Ex vitro rooting method for siraitia grosvenorii tissue culture seedlings

Abstract

The invention discloses a siraitia grosvenorii tissue culture seedling ex-vitro rooting method, belongs to the technical field of plant tissue culture, and mainly solves the problems of long rooting time, untidy root development and low transplanting survival rate in the traditional tissue culture seedling ex-vitro rooting technology. The method comprises the following steps: selecting young branches to prepare sterile explants, and performing hormone-induced culture to obtain robust tissue culture seedlings; a growth hormone combination with a specific ratio is adopted in the rooting pre-culture stage; humidity change is controlled through three-stage gradient seedling hardening; the tissue culture seedlings are transplanted to a matrix after being soaked in a 200-350 mg / L ABT rooting solution. The method remarkably shortens the rooting time, improves the root strength, improves the transplanting survival rate, and can be efficiently used for large-scale production of the virus-free seedlings of momordica grosvenori and resist environmental stress such as late spring coldness.

Description

Technical Field

[0001] The invention belongs to the technical field of momordica grosvenori tissue culture, and specifically relates to a method for rooting momordica grosvenori tissue culture seedlings outside a bottle. Background Art

[0002] Momordica grosvenori, scientific name "Mogrosvenori" ( Siraitia grosvenorii (Swingle. )C. Jeffrey), is the fruit of a perennial vine of the Cucurbitaceae family. It is one of the authentic medicinal materials of Guangxi's "Ten Flavors of Gui" and a key development variety in Guangxi. It is also one of the first plants in China to be listed as both a medicine and food, and has extremely high medicinal and nutritional value. In recent years, mogroside, as a high-sweetness, low-calorie functional natural sweetener, has been widely used in food, beverage and health food industries. Therefore, the monk fruit industry has a good development prospect. However, due to the mixed varieties of traditionally planted pressed potato seedlings, they are easily infected with mosaic viruses in a natural state, resulting in virus accumulation and serious degradation of germplasm, and low yield, which seriously restricts the expansion of monk fruit production. In recent years, the application of monk fruit tissue culture and virus-free seedlings can greatly increase the yield, making large-scale and professional production of monk fruit possible.

[0003] There are the following technical problems in the rooting process of Momordica grosvenori tissue culture seedlings in bottles: 1. Long rooting period and uneven root development In traditional methods, tissue culture seedlings need to be cultured for 18-24 days to develop roots in a closed culture container. Due to the uneven distribution of hormone gradients in the container, there are significant differences in the start time of lateral root growth in different stem segments, and the development time difference between adjacent stem segments often exceeds 72 hours. This leads to asynchronous root growth. When some plants have formed a complete root system, the rest of the plants only have primary lateral roots. The resulting transplanted seedlings have different root strengths, and the number of lateral roots is generally less than 4 per plant, resulting in insufficient mechanical support after transplantation.

[0004] 2. Poor environmental adaptability during hardening Conventional seedling hardening is performed in one step before transplanting, which causes the humidity inside and outside the bottle to drop sharply in a short period of time, even exceeding 60%. The drastic change in humidity causes the opening and closing function of the leaf stomata to malfunction, and the epidermal cells of the new roots rupture due to the sudden change in osmotic pressure. At the same time, the light intensity increases sharply after the tissue culture seedlings are taken out of the bottle, but the reconstruction of chloroplasts lags behind, and the new leaves unfold slowly. The coupling of the above problems leads to an increase in the seedling loss rate in the early stage of transplanting.

[0005] Main difficulties in solving the above problems: First, the synergistic problem of humidity control and light adaptation. Gradually reducing humidity requires precisely matching the change in light intensity, but it is difficult for existing technologies to synchronously regulate the two. Rapid dehumidification easily causes leaf dehydration, while slow dehumidification delays the establishment of the photosynthetic capacity of new leaves and affects the photosynthetic rate. Second, a high-density lignified structure is formed at the base of the stem of Siraitia grosvenorii tissue culture seedlings during the late rooting stage. The high-density structure on the stem segment epidermis forms a physical barrier, and conventional soaking treatments cannot effectively reach the target sites with rooting active substances. Increasing the concentration of the liquid medicine easily causes toxicity, and prolonging the soaking time increases the risk of root damage. Summary of the Invention

[0006] To solve the problem of low transplanting survival rate caused by the long in-vitro rooting cycle and uneven root development of Siraitia grosvenorii tissue culture seedlings. In traditional methods, due to uneven hormone distribution, there are significant differences in the initiation of lateral root growth, and sudden changes in humidity during the acclimatization stage cause cell damage. The present invention provides a method for in-vitro rooting of Siraitia grosvenorii tissue culture seedlings.

