Method for cultivating radix curcumae in liquid medium and application thereof

Abstract

The application provides a method for breeding radix curcumae in liquid medium and application thereof, and belongs to the technical field of plant culture. The method comprises the following steps: (1) taking radix curcumae seeds, washing, and then sequentially using alcohol disinfection, sodium hypochlorite solution immersion, sterile water immersion and washing to obtain disinfected seeds; (2) inoculating the disinfected seeds into liquid medium, and culturing the sterile seeds on the liquid medium to obtain sterile tissue culture seedlings; (3) cutting the sterile tissue culture seedlings into stem segments with a length of 2-3 cm, each of the stem segments having at least one pair of axillary buds, and inoculating the stem segments into the liquid medium of the same composition in step (2) to carry out tissue culture seedling subculture. The method has good tissue culture effect on radix curcumae, can simultaneously achieve excellent cultivation effects in the germination rate of seeds, the average germination rate of tissue culture seedlings, the growth height and the differentiation rate, and has high research value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant culture technology, specifically relating to a method for breeding turmeric in liquid culture medium and its application. Background Technology

[0002] Turmeric (Curcuma aromatica), also known as yellow turmeric, broad turmeric, and heavenly bamboo yellow, is a perennial herb belonging to the ginger family (Zingiberaceae). Turmeric has extremely high medicinal value. In Traditional Chinese Medicine, it is mainly used to promote blood circulation and relieve pain, regulate qi and relieve depression, and clear heat and cool the blood, treating symptoms such as chest and abdominal pain, irregular menstruation, and injuries from falls and blows. Its main active ingredient is curcumin, which possesses various biological activities including anti-inflammatory, antioxidant, antibacterial, and anticancer properties. Turmeric roots and stems are edible and commonly used in cooking, especially in Southeast Asian cuisine, adding a unique aroma and yellow color to food. Ground turmeric roots and stems can be used to make a natural dye for textile dyeing, especially for yellow tones. Turmeric extract is also used in skincare products due to its antioxidant and anti-inflammatory properties, contributing to skin health, reducing inflammation, and promoting wound healing.

[0003] Tissue culture is a highly efficient method of asexual reproduction, which can improve propagation efficiency while preserving the superior traits of the mother plant, playing an important role in rapid seedling propagation and superior variety selection. Developing turmeric tissue culture technology is a quick and effective way to solve the problem of turmeric propagation. In current technologies, MS medium is usually used as the basic medium for turmeric tissue culture, but the effect on turmeric tissue culture is not good. It is impossible to achieve good cultivation results in four aspects at the same time: seed germination rate, average germination rate of tissue culture seedlings, growth height, and differentiation rate. This leads to various types of waste, not only reflected in economic benefits but also in the loss of resources, time, and opportunity costs. Therefore, there is an urgent need for a highly efficient breeding method for turmeric, which has extremely high research value and application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a method for breeding turmeric in liquid culture medium and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for breeding turmeric in a liquid culture medium, the method comprising the following steps:

[0007] (1) Take turmeric seeds, wash them, and then disinfect them with alcohol, soak them in sodium hypochlorite solution, soak them in sterile water and rinse them in turn to obtain disinfected seeds;

[0008] (2) Inoculate the sterilized seeds onto a liquid culture medium and germinate the sterile seeds on the liquid culture medium to obtain sterile tissue culture seedlings.

[0009] The liquid culture medium comprises at least ulmoides ferment and banana hydrolysate.

