A method for raising seedlings of Pinus davidiana tissue culture in factories

Abstract

The invention provides a method for raising seedlings of Pinus gracile by tissue culture in a factory, comprising the following steps: S1: collection and treatment of explants, S2: disinfection and inoculation, S3: culture, S4: differentiation and proliferation, S5: rooting culture, S6: transplanting and acclimation, etc. The tissue culture technology includes: through strict aseptic operation and environmental control, the explants are disinfected and then inoculated; during the culture process, the culture environment is strictly controlled, and the formula of the culture medium and other conditions are adjusted, so that necessary nutrition and growth conditions are effectively provided, rooting of the explants is promoted, and the number and length of roots are increased, thereby improving the rooting rate and the survival rate of transplanting; the tissue culture method of Pinus gracile has a fast propagation speed and is not restricted by seasons, thereby greatly improving the seedling raising efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for factory-based seedling cultivation of Pinus davidiana tissue culture. Background Art

[0002] Baeckea frutescens L. is a small tree or shrub of the Myrtaceae family. The young branches are slender and branched; the leaves are small and sessile, or have short petioles, and the leaf blades are narrowly linear or linear, with pointed tips, grooves on the upper side, protrusions on the lower side, and transparent oil glands. They are brown after drying; the flowers are small, white, solitary in the leaf axils, the bracts fall off early, the calyx tube is bell-shaped, the calyx teeth are small and triangular, with acute tips, and the petals are round and narrow at the base into short petioles; the capsule is small; the seeds are flat and angular; the flowering period is July-August; the fruiting period is September-November. It is named because its leaves are shorter like pine needles. It is a positive plant that likes high temperature, humid, sunny places. The suitable growth temperature is 22-32℃ and the sunshine is 70%-100%. It grows slowly and is heat-resistant, drought-resistant and wind-resistant. Humus or sandy loam is the best cultivation medium. It often grows in low hills, grass slopes and bushes on barren mountains.

[0003] The germination rate of Pinus dasyphylla seeds is relatively low, which limits the demand for seedlings in large-scale production. Due to the low germination rate, traditional breeding methods cannot meet the needs of large-scale planting. The general breeding methods include sowing, cutting propagation and high pressure method. The offspring of seed propagation may have differences in the medicinal components, which is not conducive to the promotion and planting of excellent varieties of Pinus dasyphylla. In addition, the germination rate of Pinus dasyphylla seeds is relatively low and the growth cycle is long. The rooting ability of Pinus dasyphylla is relatively weak, and it takes a certain amount of time and conditions for the cuttings to root. The seedlings propagated by cuttings are easily infected with pests and diseases during the growth process, especially if the cuttings themselves carry pathogens or the growth environment is unsanitary. The occurrence of pests and diseases may cause the growth of seedlings to be hindered or even die. Although the high pressure method is an effective breeding method for some plants, it also has some inherent disadvantages, especially when applied to plants such as Pinus dasyphylla. The ring barking process may damage the branches. Especially if the operation is improper or the plant itself is relatively fragile, the survival rate is not stable. Therefore, when developing Pinus dasyphylla resources, scientific and reasonable development and protection strategies should be adopted to ensure the sustainable use of Pinus dasyphylla resources. Summary of the invention

[0004] In view of this, the present invention proposes a method for factory-based tissue culture seedling raising of Pinus tabulaeformis to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: a method for raising seedlings of Pinus tabulaeformis tissue culture in factories: comprising the following steps:

[0006] S1. Collection and treatment of explants: Select healthy and pest-free Pinus davidiana plants, cut their tender side branches or leaves as explants, clean the explants, remove excess leaves and branches, cut into slices or small pieces, put them into a sterile container, and soak them in plant growth regulators for full absorption for 20-40 minutes;

[0007] S2, disinfection and inoculation: disinfect the surface of the explant to remove microorganisms on the surface. After disinfection, take out the explant, rinse it with sterile water, and then inoculate it into the culture medium. Each bottle of the culture medium is inoculated with 1-3 stem segments;

[0008] S3, culture: the inoculated explants are placed in a constant temperature incubator for induction culture for 2-3 weeks;

[0009] S4, differentiation and proliferation: The clustered buds produced by the induced culture are cultured for proliferation for 3-4 weeks, during which the explants are subcultured regularly;

[0010] S5. Rooting culture: Cut the 1.5 cm high clustered buds for rooting induction culture, and culture for 3-6 weeks to form rooted seedlings;

[0011] S6. Transplantation and domestication: Remove the rooted seedlings from the culture medium, clean them, and transplant them to the greenhouse or field for adaptive domestication for 2-3 months. During the domestication period, regularly observe the growth of the seedlings and adjust the culture conditions to ensure the normal growth and development of the seedlings. At the same time, provide necessary care and pruning for the seedlings.

