Tissue culture seedling raising method for siraitia grosvenorii seeds

Abstract

The invention relates to a tissue culture seedling raising method of siraitia grosvenorii seeds, and belongs to the technical field of agricultural biology. Aiming at the problems of low seed germination rate, high pollution rate, insufficient proliferation efficiency and unstable transplanting survival rate in traditional seedling culture, the method comprises the following steps: removing fruit bags of siraitia grosvenorii, air-drying, stripping seed shells to obtain shelled seeds, washing with running water, disinfecting with alcohol and HgCl2 solution and the like, sequentially inoculating the shelled seeds into starting, proliferation, seedling strengthening and rooting culture media containing different hormones, and culturing to obtain the siraitia grosvenorii seedlings. Conditions such as illumination and temperature in each stage are controlled, and tissue culture seedlings with roots are hardened and then planted in a mixed substrate for cultivation. The method can improve the germination rate, the proliferation efficiency and the transplanting survival rate of the siraitia grosvenorii seed tissue culture seedlings, and is suitable for efficient seedling culture of siraitia grosvenorii seeds.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to a tissue culture seedling method of Momordica grosvenori seeds. Technical Background

[0002] Monk fruit, scientifically known as Siraitia grosvenorii (Swingle.) C. Jeffrey, is a perennial vine of the genus Momordica in the Cucurbitaceae family. It is a medicinal and sweetener plant unique to China and a traditional Chinese medicine listed in the Pharmacopoeia. The fruit of the fruit is used as medicine and contains various saponins, which have cough-relieving, expectorant, and laxative effects. These saponins are the main active ingredient in the antitussive effect of the fruit. Furthermore, mogrosides are a low-calorie, high-sweetness natural sweetener, 300 to 400 times sweeter than sucrose, making it the world's sweetest. It is also low in calories and non-toxic, making it suitable for people with diabetes and obesity. In recent years, an increasing number of companies in my country have joined the ranks of monk fruit production, resulting in a significant market demand for monk fruit seedlings (tissue culture seedlings). Since Momordica grosvenori is a male-female plant, the ratio of male to female in seedlings is 3:7, and only female plants can bear fruit, while male plants are only used to collect pollen. Therefore, in the large-scale production of Momordica grosvenori, tissue culture is mainly used as a seedling raising method, and seed seedlings are rarely used. However, in genetic breeding, different hybrid combinations need to be selected for breeding and superior plant selection. At this time, seed seedlings need to be used for optimization. Therefore, seed seedling raising is still one of the important breeding methods in the genetic breeding of Momordica grosvenori. At present, the germination force and germination rate of conventional Momordica grosvenori seed seedling raising process are relatively low, which greatly limits the application of Momordica grosvenori seeds in breeding.

[0003] The inherent biological characteristics of Luo Han Guo seeds are the primary obstacle: the hard and dense seed shell not only physically hinders germination, but the germination inhibitors contained in it cause the seeds to be deeply dormant and have a low germination rate. Conventional physical shelling or chemical treatments attempt to overcome this obstacle, but it is difficult to balance effectiveness and safety - excessive treatment can easily damage the embryo, while insufficient treatment cannot break dormancy, and the operation has poor reproducibility. Even more difficult is the problem of endogenous microbial contamination carried by seeds. Conventional disinfection schemes (such as single disinfectants or short-term treatments) are difficult to completely eliminate stubborn microorganisms lurking in the micro-gaps of the seed shell or in the embryonic tissue, resulting in frequent outbreaks of contamination in the subsequent sterile culture stage and significant fluctuations in the seedling rate.

[0004] In the tissue culture proliferation link, existing technologies generally encounter the contradiction of efficiency and quality that are difficult to strike a balance. Although relying on a single or high-concentration cytokinin (such as 6-BA) can induce bud differentiation, it is easy to cause bud vitrification, deformity or overcrowding. This morphological abnormality is due to the imbalance of endogenous hormones and the inhibitory effects of metabolic toxins such as ethylene and phenols accumulated during the culture process. Conventional culture media lack an active clearance mechanism for these harmful substances, resulting in a low effective proliferation coefficient and insufficient bud robustness, which poses a hidden danger for subsequent separation operations and seedling growth stages.

[0005] The rooting stage is long limited by the quality of root development and functional defects. Traditional rooting programs often induce two types of problematic roots due to a single type of auxin or improper concentration ratio: one is that the roots are sparse and thin, and the conducting tissue is underdeveloped; the other is that the root tips are enlarged and calloused, and the absorption function is lost. This root defect directly weakens the adaptability to transplanting. In addition, the tissue culture seedlings are in a stable artificial environment for a long time, and their leaves have a thin wax layer and weakened stomatal regulation function. They are extremely sensitive to sudden changes in environmental temperature and humidity and pathogen infection. Existing seedling hardening methods mostly use simple open training, lacking gradual regulation of temperature, humidity and light intensity, resulting in the seedlings dying in large numbers after transplanting due to uncontrolled transpiration or damping-off disease, and it is difficult to break through the bottleneck of survival rate.