[0007] To achieve these objectives of the present invention, a method for in-vitro rooting of Siraitia grosvenorii tissue culture seedlings provided by the present invention includes the following steps: Select young branches of Siraitia grosvenorii to prepare stem segments with axillary buds, and obtain sterile explants through surface disinfection treatment; Inoculate the sterile explants into an MS medium containing 0.4 - 0.6 mg / L of 6-benzylaminopurine and 0.1 - 0.3 mg / L of naphthylacetic acid, and culture for 18 - 24 days under the conditions of 24 - 26 °C and 1200 - 1800 lx of light to obtain induced buds; Transfer the induced buds to an MS medium containing 0.2 - 0.4 mg / L of 6-benzylaminopurine, 0.03 - 0.07 mg / L of naphthylacetic acid, and 0.8 - 1.2 g / L of activated carbon for culture to obtain tissue culture seedlings; Select robust tissue culture seedlings and inoculate them into a 1 / 2MS medium containing 0.08 - 0.12 mg / L of 6-benzylaminopurine, 0.15 - 0.25 mg / L of naphthylacetic acid, 0.15 - 0.25 mg / L of indolebutyric acid, and 0.3 - 0.7 g / L of activated carbon, and conduct pre-rooting culture for 12 - 16 days under the conditions of 21 - 23 °C and 1200 - 1800 lx of light; Conduct gradient acclimatization on the tissue culture seedlings in the seedling culture glass bottles after pre-rooting culture: a) In the first stage, the opening angle of the lid is 15° - 20°, and the humidity inside the bottle is maintained at 85 - 90% for 24 hours; b) In the second stage, the opening angle of the lid is 45° - 50%, and the humidity is reduced to 75 - 80% and maintained for 48 hours; c) In the third stage, the lid is completely opened, and the surface water film is evaporated in an environment with an air flow rate of 0.3 - 0.5 m / s, and the medium is removed and washed; Soak the washed tissue culture seedlings in 200 - 350 mg / L of ABT rooting solution and transplant them into the substrate.

[0008] To solve the problem of the mismatch between humidity change and light adaptation during the acclimatization process. Conventional operations cannot synchronously regulate light intensity and humidity, resulting in stomatal dysfunction and lagging chloroplast reconstruction, affecting photosynthetic rate and transplant survival. Preferably, in step 5 of the present invention, spectral regulation is carried out synchronously during gradient acclimatization: a) In the first stage, a red-light dominant mode is adopted: the red light wavelength is 630±5nm, accounting for 80% (photosynthetic photon flux density (PPFD), the same below), and the light intensity is 800-1000lx; b) In the second stage, a red-blue composite spectral mode is adopted: the intensity ratio of the red light wavelength 630nm to the blue light wavelength 450nm is 3:1, and the total light intensity is 1500-1800lx; c) In the third stage, natural light is dynamically simulated: from 6:00 to 10:00, the proportion of blue light increases from 20% to 35%, from 10:00 to 16:00, the red-blue ratio is 2:1, and from 16:00 to 18:00, the proportion of red light is 75%.

[0009] To overcome the penetration barrier of lignified stem segments to rooting active substances. Single soaking treatment is difficult to break through the high-density epidermal structure, and active substances cannot effectively reach the target sites. Preferably, step 5 of the present invention also includes a synergistic strengthening treatment: 3 days before the end of rooting pre-culture, an intensifying solution containing 0.005% salicylic acid by mass-volume percentage and 0.01% manganese sulfate by mass-volume percentage is injected into the 1 / 2MS medium every 12 hours, and each injection lasts for 5-15 minutes; after taking out the tissue culture seedlings, a negative pressure intermittent soaking method is used for treatment, including placing the roots in the ABT rooting solution, applying a negative pressure of -0.05MPa for 20-40 seconds and then releasing it, and circulating 3-7 times; immediately after the negative pressure treatment, transfer it to a buffer solution containing 0.05% Tween-80 by volume percentage and 1% sorbitol by mass-volume percentage, and oscillate and soak for 2-5 minutes.

[0010] To improve the transport efficiency of ABT rooting solution in lignified tissues. Conventional solutions lack auxiliary components, resulting in insufficient transport capacity of active substances. Preferably, the rooting solution described in step 6 of the present invention is a composite solution of ABT No. 1 rooting powder at 200-350mg / L, 0.01% ferric citrate by mass-volume percentage, and 0.005% salicylic acid by mass-volume percentage.

[0011] To enhance the stress resistance of tissue culture seedlings during the rooting pre-culture stage. Low temperature or osmotic stress is likely to damage the cell structure of newly formed roots. Preferably, in step 4 of the present invention, 0.02% trehalose by mass-volume percentage and 0.001% nano-zinc oxide by mass-volume percentage are added to the 1 / 2MS medium.

[0012] To improve the thoroughness and safety of explant disinfection. When using disinfectants alternately, the activity of explants may be inhibited. Preferably, the disinfection treatment in step 1 of the present invention is: alternately treating with a 0.1% HgCl2 solution by mass / volume percentage and a 5% sodium hypochlorite solution by mass / volume percentage at a volume ratio of 3:1, and rinsing with sterile water in pulses for 5 - 20 seconds at intervals between each treatment.

[0013] The present invention has at least the following beneficial effects: 1. By controlling the humidity at different stages of acclimatization seedlings through gradient, the risk of sudden change in cell osmotic pressure is reduced; pre-culture and ABT soaking synergistically promote the synchronous development of roots; the seedling soil substrate provides a basic support environment, ultimately improving the robustness of roots and transplant adaptability.