[0012] Furthermore, the S1 plant growth regulator comprises the following raw materials in parts by weight: 10-50 parts of indoleacetic acid, 8-12 parts of gibberellins, 5-10 parts of cytokinins, 3-5 parts of brassinolide, and 4-6 parts of jasmonic acid.

[0013] Furthermore, the S2 disinfection treatment is first treated with alcohol, then disinfected with 0.1-0.3wt% mercuric chloride for 5-12 minutes, and 3-5 drops of coconut oil are added during the disinfection with mercuric chloride, and finally rinsed with sterile water for 4-6 times.

[0014] Furthermore, the culture medium of S2 includes MS basal culture medium, sucrose 10-30 g / L, N-acetylaspartic acid 0.1-0.3 mg / L, potassium dihydrogen phosphate 0.8-1.5 mg / L, vitamin B1 0.1-0.3 mg / L, vitamin B2 0.3-0.8 mg / L, zeatin 0.8-1.5 mg / L, and plant polypeptide solution 0.1-0.3 mg / L.

[0015] Furthermore, the plant polypeptide liquid is yam polypeptide liquid, tea polypeptide liquid and ginkgo polypeptide liquid with a volume of (8-10):(4-7):(1-3).

[0016] Furthermore, the tea polypeptide liquid is a small molecule water-soluble peptide obtained by enzymatic hydrolysis of any one of black tea polypeptide, camellia sinensis polypeptide and partridge tea polypeptide, and the mass ratio of the tea polypeptide to the protease is 25-38:2.

[0017] Furthermore, the protease is papain, neutral protease, and alkaline protease in a mass ratio of (1-3):(0.2-0.8):(5-8).

[0018] Furthermore, the induction culture conditions in S3 are: temperature of 25-30° C., light of 1500-2000 lux, light duration of 10-18 h / d, and humidity of 70-80%.

[0019] Furthermore, the conditions for proliferation culture in S4 are: temperature of 25-30°C, light of 3000-4000 lux, light time of 10-18h / d, and humidity of 60-70%.

[0020] Furthermore, the conditions for the adaptive acclimatization culture in S6 are: temperature of 20-30° C., light of 5000-6000 lux, light duration of 10-18 h / d, and humidity of 50-60%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The method for raising seedlings of Pinus tabulaeformis by tissue culture in a factory can obtain a large number of high-quality seedlings in a short time through tissue culture technology. Compared with traditional cutting or seed propagation, the tissue culture method has a fast propagation speed and is not restricted by seasons, thereby greatly improving the efficiency of raising seedlings. In the process of tissue culture, the excellent traits of the mother plant can be maintained through asexual reproduction, avoiding genetic variation that may be caused by sexual reproduction. In this way, it can be ensured that the produced seedlings have consistency in growth rate, disease resistance, yield, etc.

[0023] The present invention uses strict aseptic operation and environmental control, adds coconut oil and mercuric chloride to synergistically reduce the occurrence of diseases and insect pests during explant disinfection. Compared with traditional seedling raising methods, the incidence of diseases and insect pests in tissue culture seedlings is lower, which is beneficial to improving the survival rate and growth quality of seedlings; the present invention optimizes the growth environment of explants by adjusting the formula of the culture medium and other conditions. The quality of the growth speed, root development, stress resistance and other aspects of the seedlings can be improved, thereby improving the rooting rate and survival rate.

[0024] The factory-based tissue culture seedling raising method of Pinus glauca has the advantages of high efficiency, rapidity, improved quality, convenience for large-scale production, and reduction of pests and diseases, and is of great significance for realizing large-scale planting and industrial development of Pinus glauca. DETAILED DESCRIPTION

[0025] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0026] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0027] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0028] Example 1

[0029] A method for raising seedlings of Pinus davidiana in factory-based tissue culture comprises the following steps:

[0030] S1. Collection and treatment of explants: Select healthy and pest-free Pinus dactylifera plants, cut their tender side branches or leaves as explants, clean the explants, remove excess leaves and branches, cut into slices or small pieces, put them into a sterile container, and soak them in a plant growth regulator for full absorption for 20 minutes; the plant growth regulator includes the following raw materials by weight: 10 parts of indoleacetic acid, 8 parts of gibberellins, 5 parts of cytokinins, 3 parts of brassinolide, and 4 parts of jasmonic acid;