[0006] The deeper problem lies in the fragmented optimization of technical links. Seed germination, proliferation, seedling growth and rooting stages have very different physiological requirements, but existing methods often design the culture medium formula and environmental parameters for each stage in isolation, ignoring the physiological continuity between stages. For example, the morphology of the buds formed in the germination stage directly affects the efficiency of proliferation and separation; the quality of the buds in the proliferation stage restricts the effect of seedling growth; and the sudden conversion of hormone formulas can easily cause growth retardation. This disconnect leads to poor stability of the overall process and significant differences in seedling rates between batches, making it difficult to meet the stringent requirements of large-scale production for consistency and reliability. The above problems are closely linked and together constitute the technical barriers to the industrial application of Momordica grosvenori tissue culture seedlings. Summary of the Invention

[0007] The present invention solves the following technical problems:

[0008] Address the systemic technical deficiencies in traditional tissue culture of Momordica grosvenori seedlings, including low seed germination rates, high contamination rates, insufficient proliferation efficiency, and unstable transplant survival rates. Specifically, these deficiencies include: a hard seed shell that makes dormancy difficult to break; conventional disinfection that struggles to remove stubborn endophytes; single-hormone induction that easily produces deformed buds; poor root development during the rooting phase; and a lack of a gradual environmental acclimatization process. These issues result in long seedling production cycles and low seedling success rates, hindering industrial application.

[0009] To address the issues of uneven bud differentiation and poor germination synchronization during the seed germination stage, conventional constant temperature and constant light cultivation leads to disrupted embryo metabolism and large fluctuations in germination rates. A breakthrough is needed in the technical bottleneck of coordinated regulation of light quality and temperature to achieve precise induction of germination physiological activities.

[0010] This overcomes the drawbacks of incomplete dormancy release and insufficient bioactivity of the priming culture medium. In existing technologies, seed pre-activation is missing, and residual endogenous germination inhibitors lead to delayed germination. Furthermore, the culture medium lacks stress-inducing ingredients, making the embryo susceptible to oxidative damage.

[0011] Addressing three key pain points during the proliferation phase: ① Overcrowding of buds, leading to separation damage; ② Accumulation of metabolic toxins inhibiting lateral bud differentiation; and ③ High bud vitrification rates. Simultaneous optimization of culture medium composition, environmental control, and separation procedures is required.

[0012] Corrects the imbalance in phototropism caused by a fixed light source. In traditional culture, shoots are tilted in polarity due to unidirectional light exposure, affecting the uniformity of proliferation and the quality of subsequent seedlings.

[0013] Eliminate the technical barrier of unstable activated carbon adsorption performance. Ordinary activated carbon is prone to residual alkaline impurities, changing the pH of the culture medium, and has poor adsorption selectivity, resulting in ineffective adsorption of key growth factors.

[0014] Avoid oxidative damage and pathogen infection caused by cutting. Conventional separation has a high rate of browning on the cut surface and a high rate of contamination on the subculture, leading to loss of effective buds.

[0015] Optimize the utilization of coconut water's active ingredients. Unpurified coconut water contains a large proportion of macromolecular impurities (such as pectin and phenols), which interfere with the biological activity of small molecule growth factors and reduce the proliferation coefficient.

[0016] Alleviate the stress disorder of clustered bud growth caused by the sudden change of hormone ratio. Direct transfer to high-auxin seedling medium can easily lead to callus at the base of the bud, delaying the elongation of new buds.

[0017] To achieve the above-mentioned object of the present invention, the present invention provides a method for tissue culture seedlings of Momordica grosvenori seeds, comprising the following steps:

[0018] S1: After the fruit capsule of the monk fruit is removed and air-dried, the seed shell is peeled off to obtain shelled seeds;

[0019] S2: Rinse the seeds with running water for 25-35 minutes, transfer them to a clean bench, and immerse them in 70-80% alcohol by volume for 1-3 minutes and 0.08-0.12% HgCl2 solution for 8-12 minutes, with continuous shaking. Rinse them with sterile water 4-6 times, and dry the surface moisture with sterile filter paper.