[0010] The liquid culture medium contains the following components: 25-27 g / L of *Ulva prostrata* ferment, 12-16 g / L of banana enzymatic hydrolysate, 20-24 g / L of sucrose, 1200-1400 mg / L of potassium nitrate, 1100-1300 mg / L of ammonium nitrate, 120-150 mg / L of potassium dihydrogen phosphate, 5-7 mg / L of zinc sulfate, 0.21-0.24 mg / L of sodium molybdate, 23-24 mg / L of disodium ethylenediaminetetraacetate, and 300-350 mg / L of magnesium sulfate heptahydrate. L, calcium chloride dihydrate 400-430 mg / L, potassium iodide 0.44-0.52 mg / L, boric acid 4-5 mg / L, manganese sulfate tetrahydrate 20-21 mg / L, ferrous sulfate 19-20 mg / L, inositol 95-97 mg / L, glycine 1.3-1.7 mg / L, and a hormone, wherein the hormone is composed of 1.2-1.3 mg / L of 6-benzylaminopurine, 0.09-0.13 mg / L of indolebutyric acid, and 0.15-0.18 mg / L of naphthaleneacetic acid.

[0011] The method for preparing the *Ulva prostrata* ferment is as follows:

[0012] Step 1: Wash and dry the Ulva lactuca, then pulverize it into ultrafine powder to obtain Ulva lactuca powder;

[0013] Step 2: Sterilize the Ulva micropowder by irradiation, add 4-5 times the weight of sterile water, and add 1%-3% of the weight of the Ulva micropowder with compound bacteria. The compound bacteria are a mixture of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium lactis in a mass ratio of (1.2-1.5):1:(0.5-0.8). Perform anaerobic fermentation at 34-37℃ for 72-96 hours, then sterilize to obtain the Ulva fermented product.

[0014] The bacterial strains used in this invention are commercially available products.

[0015] Lactobacillus plantarum, accession number: CCTCC AB 2010210. Deposited at the China Center for Type Culture Collection.

[0016] Lactobacillus acidophilus, accession number: CCTCC AB 2010208. Deposited at the China Center for Type Culture Collection.

[0017] Bifidobacterium lactis, accession number: SHBCC D24675. Deposited at the Shanghai Center for Microbiological Collections.

[0018] Seed germination rates using commercially available MS medium were not ideal. Ulva, also known as sea lettuce, is a widely distributed green seaweed. Ulva plays an important role in the ecosystem, being rich in minerals, vitamins, and organic compounds, making it a potential source of nutrients in culture media. This invention prepares a liquid culture medium by compounding multiple components; however, the inventors found that directly adding crushed Ulva did not significantly improve seed germination rates. However, fermenting Ulva with specific bacterial strains improved seed germination rates. Analysis showed that a specific ratio of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Bifidobacterium lactis* produced a series of metabolites during fermentation, including organic acids, enzymes, antimicrobial peptides, vitamins, short-chain fatty acids, antioxidants, and potential plant growth regulators. When these bacteria are used to ferment Ulva lactuca, the complex components in the fermentation products, combined with the other components in the system, can degrade the complex organic matter in Ulva lactuca, such as cellulose, protein, and starch, into simpler compounds, such as monosaccharides, amino acids, and other small molecules. These simpler compounds can serve as a nutrient source for seed germination. The metabolites produced during fermentation may contain plant growth regulators, such as auxins and cytokinins. The synergistic effect of these components with the rest of the system can promote seed germination.

[0019] The method for preparing the banana enzymatic hydrolysate is as follows:

[0020] S1: Soak bananas with peel and a ripeness level of 5 in a 120-140 mg / L food-grade sodium metabisulfite aqueous solution at 30-35℃ for 10-12 hours, and then blanch them in hot water at 92-95℃ for 5-7 minutes to obtain pre-treated bananas.

[0021] S2: Peel the pre-treated bananas, cut them into 1-2 cm banana segments, soak them in a 60-70 mg / L food-grade sodium metabisulfite aqueous solution in a water bath for 80-100 min, and then use a pulping machine to pulp them to obtain banana pulp;

[0022] S3: Add a compound enzyme to the banana pulp to make the concentration of the compound enzyme in the banana pulp 120-150mg / L. The compound enzyme is a mixture of β-glucosidase, compound protease and saccharifying enzyme in a mass ratio of 1:(1.3-1.6):(0.5-0.8). Enzymatically hydrolyze in a water bath at 40-45℃ for 50-60min, then inactivate the enzyme to obtain banana enzymatic hydrolysate.

