Breeding Method of Cinnamomum camphora (L.) Presl var. borneoliferum H. W. Li and Its Application

Abstract

The present invention provides a breeding method and application of Cinnamomum camphora var. borneolifera, belonging to the technical field of plant cultivation methods. The breeding method comprises the following steps: (1) Selecting the apical bud stem segments of the current-year branches of Cinnamomum camphora var. borneolifera as explants, and culturing to obtain cluster seedlings; soaking the cluster seedlings in a compound fungicide for 10 - 15 minutes first, then soaking the bases in a growth regulator solution for 10 - 15 minutes, and then inoculating them into a rooting medium for rooting culture to obtain tissue culture seedlings; (2) Selecting soil with a pH value between 5 and 6, loosening the soil, sterilizing, fertilizing, and watering; (3) Inserting the tissue culture seedlings into the soil; (4) Disinfecting the tissue culture seedlings again after cutting, and spraying a rooting powder nutrient solution on the soil around the tissue culture seedlings; (5) Covering with a shading net, culturing for 35 - 40 days, removing the shading net, and continuing the maintenance management for 50 - 60 days. The breeding method of the present invention has a high rooting rate, average plant height, and survival rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant cultivation methods, and particularly relates to a breeding method and application of Cinnamomum camphora var. borneolifera. Background Art

[0002] Cinnamomum camphora var. borneolifera is a special variety of camphor tree. Different from other camphor trees, Cinnamomum camphora var. borneolifera is rich in d-borneol (borneol), which is one of the main sources of natural borneol. Natural borneol is a very precious natural substance with extensive medicinal value and commercial uses. Cinnamomum camphora var. borneolifera is a plant with extremely high value. Its discovery not only brings valuable natural resources to China, but also provides a new raw material source for industries such as medicine, spices, and chemical engineering. In recent years, the scale of the artificial forest of Cinnamomum camphora var. borneolifera has expanded rapidly, and the market demand for high-quality seedlings of Cinnamomum camphora var. borneolifera is increasing continuously. However, problems such as high transportation costs of Cinnamomum camphora var. borneolifera saplings, inability to be directly transported to the mountains for planting, and low planting survival rate restrict the development and exploitation of the Cinnamomum camphora var. borneolifera seedling industry.

[0003] The breeding methods of Cinnamomum camphora var. borneolifera mainly involve two methods: cutting propagation and tissue culture. Both of these methods have certain technical problems in actual operation. Technical problems in cutting propagation: (1) Unstable survival rate: Although the survival rate of cutting propagation can reach over 80%, due to the influence of environmental factors, operation techniques and other factors, the actual survival rate may fluctuate. And the high or low survival rate directly affects the quality and quantity of seedlings. (2) High requirements for operation techniques: Cutting propagation has certain requirements for the skills of operators. Improper operation may lead to difficult rooting or poor growth of cuttings. For example, the selection, treatment, disinfection of cuttings and the management of cutting beds all need to be precisely controlled. (3) Restrictions on environmental conditions: The success of cutting propagation is greatly affected by environmental conditions, such as temperature, humidity, light, etc. For example, a specific temperature range and suitable humidity are required to promote rooting. (4) Pest and disease control: Cuttings are easily invaded by pests and diseases during the cutting process, and effective control measures need to be taken. Pests and diseases will affect the survival rate of cuttings and the quality of seedlings. Technical problems in tissue culture: (1) High technical threshold: Tissue culture requires strict aseptic conditions and professional laboratory equipment. The technical threshold is relatively high and requires special training and technical support. (2) Complicated operation: The process of tissue culture is relatively complicated, including steps such as disinfection, medium preparation, inoculation, and culture. Each step needs to be strictly controlled to ensure the success rate of culture. (3) High cost: The equipment and consumables required for tissue culture are relatively expensive. Coupled with the labor cost of professional technical personnel, the entire process is costly.

[0004] The Chinese invention patent with the publication number of CN 108781957A discloses a method for factory seedling raising of Cinnamomum camphora var. borneolifera. After acclimatizing the tissue culture bottle seedlings, they are transferred to 128-hole trays or 105-hole trays for planting for 1.5 - 2.5 months, and then changed to 50-hole forest tree trays or nutrient bags for continuous planting for 2.5 - 4 months. This speeds up the seedling raising speed of Cinnamomum camphora var. borneolifera seedlings, reduces the transportation cost of Cinnamomum camphora var. borneolifera at the same time, and makes it easy to carry when Cinnamomum camphora var. borneolifera is planted on the mountain. However, the planting survival rate of the Cinnamomum camphora var. borneolifera seedlings by this method is not ideal, resulting in high seedling raising cost and unable to meet the needs of the existing market.