[0020] S3: Inoculate the sterilized seeds into a starter culture medium consisting of MS + 0.08-0.12 mg / L 6-benzylaminopurine + 0.15-0.25 mg / L gibberellin GA3, and culture under conditions of 1100-1300 lx, 14-18 hours of light per day, and 24-26°C for 7-10 days until the seeds germinate and form buds;

[0021] S4: transferring the buds to a proliferation medium containing MS + 0.30-0.40 mg / L 6-benzylaminopurine + 0.15-0.25 mg / L naphthaleneacetic acid (NAA), and culturing them under the same light and temperature conditions for 10-14 days to obtain clustered buds;

[0022] S5: inoculating the clustered buds into a seedling-strengthening medium containing MS, 0.15-0.25 mg / L 6-benzylaminopurine, and 0.40-0.60 mg / L naphthaleneacetic acid (NAA), and culturing for 10-14 days to form strong seedlings;

[0023] S6: Transfer the healthy seedlings to 1 / 2MS + naphthaleneacetic acid NAA 0.15-0.25 mg / L + indolebutyric acid IAA

[0024] 0.15-0.25 mg / L rooting medium, culture for 10-14 days to obtain seed tissue culture seedlings with roots;

[0025] S7: The rooted seed tissue culture seedlings are removed from the culture room, exposed to the air for 3-5 days in an environment with a humidity of ≥85%, and then the residual culture medium on the roots is cleaned and planted in a mixed matrix of sterilized seedling soil and vermiculite in a volume ratio of 2:1-4:1, with a shading rate of 50-70%, and the matrix humidity is maintained at 60-80%. The seedlings are cultivated for 25-35 days to obtain the amplified and propagated Momordica grosvenori seedlings.

[0026] Preferably, the illumination culture process in step S3 of the present invention is further optimized as follows:

[0027] The segmented spectrum control mode is adopted. In the daily light cycle, the first 6 hours are irradiated with a composite of far-red light with a wavelength of 660-680nm and blue light with a wavelength of 440-460nm, with a light intensity ratio of 2:1-3:1;

[0028] In the middle 6 hours, the irradiation is switched to a composite irradiation of red light with a wavelength of 620-640nm and green light with a wavelength of 520-540nm, with a light intensity ratio of 4:1-5:1;

[0029] The last 2-6 hours were returned to full-spectrum white light exposure;

[0030] Intermittent low temperature treatment is implemented once every 24 hours, and the culture temperature is instantly reduced to 18-20℃ and maintained for 30-40 minutes. During the treatment, the light intensity is increased to 1500-1800lx.

[0031] Preferably, in step S3 of the present invention, the seeds are pre-activated before inoculation: the shelled seeds are soaked in a mixed solution containing 0.5-1.5 mmol / L calcium nitrate and 0.1-0.2 mmol / L salicylic acid, and cultured with shaking at 4-6°C in the dark for 12-18 hours with an oscillation frequency of 80-100 rpm; 0.02-0.04 mg / L brassinolide and 0.1-0.3 mg / L proline composite synergist are also added to the start-up culture medium, and the synergist is aseptically added through a 0.22 μm filter membrane after the culture medium is sterilized.

[0032] Preferably, the preparation of the proliferation culture medium in step S4 of the present invention further includes the following improvements:

[0033] 0.05-0.15 g / L activated carbon powder, pretreated coconut water concentrate, and 1.5-2.5 g / L osmotic agent are added to MS basal medium, wherein the activated carbon powder has a particle size of 80-120 mesh; the concentrate is obtained by the following steps: fresh coconut water is purified by DEAE-cellulose chromatography, mixed with 0.5-1.0 g / L sodium alginate, and spray-dried to form a microcapsule powder, the addition amount of which is 0.8-1.2% of the total mass of the medium;

[0034] The proliferation culture stage adopts a day and night temperature difference control mode, specifically: the temperature is maintained at 25-26°C and the light intensity is 1100-1300lx for the first 12 hours of each day, and the temperature is lowered to 20-22°C and the light is turned off for the next 12 hours, and the control cycle is continued until the end of the culture;

[0035] The concentration ratio of 6-benzylaminopurine to naphthylacetic acid (NAA) is 1.5:1-2.0:1, and the total concentration of the two is controlled within the range of 0.45-0.65 mg / L.

[0036] The cluster bud separation operation is completed in a clean bench. During separation, a scalpel blade is used to cut the base of the bud at an angle of 45-60 degrees, retaining callus tissue with a bud length of 2-3 mm. After cutting, the buds are immediately soaked in sterile water containing 0.01-0.03% vitamin C for 5-8 minutes.

[0037] Preferably, the proliferation culture stage in step S4 of the present invention further includes the following optimization:

[0038] In the day and night temperature difference control mode, the light source uses a red and blue composite LED lamp. The light intensity ratio of the red light wavelength of 630-660nm and the blue light wavelength of 440-460nm is 3:1-5:1, and the main light source direction is automatically switched every 6 hours, with a switching angle of 15-30°.

[0039] Preferably, the activated carbon powder of the present invention is pretreated before addition: the activated carbon is immersed in a 0.1-0.3 mol / L sodium citrate solution and ultrasonically treated for 20-30 minutes at an ultrasonic frequency of 40 kHz, then rinsed three times with a pH 5.6-6.0 MS buffer solution, and vacuum dried at 60° C. to a water content of ≤5%.

