Method for improving tissue culture and rapid propagation proliferation rate of chirita biloba

Abstract

The invention provides a method for improving tissue culture and rapid propagation proliferation rate of bilochia, which comprises the following steps: by taking a bilochia plant as a female parent and taking a tender new leaf as an explant, preparing and disinfecting the explant, performing adventitious bud induction culture, performing adventitious bud proliferation culture, performing seedling strengthening and rooting culture, transplanting a tissue culture seedling and the like, thereby obtaining the bilochia tissue culture and rapid propagation proliferation rate of the bilochia. By optimizing a culture medium formula and culture conditions, the high-efficiency induction success rate and rapid proliferation of the adventitious buds in the tissue culture and rapid propagation process of the chirita biloba are realized, and the proliferation rate is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant propagation, and specifically relates to a method for improving the rapid propagation rate of tissue culture of scutellaria baicalensis. Background Art

[0002] Most Gesneriaceae plants are perennial herbs with a wide variety of species. There are about 140 genera and more than 2,000 species in the world, and 58 genera and 463 species in my country. Some species are drought-tolerant, shade-tolerant, and moisture-tolerant, while others have rich plant, leaf, and flower shapes, beautiful flowers and leaves, and high ornamental value. They have certain ornamental value development prospects for indoor potted flowers and landscape engineering applications. As people's understanding of Gesneriaceae plants gradually deepens, consumers have a strong interest in them. However, in the field of ornamental plant breeding and protection, Gesneriaceae, as a rare and ornamental plant, is particularly important for its protection and breeding because of its harsh requirements for environmental conditions, low natural reproduction rate, and decreasing population year by year. At present, the main propagation methods for Gesneriaceae include seed propagation and leaf cutting propagation, but the former is often affected by seed source restrictions and unstable germination rate, while the latter has problems such as long reproduction cycle and low coefficient. In recent years, tissue culture technology has gradually become an important means of rapid propagation of rare plants such as Gesneriaceae due to its advantages such as short cycle, high reproduction coefficient and good genetic stability. However, the existing tissue culture rapid propagation technology of Gesneriaceae still faces bottlenecks such as low proliferation rate and long culture cycle, which limits its application in large-scale production. The use of tissue culture rapid propagation technology is conducive to the commercial development and germplasm resource preservation of Gesneriaceae plants, and is also conducive to the further development of the breeding of improved varieties, artificial seed cultivation, and breeding technology research of Gesneriaceae plants. Summary of the invention

[0003] The purpose of the present invention is to provide a method for improving the proliferation rate of tissue culture rapid propagation of biloba, using young and tender new leaves of biloba as explants, and optimizing explant treatment, culture medium formula and culture conditions to significantly improve the tissue culture rapid propagation efficiency and proliferation rate of biloba.

[0004] In order to achieve the above object, the present invention provides the following technical solutions: A method for improving the proliferation rate of rapid propagation of bifolia larvae tissue culture, comprising the following steps: S1: Preparation and disinfection of explants: Using a two-leaf Gesneriaceae plant as the mother plant, taking young and tender new leaves as explants, washing them clean and moving them into a clean bench, soaking them in alcohol and washing them with sterile water; then dripping disinfectant, shaking them constantly to make the explants fully contact with the disinfectant, washing them with sterile water for 3-6 times, and then placing the explants on sterile filter paper to absorb moisture to obtain sterilized explants; S2: Adventitious bud induction culture: Cut the explant leaves disinfected in step S1, place the leaves facing upward on the induction medium for induction culture, and transfer the leaves to the proliferation medium after adventitious buds grow out of the cut positions of the leaves; S3: Proliferation culture of adventitious buds: cutting the adventitious buds from the leaves in step S2, inoculating them into a proliferation medium for proliferation and expansion culture, so as to achieve rapid proliferation of the buds, form robust bud seedlings, and obtain adventitious bud tissues; S4: Rooting and rooting culture of seedlings: the seedlings are cut from the adventitious bud tissue obtained in step S3, and inoculated into the rooting culture medium for rooting culture to promote the growth of the root system, and obtain rooted seedlings with a high rooting rate and a well-developed root system. After the roots of 2-3 cm are grown, they can be taken out of the bottle for planting; S5: Transplanting of tissue culture seedlings: Take out the seedlings in step S4 from the culture bottle, wash off the culture medium with clean water, transfer to the water curtain greenhouse, and plant them in pots; after cultivation, place the leaves of the seedlings parallel to the diagonal of the pot; sprinkle with 2000 times zinc after transplanting, the humidity of the planting environment is 75-90%, the temperature is 20-30℃, the planting substrate is imported peat soil, and after the roots grow out, start to fertilize and water to complete the transplanting.

