A composite growth regulator and its application in tissue culture seedlings

Abstract

The present invention relates to a composite growth regulator and its application in tissue culture seedlings, belonging to the technical field of plant growth regulators. The composite growth regulator of the present invention comprises the following components by weight: 10-12 parts of Jerusalem artichoke stem and leaf extract, 10-15 parts of Brevibacillus laterosporus fermentation product, 3-5 parts of glabridin, 8-12 parts of α-lipoic acid, 6-10 parts of indolebutyric acid, 4-8 parts of taurine, and 20-25 parts of sodium alginate. Adding the composite growth regulator in the process of plant tissue culture can significantly improve the rooting rate of tissue culture seedlings, and high-quality regenerated adventitious roots can be obtained, which can improve the survival rate of tissue culture seedlings and prevent the vitrification phenomenon of tissue culture seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth regulators and relates to a composite growth regulator and application thereof in tissue culture seedlings. Background Art

[0002] Plant in vitro tissue regeneration is a complex process. There are problems such as difficulty in cell dedifferentiation, poor sensitivity to plant growth regulators, inhibition of growth by secondary metabolites, and high pollution rate during the rooting process, which lead to low rooting rate, poor rooting quality or deformed seedlings in some plant tissue culture seedlings. Therefore, improving the rooting rate of plant tissue culture seedlings and obtaining high-quality regenerated adventitious roots has become a research hotspot.

[0003] Hyperhydricity refers to the translucent phenomenon similar to water immersion that occurs in plant seedlings during their growth process. It is different from the "vitrification" in embedding vitrification cryopreservation and vitrification ultra-low temperature detoxification. Vitrification is a pathological phenomenon of plant physiological disorders. In tissue culture, repeated subculture will cause severe vitrification. After vitrification, seedlings will show symptoms such as poor development, low differentiation ability and difficulty in survival. Once vitrification is formed, it will be difficult to restore to normal plants, affecting the proliferation efficiency of tissue culture seedlings. Summary of the invention

[0004] The main purpose of the present invention is to provide a composite growth regulator and its application in tissue culture seedlings. Adding the composite growth regulator in the process of plant tissue culture can significantly improve the rooting rate of tissue culture seedlings, obtain high-quality regenerated adventitious roots, improve the survival rate of tissue culture seedlings, and prevent the vitrification phenomenon of tissue culture seedlings.

[0005] The present invention adopts the following technical solutions to achieve the above purpose:

[0006] A composite growth regulator, comprising the following components in parts by weight:

[0007] 10-12 parts of Jerusalem artichoke stem and leaf extract, 10-15 parts of Brevibacillus laterosporus fermentation product, 3-5 parts of glabridin, 8-12 parts of alpha-lipoic acid, 6-10 parts of indolebutyric acid, 4-8 parts of taurine and 20-25 parts of sodium alginate.

[0008] The preparation method of the Jerusalem artichoke stem and leaf extract comprises the following steps:

[0009] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, adjusting the pH to 5.0-5.5, extracting by distillation, and using the distilled product and the mixed solution after distillation for later use;

[0010] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 50-60%, adjusting the pH to 8.0-8.5, refluxing and extracting, and using the extract for later use;

[0011] Step C, concentrating the extract of step B under reduced pressure, separating with macroporous resin, eluting with ethanol solution with pH 7.5-8.0, collecting the eluate eluted with 50-70% ethanol solution, removing residual ethanol from the eluate, and mixing with the distillation product obtained in step A to obtain Jerusalem artichoke stem and leaf extract.

