 To address the issue of gradually decreasing transformation efficiency of monk fruit callus during long-term subculture, a systematic quality control procedure was introduced during the subculture stage, building upon Examples 1 to 5. This procedure included low-temperature pretreatment, selective subculture (morphological screening), low-concentration hygromycin preculture, recovery culture containing abscisic acid, and microdissection combined with Evans blue staining for activity screening. This ensured that the same batch of callus maintained high transformation activity even after long-term subculture. All operations in this example were performed under aseptic conditions.

[0069] Green, firm callus tissue from *Siraitia grosvenorii* pulp 55 days after pollination was obtained according to the method in Example 1: Pulp explants were inoculated onto callus induction medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 30 g / L sucrose + 7 g / L agar, pH 5.8, and cultured in the dark at 25±2℃ for 30 days. The induced callus tissue was subcultured every 25 to 30 days on a subculture medium with the same composition, for a total of 3 subcultures. The third-generation callus tissue was used as the starting material for this example. This starting material should have the following characteristics: bright green to emerald green color, firm, granular texture, glossy surface, no browning or yellowing areas, and a conversion efficiency of approximately 59%.

[0070] All the following culture media used MS basal medium as the substrate, with 30 g / L sucrose and 7 g / L agar added. The pH was adjusted to 5.8 ± 0.1 with 1 mol / L NaOH or HCl, and then autoclaved at 121°C for 20 minutes. Hormones and antibiotics were added after the culture media had cooled to 50 to 55°C following sterilization.

[0071] Subculture medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ.

[0072] Pre-culture medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 15 mg / L hygromycin.

[0073] Recovery medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ, hygromycin-free. For treatments containing abscisic acid, add filtered sterilized abscisic acid (ABA) stock solution to a final concentration of 0.2 mg / L after sterilization and cooling to 55°C.

[0074] Evans blue staining solution: Weigh 0.02 g of Evans blue powder and dissolve it in 100 mL of sterile distilled water to prepare a 0.02% (w / v) staining solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4°C in the dark.

[0075] Starting from the 4th generation, before each subculture, the callus blocks to be subcultured, along with the culture dishes, were transferred to a refrigerator at 4℃±1℃ for 36 hours for cryogenic treatment. After the cryogenic treatment, the culture dishes were removed and placed in a clean bench at room temperature for 30 minutes to allow the callus to return to room temperature.

[0076] The callus tissue blocks treated at low temperature were observed under a stereomicroscope. Only callus granules meeting all of the following morphological characteristics were selected: bright green to emerald green color, without yellow, white, brown, or black areas; firm, granular texture; glossy surface; not water-soaked; and only the surface layer of the callus tissue block was taken, with a thickness of approximately 0.5 to 1 mm. The central yellowish-white, loose, water-soaked, or browned parts were discarded. The selected qualified callus tissue was cut into uniform small pieces on sterile filter paper, each piece approximately 0.3 cm × 0.3 cm × 0.2 cm, weighing approximately 15 to 20 mg. The cut pieces were inoculated onto fresh subculture medium, 8 to 10 pieces per 60 mm culture dish, and cultured in the dark at 25 ± 2 °C. Subculture was performed every 25 to 30 days. The passage number (P4, P5, …) was recorded for each subculture.

[0077] When the callus tissue has been subcultured three times, starting from the fourth generation, a low-concentration hygromycin pre-culture and recovery culture step is added before each subculture.

[0078] (1) Low-concentration hygromycin pre-culture: All callus blocks to be subcultured were transferred to a pre-culture medium containing 15 mg / L hygromycin, 8 to 10 blocks per 60 mm dish, and cultured in the dark at 25±2℃ for 6 days. Observe on the 3rd and 6th days. If more than 30% of the callus blocks are severely browned, they are discarded.

[0079] (2) Recovery culture with abscisic acid: After 6 days of pre-culture, the callus was removed from the PCM, and residual culture medium on the surface was absorbed with sterile filter paper. It was then transferred to recovery culture medium containing 0.2 mg / L abscisic acid (ABA). The callus was cultured in the dark at 25±2℃ for 8 days. The addition of ABA can induce the expression of cellular stress-resistant proteins, enhance the tolerance of callus tissue to subsequent transformation stress, and promote the differentiation potential of embryogenic cells. After the recovery culture was completed, callus that was still yellowish-white, soft, or water-soaked was discarded.