[0005] Preferably, step S1 is specifically as follows: selecting a two-leaf Gesneriaceae plant that is healthy, pest-free, and growing well as the mother plant, taking young tender new leaves that have grown for 10-20 days at the top as explants, rinsing them with tap water, moving them into a clean bench, soaking them in 75% alcohol for 30-60 seconds, washing them twice with sterile water, and then treating them with a 0.1% HgCl2 solution with 2 drops of TWeen-20 for 5-8 minutes, shaking them continuously during the process to ensure that the explants are in full contact with the disinfectant, and washing them with sterile water 3-6 times; placing the explants on sterile filter paper to absorb moisture.

[0006] Preferably, the induction culture medium in step S2 is 1 / 3MS+6-BA 1.0-3.0mg / L+NAA 0.1-0.2mg / L; the sucrose content of the culture medium is 25-30g / L, and the pH is 5.8-6.0.

[0007] Preferably, the induction culture in step S2 is carried out at an ambient temperature of 24-28° C., with a light duration of 8-10 hours, a light intensity of 500-1000 lux, and culture for 40-50 days.

[0008] Preferably, the proliferation culture medium in step S3 is: 1 / 2MS + 6-BA 0.5-1.5 mg / L + NAA 0.05-0.1 mg / L, wherein the sucrose content of the culture medium is 25-30 g / L, and the pH is 5.8-6.0.

[0009] Preferably, the proliferation culture in step S3 is carried out at an ambient temperature of 24-28° C., with a lighting duration of 10-12 hours, a light intensity of 1500-2000 lux, and culture for 35-40 days.

[0010] Preferably, the rooting medium in step S4 is: 1 / 2MS+IBA 0.1-0.2mg / L+NAA0.1-0.2mg / L, the sugar content of the medium is 25-30 / L, and the pH is 5.8-6.0.

[0011] Preferably, the ambient temperature of the rooting culture in step S4 is 24-28° C., the lighting duration is 8-10 hours, the light intensity is 2000-2500 lux, the culture is 35-40 days, and the seedlings grow to 4-5 leaves.

[0012] Preferably, in step S5, after planting, 2000 times of zinc disulfide is applied once, and the planting environment humidity is 75-90% and the temperature is 20-30°C.

[0013] Preferably, the tissue culture seedling transplanting matrix in step S5 is an imported peat matrix.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are: (1) The present invention achieves high efficiency and high quality of rapid tissue culture propagation of Sedum bifolium through specific explant treatment, induction culture medium, subculture proliferation culture medium and rooting culture medium formula, as well as strict culture condition control. The culture medium formula adopted by the present invention is simple, easy to operate, and relatively low in cost, and is suitable for large-scale promotion and application.