[0012] The preparation method of the Brevibacillus laterosporus fermentation product comprises the following steps:

[0013] Step a, inoculating Brevibacillus laterosporus seed liquid into a liquid culture medium containing 40-50% of Chlorella, 10-12% of yeast extract powder, 15-20% of bran, 0.03-0.05% of potassium dihydrogen sulfate, 0.01-0.03% of magnesium sulfate, and 0.1-0.3% of cysteine, fermenting at 25-30° C. for 3-4 days, then adding lysozyme, performing enzymolysis at 40-50° C. for 1-1.5 days, performing ultrasonic treatment after the enzymolysis is completed, then placing in a homogenizer for crushing, filtering, and using the filtrate for standby use;

[0014] Step b, concentrating the filtrate obtained in step a under reduced pressure, separating by silica gel column, using ethyl acetate-n-butanol mixed solution as elution solvent, collecting the eluate, removing the organic solvent, and drying to obtain the fermentation product of Brevibacillus laterosporus.

[0015] Preferably, the OD value of the Brevibacillus laterosporus seed solution in step a is 0.3-0.5.

[0016] Preferably, the inoculation amount of the Brevibacillus laterosporus seed solution in step a is 10-15 mL / L.

[0017] Preferably, the added amount of lysozyme is 10-12 g / L.

[0018] Preferably, the lysozyme has an enzymatic activity of 100,000 U / g.

[0019] Preferably, the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol mixed solution in step b is 1:(0.04-0.06).

[0020] The present invention provides a method for preparing the composite growth regulator, which specifically comprises the following steps:

[0021] After dissolving sodium alginate in water, add Jerusalem artichoke stem and leaf extract, fermentation product of Brevibacillus laterosporus, glabridin and α-lipoic acid, stir at 40-50°C until the solution becomes viscous, add indolebutyric acid and taurine, mix well, and dry to obtain a composite growth regulator.

[0022] The present invention has the following beneficial effects:

[0023] 1. The composite growth regulator of the present invention is obtained by compounding the commonly used plant hormone indolebutyric acid and other compounds with plant extracts, microbial fermentation products, taurine and other compounds. The use of the composite growth regulator of the present invention can significantly improve the germination rate and proliferation rate in plant tissue culture; can significantly promote the root growth of tissue culture seedlings, improve the rooting rate, and the root length, root surface area, etc. are significantly better than those of tissue culture seedlings obtained by traditional plant tissue culture; at the same time, it can also effectively prevent the vitrification of tissue culture seedlings caused in the process of plant tissue culture, and has a certain recovery effect on vitrified tissue culture seedlings.

[0024] 2. The present invention optimizes the preparation process of Jerusalem artichoke extract and microbial fermentation product, and finally obtains effective active ingredients that promote the growth of tissue culture seedlings. In the preparation of Jerusalem artichoke extract, a volatile component with certain activity is obtained by distillation, and then extracted and purified in an alkaline environment to obtain an effective ingredient with biological activity; in the preparation process of microbial fermentation product, Brevibacillus laterosporus is fermented and cultured in a culture medium containing Chlorella and cysteine ​​to obtain a microbial fermentation product, and then the microorganism is destroyed by lysozyme, ultrasound, homogenization, etc., so that the intracellular and extracellular metabolites produced in the fermentation process are fully released, and at the same time, the components in Chlorella are decomposed by fermentation to release effective ingredients with biological activity.

[0025] 3. In the present invention, by effectively compounding Jerusalem artichoke extract, microbial fermentation products with glycyrrhizin, α-lipoic acid, taurine and other ingredients, combined with existing indolebutyric acid plant hormones, the synergistic effect of the various ingredients can significantly promote the growth of tissue culture seedlings, and the effect is better than that of traditional plant hormones. It can also effectively avoid the failure or poor quality of tissue culture seedlings caused by improper use of indolebutyric acid and other plant hormones. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope protected by the claims of this application. In the following implementation methods, the Brevibacillus laterosporus seed solution is obtained by independent cultivation in this laboratory, specifically, the Brevibacillus laterosporus strain with a preservation number of CGMCC 1.3097 purchased from the China General Microbiological Culture Collection Center is activated according to the instructions and expanded to OD value 0.3-0.5 to obtain; the enzyme used is an existing commercially available product, which can be purchased from online or offline channels.