[0005] Therefore, there is an urgent need for a new breeding method and its application for Cinnamomum camphora var. borneolifera. Summary of the Invention

[0006] The purpose of the present invention is to provide a breeding method and its application for Cinnamomum camphora var. borneolifera.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A breeding method for Cinnamomum camphora var. borneolifera, the breeding method comprising the following steps:

[0009] (1): Select the apical bud stem segment of the current-year branches of Cinnamomum camphora var. borneolifera as the explant, disinfect; inoculate it into the primary culture medium to induce the formation of callus;

[0010] Inoculate the callus into the proliferation subculture medium for proliferation subculture to obtain cluster seedlings;

[0011] Soak the cluster seedlings in a compound bactericide, then soak them in a growth regulator solution, and then inoculate them into the rooting medium for cultivation to obtain tissue culture seedlings;

[0012] The compound bactericide includes: carbendazim 0.5 - 0.9 μg / mL, galangin 3.2 - 3.7 μg / mL, aloe-emodin 1.4 - 1.7 μg / mL, celangulin 0.4 - 0.6 μg / mL, matrine 0.7 - 1.2 μg / mL, prepared with water;

[0013] The composition of the rooting medium: 1 / 2MS, sucrose 12 - 14 g / L, agar 20 - 22 g / L, and NAA 0.6 - 0.9 mg / L;

[0014] (2): Loosen the soil, sterilize, fertilize, and water the soil to obtain the treated soil;

[0015] (3): Transplant the tissue culture seedlings into the treated soil in step (2);

[0016] (4): Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, then water, and insert bamboo slices and cover with a film for sealing;

[0017] (5): Cover the shading net, fix the four sides, cultivate under light conditions, and continue the maintenance management after removing the shading net.

[0018] Furthermore, the breeding method of Cinnamomum camphora var. borneolifera includes the following steps:

[0019] (1) Select the apical bud stem segments of the current-year branches of Cinnamomum camphora var. borneolifera as explants, and perform disinfection treatment; then inoculate them into the primary culture medium to induce the formation of callus;

[0020] Inoculate the callus into the proliferation subculture medium for proliferation subculture to obtain cluster seedlings;

[0021] Soak the cluster seedlings in a compound fungicide for 10 - 15 minutes first, then soak the base in a growth regulator solution for 10 - 15 minutes, and then inoculate them into the rooting medium for rooting culture to obtain tissue culture seedlings;

[0022] The compound fungicide includes the following components: carbendazim 0.5 - 0.9 μg / mL, galangin 3.2 - 3.7 μg / mL, aloe-emodin 1.4 - 1.7 μg / mL, celangulin 0.4 - 0.6 μg / mL, matrine 0.7 - 1.2 μg / mL, and is prepared with water;

[0023] The composition of the rooting medium is 1 / 2MS, sucrose 12 - 14 g / L, agar 20 - 22 g / L, and NAA 0.6 - 0.9 mg / L;

[0024] (2) Select the soil with a pH value between 5 and 6, loosen the soil, sterilize, fertilize, and water to obtain the treated soil;

[0025] (3) On sunny days at 13 - 17 °C, transplant the tissue culture seedlings after rooting culture into the soil treated in step (2), with the leaves aligned in the same direction, the bud bracts placed 1 cm above the soil surface, and the row spacing and plant spacing of each plant being 6 - 8 cm;

[0026] (4) Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, spray an average of 300 - 500 mL of the rooting powder nutrient solution per tissue culture seedling, then water, insert bamboo slices and cover with a film for sealing, and control the humidity at 70% - 90%;

[0027] (5) Cover a shading net with a shading rate of 87 - 92%, fix the four sides, cultivate under light conditions of 7000 - 9000 lux for 35 - 40 days, and then remove the shading net; under light of 10000 - 12000 lux, control the growth temperature at 16 - 20 °C, and continue the maintenance management for 50 - 60 days.

[0028] At present, carbendazim solution is commonly used as a bactericide in the breeding process in the market to prevent germs from invading through wounds. However, in the breeding process of Cinnamomum camphora 'Borneol', it is found that the use effect is not ideal and the survival rate is affected. The compound bactericide prepared by compounding carbendazim with multiple components in the present invention has good bactericidal effect and improves the survival rate of Cinnamomum camphora 'Borneol' breeding. The concentration of carbendazim used in the present invention can not only play a bactericidal role but also cause no harm to the cuttings. Galangin has strong antibacterial activity and can further enhance the bactericidal effect; Aloe-emodin helps to promote wound healing and reduce the damage of cuttings during the treatment process; Celangulin has antibacterial activity and can inhibit the growth of a variety of pathogenic microorganisms; Matrine has a wide range of antibacterial and antiviral effects and can further reduce the pathogens on the cuttings. These components work together to produce a synergistic effect, that is, the components cooperate with each other to enhance the overall bactericidal effect, thereby improving the survival rate of Cinnamomum camphora 'Borneol' breeding.