[0014] 2. The spectral dynamic regulation matches the rhythm of humidity change. The red light stage promotes the expansion of mesophyll cells, the blue light temporarily enhances and optimizes stomatal responses, and the dynamic simulation accelerates the photoautotrophic conversion, realizing the coordinated adaptation of humidity and light, reducing chlorophyll degradation, and improving the photosynthetic rate and survival rate.

[0015] 3. The pulsed injection of salicylic acid activates the resistance pathway, and the negative pressure intermittent soaking breaks through the lignified epidermal barrier, strengthening the transport of rooting substances to the cambium, significantly improving the uniformity of lateral root differentiation and the degree of root lignification.

[0016] 4. Ferric citrate catalyzes the directional transport of auxin, and salicylic acid enhances cell signal transduction. The composite solution promotes the synchronous initiation of lateral and secondary roots, and improves the longitudinal migration efficiency of active ingredients in the xylem.

[0017] 5. Trehalose maintains the integrity of membrane lipid structure, and nano-zinc oxide induces the expression of stress-resistant proteins, synergistically enhancing the stability of cells under low temperature and osmotic stress, and reducing the abnormal leakage of electrolytes.

[0018] 6. The alternate use of disinfectants expands the bactericidal spectrum, and the pulsed rinsing ensures the activity of explants while controlling microbial contamination, establishing a reliable basis for aseptic culture.

[0019] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will be understood by those skilled in the art through the research and practice of the present invention. Detailed implementation manners

[0020] The following further elaborates the present invention with reference to examples, so that those skilled in the art can implement it according to the description in the specification.

[0021] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0022] Description of experimental methods I. Root System Development Detection 1. Determination of Rooting Time Steps: Starting from the transplantation date, observe the base of the tissue culture seedlings daily. Record the number of days or hours when the length of the newly grown roots of 50% of the plants is ≥ 0.5 cm.

[0023] 2. Statistics of Root Length and Lateral Root Number Steps: On the 14th day after transplantation, completely remove the root system of the plants. After rinsing with clear water: Measure the distance from the tip to the base of the main root (excluding root hairs); Count the number of lateral roots with a length ≥ 0.5 cm.

[0024] Sample size: Randomly select 30 plants per group, with 3 replicates.

[0025] II. Leaf Physiology Detection 1. Determination of SPAD Value Steps: On the 7th day after transplantation, select the third fully expanded leaf, avoid the main vein, take three points in the middle of the leaf, and measure with a SPAD-502 chlorophyll meter, then take the average value.

[0026] 2. Net Photosynthetic Rate Steps: On the 7th day after transplantation, measure with a Li-6400XT portable photosynthesis meter; Set the light intensity at 1500 μmol·m -2 ·s -1 ; The CO2 concentration is 400 ppm; The leaf chamber temperature is 25°C.

[0027] Record: Read the data 3 times after stabilization and take the average value.

[0028] III. Cell Structure and Stress Resistance Detection 1. Electrolyte Leakage Rate Steps: Take the third fully expanded leaf, avoid the main vein, punch to obtain 1.0 cm diameter leaf discs (3 per plant); Rinse 3 times with ultrapure water (conductivity ≤ 0.5 μS / cm); Load into a 50 mL centrifuge tube, add 20 mL of ultrapure water; Treat in the dark at 5 ± 0.5°C for 24 hours; Shake on a shaker (120 rpm, 25°C) for 2 hours, measure the initial conductivity C1; Boil in a water bath for 15 minutes, cool and then measure the final conductivity C2; Calculate: Leakage rate (%) = (C2 / C1) × 100.

[0029] 2. Membrane Lipid Peroxidation (MDA Content) Steps: Take 0.5 g of fresh leaves, grind in liquid nitrogen; Add 5 mL of 0.1% TCA (trichloroacetic acid) and centrifuge (10000 g, 10 minutes); Take 2 mL of the supernatant + 2 mL of 0.5% TBA (thiobarbituric acid); Heat in a water bath at 95°C for 30 minutes, cool in an ice bath; Measure the absorbance at 532 nm and 600 nm after centrifugation; Calculate according to the formula: MDA (nmol / g) =[(A 532– A 600 ) / 155]× 10 6 。

[0030] 3. Antioxidant enzyme activities (SOD / CAT / PAL) SOD (superoxide dismutase) was assayed by the photochemical reduction method of nitroblue tetrazolium (NBT): The enzyme extract and the reaction mixture (containing NBT and riboflavin) were irradiated at 4000 lx for 20 minutes; the decrease rate of absorbance at 560 nm was measured. One unit of SOD was defined as the amount of enzyme that inhibited 50% of NBT reduction.

[0031] CAT (catalase) was assayed by the ultraviolet absorption method: The reaction system contained the enzyme solution and 15 mM H2O2; a decrease in absorbance of 0.01 per minute at 240 nm was recorded as one activity unit.