[0080] After the recovery culture was completed, the callus tissue was microscopically dissected and screened for viability.

[0081] (1) Microcutting: Under a stereomicroscope of 20× to 40×, each callus particle is cut into 2 to 4 small pieces with a diameter of about 0.1 cm using a sterile microscalpel or a No. 10 scalpel blade. Each small piece contains about 50 to 100 cells. The blade should be kept sharp during cutting to avoid squeezing and damaging the cells.

[0082] (2) Evans blue staining: Place the cut pieces individually in a sterile 35 mm petri dish, add 0.02% Evans blue staining solution to immerse the tissue, and incubate at room temperature (25±2℃) for 3 minutes, gently shaking once every minute during this period. After staining, aspirate the staining solution, add sterile distilled water to rinse, gently shaking for 30 seconds each time before aspirating, repeating 4 times. After the last rinse, transfer the pieces to sterile filter paper and blot dry the surface moisture.

[0083] (3) Screening: Under a microscope equipped with a rhodamine filter, with an excitation wavelength of 540 nm and an emission wavelength of 590 nm, small patches stained deep blue with severely damaged cell membranes were completely removed; small patches with only the edges or a few cells showing blue but the center remaining green or colorless, and small patches with only slight surface staining, as well as completely unstained patches, were retained. The retained patches were combined, and the surface moisture was blotted dry with sterile filter paper for further subculture or direct transformation.

[0084] For further subculture, the small pieces retained after staining and screening are directly inoculated onto fresh subculture medium, 8 to 10 pieces per plate, each piece being considered an independent subculture unit. These are then incubated in the dark at 25±2℃ for 25 to 30 days, which will become the next generation of callus, for example, P4 obtained from P3. The above steps are repeated for each subculture.

[0085] For genetic transformation, the small pieces retained after staining and screening were directly used as recipient materials. Agrobacterium infection, co-culture, and screening were performed according to the methods described in Examples 3 and 4: The small pieces were transferred to co-culture medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 150 μM AS, and cultured in the dark at 25±2℃ for 3 days; then transferred to screening medium: MS + 2.5 mg / L PIC + 0.5 mg / L TDZ + 50 mg / L hygromycin + 300 mg / L Timentin, and cultured at 25±2℃ under 16 h / 8 h light / dark conditions. Subculture was performed every 2 weeks for a total of 4 rounds of screening; the obtained resistant callus tissue was identified by GFP fluorescence detection or GUS staining.

[0086] Following the complete steps described above, the monk fruit pulp callus was subcultured for an extended period, from P3 to P15. After each subculture, a portion of the callus was harvested for genetic transformation, and the rate of resistant callus acquisition and GFP positivity were statistically analyzed. Three independent replicate experiments were conducted, with three culture dishes per generation for each experiment, containing 8 to 10 callus fragments per dish. During transformation, 30 callus fragments were harvested from each generation, microscopically dissected, and then used for infection. The results are shown in Table 4.

[0087] Table 4. Transformation efficiency of different succession generations under the method of Example 6 As shown in Table 4, using the method of this embodiment, from P3 to P15, the resistant callus acquisition rate only slowly decreased from 59.2% to 42.0%, and the effective number of generations was extended to more than 15. Compared with conventional subculture, which does not perform low-temperature pretreatment, morphological screening, low-concentration hygromycin pre-culture, recovery culture, or Evans blue staining, and directly transfers the callus tissue into fresh subculture medium after being chopped up whole, the resistant callus acquisition rate of conventional subculture drops to 31.2% in the 6th generation and is below 10% in the 8th generation. This embodiment still maintains 49.8% in the 12th generation and 42.0% in the 15th generation, which is significantly better than the prior art.

[0088] Three independent replicate experiments were conducted for passages P6, P8, and P10, and the coefficients of variation (CV%) of resistant callus acquisition rate were calculated: 0.9% for P6, 1.0% for P8, and 0.8% for P10, indicating that the method of this embodiment has excellent reproducibility. Callus tissue subcultured to P15 using the method of this embodiment remained bright green, with uniform granules and a glossy surface under a stereomicroscope, without browning or vitrification.