[0027] Example 1

[0028] Preparation of Jerusalem artichoke stem and leaf extract:

[0029] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, adjusting the pH to 5.0, and extracting by distillation, and the distillation product and the mixed solution after distillation are reserved;

[0030] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 50% with anhydrous ethanol, adjusting the pH to 8.5, and extracting under reflux at 80° C., and setting the extract aside;

[0031] Step C, reducing the pressure and concentrating the extract of step B, loading the D101 macroporous adsorption resin, eluting with 90%, 70%, and 50% ethanol solutions with a pH of 8.0, collecting the eluates eluted with the 50% and 70% ethanol solutions, rotary evaporating the eluates to remove residual ethanol, mixing the eluates with the distillation product obtained in step A, and drying to obtain the Jerusalem artichoke stem and leaf extract.

[0032] Preparation of Brevibacillus laterosporus fermentation:

[0033] Step a, taking each component in a ratio of 50% by mass of Chlorella, 10% by mass of yeast extract powder, 20% by mass of bran, 0.05% by mass of potassium dihydrogen sulfate, 0.01% by mass of magnesium sulfate, and 0.3% by mass of cysteine, adding water to prepare a liquid culture medium, inoculating a seed solution of Brevibacillus laterosporus (OD value is 0.5) at an inoculation amount of 15 mL / L, fermenting at 30° C. for 3 days, after the fermentation is completed, adding 12 g / L of lysozyme with an enzyme activity of 100,000 U / g to the mixed fermentation solution, performing enzymolysis at 40° C. for 1.5 days, performing ultrasonic treatment after the enzymolysis is completed, and then placing the mixture in a homogenizer for crushing, filtering, and using the filtrate for standby use;

[0034] Step b, concentrating the filtrate obtained in step a under reduced pressure, loading the sample on a silica gel column, using a mixed solution of ethyl acetate: n-butanol in a volume ratio of 1:0.04 as an elution solvent, collecting the eluate, removing the organic solvent by rotary evaporation, and drying to obtain a fermentation product of Brevibacillus laterosporus.

[0035] Preparation of composite growth regulators:

[0036] After dissolving 25 parts of sodium alginate in water, add 12 parts of Jerusalem artichoke stem and leaf extract, 15 parts of Brevibacillus laterosporus fermentation product, 5 parts of glabridin, and 12 parts of α-lipoic acid, heat and stir at 50°C until the solution becomes viscous, add 6 parts of indolebutyric acid and 8 parts of taurine, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0037] Example 2

[0038] Preparation of Jerusalem artichoke stem and leaf extract:

[0039] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, adjusting the pH to 5.5, extracting by distillation, and using the distilled product and the mixed solution after distillation for later use;

[0040] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 60% with anhydrous ethanol, adjusting the pH to 8.0, and extracting under reflux at 70° C., and setting the extract aside;

[0041] Step C, concentrating the extract of step B under reduced pressure, loading D101 macroporous adsorption resin, eluting with 90%, 70%, and 50% ethanol solutions with a pH of 7.5, collecting the eluates eluted with 50% and 70% ethanol solutions, rotary evaporating the eluates to remove residual ethanol, mixing the eluates with the distillation product obtained in step A, and drying to obtain the Jerusalem artichoke stem and leaf extract.