[0023] (3) Cut the sterile tissue culture seedlings into 2-3 cm stem segments, each stem segment having at least one pair of axillary buds, and inoculate them onto the liquid culture medium of the same composition as in step (2) for subculture of tissue culture seedlings.

[0024] Bananas (Musa spp.) are a tropical fruit that, besides being a food source, are widely used in plant tissue culture. The advantage of bananas as a culture medium component lies in their rich nutritional content. However, adding banana pulp to the liquid culture medium of this invention does not ideally improve the differentiation rate. In contrast, banana hydrolysate prepared by adding enzymes in specific proportions can improve the differentiation rate of tissue culture seedlings after subculture. The analysis shows that the enzymatic hydrolysis process can break down complex macromolecules such as proteins, starch, and pectin into smaller molecules such as amino acids and sugars. These smaller molecule nutrients are more easily absorbed and utilized by plant cells, thereby promoting cell growth and differentiation. The fermented product after enzymatic hydrolysis usually has better fluidity and permeability. Specific enzymes can produce growth regulators such as cytokinins and auxins, which are crucial for promoting cell division and differentiation. When combined with other components in the system, these substances can improve the differentiation rate in plant tissue culture.

[0025] Those skilled in the art know that improper addition of hormones to the culture medium can significantly affect various aspects such as seed germination rate, differentiation rate, average number of buds, and average plant height. Hormones play a crucial role in plant growth and development, and their concentration and ratio must be precisely controlled to achieve ideal cultivation results. Since the *Ulva prostrata* ferment and banana enzymatic hydrolysate added to the liquid culture medium system of this invention both contain plant growth regulators, extensive experiments have shown that adding hormones of a specific composition to the system of this invention can synergistically enhance the effects of plant growth regulators contained in the *Ulva prostrata* ferment and banana enzymatic hydrolysate, resulting in a significant improvement in average number of buds and average plant height. Simultaneously, under these conditions, seed germination rate and differentiation rate are not inhibited, achieving coordination and balance among multiple factors.

[0026] Furthermore, the weight ratio of banana segments to food-grade sodium metabisulfite aqueous solution is 15-18:1.

[0027] Furthermore, the conditions for irradiation sterilization are: 1500~2000 kRad irradiation for 2-3 hours.

[0028] Furthermore, the particles are ultra-finely pulverized to a particle size of less than 40 μm.

[0029] Furthermore, the alcohol used for disinfection has a volume fraction of 75% and a disinfection time of 30-40 seconds.

[0030] Furthermore, the liquid culture medium is sterilized at 121°C for 18-20 minutes.

[0031] Furthermore, the conditions for seed germination culture are as follows: culture is carried out in a sterile culture room with a light intensity of 5000-5200 Lux, a light duration of 15-17 h / d, a temperature of 25-27℃ during light exposure, and a temperature of 16-18℃ during darkness.

[0032] Furthermore, the culture conditions for the subculture of the tissue culture seedlings are as follows: cultured in a sterile culture room, with a light intensity of 5300-5500 Lux, a light duration of 18-20 h / d, a temperature of 25-27℃ during light exposure, and a temperature of 16-18℃ during darkness.

[0033] Furthermore, the sodium hypochlorite solution concentration is 3-5 wt%, and the turmeric seeds are soaked for 6-8 minutes.

[0034] The present invention also provides the application of the method in breeding turmeric.

[0035] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0036] 1. The method of the present invention has good effect on the tissue culture of turmeric, and can simultaneously achieve excellent cultivation results in four aspects: seed germination rate, average germination rate of tissue culture seedlings, growth height and differentiation rate. It has extremely high research value and application prospects.

[0037] 2. Seed germination rates using commercially available MS medium are not ideal. This invention improves seed germination rates by preparing a liquid culture medium from multiple components and adding *Ulva lactuca* fermentation product to the liquid medium.

[0038] 3. The inventors further added banana enzymatic hydrolysate to the liquid culture medium system, which can improve the differentiation rate of tissue culture seedlings after subculture.