[0040] Preferably, the scalpel blade of the present invention needs to be pre-soaked in a sterile solution containing 0.1-0.2 mg / L methyl jasmonate for 10-15 minutes before cutting, and after cutting, the base of the bud is smeared with a composite protective agent composed of 1-2% trehalose and 0.05-0.1% chitosan, and the composite protective agent is filtered and sterilized.

[0041] Preferably, the preparation of the coconut water extract of the present invention adds a purification step: the filtered coconut water is passed through a DEAE-cellulose chromatography column, gradient eluted with a 0.05-0.15M NaCl solution, and the fraction with a conductivity of 2.5-3.5mS / cm is collected. The osmotic pressure is adjusted to 280-320mOsm / kg before mixing with the MS inorganic salt component.

[0042] Preferably, after the clustered buds of the present invention are separated, they are inoculated into a temporary transition culture medium supplemented with 0.02-0.04 mg / L sodium selenite, pre-cultured for 48-72 hours under the conditions of light intensity of 800-1000 lx and temperature of 22-24° C., and then transferred to a seedling-strengthening culture medium.

[0043] The present invention has at least the following beneficial effects:

[0044] Through seed shell peeling and step-by-step disinfection (alcohol + HgCl2 oscillation treatment), the contamination rate is reduced and the germination rate is greatly improved;

[0045] Precise hormone ratios for the four stages of priming / proliferation / seedling strengthening / rooting (such as 6-BA and NAA concentration gradient design) ensure robust shoots and significantly increase the proliferation coefficient;

[0046] During the seedling hardening stage, a high humidity environment and a mixed substrate (seedling soil: vermiculite = 3:1) were combined to stabilize the transplant survival rate at over 85%.

[0047] The entire process cycle is shortened to 60-70 days, which is significantly more efficient than traditional methods.

[0048] The segmented spectrum (far-red / blue light → red / green light → white light) cooperates with intermittent low temperature to activate the phytochrome PHYB and low-temperature response genes, and the bud synchronization is increased to more than 90%;

[0049] A specific wavelength combination (660nm far-red light + 450nm blue light) promoted embryonic axis elongation, and the standard deviation of germination uniformity was reduced from 2.3 days to 0.8 days.

[0050] Calcium nitrate-salicylic acid pre-activation released seed dormancy and significantly reduced the endogenous abscisic acid content;

[0051] The brassinolide and proline compound enhancer increased the activity of antioxidant enzymes and significantly reduced the germ damage rate.

[0052] The adsorption rate of the activated carbon-coconut water microcapsule system reaches over 95%, and the occurrence rate of vitrified buds is reduced to less than 7%;

[0053] When the temperature difference between day and night was controlled (25℃ / 20℃) and the hormone ratio was controlled (6-BA:NAA=1.8:1), the number of lateral buds increased to 4.2 per bud.

[0054] 45° inclined cutting preserved callus tissue, and the separation survival rate increased to 98%. Vitamin C treatment inhibited the browning of the wound surface.

[0055] The light source is deflected periodically by 15-30 degrees to eliminate phototropic bending, the deviation angle of bud uprightness is less than 5 degrees, and the proliferation uniformity is improved by 32%.

[0056] Sodium citrate pretreatment removed metal impurities from activated carbon, improved pH stability (fluctuation ≤ 0.2), and reduced auxin adsorption by 40%.

[0057] Soaking in methyl jasmonate enhanced the antibacterial property of the blade, and the composite protective agent (trehalose + chitosan) formed a physical barrier, reducing the wound infection rate to 3%.

[0058] DEAE chromatography accurately removes large molecular impurities, with a retention rate of >88% for active small molecules, and osmotic pressure regulation avoids cell osmotic stress.

[0059] Sodium selenite transition culture induced the expression of antioxidant enzymes (glutathione peroxidase activity increased 2.3 times), and the elongation rate of new shoots accelerated by 41% after transfer to seedling growth medium. DETAILED DESCRIPTION

[0060] The present invention is described in further detail below so that those skilled in the art can implement the invention with reference to the description.

[0061] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0062] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0063] <Example 1>

[0064] A method for tissue culture seedlings of Momordica grosvenori seeds comprises the following steps:

[0065] S1: After the fruit capsule of the monk fruit is removed and air-dried, the seed shell is peeled off to obtain shelled seeds;

[0066] S2: Rinse the seeds with running water for 30 minutes, transfer them to a clean bench, and immerse them in 75% alcohol for 2 minutes and 0.08-0.12% HgCl2 solution for 10 minutes, with continuous shaking. Rinse them with sterile water five times, and dry the surface moisture with sterile filter paper.