[0042] Preparation of Brevibacillus laterosporus fermentation:

[0043] Step a, taking each component in a ratio of 40% by mass of Chlorella, 12% by mass of yeast extract powder, 15% by mass of bran, 0.03% by mass of potassium dihydrogen sulfate, 0.03% by mass of magnesium sulfate, and 0.1% by mass of cysteine, adding water to prepare a liquid culture medium, inoculating a seed solution of Brevibacillus laterosporus (OD value is 0.3) at an inoculation amount of 10 mL / L, fermenting at 25° C. for 4 days, after the fermentation is completed, adding 10 g / L of lysozyme with an enzyme activity of 100,000 U / g to the mixed fermentation solution, performing enzymolysis at 50° C. for 1 day, performing ultrasonic treatment after the enzymolysis is completed, and then placing the mixture in a homogenizer for crushing, filtering, and using the filtrate for standby use;

[0044] Step b, concentrating the filtrate obtained in step a under reduced pressure, loading the sample on a silica gel column, using a mixed solution of ethyl acetate: n-butanol in a volume ratio of 1:0.06 as an elution solvent, collecting the eluate, removing the organic solvent by rotary evaporation, and drying to obtain a fermentation product of Brevibacillus laterosporus.

[0045] Preparation of composite growth regulators:

[0046] After dissolving 20 parts of sodium alginate in water, add 10 parts of Jerusalem artichoke stem and leaf extract, 10 parts of Brevibacillus laterosporus fermentation product, 3 parts of glabridin, and 8 parts of α-lipoic acid, heat and stir at 40°C until the solution becomes viscous, add 10 parts of indolebutyric acid and 4 parts of taurine, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0047] Example 3

[0048] Preparation of Jerusalem artichoke stem and leaf extract:

[0049] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, adjusting the pH to 5.0, and extracting by distillation, and the distillation product and the mixed solution after distillation are reserved;

[0050] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 55% with anhydrous ethanol, adjusting the pH to 8.5, and extracting under reflux at 75° C., and setting the extract aside;

[0051] Step C, reducing the pressure and concentrating the extract of step B, loading the D101 macroporous adsorption resin, eluting with 90%, 70%, and 50% ethanol solutions with a pH of 8.0, collecting the eluate eluted with the 50-70% ethanol solution, rotary evaporating the eluate to remove residual ethanol, mixing the eluate with the distillation product obtained in step A, and drying to obtain the Jerusalem artichoke stem and leaf extract.

[0052] Preparation of Brevibacillus laterosporus fermentation:

[0053] Step a, taking each component in a ratio of 45% of Chlorella, 11% of yeast extract powder, 18% of bran, 0.04% of potassium dihydrogen sulfate, 0.02% of magnesium sulfate, and 0.2% of cysteine ​​according to mass fractions, adding water to prepare a liquid culture medium, inoculating Brevibacillus laterosporus seed solution (OD value is 0.43) according to an inoculation amount of 13 mL / L, fermenting at 30° C. for 3 days, after the fermentation is completed, adding 11 g / L of lysozyme with an enzyme activity of 100,000 U / g to the mixed fermentation solution, enzymolysis at 40° C. for 1 day, ultrasonic treatment after the enzymolysis is completed, and then crushing in a homogenizer, filtering, and using the filtrate for standby use;

[0054] Step b, concentrating the filtrate obtained in step a under reduced pressure, loading the sample on a silica gel column, using a mixed solution of ethyl acetate: n-butanol in a volume ratio of 1:0.05 as an elution solvent, collecting the eluate, removing the organic solvent by rotary evaporation, and drying to obtain a fermentation product of Brevibacillus laterosporus.

[0055] Preparation of composite growth regulators:

[0056] After dissolving 23 parts of sodium alginate in water, add 11 parts of Jerusalem artichoke stem and leaf extract, 13 parts of Brevibacillus laterosporus fermentation product, 4 parts of glabridin, and 10 parts of α-lipoic acid, heat and stir at 50°C until the solution becomes viscous, add 8 parts of indolebutyric acid and 6 parts of taurine, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0057] Comparative Example 1

[0058] Preparation of Jerusalem artichoke stem and leaf extract: Take fresh Jerusalem artichoke stem and leaf, add water to make a homogenate, use anhydrous ethanol to adjust the alcohol content to 55%, and adjust the pH to 8.5, reflux extraction at 75°C, concentrate the extract under reduced pressure and then dry to obtain the Jerusalem artichoke stem and leaf extract.