[0015] (2) The present invention uses a low-concentration nutrient component of 1 / 3MS in the induction culture step to induce adventitious buds, reduce the browning degree of the explant material, and achieves a high-efficiency induction success rate and rapid proliferation of adventitious buds in the rapid propagation process of the double-leaf chrysanthemum tissue culture by optimizing the culture medium formula and culture conditions, and significantly improves the proliferation rate; in the proliferation culture step, a low-concentration nutrient component of 1 / 2MS and a low-concentration hormone ratio of 0.5-1.5 mg / L of 6-benzylaminopurine and 0.05-0.1 mg / L of naphthaleneacetic acid are used to effectively reduce the vitrification degree of the adventitious buds, and the proliferation coefficient reaches 3.0-4.0. Compared with the general division propagation method, the quality of the seedlings is greatly improved. The rooting culture environment temperature is 24-28°C, the light duration is 10-12 hours, and the light intensity is 2000-2500 lux. It can stably propagate robust tissue culture seedlings for a long time, and the root induction rate reaches 98%. After optimized rooting and cultivation steps, the planting medium is imported peat soil; the root system grows robustly, and the survival rate of seedlings after transplanting is high, reaching more than 95%. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Example 1 This embodiment provides a method for improving the proliferation rate of rapid propagation of Scutellaria baicalensis through tissue culture, comprising the following steps: S1: Preparation and disinfection of explants: Select healthy, pest-free, and well-growing two-leaf Gesneriaceae plants as the mother plant, take the young and tender new leaves of the top that have grown for 10-20 days as the explants, rinse them with tap water, move them to the clean bench, soak them in 75% alcohol for 30-60 seconds, wash them with sterile water twice, and then rinse them with 0.1% HgCl with 2 drops of TWeen-20 2 The solution is treated for 5-8 minutes, during which the explant is shaken continuously to allow full contact between the explant and the disinfectant, and then washed with sterile water for 3-6 times; the explant is placed on sterile filter paper to absorb the water, thereby obtaining the sterilized explant; S2: Adventitious bud induction culture: Cut the explant leaves disinfected in step S1 into 10 mm × 10 mm size, place the leaves facing upward on the induction medium, culture at 24-28°C, light duration 8-10 hours, light intensity 500-1000 lux, culture for 40-50 days, and transfer to the proliferation medium after adventitious buds grow at the leaf cut; the induction medium is composed of: NH 4 NO 3 550mg / L, KNO 3 633.3mg / L, MgSO 4 7H 2 O 123.3mg / L, KH 2 PO 4 56.6mg / L, CaCl 2 ·2H 2 O 146.6mg / L, KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O8.6mg / L, Na 2 MoO 2 ·2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O 0.025mg / L CoCl2 6H 2 O 0.025mg / L, FeSO 4 7H 2 O 27.8mg / L, Na 2 EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2mg / L, inositol 100mg / L, sugar 25g / L, agar 6.2g / L, 6-benzylaminopurine 1.0mg / L, naphthaleneacetic acid 0.1mg / L; adjust the pH of the culture medium to 5.8-6.0 before sterilization; S3: Proliferation culture of adventitious buds: The adventitious buds are cut off from the leaves in step S2 and inoculated into a proliferation medium for proliferation and expansion. The culture environment temperature is 24-28°C, the light duration is 10-12 hours, and the light intensity is 1500-2000 lux. The culture is continued for 35-40 days under the same temperature, light intensity and light duration conditions to achieve rapid proliferation of buds, form robust bud seedlings, and obtain a large number of adventitious bud tissues; wherein the components of the proliferation medium are: NH 4 NO 3 825mg / L, KNO 3 950mg / L, MgSO 4 7H 2 O 185mg / L, KH 2 PO 4 85mg / L, CaCl 2 ·2H 2 O 220mg / L, KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 2 ·2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O0.025mg / L CoCl 2 6H 2 O 0.025mg / L, FeSO 4 7H 2 O 27.8mg / L, Na 2EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2mg / L, inositol 100mg / L, sugar 25g / L, agar 6.2g / L, 6-benzylaminopurine 0.5mg / L, naphthaleneacetic acid 0.05mg / L; adjust the pH of the culture medium to 5.8-6.0 before sterilization; S4: Seedling rooting culture: The seedlings are cut from the adventitious bud tissue obtained in step S3 and inoculated into the seedling rooting medium. The culture environment temperature is 24-28°C, the light duration is 8-10 hours, the light intensity is 2000-2500 lux, and the seedlings are cultured for 35-40 days. When the seedlings grow to 4-5 leaves, the root growth is promoted to obtain rooted seedlings with high rooting rate and well-developed root system. After the roots of 2-3 cm are grown, they can be taken out of the bottle for planting; wherein the components of the rooting medium are: NH 4 NO 3 825mg / L, KNO 3 950mg / L, MgSO 4 7H 2 O 185mg / L, KH 2 PO 4 85mg / L, CaCl 2 ·2H 2 O 220mg / L, KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 2 ·2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O 0.025mg / L CoCl 2 6H 2 O0.025mg / L, FeSO 4 7H 2 O 27.8mg / L, Na 2 EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2mg / L, inositol 100mg / L, sugar 25g / L, agar 6.2g / L, indolebutyric acid 0.1mg / L, naphthaleneacetic acid 0.1mg / L; adjust the pH of the culture medium to 5.8-6.0 before sterilization; S5: Transplanting of tissue culture seedlings: Take the seedlings from step S4 out of the culture bottle, wash off the culture medium with clean water, transfer to the water curtain greenhouse, and plant them in 72-hole pots; after planting, place the leaves of the seedlings parallel to the diagonal of the pots. After planting, sprinkle once with 2000 times of zinc disulfide, the planting environment humidity is 75%, the temperature is 20℃, the planting substrate is imported peat soil, and after the roots grow, start to fertilize and water.