[0032] PAL (phenylalanine ammonia-lyase): The enzyme solution was incubated with 0.02 M L-phenylalanine for 1 hour; the reaction was terminated by adding 6 M HCl, and the increase in absorbance at 290 nm was measured.

[0033] IV. Disinfection and detection 1. Contamination rate of explants Procedure: The disinfected explants were inoculated onto hormone-free MS medium; cultured at 25°C for 7 days, and the proportion of samples with colony growth was counted.

[0034] 2. Browning rate: Cultured at 25°C for 7 days, and the proportion of browning samples was counted.

[0035] 3. Callus induction rate Procedure: 7 days after inoculation of the explants, observed under a stereomicroscope; the proportion of samples forming callus ≥2 mm was counted.

[0036] Example 1 An example of the method for rooting in vitro of Siraitia grosvenorii tissue culture seedlings of the present invention includes the following steps: Preparation of aseptic explants: Take young shoots of Siraitia grosvenorii, cut them into 1-cm stem segments with axillary buds, soak them in a 1% (m / v) washing powder solution for 10 minutes, and rinse them with running water for 20 minutes. After rinsing 3 times with sterile water, treat them with 75% (v / v) alcohol for 30 seconds, and then treat them with 0.1% HgCl2 (containing 0.5 mL / 100 L Tween-20) for 7 minutes, rinse 3 times with sterile water, and dry them with sterile filter paper.

[0037] Induced bud culture: Inoculate the explants onto MS medium containing 0.5 mg / L 6-benzylaminopurine and 0.2 mg / L naphthaleneacetic acid, and culture them at 25 ± 1°C under 1500 lx illumination (11 hours / day) for 21 days.

[0038] Tissue culture seedling cultivation: Induce the buds to be transferred to MS medium containing 0.3 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic acid, and 1 g / L activated carbon, and cultivate under the same conditions until the seedling height ≥ 3 cm.

[0039] Rooting pre-cultivation: Inoculate the healthy tissue culture seedlings into 1 / 2 MS medium containing 0.1 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 0.2 mg / L indolebutyric acid, and 0.5 g / L activated carbon, and cultivate at 22 ± 1 °C and 1500 lx light (9 hours / day) for 14 days.

[0040] During gradient acclimatization, in the first stage, the opening angle of the lid is 18°, and the humidity inside the bottle is maintained at 87 ± 3% for 24 hours; in the second stage, the opening angle of the lid is 48°, and the humidity is reduced to 78 ± 3% and maintained for 48 hours; in the third stage, the lid is completely opened, and the air flow rate of 0.4 m / s is used to blow for 10 minutes, and then take out and wash the medium.

[0041] Transplanting: Soak the tissue culture seedlings in 280 mg / L ABT No. 1 rooting solution for 8 minutes, and transplant them into the substrate (using PINDSTRUP moss peat organic substrate) (humidity 45 ± 5%, shading rate 55 ± 5%).

[0042] Comparative Example 1 The difference from Example 1 is only in the acclimatization operation: Delete the gradient acclimatization in step 5; directly open the lid after rooting pre-cultivation, place it at room temperature for 48 hours, wash the medium and transplant (other steps are the same as in Example 1).

[0043] Table 1 It can be seen that the traditional one-step opening of the lid for acclimatization in Comparative Example 1 leads to a sudden drop in humidity > 60%, causing the collapse of leaf stomata and the rupture of the osmotic pressure of root cells. In Example 1 of the present invention, three-stage gradient acclimatization is adopted. In the first stage, the lid is opened at 18° and high humidity (87%) is maintained for 24 hours; in the second stage, the lid is opened at 48° and the humidity is reduced to 78%; in the third stage, the lid is fully opened and combined with a circulating air speed of 0.4 m / s to evaporate the water film, effectively reducing the extracellular leakage rate of cells, significantly improving cell integrity; significantly increasing the number of lateral roots and enhancing the root grip; the transplanting survival rate is also increased from 68.2% to 88.5%.

[0044] Example 2 An example of the method for rooting tissue culture seedlings of Momordica grosvenori outside the bottle in the present invention. On the basis of Example 1, spectral regulation is carried out synchronously during the gradient acclimatization in step 5: a) In the first stage, a red light-dominated mode is adopted: the wavelength of 630 ± 5 nm accounts for 80%, and the light intensity is 800 - 1000 lx; b) In the second stage, a red-blue composite spectrum mode is adopted: the intensity ratio of red light at 630 nm to blue light at 450 nm is 3:1, and the total light intensity is 1500 - 1800 lx; c) In the third stage, natural light is dynamically simulated: from 6:00 to 10:00, the proportion of blue light is 20% to 35%, from 10:00 to 16:00, the red-blue ratio is 2:1, and from 16:00 to 18:00, the proportion of red light is 75%.

[0045] Specifically, the pretreatment steps are the same as steps 1 - 4 of Example 1 (preparation of sterile explants to rooting pre-culture).