[0059] Preparation of Brevibacillus laterosporus fermentation product: taking each component in a proportion of 11% by mass of yeast extract powder, 18% by mass of bran, 0.04% by mass of potassium dihydrogen sulfate, 0.02% by mass of magnesium sulfate, and 0.2% by mass of cysteine, adding water to prepare a liquid culture medium, inoculating Brevibacillus laterosporus seed solution (OD value is 0.4) at an inoculum volume of 13 mL / L, fermenting at 30°C for 3 days, after the fermentation is completed, adding 11 g / L of lysozyme with an enzyme activity of 100,000 U / g to the mixed fermentation solution, performing enzymolysis at 40°C for 1 day, performing ultrasonic treatment after the enzymolysis is completed, and then crushing in a homogenizer, filtering, and drying the filtrate to obtain the Brevibacillus laterosporus fermentation product.

[0060] Preparation of composite growth regulators:

[0061] After dissolving 23 parts of sodium alginate in water, add 11 parts of Jerusalem artichoke stem and leaf extract, 13 parts of Brevibacillus laterosporus fermentation product, 4 parts of glabridin, and 10 parts of α-lipoic acid, heat and stir at 50°C until the solution becomes viscous, add 8 parts of indolebutyric acid and 6 parts of taurine, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0062] Comparative Example 2

[0063] Preparation of Jerusalem artichoke stem and leaf extract:

[0064] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, distilling and extracting, and using the distilled mixed solution for later use;

[0065] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 55% with anhydrous ethanol, extracting under reflux at 75° C., and setting the extract aside;

[0066] Step C, reducing the pressure and concentrating the extract of step B, loading the D101 macroporous adsorption resin, eluting with ethanol solutions with mass fractions of 90%, 70%, 50%, 30%, and 10%, collecting the eluate eluted with 10-30% ethanol solution, rotary evaporating the eluate to remove residual ethanol, and drying to obtain the Jerusalem artichoke stem and leaf extract.

[0067] Preparation of Chlorella Extract:

[0068] Chlorella is added with water to prepare a homogenate, subjected to reflux extraction, filtered to obtain an extract, and dried to obtain a Chlorella extract.

[0069] Preparation of composite growth regulators:

[0070] After dissolving 23 parts of sodium alginate in water, add 11 parts of Jerusalem artichoke stem and leaf extract, 13 parts of chlorella extract, 4 parts of glabridin, and 10 parts of α-lipoic acid, heat and stir at 50°C until the solution becomes viscous, add 8 parts of indolebutyric acid and 6 parts of taurine, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0071] Comparative Example 3

[0072] Preparation of Jerusalem artichoke stem and leaf extract:

[0073] Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, distilling and extracting, and using the distilled product and the mixed solution after distillation for later use;

[0074] Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 55% with anhydrous ethanol, extracting under reflux at 75° C., and setting the extract aside;

[0075] Step C, concentrating the extract of step B under reduced pressure, loading D101 macroporous adsorption resin, eluting with ethanol solutions with mass fractions of 90% and 70%, collecting the eluate eluted with 70% ethanol solution, rotary evaporating the eluate to remove residual ethanol, mixing with the distillation product obtained in step A, and drying to obtain the Jerusalem artichoke stem and leaf extract.

[0076] Preparation of Brevibacillus laterosporus fermentation:

[0077] Step a, taking each component according to the mass fraction of 45% chlorella, 11% yeast extract powder, 18% bran, 0.04% potassium dihydrogen sulfate, 0.02% magnesium sulfate, and 0.2% cysteine, adding water to prepare a liquid culture medium, inoculating Brevibacillus laterosporus seed solution (OD value is 0.4) according to the inoculation amount of 13mL / L, fermenting at 30°C for 3 days, after the fermentation is completed, adding 11g / L of lysozyme with an enzyme activity of 100,000U / g to the mixed fermentation solution, enzymolysis at 40°C for 1 day, ultrasonic treatment after the enzymolysis is completed, and then crushing in a homogenizer, filtering, and using the filtrate for standby use;

[0078] Step b, concentrating the filtrate obtained in step a under reduced pressure, loading the sample on a silica gel column, using ethyl acetate as an eluting solvent, collecting the eluate, rotary evaporating to remove the organic solvent, and drying to obtain a fermentation product of Brevibacillus laterosporus.