[0029] Furthermore, the preparation method of the rooting powder nutrient solution is as follows:

[0030] (1) Mix burdock root, astragalus, and eucalyptus leaves in a mass ratio of 1:(0.5 - 0.7):(1.2 - 1.4), crush them to less than 80 - 100 mesh to obtain a mixture. Add 10 - 13 parts by mass of water to 1 part by mass of the mixture to obtain a mixed solution. Continuously add Lactobacillus brevis and carry out anaerobic fermentation. The fermentation temperature is 32 - 35 °C and the fermentation time is 15 - 20 h. After sterilization, obtain a sterilized product. Continuously add Lactobacillus plantarum and carry out anaerobic fermentation. The fermentation temperature is 32 - 35 °C and the fermentation time is 10 - 12 h. After sterilization, filter through a 0.22 μm filter membrane to obtain a filtrate. Concentrate and dry the filtrate to obtain a fermentation powder;

[0031] (2) Mix potassium α-naphthylacetate (Shanghai Yuanye Bio-Technology Co., Ltd.), potassium 3-indolebutyrate (Shanghai Yuanye Bio-Technology Co., Ltd.), and the fermentation powder to obtain a mixed powder. Dilute the mixed powder and water in a ratio of 400 - 500 mg:1 L to obtain the rooting powder nutrient solution.

[0032] Furthermore, the concentration of Lactobacillus brevis in the mixed solution is 10 6 -10 7 CFU / mL.

[0033] Furthermore, the concentration of Lactobacillus plantarum in the sterilized product is 10 7 -10 8 CFU / mL.

[0034] Furthermore, the mass ratio of potassium α-naphthylacetate, potassium 3-indolebutyrate, and the fermentation powder is (0.2 - 0.5):1:(1.4 - 1.7).

[0035] In the rooting powder nutrient solution of the present invention, potassium α-naphthalene acetate, potassium 3-indolebutyrate and baking powder are added simultaneously, which can improve the rooting rate of breeding Cinnamomum camphora. Potassium α-naphthalene acetate can promote the growth of the roots of Cinnamomum camphora, especially effective in promoting the formation of lateral roots and adventitious roots. Appropriate concentration can significantly improve the rooting rate, while too high or too low concentration may inhibit rooting. Potassium 3-indolebutyrate is also a plant growth regulator of auxin type, which is particularly effective in promoting rooting and helps to improve the success rate of cutting rooting. The active components in the baking powder can stimulate the growth of plant roots and improve the rooting rate. The synergistic effect of these components can promote the rapid formation and growth of roots, thus improving the success rate of cutting. And when these three components are compounded in a specific ratio, the average number of rooted cuttings in breeding can be improved.

[0036] Further, the growth regulator solution comprises the following components: 6-BA 6.5 - 8.6 mg / L, NAA 1.2 - 3.5 mg / L, IBA 1.3 - 2.6 mg / L, IAA 2.4 - 3.5 mg / L, plant extract 2.4 - 4.6 mg / L, and is prepared with water.

[0037] 6-Benzylaminopurine (6-BA), Naphthaleneacetic acid (NAA), Indolebutyric acid (IBA) and Indoleacetic acid (IAA) are all commercially available products.

[0038] Further, the preparation method of the plant extract is as follows:

[0039] (1) Mix Platycladus orientalis leaves, Gelidium amansii and Astragalus membranaceus, and crush them to a particle size less than 80 - 100 mesh to obtain a mixed material;

[0040] (2) Perform supercritical extraction on the mixed material. First, extract at 40 - 45 °C, 30 - 35 MPa, and a CO2 flow rate of 17 - 20 L / h for 100 - 120 min to obtain a first fraction, and continue to extract at 25 - 30 °C, 25 - 30 MPa, and a CO2 flow rate of 17 - 20 L / h for 60 - 80 min to obtain a second fraction;

[0041] (3) Mix the first fraction and the second fraction to obtain the plant extract.

[0042] Further, the mass ratio of Platycladus orientalis leaves, Gelidium amansii and Astragalus membranaceus is 1:(1.3 - 1.5):(0.3 - 0.7).