[0046] Gradient acclimatization and spectrum regulation: First stage: the opening angle of the lid is 18°, red light mode (80% of wavelength 632 nm, light intensity 900 lx), maintaining the humidity at 87 ± 3% for 24 hours; Second stage: the opening angle of the lid is 48°, red-blue composite mode (red light at 630 nm: blue light at 450 nm = 3:1, total light intensity 1700 lx), reducing the humidity to 78 ± 3%. The proportion of blue light is temporarily increased to 40% within 5 minutes after each atomization (atomization is carried out 3 times a day) and maintained for 48 hours; Third stage: fully open the lid, the circulating air speed is 0.4 m / s, dynamic spectrum (blue light is 25% at 7:00, increased to 35% at 10:00, red:blue = 2:1 at 12:00, red light is 75% at 17:00).

[0047] Transplanting is the same as step 6 of Example 1 (transplanting after soaking in ABT rooting solution).

[0048] Comparative Example 2 The difference from Example 2 is only in spectrum regulation: a fixed white light (spectrum range 400 - 700 nm, light intensity 1500 lx) is used in the gradient acclimatization stage, and other parameters are exactly the same as those in Example 2.

[0049] Table 2 As can be seen from the above table, the fixed white light in Comparative Example 2 cannot match the rhythm of humidity change. In the examples of the present invention, the spectrum is dynamically adjusted. In the red light stage (80% proportion), cell expansion is activated; the proportion of blue light is temporarily increased to 40% within 5 minutes after atomization to stimulate the opening of stomata; in the third stage, the change of natural morning light is simulated (the proportion of blue light is 20% to 35%), which significantly improves the photosynthetic ability of the leaves, shortens the new leaf unfolding time to 5.1 days, and further improves the transplanting survival rate.

[0050] Example 3 An example of the method for rooting in vitro of Siraitia grosvenorii tissue culture seedlings of the present invention, and step 5 further includes a synergistic strengthening treatment: Three days before the end of rooting pre-culture, a strengthening solution containing 0.005% salicylic acid and 0.01% manganese sulfate was injected into the 1 / 2MS medium (containing 0.08 - 0.12 mg / L of 6-benzylaminopurine, 0.15 - 0.25 mg / L of naphthaleneacetic acid, 0.15 - 0.25 mg / L of indolebutyric acid and 0.3 - 0.7 g / L of activated carbon) every 12 hours, and each injection lasted for 10 minutes; After taking out the tissue culture seedlings, the negative pressure intermittent soaking method was used for treatment: the roots were placed in the ABT rooting solution, and after applying a negative pressure of -0.05 MPa for 30 seconds, it was released, and the cycle was repeated 5 times; Immediately after the negative pressure treatment, it was transferred to a buffer solution containing 0.05% Tween - 80 and 1% sorbitol, and soaked with shaking for 2 - 5 minutes. Finally, it was transplanted into the substrate.

[0051] Specifically, the pretreatment steps were the same as those in Step 1 - 4 of Example 1.

[0052] The difference is that three days before the end of rooting pre-culture, a strengthening solution (0.005% salicylic acid + 0.01% manganese sulfate aqueous solution) was injected every 12 hours. A sterile syringe was used to slowly inject it into the surface layer of the medium (the injection volume was 1 mL / bottle), and each injection lasted for 10 minutes. After injection, static culture was carried out. The operation time was carried out punctually at 8:00 and 20:00 every day.

[0053] The acclimatization of seedlings was the same as that in Example 1, and the three-stage humidity control was also adopted.

[0054] After washing the root medium of the tissue culture seedlings, the roots were immersed in the ABT rooting solution (300 mg / L of ABT No. 1 rooting powder) in a sealed container, and then negative pressure intermittent soaking was carried out: a vacuum dryer was connected to a pressure gauge and a sealed container to provide a pressure of -0.05 MPa for 30 seconds, and then released, and the cycle was repeated 5 times. After completion, it was immediately transferred to a phosphate buffer solution (pH 6.0) containing 0.05% Tween - 80 and 1% sorbitol, and soaked with shaking (120 rpm) for 3 minutes.

[0055] Finally, transplantation was carried out, and the transplantation was the same as Step 6 of Example 1.

[0056] Comparative Example 3 The difference from Example 3 was that the injection of the strengthening solution during rooting pre-culture was deleted; the negative pressure treatment and buffer solution soaking were deleted; the other steps were exactly the same as those in Example 3. The ABT soaking operation was that the tissue culture seedlings were statically soaked in the 300 mg / L ABT rooting solution for 10 minutes.

[0057] The test results were as follows 14 days after transplantation: Table 3 The epidermis of the lignified stem segment of Siraitia grosvenorii is dense, and the permeability of a single ABT treatment is low, making it difficult for active substances to reach the cambium. It can be seen that compared with Comparative Document 3 which only uses a single ABT without using a strengthening solution, negative pressure treatment, and buffer solution, the PAL enzyme activity in Example 3 is significantly improved, which can induce cell cycle synchronization, the number of lateral roots reaches more than 7 per plant, and the survival rate breaks through 96.5%, showing a significant improvement.

[0058] Example 4 An example of the method for rooting in vitro of tissue-cultured seedlings of Siraitia grosvenorii according to the present invention. In step 6, the rooting solution is a composite solution of 200 - 350 mg / L of ABT No. 1 rooting powder, 0.01% ferric citrate, and 0.005% salicylic acid.