[0079] Preparation of composite growth regulators:

[0080] After dissolving 23 parts of sodium alginate in water, add 11 parts of Jerusalem artichoke stem and leaf extract and 13 parts of Brevibacillus laterosporus fermentation product, heat and stir at 50°C until the solution becomes viscous, add 8 parts of indolebutyric acid, mix well, and dry to obtain a composite growth regulator. The 1 part can be a conventional weight unit such as 1g or 1kg.

[0081] 1. Effects of compound growth regulators on vitrification of tissue culture seedlings

[0082] 1. Materials and Methods

[0083] 1.1 Experimental Materials

[0084] In this experiment, "Grande" asparagus and "Hongyan" strawberry were selected as experimental materials. The experiment was carried out in the tissue culture room of our school's experimental base.

[0085] 1.2 Experimental process

[0086] Experiment 1: After routine disinfection and other operations, the collected strawberry stem tips were peeled off under a dissecting microscope and inoculated into conventional culture medium. Primary culture was carried out at 25±2℃ and 12h / d of light. The test tube seedlings with consistent growth and no contamination in the primary culture were inoculated in different culture media for 30 days. Two seedlings were inoculated in each bottle, and 20 bottles were grouped. The culture was continued at 25±2℃ and 12h / d of light. The growth differences of strawberry seedlings in different culture media were observed, and the vitrification rate of strawberry tissue culture seedlings was calculated.

[0087] Vitrification rate of strawberry tissue culture seedlings (%) = number of vitrified seedlings after 30 days of culture / total number of tissue culture seedlings × 100%

[0088] Experiment 2: After routine disinfection, the collected asparagus stem tips were cut into pieces with an area of ​​about 5 mm under a dissecting microscope. 2 Small pieces of asparagus were inoculated in conventional culture medium for subculture. Callus tissues after three generations of subculture were taken and inoculated into different culture media respectively, with 2 inoculated in each bottle. 20 bottles made a group. They were cultured at 25±2℃ and 12h / d of light. The differences in the growth of asparagus seedlings in different culture media were observed, and the vitrification rate of asparagus tissue culture seedlings was calculated.

[0089] Asparagus tissue culture seedling vitrification rate (%) = number of vitrified seedlings after 30 days of culture / total number of tissue culture seedlings × 100%

[0090] Experiment 3: The vitrified tissue culture seedlings produced during the tissue culture proliferation culture were taken and inoculated into different culture media, with 2 strains in each bottle, and each 10 bottles formed a group. They were cultured at 25±2℃ and 12h / d of light for 40 days. After the culture was completed, the growth status of the tissue culture seedlings was analyzed and the vitrification recovery rate was calculated.

[0091] Recovery rate of vitrification of strawberry tissue culture seedlings (%) = (total number of seedlings - number of vitrified seedlings) / total number of seedlings × 100%

[0092] The following are the culture medium recipes used in each experiment:

[0093] Medium 1: conventional medium + compound growth regulator of Example 1 (0.5 mg / L);

[0094] Medium 2: conventional medium + compound growth regulator of Example 2 (0.5 mg / L);

[0095] Medium 3: conventional medium + compound growth regulator of Example 3 (0.5 mg / L);

[0096] Medium 4: conventional medium + compound growth regulator of comparative example 1 (0.5 mg / L);

[0097] Medium 5: conventional medium + composite growth regulator of comparative example 2 (0.5 mg / L);

[0098] Medium 6: conventional medium + composite growth regulator of comparative example 3 (0.5 mg / L);

[0099] Medium 7: conventional medium + indolebutyric acid (0.3 mg / L) + naphthaleneacetic acid (0.2 mg / L);

[0100] Medium 8: Conventional medium.