[0031] S2, disinfection and inoculation: the explants are surface disinfected, first treated with alcohol, then disinfected with 0.1wt% mercuric chloride for 5 minutes, and 3 drops of coconut oil are added to the mercuric chloride when using mercuric chloride for disinfection, and finally rinsed with sterile water for 4 times to remove surface microorganisms. After disinfection, the explants are taken out, rinsed with sterile water, and then inoculated into culture medium, and 3 stem segments are inoculated into each bottle of the culture medium; the culture medium includes MS basal culture medium, 10g / L sucrose, 0.1mg / L N-acetylaspartic acid, 0.8mg / L potassium dihydrogen phosphate, 0.1mg / L vitamin B1, 0.3mg / L vitamin B2, 0.8mg / L zeatin, and 0.1mg / L plant polypeptide liquid; the plant polypeptide liquid is yam peptide liquid, tea polypeptide liquid and ginkgo peptide liquid with a volume ratio of 8:4:1;

[0032] S3. Cultivation: Place the inoculated explants in a constant temperature incubator at 25°C, 1500 lux, 10 h / d, and 70% humidity for induced culture for 2 weeks;

[0033] S4. Differentiation and proliferation: The clustered buds produced by the induced culture were cultured for proliferation at a temperature of 25°C, a light intensity of 3000 lux, a light intensity of 10 h / d, and a humidity of 60% for 3 weeks. During the culture period, the explants were subcultured regularly.

[0034] S5, rooting culture: cut the 1.5 cm high clustered buds for rooting induction culture, culture for 3 weeks to form rooted seedlings;

[0035] S6. Transplantation and acclimatization: Remove the rooted seedlings from the culture medium, clean them, and transplant them to a greenhouse or field for acclimatization for 2 months at a temperature of 20°C, a light of 5000 lux, a lighting time of 10 h / d, and a humidity of 50%. During the acclimatization period, regularly observe the growth of the seedlings and adjust the culture conditions to ensure the normal growth and development of the seedlings. At the same time, provide necessary care and pruning for the seedlings.

[0036] Example 2

[0037] A method for raising seedlings of Pinus davidiana in factory-based tissue culture comprises the following steps:

[0038] S1. Collection and treatment of explants: Select healthy and pest-free Pinus dactylifera plants, cut their tender side branches or leaves as explants, clean the explants, remove excess leaves and branches, cut into slices or small pieces, put them into a sterile container, and soak them in a plant growth regulator for full absorption for 40 minutes; the plant growth regulator includes the following raw materials by weight: 50 parts of indoleacetic acid, 12 parts of gibberellins, 10 parts of cytokinins, 5 parts of brassinolide, and 6 parts of jasmonic acid;

[0039] S2, disinfection and inoculation: the explants are surface disinfected, first treated with alcohol, and then disinfected with 0.3wt% mercuric chloride for 12 minutes, and 5 drops of coconut oil are added to the mercuric chloride when using mercuric chloride for disinfection, and finally rinsed with sterile water for 6 times to remove surface microorganisms. After disinfection, the explants are taken out, rinsed with sterile water, and then inoculated into the culture medium, and 3 stem segments are inoculated into each bottle of the culture medium; the culture medium includes MS basal culture medium, 30g / L sucrose, 0.3mg / L N-acetylaspartic acid, 1.5mg / L potassium dihydrogen phosphate, 0.3mg / L vitamin B1, 0.8mg / L vitamin B2, 1.5mg / L zeatin, and 0.3mg / L plant polypeptide liquid; the plant polypeptide liquid is yam peptide liquid, tea polypeptide liquid and ginkgo peptide liquid with a volume ratio of 10:7:3;

[0040] S3. Cultivation: Place the inoculated explants in a constant temperature incubator at 30°C, 2000 lux, 18 h / d, and 80% humidity for induction culture for 3 weeks;

[0041] S4. Differentiation and proliferation: The clustered buds produced by the induced culture were cultured for proliferation at a temperature of 30°C, a light intensity of 4000 lux, a light intensity of 18 h / d, and a humidity of 70% for 4 weeks. During the culture period, the explants were subcultured regularly.