[0059] Specifically, the pretreatment completely adopts the steps of Example 3 (including the co - strengthening treatment, gradient acclimatization, and negative pressure treatment).

[0060] In step 6, the rooting solution is modified. Use a composite solution of 280 mg / L of ABT No. 1 + 0.01% ferric citrate + 0.005% salicylic acid. After the tissue - cultured seedlings are washed with the root medium, the roots are immersed in the composite solution in a sealed container, and the negative pressure intermittent soaking method and subsequent treatments are carried out as in Example 3.

[0061] Comparative Example 4 Two groups of controls are set. Among them, the rooting solution of Comparative Example 4A is 280 mg / L of ABT No. 1 + 0.01% ferric citrate, and other steps are exactly the same as those of Example 4. The rooting solution of Comparative Example 4B uses a composite solution of 280 mg / L of ABT No. 1 + 0.005% salicylic acid, and other steps are exactly the same as those of Example 4.

[0062] The detection results 7 days after transplantation are as follows in the table: Table 4 It can be seen that the lack of salicylic acid in Comparative Example 4A leads to a significant decrease in the number of lateral roots, indicating that the lack of salicylic acid causes asynchronous lateral root differentiation. The lack of ferric citrate in Comparative Example 4B shortens the length of the newly grown roots, indicating that the lack of ferric citrate accelerates the degradation of auxin, resulting in shorter root lengths. In this example, a composite solution of 280 mg / L of ABT + 0.01% ferric citrate + 0.005% salicylic acid ternary combination is used, effectively increasing the number of lateral roots, promoting root growth, and the survival rate after 7 - day transplantation is as high as over 97%.

[0063] Example 5 An example of the method for rooting in vitro of tissue - cultured seedlings of Siraitia grosvenorii according to the present invention. In step 4, 0.02% trehalose and 0.001% nano - zinc oxide are added to the 1 / 2MS medium.

[0064] Specifically, the culture medium was improved. The rooting pre-culture medium included: 1 / 2MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L IBA + 0.5 g / L activated carbon + 0.02% trehalose + 0.001% nano-zinc oxide (particle size 50 nm). During rooting pre-culture, robust tissue culture seedlings were inoculated into the improved medium and cultured for 14 days at 22 ± 1 °C under 1500 lx light (9 hours / day).

[0065] The pretreatment steps were the same as those in Step 1-3 of Example 1 (culturing from sterile explants to tissue culture seedlings).

[0066] The subsequent treatment was the same as that in Step 5-6 of Example 1 (gradient acclimatization and transplantation).

[0067] Comparative Example 5 Three groups were set as the control group: Comparative Example 5A: 1 / 2MS medium without any additives (basic formula); Comparative Example 5B: Only 0.02% trehalose was added; Comparative Example 5C: Only 0.001% nano-zinc oxide was added.

[0068] Other steps were exactly the same as those in Example 5.

[0069] The test results were as follows: Table 5 Among them, the lower the electrolyte leakage rate, the better the cell membrane integrity; MDA is a marker of membrane lipid peroxidation, and the lower the value, the less the damage; SOD (superoxide dismutase) scavenges free radicals; proline maintains cell osmotic balance.

[0070] Trehalose can form a hydrogen bond network to protect membrane proteins, reduce the electrolyte leakage rate, maintain osmotic balance at the same time, and increase the proline accumulation. Nano-zinc oxide induces the expression of antioxidant enzymes and increases the SOD activity. Zinc ions can activate cold-resistant genes and improve the survival rate at low temperatures.

[0071] It can be seen that low temperature induces membrane lipid peroxidation. After treatment at 5 °C for 24 h, the survival rate of Comparative Example 6A was only 43.6%. By adding 0.02% trehalose to maintain the membrane structure and 0.001% nano-zinc oxide to induce antioxidant enzymes during rooting pre-culture in the present invention, the membrane lipid peroxidation product (MDA) was significantly reduced, and the electrolyte leakage rate decreased by 49.2%; the simulated survival rate during the late spring cold snap was increased to 88.3%, and the recovery growth time (new root elongation ≥ 1 mm or new leaf unfolding) was shortened to 2.5 days after experiencing the simulated late spring cold snap experiment.

[0072] Example 6 Select the robust tissue culture seedlings obtained in step 3 of Example 1 (seedling height 3.0 ± 0.5 cm, with 4 - 5 leaves), place them in MS buffer solution (pH 5.8, containing 0.01% Tween - 80) with 0.03 μM methyl jasmonate (MeJA), and soak them with shaking at 25°C for 45 minutes (rotation speed 120 rpm).