[0101] The conventional culture medium formula is: using MS as the basic culture medium, adding 7 g / L agar and 30 g / L sucrose, and adjusting the pH to about 5.8.

[0102] 2. Results and Analysis

[0103] From the results in Table 1 and Table 2, it can be seen that adding the composite growth regulator of the present invention can significantly reduce the incidence of vitrified seedlings in strawberry and asparagus tissue culture, which is beneficial to the normal growth of tissue culture seedlings.

[0104] From the results in Table 3, it can be seen that the use of the composite growth regulator of the present invention can significantly improve the recovery rate of strawberry vitrified seedlings.

[0105] Table 1 Comparison of vitrification of strawberry tissue culture seedlings in Experiment 1

[0106]

[0107] Table 2 Comparison of vitrification of asparagus tissue culture seedlings in Experiment 2

[0108]

[0109] Table 3 Comparison of vitrification recovery of strawberry tissue culture seedlings in Experiment 3

[0110]

[0111] 2. Effect of compound growth agent on the growth of strawberry tissue culture seedlings

[0112] 1. Materials and Methods

[0113] 1.1 Materials

[0114] In this experiment, "Hongyan" strawberry was selected as the experimental material, and the experiment was carried out in the tissue culture room of our school's experimental base.

[0115] 1.2 Experimental process

[0116] Experiment 4: Select the stem tips of freshly picked strawberry runners, sterilize them and inoculate them in different culture media, 3 in each bottle, 1 group of 20 bottles, and culture them at 25±2℃ and 12h / d of light for 30 days. Count the number of sprouts, calculate the germination rate and proliferation coefficient, and observe the growth.

[0117] Germination rate (%) = number of germinations / number of inoculated seeds × 100%

[0118] Proliferation coefficient = total number of proliferation and differentiation / number of inoculations

[0119] Experiment 5: Select the stem tips of freshly harvested strawberry runners, and after routine disinfection and other operations, inoculate them in conventional culture medium, and culture them at 25±2℃ and 12h / d of light for 30 days. Select tissue culture seedlings with basically the same growth conditions and transfer them to different culture media. Inoculate 3 plants in each bottle, and 1 group of 20 bottles. Culture them at 25±2℃ and 12h / d of light for 30 days. Randomly select 10 plants from each group, count the number of rooted plants, measure the root length, root surface area, and number of root tips of the tissue culture seedlings, and calculate the rooting coefficient.

[0120] Rooting coefficient = number of roots / number of inoculated plants

[0121] The following are the culture medium recipes used in each experiment:

[0122] Medium 1: conventional medium + compound growth regulator of Example 1 (0.5 mg / L);

[0123] Medium 2: conventional medium + compound growth regulator of Example 2 (0.5 mg / L);

[0124] Medium 3: conventional medium + compound growth regulator of Example 3 (0.5 mg / L);

[0125] Medium 4: conventional medium + compound growth regulator of comparative example 1 (0.5 mg / L);

[0126] Medium 5: conventional medium + composite growth regulator of comparative example 2 (0.5 mg / L);

[0127] Medium 6: conventional medium + composite growth regulator of comparative example 3 (0.5 mg / L);

[0128] Medium 7: conventional medium + indolebutyric acid (0.3 mg / L) + naphthaleneacetic acid (0.2 mg / L);

[0129] Medium 8: Conventional medium.

[0130] The conventional culture medium formula described in the above culture medium is: MS is used as the basic culture medium, 7 g / L agar and 30 g / L sucrose are added, and the pH is adjusted to about 5.8.