[0039] 4. The addition of hormones with specific compositions to the system of this invention can synergistically enhance the effects of plant growth regulators contained in both Ulva ferment and banana enzymatic hydrolysate, resulting in a significant improvement in average bud count and average plant height. Simultaneously, under these conditions, seed germination rate and differentiation rate are not inhibited, achieving coordination and balance among multiple factors. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Some of the raw materials used in this invention were purchased from the following manufacturers; all others, unless otherwise specified, are commercially available products.

[0042] Glycoamylase, 50U / mg, Hebei Jiuyu Biotechnology Co., Ltd.

[0043] β-glucosidase, 50 U / mg, Jiangsu Aofu Biotechnology Co., Ltd.

[0044] Complex protease, 100 U / mg, Hunan Yunbang Biotechnology Co., Ltd.

[0045] Pectinase, 50 U / mg, Hunan Yunbang Biotechnology Co., Ltd.

[0046] Neutral protease, 50 U / mg, Hunan Yunbang Biotechnology Co., Ltd.

[0047] Trypsin, 50 U / mg, Jiangsu Jiujia Biotechnology Co., Ltd.

[0048] The strains used for fermentation are Bacillus amyloliquefaciens, Bacillus licheniformis; Saccharomyces boulardii, Saccharomyces cerevisiae, and Saccharomyces flavoringus; Lactobacillus acidophilus and Lactobacillus exigua.

[0049] Lactobacillus plantarum, viable count 10 billion CFU / g. Accession number: CCTCC AB 2010210. Deposited at the China Center for Type Culture Collection.

[0050] Lactobacillus acidophilus, viable count 20 billion CFU / g. Accession number: CCTCC AB 2010208. Deposited at the China Center for Type Culture Collection.

[0051] Bifidobacterium lactis, with a bacterial count of 10 billion CFU / g. Accession number: SHBCC D24675. Deposited at the Shanghai Center for Microbiological Preservation.

[0052] Bifidobacterium longum, viable count 10 billion CFU / g. Accession number: CCTCCHB20082718. Deposited at the China Center for Type Culture Collection.

[0053] Lactobacillus bulgaricus, viable count 20 billion CFU / g. Accession number: CCTCCB20082295; deposited at the China Center for Type Culture Collection.

[0054] Lactobacillus helveticus, viable count 20 billion CFU / g. Accession number: CCTCAB2010205; deposited at the China Center for Type Culture Collection.

[0055] Example 1

[0056] This embodiment provides a method for breeding turmeric in a liquid culture medium, the method comprising the following steps:

[0057] (1) Take turmeric seeds, wash them, and then disinfect them with alcohol, soak them in sodium hypochlorite solution, soak them in sterile water and rinse them in sequence to obtain disinfected seeds; the alcohol volume concentration used for alcohol disinfection is 75% and the disinfection time is 35s; the sodium hypochlorite solution concentration is 4wt% and the soaking time of the turmeric seeds is 7min.

[0058] (2) Inoculate the sterilized seeds onto liquid culture medium and germinate the sterile seeds on the liquid culture medium. The conditions for seed germination culture are: culture in a sterile culture room, light intensity 5100 Lux, light duration 16 h / d, temperature during light 26℃, and temperature during darkness 17℃ to obtain sterile tissue culture seedlings.

[0059] (3) Cut the sterile tissue culture seedlings into 2-3cm stem segments, each stem segment having a pair of axillary buds, and inoculate them onto the liquid culture medium of the same composition as in step (2) for subculture. The culture conditions for subculture of tissue culture seedlings are: cultured in a sterile culture room, light intensity 5400 Lux, light duration 19h / d, temperature during light 26℃, and temperature during darkness 17℃.