[0043] By using the growth regulator solution, the present invention can increase the average plant height of Cinnamomum camphora. By reasonably adding 6-BA, NAA, IBA, IAA and plant extracts to the growth regulator solution, the height of the plants obtained in the breeding process can be effectively increased. These components act on the growth and development of the plants through various mechanisms such as promoting cell division, cell elongation, bud formation and root growth, so as to achieve the purpose of increasing the plant height.

[0044] The present invention also provides an application of the breeding method of Cinnamomum camphora.

[0045] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0046] 1. The compound bactericide prepared by compounding carbendazim and various components in the present invention has good bactericidal effect and improves the survival rate of Cinnamomum camphora breeding.

[0047] 2. Potassium α-naphthylacetate, potassium 3-indolebutyrate and baking powder are simultaneously added to the rooting powder nutrient solution of the present invention, which can improve the rooting rate of Cinnamomum camphora breeding. And when these three components are compounded in a specific ratio, the average number of roots in breeding can be improved.

[0048] 3. By using the growth regulator solution, the present invention can increase the average plant height of Cinnamomum camphora. Specific embodiments

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0050] Some raw materials used in the present invention are purchased from the following manufacturers:

[0051] Carbendazim, purchased from Hubei Qinniu Chemical Technology Co., Ltd.;

[0052] Galangin, Sichuan Jingcuibao Biotechnology Co., Ltd.;

[0053] Aloe-emodin, Shanghai Yuanye Biotechnology Co., Ltd.;

[0054] Celangulin, Shanghai Yuanye Biotechnology Co., Ltd.;

[0055] Matrine, Shanghai Yuanye Biotechnology Co., Ltd.;

[0056] Potassium α-naphthylacetate, Shanghai Yuanye Biotechnology Co., Ltd.;

[0057] Potassium 3-indolebutyrate, Shanghai Yuanye Biotechnology Co., Ltd.;

[0058] Lactobacillus brevis, preservation number: SHBCCD14346, purchased from Shanghai Center for Microbial Preservation.

[0059] Lactobacillus plantarum, preservation number: CCTCC AB2010210, purchased from China Center for Type Culture Collection.

[0060] The primary culture medium consists of MS medium, 25 g / L of sucrose, 25 g / L of agar, 1.0 mg / L of 6-BA, and 0.8 mg / L of NAA.

[0061] The multiplication subculture medium consists of MS medium, 20 g / L of sucrose, 25 g / L of agar, 2 mg / L of 6-BA, and 2 mg / L of NAA.

[0062] Example 1

[0063] This example provides a breeding method for Cinnamomum camphora var. borneolifera, and the breeding method includes the following steps:

[0064] (1) Select the apical bud stem segment of the current-year branches of Cinnamomum camphora var. borneolifera as the explant and perform disinfection treatment; inoculate the explant into the primary culture medium to induce the formation of callus; inoculate the callus into the multiplication subculture medium for multiplication subculture to obtain cluster seedlings; soak the cluster seedlings in a compound fungicide for 12 min first, then soak the base in a growth regulator solution for 12 minutes, and then inoculate them into the rooting medium for rooting culture to obtain tissue culture seedlings;

[0065] The rooting medium is 1 / 2MS, 13 g / L of sucrose, 21 g / L of agar, and 0.8 mg / L of NAA;

[0066] (2) Select the soil with a pH value of 5.5, loosen the soil, sterilize it, apply fertilizer, and water it to obtain the treated soil;

[0067] (3) On a sunny day at 15°C, transplant the tissue culture seedlings after rooting culture into the soil treated in step (2), with the leaves aligned in the same direction and the bud bracts placed 1 cm above the soil surface, and the row spacing and plant spacing of each plant being 7 cm;

[0068] (4) Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, with an average of 400 mL sprayed per tissue culture seedling, then water it, and insert bamboo slices and cover it with a white film to seal it, ensuring that the humidity is controlled at 80%;

[0069] (5) Cover with a shading net, the shading rate of the shading net is 90%, fix the four sides, with a light intensity of 8000 lux, culture for 36 days, remove the shading net, with a light intensity of 11000 lux, and control the growth temperature at 18°C, and continue the maintenance management for 55 days.

[0070] The compound bactericide comprises the following components: carbendazim 0.7 μg / mL, galangin 3.4 μg / mL, aloe-emodin 1.5 μg / mL, celangulin 0.5 μg / mL, matrine 0.9 μg / mL, and is prepared with water.