[0042] S5, rooting culture: cut the 1.5 cm high clustered buds for rooting induction culture, culture for 6 weeks to form rooted seedlings;

[0043] S6. Transplantation and domestication: Remove the rooted seedlings from the culture medium, clean them, and transplant them to a greenhouse or field for domestication for 3 months at a temperature of 30°C, a light of 6000 lux, a light duration of 18 h / d, and a humidity of 60%. During the domestication period, regularly observe the growth of the seedlings and adjust the culture conditions to ensure the normal growth and development of the seedlings. At the same time, provide necessary care and pruning for the seedlings.

[0044] Example 3

[0045] A method for raising seedlings of Pinus davidiana in factory-based tissue culture comprises the following steps:

[0046] S1. Collection and treatment of explants: Select healthy and pest-free Pinus dactylifera plants, cut their tender side branches or leaves as explants, clean the explants, remove excess leaves and branches, cut into slices or small pieces, put them into a sterile container, and soak them in a plant growth regulator for full absorption for 30 minutes; the plant growth regulator includes the following raw materials by weight: 30 parts of indoleacetic acid, 10 parts of gibberellins, 8 parts of cytokinins, 4 parts of brassinolide, and 5 parts of jasmonic acid;

[0047] S2, disinfection and inoculation: the explants are surface disinfected, first treated with alcohol, then disinfected with 0.2wt% mercuric chloride for 8min, and 4 drops of coconut oil are added to the mercuric chloride when using mercuric chloride for disinfection, and finally rinsed with sterile water for 5 times to remove surface microorganisms. After disinfection, the explants are taken out, rinsed with sterile water, and then inoculated into culture medium, and 3 stem segments are inoculated into each bottle of the culture medium; the culture medium includes MS basal culture medium, sucrose 20g / L, N-acetylaspartic acid 0.2mg / L, potassium dihydrogen phosphate 1.2mg / L, vitamin B1 0.2mg / L, vitamin B2 0.5mg / L, zeatin 1.3mg / L, and plant polypeptide liquid 0.2mg / L; the plant polypeptide liquid is yam peptide liquid, tea polypeptide liquid and ginkgo peptide liquid with a volume ratio of 9:5:2;

[0048] S3. Cultivation: Place the inoculated explants in a constant temperature incubator at 28°C, 1800 lux, 15 h / d, and 75% humidity for induction culture 2;

[0049] S4. Differentiation and proliferation: The clustered buds produced by the induced culture were cultured for proliferation at a temperature of 28°C, a light intensity of 3500 lux, a light intensity of 15 h / d, and a humidity of 65% for 3 weeks. During the culture period, the explants were subcultured regularly.

[0050] S5, rooting culture: cut the 1.5 cm high clustered buds for rooting induction culture, culture for 5 weeks to form rooted seedlings;

[0051] S6. Transplantation and acclimatization: Remove the rooted seedlings from the culture medium, clean them, and transplant them to a greenhouse or field for acclimatization for 2.5 months at a temperature of 25°C, a light of 5500 lux, a light time of 15 h / d, and a humidity of 55%. During the acclimatization period, regularly observe the growth of the seedlings and adjust the culture conditions to ensure the normal growth and development of the seedlings. At the same time, provide necessary care and pruning for the seedlings.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 3 is that in the S2 disinfection treatment, mercuric chloride is replaced by an equal amount of sodium chlorate.

[0054] Comparative Example 2

[0055] The difference between this comparative example and Example 3 is that no coconut oil is added during the disinfection treatment of S2.

[0056] Comparative Example 3

[0057] The difference between this comparative example and Example 3 is that the culture medium does not contain plant polypeptide liquid.

[0058] 1. Effect measurement

[0059] The rooting rate and survival rate of the seedlings cultivated in the above Examples 1-3 and Comparative Examples 1-3 were calculated according to the following formula 1 and formula 2:

[0060] Rooting rate = (number of rooted seedlings / total number of seedlings) × 100%, (1);

[0061] Survival rate = (number of surviving seedlings / total number of seedlings) × 100%, (2);

[0062] Rooting rate% Survival rate% Example 1 98.1 91.2 Example 2 98.3 91.8 Example 3 98.8 92.3 Comparative Example 1 82.6 79.5 Comparative Example 2 76.7 78.9 Comparative Example 3 75.2 65.4

[0063] It can be seen from the above results that the method for raising seedlings in a factory by tissue culture of Pinus tabulaeformis of the present invention has good rooting rate and survival rate, with the rooting rate being above 98% and the survival rate being above 91%. The rooting rate of the embodiment group is 98.1-98.8% and the survival rate is 91.2-92.3%. Compared with Comparative Example 1, the use of mercuric chloride is beneficial to reducing or eliminating microorganisms that may cause pollution or infection; compared with Comparative Example 2, the addition of coconut oil and mercuric chloride synergizes to achieve the killing efficiency of microorganisms, which helps to achieve better disinfection effect in a shorter time. Coconut oil has a high solubility in water and is easily washed away after disinfection treatment, reducing the residual HgCl2 on the plant material; coconut oil is relatively mild and has less irritation to many plant materials, so it can reduce the potential damage to the plant material.