[0060] The contents of each component of the liquid culture medium in steps (2) and (3) are as follows: 26 g / L of Ulva ferment, 14 g / L of banana hydrolysate, 23 g / L of sucrose, 1300 mg / L of potassium nitrate, 1200 mg / L of ammonium nitrate, 140 mg / L of potassium dihydrogen phosphate, 6 mg / L of zinc sulfate, 0.23 mg / L of sodium molybdate, 23.4 mg / L of disodium EDTA, 320 mg / L of magnesium sulfate heptahydrate, 420 mg / L of calcium chloride dihydrate, 0.48 mg / L of potassium iodide, 4.7 mg / L of boric acid, 20.5 mg / L of manganese sulfate tetrahydrate, 19.7 mg / L of ferrous sulfate, 96 mg / L of inositol, 1.5 mg / L of glycine, and a hormone composed of 1.25 mg / L of 6-benzylaminopurine, 0.11 mg / L of indolebutyric acid, and 0.17 mg / L of naphthaleneacetic acid.

[0061] The method for preparing the *Ulva prostrata* ferment is as follows:

[0062] Step 1: Wash and dry the Ulva lactuca, then perform ultra-fine pulverization until the particle size is less than 40μm to obtain Ulva lactuca powder;

[0063] Step 2: Sterilize the Ulva micropowder by irradiation at 1800 kRad for 2.5 h, add 5 times the mass of sterile water containing the Ulva micropowder, add 2% of the mass of the Ulva micropowder containing a compound bacteria, which is a mixture of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium lactis in a mass ratio of 1.4:1:0.7, anaerobic ferment at 36℃ for 80 h, sterilize, and obtain the Ulva fermented product;

[0064] The method for preparing the banana enzymatic hydrolysate is as follows:

[0065] S1: Bananas with peel and a ripeness level of 5 are immersed in a 130 mg / L food-grade sodium metabisulfite aqueous solution at 33°C for 11 hours, and then blanched in hot water at 94°C for 6 minutes to obtain pretreated bananas.

[0066] S2: Peel the pre-treated bananas, cut them into 1-2 cm banana segments, and soak them in a 65 mg / L food-grade sodium metabisulfite aqueous solution for 90 min in a water bath. The weight ratio of banana segments to food-grade sodium metabisulfite aqueous solution is 17:1. Then, use a pulping machine to pulp the bananas to obtain banana pulp.

[0067] S3: Add a compound enzyme to the banana pulp to make the concentration of the compound enzyme in the banana pulp 140 mg / L. The compound enzyme is a mixture of β-glucosidase, compound protease and saccharifying enzyme in a mass ratio of 1:1.4:0.7. Incubate the mixture in a water bath at 42℃ for 55 min to inactivate the enzyme and obtain the banana enzymatic hydrolysate.

[0068] The liquid culture medium was sterilized at 121°C for 18 minutes.

[0069] Example 2

[0070] This embodiment provides a method for breeding turmeric in a liquid culture medium, the method comprising the following steps:

[0071] (1) Take turmeric seeds, wash them, and then disinfect them with alcohol, soak them in sodium hypochlorite solution, soak them in sterile water and rinse them in sequence to obtain disinfected seeds; the alcohol concentration used for disinfection is 75% by volume and the disinfection time is 30s; the sodium hypochlorite solution concentration is 3wt% and the soaking time of the turmeric seeds is 6min.

[0072] (2) Inoculate the sterilized seeds onto liquid culture medium and carry out germination culture of the sterile seeds on liquid culture medium. The conditions for seed germination culture are: culture in a sterile culture room, light intensity 5000 Lux, light time 15h / d, temperature during light 27℃, and temperature during darkness 8℃ to obtain sterile tissue culture seedlings.

[0073] (3) Cut the sterile tissue culture seedlings into 2-3cm stem segments, each stem segment having a pair of axillary buds, and inoculate them onto the liquid culture medium of the same composition as in step (2) for subculture. The culture conditions for subculture of tissue culture seedlings are: cultured in a sterile culture room, light intensity 5300 Lux, light duration 20h / d, temperature during light exposure 25℃, and temperature during darkness 18℃.