[0071] The preparation method of the rooting powder nutrient solution is as follows:

[0072] (1) Mix burdock root, astragalus root, and eucalyptus leaves in a mass ratio of 1:0.6:1.3, crush them to less than 100 mesh to obtain a mixture. Add 12 parts by mass of water to 1 part by mass of the mixture to obtain a mixed solution. Then continue to add Lactobacillus brevis, and the concentration of Lactobacillus brevis in the mixed solution is 10 6 CFU / mL, and carry out anaerobic fermentation at a fermentation temperature of 33 °C and a fermentation time of 17 h. After sterilization, obtain a sterilized product. Then continue to add Lactobacillus plantarum, and the concentration of Lactobacillus plantarum in the sterilized product is 10 8 CFU / mL, carry out anaerobic fermentation at a fermentation temperature of 33 °C and a fermentation time of 11 h. After sterilization, filter through a 0.22 μm filter membrane to obtain a filtrate. Concentrate and dry the filtrate to obtain a fermentation powder;

[0073] (2) Mix potassium α-naphthylacetate, potassium 3-indolebutyrate, and the fermentation powder in a mass ratio of 0.4:1:1.5 to obtain a mixed powder. Dilute the mixed powder and water in a ratio of 450 mg:1 L to obtain the rooting powder nutrient solution.

[0074] The growth regulator solution comprises the following components: 6-BA 7.5 mg / L, NAA 2.4 mg / L, IBA 2.2 mg / L, IAA 3.1 mg / L, plant extract 4.0 mg / L, and is prepared with water.

[0075] The preparation method of the plant extract is as follows:

[0076] (1) Mix Platycladus orientalis leaves, Gelidium amansii, and astragalus root in a mass ratio of 1:1.4:0.5, crush them to a particle size of less than 100 mesh to obtain a mixed material;

[0077] (2) Carry out supercritical extraction on the mixed material. First, extract at 42 °C, 32 MPa, and a CO2 flow rate of 18 L / h for 110 min to obtain a first fraction. Then continue to extract at 27 °C, 28 MPa, and a CO2 flow rate of 18 L / h for 70 min to obtain a second fraction;

[0078] (3) Mix the first fraction and the second fraction to obtain the plant extract.

[0079] Example 2

[0080] This embodiment provides a breeding method for Cinnamomum camphora var. borneolifera, and the breeding method includes the following steps:

[0081] (1) Select the apical bud stem segments of the current-year branches of Cinnamomum camphora var. borneolifera as explants, and perform disinfection treatment; inoculate the explants into the primary culture medium to induce the formation of callus; inoculate the callus into the proliferation subculture medium for proliferation subculture to obtain cluster seedlings; soak the cluster seedlings in a compound fungicide for 10 min first, then soak the base in a growth regulator solution for 10 minutes, and then inoculate them into the rooting medium for rooting culture to obtain tissue culture seedlings;

[0082] The rooting medium is 1 / 2MS, sucrose 12 g / L, agar 20 g / L, NAA 0.6 mg / L;

[0083] (2) Select soil with a pH value of 5, loosen the soil, sterilize it, apply fertilizer, and water it to obtain the treated soil;

[0084] (3) On a sunny day at 13 °C, transplant the tissue culture seedlings after rooting culture into the soil treated in step (2), with the leaves aligned in the same direction, and the buds placed 1 cm below the soil surface. The row spacing and plant spacing are both 6 cm;

[0085] (4) Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, with an average of 300 mL sprayed per tissue culture seedling, then water, and insert bamboo slices and cover with a white film to seal it, ensuring that the humidity is controlled at 70%;

[0086] (5) Cover with a shading net, the shading rate of the shading net is 87%, fix the four sides, with a light intensity of 7000 lux, culture for 35 days, remove the shading net, with a light intensity of 10000 lux, and control the growth temperature at 16 °C, and continue the maintenance management for 50 days.

[0087] The compound fungicide includes the following components: carbendazim 0.5 μg / mL, galangin 3.2 μg / mL, aloe-emodin 1.4 μg / mL, celangulin 0.4 μg / mL, matrine 0.7 μg / mL, and is prepared with water.