[0089] To verify the necessity and synergistic effect of low-temperature pretreatment, abscisic acid recovery culture, and microdissection, the following comparative examples were set up. All comparative examples used third-generation monk fruit pulp callus from the same source as in Example 6, and the transformation operations were uniformly referred to in Examples 3 to 4.

[0090] Comparative Example 10 was performed without cryogenic pretreatment: except for the absence of 4°C cryogenic treatment, all other steps were identical to Example 6, including morphological screening, low-concentration hygromycin pre-culture, ABA-containing recovery culture, and Evans blue staining. The results are shown in Table 5. The resistant callus acquisition rate was 50.5% in the 6th generation, 42.5% in the 10th generation, and 38.0% in the 12th generation. Although better than conventional subculturing, it was significantly lower than in Example 6. This indicates that cryogenic pretreatment can actively eliminate low-viability cells and enhance the subsequent screening effect.

[0091] Comparative Example 11 involved recovery culture without abscisic acid: except for the absence of abscisic acid in the recovery medium, the remaining steps were the same as in Example 6, including low-temperature pretreatment, morphological screening, hygromycin pre-culture, microdissection, and Evans blue staining. The results are shown in Table 5. The resistant callus acquisition rate was 52.0% in the 6th generation, 45.5% in the 10th generation, and 40.2% in the 12th generation. This is lower than in Example 6, indicating that the addition of ABA helps improve the stress resistance and transformation potential of the callus tissue.

[0092] Comparative Example 12 involved direct staining of the entire callus without microdissection: except for the absence of microdissection (i.e., staining the entire callus granules with Evans blue directly after recovery culture), the remaining steps were the same as in Example 6, including low-temperature pretreatment, morphological screening, hygromycin pre-culture, and ABA-containing recovery culture. The results are shown in Table 5. The resistant callus acquisition rate was 51.8% in the 6th generation, 44.2% in the 10th generation, and 38.5% in the 12th generation. This was significantly lower than in Example 6, indicating that microdissection into small clumps can break down the heterogeneity within the callus, making staining more precise, while also increasing the uniformity of surviving cell clusters after screening, thus improving the stability of transformation efficiency after long-term subculturing.

[0093] Table 5 Comparison of resistant callus acquisition rate (%) between different comparative examples and Example 6 Note: "—" indicates that the conversion efficiency has fallen below 10%, and the experiment has been terminated.

[0094] As shown in Table 5, the transformation efficiencies of Comparative Examples 10, 11, and 12 after long-term subculture were significantly lower than those of Example 6. For example, in the 12th generation, the resistant callus acquisition rate of Example 6 was 49.8%, while that of Comparative Examples 10, 11, and 12 were 38.0%, 40.2%, and 38.5%, respectively. This indicates a synergistic effect among low-temperature pretreatment, ABA recovery culture, and microdissection, and none of these factors can be omitted. Particularly noteworthy is that the conversion rate of Comparative Example 10 decreased to 50.5% in the 6th generation, while that of Example 6 was 56.8% in the 6th generation; the difference increased with increasing generation, indicating that low-temperature pretreatment plays a crucial role in maintaining cell viability during long-term culture.

[0095] Three independent replicate experiments were conducted using Comparative Examples 10, 11, 12 and 6 respectively. The resistant callus acquisition rate of the 6th and 10th generations was statistically analyzed, and the coefficient of variation (CV%) was calculated. The results are shown in Table 6.

[0096] Table 6. Comparison of batch-to-batch stability of conversion efficiency under different schemes The batch-to-batch coefficient of variation (CV<2%) of Example 6 was significantly lower than that of other comparative examples (CV≥6.8%), indicating that the standardized and objective operations of this example, such as low-temperature treatment, microdissection, and staining screening, eliminated the influence of human factors and the results were highly reproducible.