[0067] S3: The sterilized seeds were inoculated into a starter culture medium consisting of MS + 0.1 mg / L 6-benzylaminopurine + 0.2 mg / L gibberellin GA3, and cultured at a light intensity of 1200 lx, 16 hours of light per day, and a temperature of 25°C for 8 days until the seeds germinated and formed buds;

[0068] S4: transferring the buds to a proliferation medium containing MS, 0.35 mg / L 6-benzylaminopurine, and 0.20 mg / L naphthaleneacetic acid (NAA), and culturing them under the same light and temperature conditions for 12 days to obtain clustered buds;

[0069] S5: inoculating the clustered buds into a seedling-strengthening medium containing MS, 0.2 mg / L 6-benzylaminopurine, and 0.5 mg / L naphthaleneacetic acid (NAA), and culturing for 12 days to form strong seedlings;

[0070] S6: The healthy seedlings were transferred to a rooting medium containing 1 / 2MS, 0.2 mg / L naphthaleneacetic acid (NAA), and 0.2 mg / L indolebutyric acid (IAA), and cultured for 12 days to obtain seed tissue culture seedlings with roots;

[0071] S7: The rooted seed tissue culture seedlings are moved out of the culture room, exposed to the air for 4 days in an environment with a humidity of ≥85%, and then the residual culture medium on the roots is cleaned and planted in a mixed matrix of sterilized seedling soil and vermiculite in a volume ratio of 3:1, with a shading rate of 60%, and the matrix humidity is maintained at 70%. The seedlings are cultivated for 30 days to obtain the amplified and propagated Momordica grosvenori seedlings.

[0072] <Example 2>

[0073] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 1>, except that the illumination culture process in step S3 is further optimized as follows:

[0074] The system uses a segmented spectrum control mode. During the daily light cycle, the first 6 hours of the system use a combination of far-red light with a wavelength of 660-680nm and blue light with a wavelength of 440-460nm, with a light intensity ratio of 2.5:1.

[0075] In the middle 6 hours, the irradiation was switched to a composite irradiation of red light with a wavelength of 620-640nm and green light with a wavelength of 520-540nm, with a light intensity ratio of 4.5:1;

[0076] The last 4 hours were returned to full-spectrum white light exposure;

[0077] Intermittent low temperature treatment was implemented once every 24 hours, and the culture temperature was instantly reduced to 19°C and maintained for 35 minutes. During the treatment, the light intensity was increased to 1650 lx.

[0078] <Example 3>

[0079] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 2>, except that, in step S3, the seeds are pre-activated before inoculation: the shelled seeds are immersed in a mixed solution containing 1.0 mmol / L calcium nitrate and 0.15 mmol / L salicylic acid, and cultured with shaking at 5°C in the dark for 15 hours at an shaking frequency of 90 rpm; and a composite synergist of 0.03 mg / L brassinolide and 0.2 mg / L proline is further added to the start-up culture medium, and the synergist is aseptically added through a 0.22 μm filter membrane after the culture medium is sterilized.

[0080] <Example 4>

[0081] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 3>, except that the preparation of the proliferation culture medium in step S4 further includes the following improvements:

[0082] 0.1 g / L activated carbon powder, pretreated coconut water concentrate, and 2.0 g / L osmotic agent were added to MS basal medium, wherein the activated carbon powder had a particle size of 100 mesh; the concentrate was obtained by the following steps: fresh coconut water was purified by DEAE-cellulose chromatography, mixed with 0.75 g / L sodium alginate, and spray-dried to form a microcapsule powder, the addition amount of which was 1% of the total mass of the medium;

[0083] The proliferation culture stage adopts a day and night temperature difference control mode, specifically: the temperature is maintained at 25°C and the light intensity is 1200lx for the first 12 hours of each day, and the temperature is lowered to 21°C and the light is turned off for the next 12 hours, and the control cycle is continued until the end of the culture;

[0084] The concentration ratio of 6-benzylaminopurine to naphthylacetic acid (NAA) is 1.7:1, and the concentration of 6-benzylaminopurine is within the range of 0.35 mg / L.

[0085] The clustered bud separation operation is completed in a clean bench. During separation, a scalpel blade is used to cut the base of the bud at a 55° tilt angle, retaining callus tissue with a bud length of 2-3 mm. After cutting, the buds are immediately soaked in sterile water containing 0.02% vitamin C for 6 minutes.

[0086] <Example 5>

[0087] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 4>, except that the proliferation culture stage in step S4 further includes the following optimization:

[0088] In the day and night temperature difference control mode, the light source uses a red and blue composite LED lamp. The light intensity ratio of the red light wavelength of 630-660nm and the blue light wavelength of 440-460nm is 4:1, and the main light source direction is automatically switched every 6 hours with a switching angle of 23°.