[0088] The preparation method of the rooting powder nutrient solution is as follows:

[0089] (1) Mix burdock root, astragalus, and eucalyptus leaves in a mass ratio of 1:0.5:1.2, crush them to less than 80 mesh to obtain a mixture, add 10 parts by mass of water to 1 part by mass of the mixture to obtain a mixed solution, and continue to add Lactobacillus brevis. The concentration of Lactobacillus brevis in the mixed solution is 10 6 CFU / mL, perform anaerobic fermentation, the fermentation temperature is 32 °C, the fermentation time is 15 h, sterilize to obtain a sterilized product, and continue to add Lactobacillus plantarum. The concentration of Lactobacillus plantarum in the sterilized product is 107 CFU / mL, anaerobic fermentation was carried out at a fermentation temperature of 32 °C for 10 h. After sterilization, the filtrate was obtained by filtration through a 0.22-μm filter membrane, and the filtrate was concentrated and dried to obtain fermented powder;

[0090] (2) Potassium α-naphthylacetate, potassium 3-indolebutyrate and fermented powder were mixed at a mass ratio of 0.2:1:1.4 to obtain a mixed powder, and the mixed powder and water were diluted at a ratio of 400 mg:1 L to obtain a rooting powder nutrient solution.

[0091] The growth regulator solution includes the following components: 6-BA 6.5 mg / L, NAA 1.2 mg / L, IBA 1.3 mg / L, IAA 2.4 mg / L, plant extract 2.4 mg / L, and is prepared with water.

[0092] The preparation method of the plant extract is as follows:

[0093] (1) Platycladus orientalis leaves, Gelidium amansii, and Astragalus membranaceus were mixed at a mass ratio of 1:1.3:0.3, and pulverized to a particle size less than 80 mesh to obtain a mixed material;

[0094] (2) The mixed material was subjected to supercritical extraction. First, it was extracted at 40 °C, 30 MPa, and a CO2 flow rate of 17 L / h for 100 min to obtain the first fraction, and then it was continuously extracted at 25 °C, 25 MPa, and a CO2 flow rate of 17 L / h for 60 min to obtain the second fraction;

[0095] (3) The first fraction and the second fraction were mixed to obtain a plant extract.

[0096] Example 3

[0097] This example provides a breeding method for Cinnamomum camphora var. borneolifera, and the breeding method includes the following steps:

[0098] (1) Select the apical bud stem segment of the current-year branches of Cinnamomum camphora var. borneolifera as the explant and perform disinfection treatment; inoculate the explant into the primary culture medium to induce the formation of callus; inoculate the callus into the proliferation subculture medium for proliferation subculture to obtain cluster seedlings; first soak the cluster seedlings in a compound fungicide for 15 min, then soak the base in the growth regulator solution for 15 minutes, and then inoculate them into the rooting medium for rooting culture to obtain tissue culture seedlings;

[0099] The rooting medium is 1 / 2MS, sucrose 14 g / L, agar 22 g / L, NAA 0.9 mg / L;

[0100] (2) Select the soil with a pH value of 6, loosen the soil, sterilize, fertilize, and water to obtain the treated soil;

[0101] (3) On a sunny day at 17°C, transplant the tissue culture seedlings after rooting culture into the soil treated in step (2), align the leaves in the same direction, place the buds 1 cm below the soil surface, and set the row spacing and plant spacing at 8 cm for each plant;

[0102] (4) Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, spray 500 mL on average for each tissue culture seedling, then water, insert bamboo slices and cover with a white film to seal it, and ensure that the humidity is controlled at 90%;

[0103] (5) Cover with a shading net, the shading rate of the shading net is 92%, fix the four sides, the light is 9000 lux, culture for 40 days, remove the shading net, the light is 12000 lux, control the growth temperature at 20°C, and continue the maintenance management for 60 days.

[0104] The compound bactericide comprises the following components: carbendazim 0.9 μg / mL, galangin 3.7 μg / mL, aloe-emodin 1.7 μg / mL, celangulin 0.6 μg / mL, matrine 1.2 μg / mL, and is prepared with water.

[0105] The preparation method of the rooting powder nutrient solution is as follows:

[0106] (1) Mix burdock root, astragalus, and eucalyptus leaves in a mass ratio of 1:0.5:1.2, crush to less than 100 mesh to obtain a mixture, add 13 parts by mass of water to 1 part by mass of the mixture to obtain a mixed solution, and continue to add Lactobacillus brevis. The concentration of Lactobacillus brevis in the mixed solution is 10 7 CFU / mL, carry out anaerobic fermentation, the fermentation temperature is 35°C, the fermentation time is 20 h, sterilize to obtain a sterilized product, continue to add Lactobacillus plantarum, the concentration of Lactobacillus plantarum in the sterilized product is 10 8 CFU / mL, carry out anaerobic fermentation, the fermentation temperature is 35°C, the fermentation time is 12 h, after sterilization, filter through a 0.22 μm filter membrane to obtain a filtrate, concentrate and dry the filtrate to obtain a fermentation powder;

[0107] (2) Mix potassium α-naphthylacetate, potassium 3-indolebutyrate, and the fermentation powder in a mass ratio of 0.5:1:1.7 to obtain a mixed powder, and dilute the mixed powder and water in a ratio of 500 mg:1 L to obtain the rooting powder nutrient solution.