[0074] The contents of each component of the liquid culture medium in steps (2) and (3) are as follows: 27 g / L of Ulva ferment, 12 g / L of banana hydrolysate, 24 g / L of sucrose, 1200 mg / L of potassium nitrate, 1300 mg / L of ammonium nitrate, 120 mg / L of potassium dihydrogen phosphate, 7 mg / L of zinc sulfate, 0.21 mg / L of sodium molybdate, 24 mg / L of disodium EDTA, 300 mg / L of magnesium sulfate heptahydrate, 430 mg / L of calcium chloride dihydrate, 0.44 mg / L of potassium iodide, 5 mg / L of boric acid, 20 mg / L of manganese sulfate tetrahydrate, 20 mg / L of ferrous sulfate, 95 mg / L of inositol, 1.7 mg / L of glycine, and a hormone composed of 1.2 mg / L of 6-benzylaminopurine, 0.13 mg / L of indolebutyric acid, and 0.15 mg / L of naphthaleneacetic acid.

[0075] The method for preparing the *Ulva prostrata* ferment is as follows:

[0076] Step 1: Wash and dry the Ulva lactuca, then perform ultra-fine pulverization until the particle size is less than 40μm to obtain Ulva lactuca powder;

[0077] Step 2: Sterilize the Ulva micropowder by irradiation at 1500 kRad for 3 hours, add sterile water with 4 times the mass of the Ulva micropowder, add a compound bacteria accounting for 1% of the mass of the Ulva micropowder, the compound bacteria being a mixture of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium lactis in a mass ratio of 1.2:1:0.8, anaerobic ferment at 34℃ for 96 hours, sterilize, and obtain the Ulva fermented product;

[0078] The method for preparing the banana enzymatic hydrolysate is as follows:

[0079] S1: Immerse bananas with peel and a ripeness level of 5 in a 30℃ 140mg / L food-grade sodium metabisulfite aqueous solution for 10h, then blanch them in 95℃ hot water for 5min to obtain pretreated bananas.

[0080] S2: Peel the pre-treated bananas, cut them into 1-2 cm banana segments, and soak them in a 70 mg / L food-grade sodium metabisulfite aqueous solution for 80 min in a water bath. The weight ratio of banana segments to food-grade sodium metabisulfite aqueous solution is 18:1. Then, use a pulping machine to pulp the bananas to obtain banana pulp.

[0081] S3: Add a compound enzyme to the banana pulp to make the concentration of the compound enzyme in the banana pulp 150 mg / L. The compound enzyme is a mixture of β-glucosidase, compound protease and saccharifying enzyme in a mass ratio of 1:1.3:0.8. Incubate the mixture in a water bath at 45℃ for 50 min to inactivate the enzyme and obtain the banana enzymatic hydrolysate.

[0082] The liquid culture medium was sterilized at 121°C for 20 minutes.

[0083] Comparative Example 1

[0084] The difference between this comparative example and Example 1 is that the liquid culture medium was replaced with MS liquid culture medium. This medium was purchased from Kingclone (Beijing) Biotechnology Co., Ltd., model: WP8670.

[0085] Comparative Example 2

[0086] The difference between this comparative example and Example 1 is that the liquid culture medium does not contain Ulva lactuca ferment and the sucrose content is 49 g / L.

[0087] Comparative Example 3

[0088] The difference between this comparative example and Example 1 is that the *Ulva prostrata* fermentation product is replaced with *Ulva prostrata* extract. The preparation method of the *Ulva prostrata* extract is as follows: *Ulva prostrata* is washed, dried, pulverized, passed through a 200-mesh sieve, and 5 times its weight of water is added. The mixture is then soaked at 40°C for 12 hours to obtain the *Ulva prostrata* extract.

[0089] Comparative Example 4

[0090] The difference between this comparative example and Example 1 is that the compound bacteria is a mixture of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium lactis in a mass ratio of 1:1:1.

[0091] Comparative Example 5

[0092] The difference between this comparative example and Example 1 is that the compound bacteria is a mixture of Bifidobacterium longum, Lactobacillus bulgaricus, and Lactobacillus helveticus in a mass ratio of 1.4:1:0.7.