[0073] Prepare the cold - hardening enhanced medium: containing 1 / 2 MS basal salts, 0.1 mg / L 6 - BA, 0.2 mg / L NAA, 0.2 mg / L IBA, 0.5 g activated carbon, 0.02% trehalose and 0.003% nano - zinc oxide (particle size 40 ± 5 nm) per liter. Dispense it into 200 mL tissue culture bottles (50 mL per bottle) and sterilize at 121°C for 20 minutes. Inoculate the treated tissue culture seedlings into this medium and pre - culture them at 22 ± 1°C, under a light intensity of 1500 μmol·m -2 ·s -1 illumination (red - blue light ratio 3:1), with a photoperiod of 9 hours per day. Start low - temperature cycling acclimation at 14:00 - 16:00 on the 3rd, 5th, and 7th days of pre - culture: the incubator is cooled from 22°C to 4 ± 0.5°C at a rate of 1°C / min, maintained for 2 hours, and then warmed back to 22°C at a rate of 0.5°C / min, for a total of 3 cycles.

[0074] The tissue culture seedlings that have completed pre - culture are subjected to three - stage gradient hardening (simultaneous spectral regulation of the opening angle of the lid and humidity control) according to step 5 of Example 1. After washing the root medium, soak them in a composite solution of 280 mg / L ABT rooting powder + 0.01% ferric citrate + 0.005% salicylic acid for 10 minutes. Transplant them into a substrate of peat moss: vermiculite = 3:1 (initial humidity 45 ± 5%), and cover with a blue sunshade net with a light transmittance of 75% 50 cm above the seedling bed. Turn on the LED supplementary lighting system at 6:00 - 8:00 and 16:00 - 18:00 every day to provide light with a ratio of red light (630 ± 5 nm) to far - red light (730 ± 5 nm) of 2:1 and a total light intensity of 400 ± 50 μmol·m -2 ·s -1 illumination. During the supplementary lighting period, maintain the air humidity at 80 ± 5% through an atomizer.

[0075] Use an artificial climate chamber to simulate 5°C for 24 hours (humidity 85%) to conduct the late spring cold snap response test. The results are as follows: Table 6 At the same time, use natural weak light ≤ 100 μmol·m -2 ·s -1 to simulate the rainy environment under the regulation of the late spring cold snap, and conduct the continuous rainy adaptability test. Use no supplementary lighting as the control group. The results are as follows: Table 7 Among them, the anthrax incidence rate is the proportion of the diseased spot area observed 7 days after inoculating with anthrax bacterial spores.

[0076] It can be seen that in Example 6 under the unified stress condition (5°C / 24 h), the cold resistance is comprehensively improved. Its survival rate is increased to 93.5%. Its MDA is decreased by 40% compared with Example 5, SOD is increased by 59% compared with Example 5, and the recovery growth is shortened to 45 hours. The weak light adaptation is also significantly improved. The photosynthetic efficiency is increased by 64% compared with no supplementary lighting, and the anthrax inhibition is increased by 73%.

[0077] Example 7 An example of the method for rooting in vitro of Siraitia grosvenorii tissue culture seedlings of the present invention. The disinfection treatment in Step 1 is as follows: Alternately treat with 0.1% HgCl2 solution and 5% sodium hypochlorite solution at a volume ratio of 3:1, and rinse with sterile water pulse for 10 seconds (pulse water pressure 0.12 MPa) at intervals of each treatment.

[0078] Specifically, the disinfection treatment: The young stem segments are soaked in 1% washing powder for 10 minutes and rinsed with running water for 20 minutes; Alternate treatment: The first round: soak in 0.1% HgCl2 solution (containing 0.5 mL / 100 L Tween-20) for 3 minutes and then pulse rinse (sterile water 2 L / min, 10 seconds); The second round: soak in 5% sodium hypochlorite solution for 1 minute and then pulse rinse (the same as above); Repeat the above cycle 2 times (total sterilization time 8 minutes); Finally, rinse with sterile water 3 times and blot dry with sterile filter paper.

[0079] Subsequent steps: The same as Steps 2-6 of Example 1 (inducing culture to transplanting).

[0080] Control Example 7 Divided into two groups for comparison: Control Example 7A: Single 0.1% HgCl2 treatment for 8 minutes (without alternation and without pulse rinse); Control Example 7B: The alternate treatment is the same, and the pulse rinse is deleted (only static soaking and rinsing).

[0081] Other steps are exactly the same as those of Example 7.

[0082] Table 8 The residual of a single disinfectant inhibits the activity of explants, and the browning rate exceeds 40%. For the 3:1 alternation of HgCl2 and sodium hypochlorite and 2 L / min pulse rinse (10 seconds each time) in the examples of the present invention, the residual mercury of the disinfectant is reduced by 79%, and the browning rate of the explants is reduced to 8.3%; the contamination rate is controlled at 5.2%, and the survival seedling rate reaches 96.4%.

[0083] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved.