[0064] Compared with Comparative Example 3, the plant polypeptide liquid as a culture medium provides the necessary nutrition and growth conditions, promotes the rooting of explants, increases the number and length of roots, and thus improves its rooting rate and transplant survival rate. Among them, the yam peptide contains rich amino acids and bioactive substances, which can enhance the resistance of explants to adverse environmental conditions and improve their survival rate and growth quality; the tea polypeptide liquid contains rich amino acids, peptides and other bioactive substances, which can provide rich nutrition for explants, promote cell division and amplification, and thus accelerate the growth and development of explants; some components in the ginkgo peptide liquid have antioxidant, anti-inflammatory and other biological activities, which can enhance the resistance of explants to adverse environmental conditions. This helps to reduce the damage to the explants during the culture process and improve their survival rate and growth quality.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for raising seedlings of Pinus davidiana tissue culture in an industrial scale, characterized in that: The following steps are involved: S1. Collection and treatment of explants: Select healthy and pest-free Pinus davidiana plants, cut their tender side branches as explants, clean the explants, remove excess leaves and branches, cut into small pieces, put them into a sterile container, and soak them in a plant growth regulator for full absorption for 20-40 minutes; the plant growth regulator comprises the following raw materials by weight: 10-50 parts of indoleacetic acid, 8-12 parts of gibberellins, 5-10 parts of cytokinins, 3-5 parts of brassinolide, and 4-6 parts of jasmonic acid; S2, disinfection and inoculation: the explants are surface disinfected, the disinfection is first treated with alcohol, and then disinfected with 0.1-0.3wt% HgCl2 for 5-12min, and when using mercuric chloride for disinfection, 3-5 drops of coconut oil are added to the mercuric chloride, and finally rinsed with sterile water for 4-6 times to remove surface microorganisms. After disinfection, the explants are taken out, rinsed with sterile water, and then inoculated into the culture medium, and 1-3 stem segments are inoculated in each bottle of the culture medium; the culture medium includes MS basal culture medium, sucrose 10-30g / L, N-acetylaspartic acid 0.1-0.3mg / L, potassium dihydrogen phosphate 0.8-1.5mg / L, vitamin B10.1-0.3mg / L, vitamin B2 0.3-0.8mg / L, zeatin 0.8-1.5mg / L, plant polypeptide liquid 0.1-0.3mg / L, the plant polypeptide liquid is yam peptide liquid, tea polypeptide liquid and ginkgo peptide liquid in a volume ratio of (8-10):(4-7):(1-3), the tea polypeptide liquid is a small molecule water-soluble peptide obtained by protease hydrolysis of any one of black tea polypeptide, camellia polypeptide and partridge tea polypeptide, and the mass ratio of tea polypeptide to protease is 25-38:2; S3. Cultivation: Place the inoculated explants in a constant temperature incubator for induction culture for 2-3 weeks. The induction culture conditions are: temperature 25-30°C, light 1500-2000 lux, light time 10-18h / d, humidity 70-80%; S4. Differentiation and proliferation: The clustered buds produced by the induced culture were cultured for proliferation for 3-4 weeks. The conditions for proliferation culture were: temperature 25-30°C, light 3000-4000 lux, light duration 10-18h / d, humidity 60-70%. During the culture period, the explants were subcultured regularly. S5. Rooting culture: Cut the 1.5 cm high clustered buds for rooting induction culture, and culture for 3-6 weeks to form rooted seedlings; S6. Transplantation and domestication: Remove the rooted seedlings from the culture medium, clean them, and transplant them to the greenhouse or field for adaptive domestication for 2-3 months. The conditions for adaptive domestication are: temperature 20-30℃, light 5000-6000lux, light time 10-18h / d, humidity 50-60%. During the domestication period, regularly observe the growth of the seedlings and adjust the culture conditions to ensure the normal growth and development of the seedlings. At the same time, provide necessary care and pruning for the seedlings.

2. A method for raising seedlings of Pinus davidiana tissue culture in factories as claimed in claim 1, characterized in that: The protease of S2 is papain, neutral protease and alkaline protease in a mass ratio of (1-3):(0.2-0.8):(5-8).