[0108] The growth regulator solution comprises the following components: 6-BA 8.6 mg / L, NAA 3.5 mg / L, IBA 2.6 mg / L, IAA 3.5 mg / L, plant extract 4.6 mg / L, and is prepared with water.

[0109] The preparation method of the plant extract is as follows:

[0110] (1)Mix Platycladus orientalis leaves, Gelidium amansii, and Astragalus membranaceus in a mass ratio of 1:1.5:0.7, and crush them to a particle size less than 100 mesh to obtain a mixed material;

[0111] (2)Perform supercritical extraction on the mixed material. First, extract for 120 min at 45 °C, 35 MPa, and a CO2 flow rate of 20 L / h to obtain the first fraction, and then continue to extract for 80 min at 30 °C, 30 MPa, and a CO2 flow rate of 20 L / h to obtain the second fraction;

[0112] (3)Mix the first fraction and the second fraction to obtain a plant extract.

[0113] Comparative Example 1

[0114] The difference between this comparative example and Example 1 is that the compound bactericide is replaced with a carbendazim aqueous solution with a concentration of 7 μg / mL.

[0115] Comparative Example 2

[0116] The difference between this comparative example and Example 1 is that the compound bactericide includes the following components: carbendazim 3.4 μg / mL, galangin 0.7 μg / mL, aloe-emodin 0.5 μg / mL, celangulin 2.0 μg / mL, matrine 0.4 μg / mL, and is prepared with water.

[0117] Comparative Example 3

[0118] The difference between this comparative example and Example 1 is that the preparation method of the rooting powder nutrient solution is as follows: Mix potassium α-naphthylacetate and potassium 3-indolebutyrate in a mass ratio of 0.4:1 to obtain a mixed powder, and dilute the mixed powder and water in a ratio of 450 mg:1 L to obtain the rooting powder nutrient solution.

[0119] Comparative Example 4

[0120] The difference between this comparative example and Example 1 is that potassium α-naphthylacetate, potassium 3-indolebutyrate, and baking powder are mixed in a mass ratio of 1:1:1.

[0121] Comparative Example 5

[0122] The difference between this comparative example and Example 1 is that the growth regulator solution includes the following components: 6-BA 2.4 mg / L, NAA 9.5 mg / L, IBA 3.1 mg / L, IAA 2.2 mg / L, and plant extract 2.0 mg / L, and is prepared with water.

[0123] Comparative Example 6

[0124] The difference between this comparative example and Example 1 is that the growth regulator solution comprises the following components: 6-BA 7.5 mg / L, NAA 4.4 mg / L, IBA 4.2 mg / L, IAA 3.1 mg / L, and is prepared with water.

[0125] Comparative Example 7

[0126] The difference between this comparative example and Example 1 is that burdock roots, astragalus roots, and eucalyptus leaves with a mass ratio of 1:1:1 are mixed.

[0127] Comparative Example 8

[0128] The difference between this comparative example and Example 1 is that oriental arborvitae leaves, gracilaria, and astragalus roots with a mass ratio of 1:1:1 are mixed.

[0129] Performance Test

[0130] Cultivation is carried out according to the breeding methods of Examples 1-3 and Comparative Examples 1-8. 100 plants are cultivated in each group, and the growth conditions of Cinnamomum camphora in each treatment are investigated. The results are shown in Table 1 below;

[0131] Average number of roots = total number of roots of all plants / number of plants.

[0132] Table 1 Performance Test Results

[0133]

[0134] It can be seen from the above performance test results that the breeding effects of Examples 1-3 are good, especially the comprehensive performance of Example 1 is the most prominent. This is mainly because the culture conditions such as the growth regulator, rooting powder nutrient solution, and compound bactericide produce a synergistic effect.

[0135] In the comparative examples, because the necessary technical solutions are not adopted, their performance in the corresponding performance tests is significantly worse than that of the examples. In Comparative Examples 1-2, the composition of the compound bactericide is changed, and the survival rate of Cinnamomum camphora breeding decreases. In Comparative Examples 3-4 and 7, the composition of the rooting powder nutrient solution is changed, and the rooting rate of Cinnamomum camphora breeding decreases. In Comparative Examples 5-6 and 8, the composition of the growth regulator solution is changed, and the average plant height of Cinnamomum camphora decreases. The above experimental results further prove the importance of the technical solutions defined in the present invention for its technical effects.