[0093] Comparative Example 6

[0094] The difference between this comparative example and Example 1 is that the liquid culture medium does not contain banana hydrolysate and the sucrose content is 37 g / L.

[0095] Comparative Example 7

[0096] The difference between this comparative example and Example 1 is that the banana enzymatic hydrolysate is replaced with banana pulp.

[0097] The preparation method of banana pulp is as follows:

[0098] S1: Immerse bananas with peel and a ripeness level of 5 in a 30℃ 140mg / L food-grade sodium metabisulfite aqueous solution for 10h, then blanch them in 95℃ hot water for 5min to obtain pretreated bananas.

[0099] S2: Peel the pre-treated bananas, cut them into 1-2 cm banana segments, and soak them in a 70 mg / L food-grade sodium metabisulfite aqueous solution for 80 min in a water bath. The weight ratio of banana segments to food-grade sodium metabisulfite aqueous solution is 18:1. Then, use a pulping machine to pulp the bananas to obtain banana pulp.

[0100] Comparative Example 8

[0101] The difference between this comparative example and Example 1 is that the complex enzyme is a combination of β-glucosidase, complex protease and saccharifying enzyme in a mass ratio of 1:1:1.

[0102] Comparative Example 9

[0103] The difference between this comparative example and Example 1 is that the complex enzyme is a combination of pectinase, neutral protease and trypsin in a mass ratio of 1:1.4:0.7.

[0104] Comparative Example 10

[0105] The difference between this comparative example and Example 1 is that the hormone consists of 0.17 mg / L of 6-benzylaminopurine, 1.25 mg / L of indolebutyric acid and 0.011 mg / L of naphthaleneacetic acid.

[0106] Comparative Example 11

[0107] The difference between this comparative example and Example 1 is that the hormone consists of 1.25 mg / L of zeatin nucleoside, 0.011 mg / L of 4-chloro-3-indoleacetic acid, and 0.17 mg / L of kinetin.

[0108] Performance testing

[0109] Following the methods of Examples 1-2 and Comparative Examples 1-11, 100 sterile turmeric seeds were germinated on liquid culture medium. After 30 days, the germination rate was calculated (germination rate = number of germinated seeds / total number of seeds * 100%). The tissue culture seedlings were then subcultured, and the differentiation rate (differentiation rate = number of differentiated explants / total number of inoculated explants * 100%), average number of buds, and average plant height were calculated. The results are shown in Table 1.

[0110] Table 1 Performance Test Results

[0111]

[0112] The results show that Examples 1-2 exhibited high seed germination and differentiation rates, and excellent average number of buds and average plant height. Comparative Examples 2-5 indicate that *Ulva prostrata* fermentation product affects seed germination rate; Comparative Examples 6-9 show that banana enzymatic hydrolysate affects the differentiation rate of tissue culture seedlings after subculture; Comparative Examples 10-11 show that hormones with specific compositions can synergistically enhance the effects of plant growth regulators found in both *Ulva prostrata* fermentation product and banana enzymatic hydrolysate, affecting the average number of buds and average plant height. Improper addition can inhibit high seed germination and differentiation rates.