[0089] <Example 6>

[0090] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 5>, except that the activated carbon powder is pretreated before addition: the activated carbon is immersed in a 0.2 mol / L sodium citrate solution and ultrasonically treated for 25 minutes at an ultrasonic frequency of 40 kHz, followed by rinsing three times with a pH 5.8 MS buffer solution, and vacuum drying at 60°C to a moisture content of ≤5%.

[0091] <Example 7>

[0092] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 6>, except that the scalpel blade needs to be pre-soaked in a sterile solution containing 0.15 mg / L methyl jasmonate for 2 minutes before cutting, and after cutting, a composite protective agent composed of 1.5% trehalose and 0.075% chitosan is applied to the base of the bud, and the composite protective agent is filtered and sterilized.

[0093] <Example 8>

[0094] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 7>, except that a purification step is added to the preparation of the coconut water extract: the filtered coconut water is passed through a DEAE-cellulose chromatography column, gradient eluted with a 0.10M NaCl solution, and a fraction with a conductivity of 3.0 mS / cm is collected. The osmotic pressure is adjusted to 300 mOsm / kg before mixing with the MS inorganic salt component.

[0095] <Example 9>

[0096] A method for tissue culture seedlings of Momordica grosvenori seeds adopts the method of <Example 9>, except that after the clustered buds are separated, they are inoculated into a temporary transition culture medium supplemented with 0.03 mg / L sodium selenite, pre-cultured for 60 hours under a light intensity of 900 lx and a temperature of 23°C, and then transferred to a seedling-strengthening culture medium.

[0097] <Comparative Example 1>

[0098] A method for tissue culture seedlings of Momordica grosvenori seeds comprises the following steps:

[0099] S1: After the fruit capsule of the monk fruit is removed and air-dried, the seed shell is peeled off to obtain shelled seeds;

[0100] S2: Rinse the seeds with running water for 30 minutes, transfer them to a clean bench, and immerse them in 75% alcohol for 2 minutes and 0.1% HgCl2 solution for 10 minutes, with continuous shaking. Rinse them with sterile water five times, and dry the surface moisture with sterile filter paper.

[0101] S3: The sterilized seeds were inoculated into a starter culture medium consisting of MS + 0.1 mg / L 6-benzylaminopurine and cultured at a light intensity of 1200 lx, 16 hours of light per day, and a temperature of 25°C for 8 days until the seeds germinated and formed buds;

[0102] S4: transferring the buds to a proliferation medium containing MS and 0.35 mg / L 6-benzylaminopurine, and culturing them for 12 days under the same light and temperature conditions to obtain clustered buds;

[0103] S5: inoculating the clustered buds into a seedling-strengthening medium containing MS and 0.5 mg / L naphthaleneacetic acid (NAA), and culturing for 12 days to form strong seedlings;

[0104] S6: The healthy seedlings were transferred to a rooting medium containing 1 / 2MS and 0.2 mg / L indolebutyric acid (IAA) and cultured for 12 days to obtain seed tissue culture seedlings with roots;

[0105] S7: The rooted seed tissue culture seedlings are moved out of the culture room, exposed to the air for 4 days in an environment with a humidity of ≥85%, and then the residual culture medium on the roots is cleaned and planted in a mixed matrix of sterilized seedling soil and vermiculite in a volume ratio of 3:1, with a shading rate of 60%, and the matrix humidity is maintained at 70%. The seedlings are cultivated for 30 days to obtain the amplified and propagated Momordica grosvenori seedlings.

[0106] The data of the germination stage, proliferation stage, rooting and transplanting are shown in Tables 1-3 below.

[0107] Table 1 Data of the budding stage

[0108]

[0109]

[0110] As can be seen from the results in Table 1, the contamination rate of Comparative Example 1 reached 32.1±3.2%, while in the present invention, after the Momordica grosvenori seeds were rinsed with running water in step S2, they were disinfected with 70-80% alcohol by volume and 0.08-0.12% HgCl2 solution by mass concentration and continuous oscillation in a stepwise disinfection manner. The contamination rate of Example 9 was reduced to 4.2±0.5%, effectively removing the endophytic microorganisms carried by the seeds, solving the problem that conventional disinfection was difficult to completely remove stubborn endophytes. In comparative example 1, the germination rate was only 58.3±4.1%, and the standard deviation of germination synchronization was 2.31±0.25 days. In step S3, the present invention adopts a segmented spectrum control mode, uses specific wavelength light for composite irradiation at different time periods during the daily light cycle, and performs intermittent low-temperature treatment every 24 hours. At the same time, the seeds are pre-activated with a mixed solution containing calcium nitrate and salicylic acid before inoculation, and the start-up culture medium is added with a composite synergist of brassinolide and proline. In example 9, the germination rate increased to 96.7±1.4%, and the standard deviation of germination synchronization decreased to 0.79±0.05 days, activating the photosensitive pigment PHYB and the low-temperature response gene, releasing seed dormancy, improving germination synchronization, and solving the problems of low seed germination rate and poor germination synchronization.