[0131] 2. Results and Analysis

[0132] From the results in Table 4, it can be seen that the composite growth regulator of the present invention can significantly improve the germination rate and proliferation rate of strawberry tissue culture seeds, and the tissue culture seedlings are in good growth state.

[0133] Table 4 Comparison of the growth of strawberry tissue culture seedlings in Experiment 4

[0134]

[0135] From the results in Table 5, it can be seen that the composite growth regulator of the present invention can significantly promote the root growth of tissue culture seedlings, improve the rooting rate, and the root length, root surface area, and number of root tips are significantly higher, and the rooting of tissue culture seedlings is good.

[0136] Table 5 Growth of tissue culture seedlings in Experiment 5

[0137]

Claims

1. A composite growth regulator, characterized in that: In parts by weight, it includes the following ingredients: 10-12 parts of Jerusalem artichoke stem and leaf extract, 10-15 parts of Brevibacillus laterosporus fermentation product, 3-5 parts of glabridin, 8-12 parts of α-lipoic acid, 6-10 parts of indolebutyric acid, 4-8 parts of taurine, and 20-25 parts of sodium alginate; The preparation method of the Jerusalem artichoke stem and leaf extract comprises the following steps: Step A, taking fresh Jerusalem artichoke stems and leaves, adding water to prepare a homogenate, adjusting the pH to 5.0-5.5, extracting by distillation, and using the distilled product and the mixed solution after distillation for later use; Step B, adjusting the alcohol content of the mixed solution after distillation in step A to 50-60%, adjusting the pH to 8.0-8.5, refluxing and extracting, and using the extract for later use; Step C, concentrating the extract of step B under reduced pressure, separating with a macroporous resin, eluting with an ethanol solution with a pH of 7.5-8.0, collecting the eluate eluted with 50-70% ethanol solution, removing residual ethanol from the eluate, and mixing with the distillation product obtained in step A to obtain a Jerusalem artichoke stem and leaf extract; The preparation method of the Brevibacillus laterosporus fermentation product comprises the following steps: Step a, inoculating a Brevibacillus laterosporus seed solution at an inoculation amount of 10-15 mL / L into a liquid culture medium containing 40-50% of Chlorella, 10-12% of yeast extract powder, 15-20% of bran, 0.03-0.05% of potassium dihydrogen sulfate, 0.01-0.03% of magnesium sulfate, and 0.1-0.3% of cysteine, fermenting at 25-30° C. for 3-4 days, then adding 10-12 g / L of lysozyme, performing enzymolysis at 40-50° C. for 1-1.5 days, performing ultrasonic treatment after the enzymolysis is completed, then placing in a homogenizer for crushing, filtering, and using the filtrate for standby use; the Brevibacillus laterosporus seed solution is obtained by expanding and culturing the Brevibacillus laterosporus strain with a preservation number of CGMCC 1.3097 of the China General Microbiological Culture Collection Center; Step b, concentrating the filtrate obtained in step a under reduced pressure, separating by silica gel column, using a mixed solution of ethyl acetate and n-butanol in a volume ratio of 1:(0.04-0.06) as an elution solvent, collecting the eluate, removing the organic solvent, and drying to obtain a fermentation product of Brevibacillus laterosporus.

2. The composite growth regulator according to claim 1, characterized in that The OD value of the Brevibacillus laterosporus seed solution in step a is 0.3-0.

5.

3. The composite growth regulator according to claim 1, characterized in that The enzyme activity of the lysozyme is 100,000 U / g.

4. The composite growth regulator according to claim 1, characterized in that The preparation method of the composite growth regulator comprises the following steps: After dissolving sodium alginate in water, add Jerusalem artichoke stem and leaf extract, fermentation product of Brevibacillus laterosporus, glabridin and α-lipoic acid, stir at 40-50°C until the solution becomes viscous, add indolebutyric acid and taurine, mix well, and dry to obtain a composite growth regulator.

5. Use of the composite growth regulator according to any one of claims 1 to 4 in plant tissue culture.

Images