[0113] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for breeding turmeric in a liquid culture medium, characterized in that, The method includes the following steps: (1) Take turmeric seeds, wash them, and then disinfect them with alcohol, soak them in sodium hypochlorite solution, soak them in sterile water and rinse them in turn to obtain disinfected seeds; (2) Inoculate the sterilized seeds onto a liquid culture medium and germinate the sterile seeds on the liquid culture medium to obtain sterile tissue culture seedlings; The components of the liquid culture medium are as follows: 25-27 g / L of *Ulva prostrata* ferment broth, 12-16 g / L of banana enzymatic hydrolysate, 20-24 g / L of sucrose, 1200-1400 mg / L of potassium nitrate, 1100-1300 mg / L of ammonium nitrate, 120-150 mg / L of potassium dihydrogen phosphate, 5-7 mg / L of zinc sulfate, 0.21-0.24 mg / L of sodium molybdate, 23-24 mg / L of disodium EDTA, 300-350 mg / L of magnesium sulfate heptahydrate, 400-430 mg / L of calcium chloride dihydrate, 0.44-0.52 mg / L of potassium iodide, 4-5 mg / L of boric acid, 20-21 mg / L of manganese sulfate tetrahydrate, 19-20 mg / L of ferrous sulfate, 95-97 mg / L of inositol, 1.3-1.7 mg / L of glycine, and hormones; The hormone consists of 1.2-1.3 mg / L of 6-benzylaminopurine, 0.09-0.13 mg / L of indolebutyric acid, and 0.15-0.18 mg / L of naphthaleneacetic acid; The preparation method of Ulva ferment is as follows: Step 1: Wash and dry the Ulva lactuca, then pulverize it into ultrafine powder to obtain Ulva lactuca powder; Step 2: Sterilize the Ulva micropowder by irradiation, add 4-5 times the weight of sterile water, and add 1%-3% of the weight of the Ulva micropowder with compound bacteria. The compound bacteria are a mixture of Lactobacillus plantarum, Lactobacillus acidophilus and Bifidobacterium lactis in a mass ratio of (1.2-1.5):1:(0.5-0.8). Perform anaerobic fermentation at 34-37℃ for 72-96 hours, then sterilize to obtain the Ulva fermented product. The preservation number of Lactobacillus plantarum is CCTCC AB 2010210, the preservation number of Lactobacillus acidophilus is CCTCC AB2010208, and the preservation number of Bifidobacterium lactis is SHBCC D24675. The preparation method of banana enzymatic hydrolysate is as follows: S1: Soak bananas with peel in a 120-140 mg / L food-grade sodium metabisulfite aqueous solution at 30-35℃ for 10-12 hours, then blanch them in hot water at 92-95℃ for 5-7 minutes to obtain pretreated bananas. S2: Peel the pre-treated bananas, cut them into 1-2 cm banana segments, soak them in a 60-70 mg / L food-grade sodium metabisulfite aqueous solution in a water bath for 80-100 min, and then use a pulping machine to pulp them to obtain banana pulp; S3: Add a compound enzyme to the banana pulp to make the concentration of the compound enzyme in the banana pulp 120-150mg / L. The compound enzyme is a mixture of β-glucosidase, compound protease and saccharifying enzyme in a mass ratio of 1:(1.3-1.6):(0.5-0.8). Enzymatically hydrolyze in a water bath at 40-45℃ for 50-60min, then inactivate the enzyme to obtain banana enzymatic hydrolysate. (3) Cut the sterile tissue culture seedlings into 2-3 cm stem segments, each stem segment having at least one pair of axillary buds, and inoculate them onto the liquid culture medium of the same composition as in step (2) for subculture of tissue culture seedlings.

2. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The weight ratio of banana segments to food-grade sodium metabisulfite aqueous solution is (15-18):

1.

3. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The conditions for irradiation sterilization are: 1500~2000 kRad irradiation for 2-3 hours.

4. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, Ultrafine grinding to a particle size of less than 40μm.

5. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The alcohol used for disinfection has a volume fraction of 75% and a disinfection time of 30-40 seconds.

6. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The liquid culture medium is sterilized at 120-123℃ for 18-20 minutes.

7. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The germination culture conditions are as follows: culture is carried out in a sterile culture room with a light intensity of 5000-5200 Lux, a light duration of 15-17 h / d, a temperature of 25-27℃ during light exposure, and a temperature of 16-18℃ during darkness.

8. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The culture conditions for the subculture of tissue culture seedlings are as follows: cultured in a sterile culture room, with a light intensity of 5300-5500 Lux, a light duration of 18-20 h / d, a temperature of 25-27℃ during light exposure, and a temperature of 16-18℃ during darkness.

9. The method for breeding turmeric in liquid culture medium according to claim 1, characterized in that, The sodium hypochlorite solution concentration is 3-5 wt%, and the turmeric seeds are soaked for 6-8 minutes.

10. The application of the method according to any one of claims 1-9 in tulip breeding.

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