[0136] The above is the preferred implementation mode of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A breeding method of Cinnamomum camphora var. borneolifera, characterized in that, The breeding method includes the following steps: (1): Select the apical bud stem segments of the current-year branches of Cinnamomum camphora var. borneolifera as explants, and disinfect them; inoculate them into the primary culture medium to induce the formation of callus; inoculate the callus into the proliferation subculture medium for proliferation subculture to obtain cluster seedlings; soak the cluster seedlings with a compound bactericide, then soak them with a growth regulator solution, and then inoculate them into the rooting culture medium for cultivation to obtain tissue culture seedlings; The compound bactericide is: carbendazim 0.5 - 0.9 μg / mL, galangin 3.2 - 3.7 μg / mL, aloe-emodin 1.4 - 1.7 μg / mL, celangulin 0.4 - 0.6 μg / mL, and matrine 0.7 - 1.2 μg / mL, prepared with water; The rooting culture medium is: 1 / 2MS, sucrose 12 - 14 g / L, agar 20 - 22 g / L, and NAA 0.6 - 0.9 mg / L; The growth regulator solution is: 6-BA 6.5 - 8.6 mg / L, NAA 1.2 - 3.5 mg / L, IBA 1.3 - 2.6 mg / L, IAA 2.4 - 3.5 mg / L, and plant extract 2.4 - 4.6 mg / L, prepared with water; (2): Loosen the soil, sterilize it, fertilize it, and water it to obtain the treated soil; (3): Transplant the tissue culture seedlings into the soil treated in step (2); (4): Disinfect the tissue culture seedlings again after cutting, spray the rooting powder nutrient solution on the soil around the tissue culture seedlings, then water them, and insert bamboo slices and cover with a film for sealing; (5): Cover with a shading net, fix the four sides, cultivate under light conditions, and continue the maintenance management after removing the shading net; The preparation method of the plant extract is: (1) Mix Chinese arborvitae leaves, Gelidium amansii, and Astragalus membranaceus in a mass ratio of 1:(1.3 - 1.5):(0.3 - 0.7), and crush them to a particle size less than 80 - 100 mesh to obtain a mixture; (2) Perform supercritical extraction on the mixture. First, extract for 100 - 120 min at 40 - 45 °C, 30 - 35 MPa, and a CO2 flow rate of 17 - 20 L / h to obtain the first fraction, and continue to extract for 60 - 80 min at 25 - 30 °C, 25 - 30 MPa, and a CO2 flow rate of 17 - 20 L / h to obtain the second fraction; (3) Mix the first fraction and the second fraction to obtain the plant extract; The preparation method of the rooting powder nutrient solution is: (1) Mix burdock roots, Astragalus membranaceus, and eucalyptus leaves in a mass ratio of 1:(0.5 - 0.7):(1.2 - 1.4), crush them to less than 80 - 100 mesh to obtain a mixture, add 10 - 13 parts by mass of water to 1 part by mass of the mixture to obtain a mixed solution, continue to add Lactobacillus brevis, and perform anaerobic fermentation. The fermentation temperature is 32 - 35 °C and the fermentation time is 15 - 20 h. After sterilization, obtain a sterilized product. Continue to add Lactobacillus plantarum and perform anaerobic fermentation. The fermentation temperature is 32 - 35 °C and the fermentation time is 10 - 12 h. After sterilization, filter through a 0.22 μm filter membrane to obtain a filtrate, concentrate and dry the filtrate to obtain a fermentation powder; (2) Mix potassium α-naphthylacetate, potassium 3-indolebutyrate and baking powder in a mass ratio of (0.2 - 0.5) : 1 : (1.4 - 1.7) to obtain a mixed powder. Dilute the mixed powder and water in a ratio of 400 - 500 mg : 1 L to obtain a rooting powder nutrient solution.

2. The breeding method of Cinnamomum camphora var. borneolifera according to claim 1, characterized in that, The concentration of Lactobacillus brevis in the mixed solution is 10 6 -10 7 CFU / mL.

3. The breeding method of Cinnamomum camphora var. borneolifera according to claim 2, characterized in that The concentration of Lactobacillus plantarum in the sterilized product is 10 7 -10 8 CFU / mL.

4. The breeding method of Cinnamomum camphora var. borneolifera according to claim 3, characterized in that, Select tissue culture seedlings with white dots after cutting and prune them.

5. Application of the breeding method according to any one of claims 1 - 4 in the breeding of Cinnamomum camphora var. borneolifera.

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