Claims

1. A method for rooting in vitro of Siraitia grosvenorii tissue culture seedlings, characterized in that, It includes the following steps: Step 1: Select young branches of Siraitia grosvenorii to prepare stem segments with axillary buds, and obtain aseptic explants through surface disinfection treatment; Step 2: Inoculate the aseptic explants into an MS medium containing 0.4 - 0.6 mg / L of 6-benzylaminopurine and 0.1 - 0.3 mg / L of naphthaleneacetic acid, and culture for 18 - 24 days at 24 - 26 °C under a light intensity of 1200 - 1800 lx to obtain induced buds; Step 3: Transfer the induced buds to an MS medium containing 0.2 - 0.4 mg / L of 6-benzylaminopurine, 0.03 - 0.07 mg / L of naphthaleneacetic acid, and 0.8 - 1.2 g / L of activated carbon for culture to obtain tissue culture seedlings; Step 4: Select strong tissue culture seedlings and inoculate them into a 1 / 2MS medium containing 0.08 - 0.12 mg / L of 6-benzylaminopurine, 0.15 - 0.25 mg / L of naphthaleneacetic acid, 0.15 - 0.25 mg / L of indolebutyric acid, and 0.3 - 0.7 g / L of activated carbon, and perform pre-rooting culture for 12 - 16 days at 21 - 23 °C under a light intensity of 1200 - 1800 lx; Step 5: Gradient acclimatization of the tissue culture seedlings after pre-rooting culture is carried out in a seedling-raising bottle, including: a) In the first stage, the opening angle of the lid is 15° - 20°, and the humidity inside the bottle is maintained at 85 - 90% for 24 hours; b) In the second stage, the opening angle of the lid is 45° - 50%, and the humidity is reduced to 75 - 80% and maintained for 48 hours; c) In the third stage, the lid is completely opened, and the surface water film is evaporated in an environment with an air flow rate of 0.3 - 0.5 m / s, and the medium is washed off; Step 6: Immerse the washed tissue culture seedlings in 200 - 350 mg / L of ABT rooting solution and transplant them into the substrate.

2. The method according to claim 1, wherein Spectral regulation is carried out synchronously during the gradient acclimatization in Step 5: a) In the first stage, a red light-dominated mode is adopted, where the proportion of the wavelength 630 ± 5 nm is 80%, and the light intensity is 800 - 1000 lx; b) In the second stage, a red and blue composite spectral mode is adopted: the intensity ratio of the red light wavelength 630 nm to the blue light wavelength 450 nm is 3:1, and the total light intensity is 1500 - 1800 lx; c) In the third stage, natural light is dynamically simulated: from 6:00 to 10:00, the proportion of blue light is 20% - 35%, from 10:00 to 16:00, the red and blue ratio is 2:1, and from 16:00 to 18:00, the proportion of red light is 75%.

3. The method according to claim 1, wherein Synergistic strengthening treatment is also included in Step 5: Three days before the end of pre-rooting culture, an intensifying solution containing 0.005% salicylic acid and 0.01% manganese sulfate by mass-volume percentage is injected into the 1 / 2MS medium every 12 hours, and each injection lasts for 5 - 15 minutes; after taking out the tissue culture seedlings, a negative pressure intermittent soaking method is used for treatment, including placing the roots in the ABT rooting solution, applying a negative pressure of -0.05 MPa for 20 - 40 seconds and then releasing, and repeating 3 - 7 times; immediately after the negative pressure treatment, transfer them to a buffer solution containing 0.05% Tween-80 by volume percentage and 1% sorbitol by mass-volume percentage, and oscillate and soak for 2 - 5 minutes.

4. The method according to claim 3, characterized in that The ABT rooting solution in Step 6 is a composite solution of ABT No. 1 rooting powder at 200 - 350 mg / L, ferric citrate at 0.01% (mass / volume percentage), and salicylic acid at 0.005% (mass / volume percentage).

5. The method according to claim 1, wherein In Step 4, directly add trehalose at 0.02% (mass / volume percentage) and nano-zinc oxide at 0.001% (mass / volume percentage) to the 1 / 2MS medium.

6. The method according to any one of claims 1-5, characterized in that During the rooting pre-culture stage in Step 4, cold resistance induction is carried out simultaneously, including: Before pre-culture, soak the tissue culture seedlings in an MS buffer solution containing 0.01 - 0.03 μM methyl jasmonate and oscillate for 30 - 60 minutes; add a composite of nano-zinc oxide at 0.001 - 0.003% (mass / volume percentage) and trehalose at 0.02% (mass / volume percentage) to the 1 / 2MS medium; starting from the 3rd day of pre-culture, apply low-temperature stress at 4°C for 2 hours every day, and then resume culturing at 22°C.

7. The method according to claim 6, wherein Weak light compensation is implemented after transplantation in Step 6: a) Cover the surface of the transplanting substrate with a blue sunshade net with a light transmittance of 70% - 80%. b) Turn on the LED supplementary lighting system from 6:00 to 8:00 and from 16:00 to 18:00 every day, providing a ratio of red light intensity at 630 ± 5 nm to far-red light intensity at 730 ± 5 nm of 2:1, with a total light intensity of 300 - 500 μmol·m -2 ·s -1 ; maintain the air humidity at 80 ± 5% during the supplementary lighting period.

8. The method according to claim 1, wherein The disinfection treatment in Step 1 is to alternately treat with a 0.1% (mass / volume percentage) HgCl2 solution and a 5% (mass / volume percentage) sodium hypochlorite solution at a volume ratio of 3:1, and rinse with sterile water for 5 - 20 seconds between each treatment.

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