[0111] Table 2 Data of the value-added stage

[0112]

[0113]

[0114] As shown in Table 2, the proliferation coefficient of Comparative Example 1 was 2.1 ± 0.3 per bud, the vitrification rate was 35.6 ± 4.2%, and the ethylene concentration was 0.28 ± 0.03 μL / L. In step S4 of the present invention, pretreated activated carbon powder, purified coconut water concentrate, etc. were added to the proliferation culture medium, a day and night temperature difference control mode was adopted to control the concentration ratio and total concentration of 6-benzylaminopurine and naphthaleneacetic acid NAA, the cluster bud separation operation was optimized, a red and blue light composite LED lamp was used and the main light source direction was switched regularly, and an ethylene suppression slow-release system was added. In Example 9, the proliferation coefficient increased to 4.6 ± 0.6 per bud, the vitrification rate decreased to 6.1 ± 0.8%, and the ethylene concentration decreased to 0.04 ± 0.005 μL / L. These measures absorb metabolic toxins, retain the active ingredients of coconut water, inhibit ethylene toxicity, eliminate the imbalance in phototropic growth of buds caused by the direction of light source, solve problems such as excessive clustering of buds, accumulation of metabolic toxins, high vitrification rate of buds and imbalance in phototropic growth during the proliferation stage, and improve proliferation efficiency and bud quality.

[0115] Table 3 Rooting and transplanting data

[0116]

[0117]

[0118] As shown in Table 3, in Comparative Example 1, the number of roots was 3.2 ± 0.4, the root length was 1.8 ± 0.3 cm, and the transplant survival rate was 65.2 ± 4.3%. In step S6, the robust seedlings were transferred to a 1 / 2 MS rooting medium containing naphthaleneacetic acid (NAA) and indolebutyric acid (IAA) for cultivation. In step S7, the rooted seedlings were hardened in an open environment with a humidity of ≥ 85% and then planted in a mixed matrix of sterilized nursery soil and vermiculite in a specific ratio. In Example 9, the number of roots increased to 6.2 ± 0.7, the root length increased to 4.6 ± 0.5 cm, and the transplant survival rate rose to 93.8 ± 1.4%. The optimized auxin ratio promoted root development. The environmental regulation during the hardening stage and the use of the mixed matrix enhanced the adaptability of the seedlings to the environment, solving the problems of poor root development during the rooting stage and the easy death of seedlings due to sudden environmental changes during the hardening process, thereby improving root quality and transplant survival rate.

[0119] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A tissue culture seedling method for Momordica grosvenori seeds, characterized in that: The following steps are involved: S1: After the fruit capsule of the monk fruit is removed and air-dried, the seed shell is peeled off to obtain shelled seeds; S2: Rinse the seeds with running water for 25-35 minutes, transfer them to a clean bench, and immerse them in 70-80% alcohol by volume for 1-3 minutes and 0.08-0.12% HgCl2 solution for 8-12 minutes, with continuous shaking. Rinse them with sterile water 4-6 times, and dry the surface moisture with sterile filter paper. S3: Inoculate the sterilized seeds into a starter culture medium consisting of MS + 0.08-0.12 mg / L 6-benzylaminopurine + 0.15-0.25 mg / L gibberellin GA3, and culture under conditions of 1100-1300 lx, 14-18 hours of light per day, and 24-26°C for 7-10 days until the seeds germinate and form buds; S4: transferring the buds to a proliferation medium containing MS + 0.30-0.40 mg / L 6-benzylaminopurine + 0.15-0.25 mg / L naphthaleneacetic acid (NAA), and culturing them under the same light and temperature conditions for 10-14 days to obtain clustered buds; S5: inoculating the clustered buds into a seedling-strengthening medium containing MS, 0.15-0.25 mg / L 6-benzylaminopurine, and 0.40-0.60 mg / L naphthaleneacetic acid (NAA), and culturing for 10-14 days to form strong seedlings; S6: transferring the healthy seedlings to a rooting medium containing 1 / 2MS, 0.15-0.25 mg / L naphthaleneacetic acid (NAA), and 0.15-0.25 mg / L indolebutyric acid (IAA), and culturing for 10-14 days to obtain rooted seedlings; S7: The rooted seed tissue culture seedlings are removed from the culture room, exposed to the air for 3-5 days in an environment with a humidity of ≥85%, and then the residual culture medium on the roots is cleaned and planted in a mixed matrix of sterilized seedling soil and vermiculite in a volume ratio of 2:1-4:1, with a shading rate of 50-70%, and the matrix humidity is maintained at 60-80%. The seedlings are cultivated for 25-35 days to obtain the amplified and propagated Momordica grosvenori seedlings.

2. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 1, wherein The illumination culture process in step S3 is further optimized as follows: The segmented spectrum control mode is adopted. In the daily light cycle, the first 6 hours are irradiated with a composite of far-red light with a wavelength of 660-680nm and blue light with a wavelength of 440-460nm, with a light intensity ratio of 2:1-3:1; In the middle 6 hours, the irradiation is switched to a composite irradiation of red light with a wavelength of 620-640nm and green light with a wavelength of 520-540nm, with a light intensity ratio of 4:1-5:1; The last 2-6 hours were returned to full-spectrum white light exposure; Intermittent low temperature treatment is implemented once every 24 hours, and the culture temperature is instantly reduced to 18-20℃ and maintained for 30-40 minutes. During the treatment, the light intensity is increased to 1500-1800lx.

3. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 2, wherein In step S3, the seeds are pre-activated before inoculation: the shelled seeds are soaked in a mixed solution containing 0.5-1.5 mmol / L calcium nitrate and 0.1-0.2 mmol / L salicylic acid, and cultured with shaking at 4-6°C in the dark for 12-18 hours with an shaking frequency of 80-100 rpm; 0.02-0.04 mg / L brassinolide and 0.1-0.3 mg / L proline composite synergist are also added to the start-up culture medium, and the synergist is aseptically added through a 0.22 μm filter membrane after the culture medium is sterilized.

4. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 1, wherein The preparation of the proliferation culture medium in step S4 also includes the following improvements: 0.05-0.15 g / L activated carbon powder, pretreated coconut water concentrate, and 1.5-2.5 g / L osmotic agent are added to MS basal medium, wherein the activated carbon powder has a particle size of 80-120 mesh; the concentrate is obtained by the following steps: fresh coconut water is purified by DEAE-cellulose chromatography, mixed with 0.5-1.0 g / L sodium alginate, and spray-dried to form a microcapsule powder, the addition amount of which is 0.8-1.2% of the total mass of the medium; The proliferation culture stage adopts a day and night temperature difference control mode, specifically: the temperature is maintained at 25-26°C and the light intensity is 1100-1300lx for the first 12 hours of each day, and the temperature is lowered to 20-22°C and the light is turned off for the next 12 hours, and the control cycle is continued until the end of the culture; The concentration ratio of 6-benzylaminopurine to naphthylacetic acid (NAA) is 1.5:1-2.0:1, and the total concentration of the two is controlled within the range of 0.45-0.65 mg / L. The cluster bud separation operation is completed in a clean bench. During separation, a scalpel blade is used to cut the base of the bud at an angle of 45-60 degrees, retaining callus tissue with a bud length of 2-3 mm. After cutting, the buds are immediately soaked in sterile water containing 0.01-0.03% vitamin C for 5-8 minutes.

5. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 4, wherein The proliferation culture stage in step S4 further includes the following optimizations: In the day and night temperature difference control mode, the light source uses a red and blue composite LED lamp. The light intensity ratio of the red light wavelength of 630-660nm and the blue light wavelength of 440-460nm is 3:1-5:1, and the main light source direction is automatically switched every 6 hours, with a switching angle of 15-30°.

6. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 4, wherein: The activated carbon powder was pretreated before addition: the activated carbon was immersed in a 0.1-0.3 mol / L sodium citrate solution and ultrasonically treated for 20-30 minutes at an ultrasonic frequency of 40 kHz, then rinsed three times with a pH 5.6-6.0 MS buffer solution, and vacuum dried at 60° C. to a water content of ≤5%.

7. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 4, wherein: The scalpel blade needs to be pre-soaked in a sterile solution containing 0.1-0.2 mg / L methyl jasmonate for 10-15 minutes before cutting. After cutting, the base of the bud is smeared with a composite protective agent composed of 1-2% trehalose and 0.05-0.1% chitosan, and the composite protective agent is filtered and sterilized.

8. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 4, wherein: The preparation of the coconut water extract includes an additional purification step: passing the filtered coconut water through a DEAE-cellulose chromatography column, gradient eluting with a 0.05-0.15M NaCl solution, collecting fractions with a conductivity of 2.5-3.5mS / cm, and adjusting the osmotic pressure to 280-320mOsm / kg before mixing with the MS inorganic salt component.

9. The tissue culture seedling method of Momordica grosvenori seeds as claimed in claim 4, wherein: After the clustered buds are separated, they are inoculated into a temporary transition culture medium supplemented with 0.02-0.04 mg / L sodium selenite, pre-cultured for 48-72 hours under the conditions of light intensity of 800-1000 lx and temperature of 22-24° C., and then transferred into a seedling-strengthening culture medium.

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