[0097] In summary, Example 6, by introducing low-temperature pretreatment, morphological screening, low-concentration hygromycin pre-culture, ABA-containing recovery culture, cutting, and Evans blue staining activity screening, extended the effective generation number of monk fruit callus tissue from the conventional 5 generations to over 15 generations. The transformation efficiency increased from 31.2% in the 6th generation to 56.8%, with a batch-to-batch coefficient of variation below 2%. This approach solves the technical challenge of transformation efficiency decay during long-term subculturing, providing a reliable and stable method for maintaining recipient material for monk fruit genetic transformation.

[0098] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for culturing resistant callus tissue from monk fruit pulp, characterized in that, Includes the following steps: Step 1: Take the fruit of the monk fruit 50 to 60 days after pollination and extract the pulp tissue; Step 2: Inoculate the pulp tissue onto a callus induction medium containing thiamethoxam and thiamethoxam, wherein the concentration of thiamethoxam is 1.0 to 4.0 mg / L and the concentration of thiamethoxam is 0.3 to 0.8 mg / L, and culture to obtain callus tissue. Step 3: Immerse the callus tissue from Step 2 in Agrobacterium-mediated bacterial solution. The OD of the Agrobacterium-mediated bacterial solution... 600 The value is 0.5 to 0.8, and the Agrobacterium bacterial suspension contains 100 to 200 μmol / L acetylsylgenone, and the infection time is 15 to 25 minutes; Step 4: Culture the infected callus tissue for 2 to 4 days. Step 5: Transfer the co-cultured callus tissue into a screening medium containing screening agents and antibacterial agents to screen and obtain resistant callus tissue.

2. The method according to claim 1, characterized in that, In step 2, the concentration of atrazine is 2.0 to 3.0 mg / L, and the concentration of thiabendazole is 0.4 to 0.6 mg / L.

3. The method according to claim 2, characterized in that, OD in step 3 600 The value was 0.6, the acetylsyl syringone concentration was 150 μmol / L, and the infection time was 20 minutes; the co-culture time in step 4 was 3 days.

4. The method according to claim 3, characterized in that, The screening agent in step 5 is hygromycin at a concentration of 40 to 60 mg / L; the antibacterial agent is ticarcillin-clavulanate potassium at a concentration of 200 to 400 mg / L.

5. The method according to claim 4, characterized in that, The concentration of hygromycin was 50 mg / L, and the concentration of ticarcillin-clavulanate potassium was 300 mg / L.

6. The method according to claim 5, characterized in that, In step 5, the screening process is carried out in 2 to 6 rounds.

7. The method according to claim 6, characterized in that, In step 2, the callus tissue is green and compact.

8. The method according to claim 1, characterized in that, In step 2, the concentration of atrazine was 2.5 mg / L, and the concentration of thiamethoxam was 0.5 mg / L; in step 3, OD 600 The value was 0.6, the concentration of acetylsuccinone was 150 μmol / L, and the infection time was 20 minutes; the co-culture time in step 4 was 3 days; in step 5, the screening agent was hygromycin with a concentration of 50 mg / L, and the antibacterial agent was ticarcillin-clavulanate potassium with a concentration of 300 mg / L.

9. The method according to any one of claims 1 to 8, characterized in that, The subculture process in step 2 also includes the following steps to maintain the high transformation activity of the resistant callus: a) Before each subculture, the callus tissue was subjected to low-temperature treatment at 4℃±1℃ for 24 to 48 hours. Then, the surface part that still retains a bright green color and firm texture was transferred to fresh culture medium, and the yellowish-white, loose or browned central part was discarded. b) After three subcultures, before each subculture, the callus tissue was pre-cultured in MS medium containing 10 to 20 mg / L hygromycin for 5 to 8 days, and then transferred to MS medium without hygromycin and supplemented with 0.1 to 0.3 mg / L abscisic acid, and cultured in the dark at 25±2℃ for 7 to 10 days for recovery culture. c) After the recovery culture is completed, cut the callus particles into 2-4 small pieces with a diameter of 0.1 to 0.2 cm, soak them in an Evans blue aqueous solution with a mass-volume ratio of 0.01% to 0.05% for 2 to 5 minutes, pour off the staining solution and rinse with sterile water 3 to 5 times, remove the blue-stained pieces, retain the unstained or only slightly stained pieces and combine them for subsequent subculture or steps 3 to 5.

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