[0018] Example 2 The difference with Example 1 is: In step S1, the samples were soaked in 75% alcohol for 60 seconds, washed twice with sterile water, and then washed with 0.1% HgCl2 solution with 2 drops of TWeen-20. 2 The solution was treated for 8 min, during which the explants were shaken constantly to ensure full contact with the disinfectant, and then washed with sterile water 6 times; In step S2, the culture environment temperature is 28° C., the illumination duration is 10 hours, the illumination intensity is 1000 lux, and the culture is carried out for 50 days; the components of the induction medium include 30 g / L white sugar, 3.0 mg / L 6-benzylaminopurine, and 0.2 mg / L naphthaleneacetic acid; In step S3, the culture environment temperature is 28° C., the illumination time is 12 hours, and the illumination intensity is 2000 lux. The culture is continued for 40 days under the same temperature, illumination intensity and illumination time conditions; the components of the growth medium include 30 g / L white sugar, 1.5 mg / L 6-benzylaminopurine, and 0.1 mg / L naphthaleneacetic acid; In step S4, the culture environment temperature is 28° C., the illumination duration is 10 hours, the illumination intensity is 2500 lux, and the culture is carried out for 40 days; the components of the rooting medium include 30 g / L white sugar, 0.2 mg / L indolebutyric acid, and 0.2 mg / L naphthaleneacetic acid; In step S5, after planting, 2000 times of zinc disulfide is applied once, and the humidity of the planting environment is 90% and the temperature is 30° C.; the other steps are the same.

[0019] Example 3 The difference with Example 1 is: In step S1, soak the sample in 75% alcohol for 45 seconds, wash twice with sterile water, and then rinse with 0.1% HgCl2 solution with 2 drops of TWeen-20. 2 The solution was treated for 6 min, during which the explants were shaken constantly to ensure full contact with the disinfectant, and then washed with sterile water 5 times; In step S2, the culture environment temperature is 26° C., the illumination time is 9 hours, the illumination intensity is 800 lux, and the culture is carried out for 46 days; the components of the induction medium include 28 g / L white sugar, 2 mg / L 6-benzylaminopurine, and 0.15 mg / L naphthaleneacetic acid; In step S3, the culture environment temperature is 25° C., the illumination time is 11 hours, and the illumination intensity is 1800 lux. The culture is continued for 38 days under the same temperature, illumination intensity and illumination time conditions; the components of the growth medium include 28 g / L white sugar, 1 mg / L 6-benzylaminopurine, and 0.07 mg / L naphthaleneacetic acid; In step S4, the culture environment temperature is 26° C., the light duration is 9 hours, the light intensity is 2300 lux, and the culture is carried out for 37 days; the components of the rooting medium include 26 g / L white sugar, 0.14 mg / L indolebutyric acid, and 0.17 mg / L naphthaleneacetic acid; In step S5, after planting, 2000 times of zinc disulfide is applied once, and the humidity of the planting environment is 85% and the temperature is 25° C.; the other steps are the same.

[0020] Comparative Example 1 The difference between this comparative example and Example 1 is that the induction medium in step S2 is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L, and the other steps are the same.

[0021] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S2, the induction environment temperature is 15° C., the illumination time is 3 hours, the illumination intensity is 200 lux, and the culture is 10 days. The other steps are the same.

[0022] Comparative Example 3 The difference between this comparative example and Example 1 is that the proliferation culture medium in step S3 is MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L, and the other steps are the same.

[0023] Comparative Example 4 The difference between this comparative example and Example 1 is that in step S3, the proliferation environment temperature is 35° C., the illumination time is 20 hours, the illumination intensity is 2500 lux, and the culture is 60 days. The other steps are the same.

[0024] Comparative Example 5 The difference between this comparative example and Example 1 is that the rooting medium in step S4 is MS+IBA 0.1 mg / L+NAA 0.1 mg / L, and the other steps are the same.

[0025] Comparative Example 6 The difference between this comparative example and Example 1 is that the ambient temperature of the rooting culture in step S4 is 10° C., the illumination time is 3 hours, the illumination intensity is 1500 lux, and the culture is 20 days. The other steps are the same.

[0026] The root induction rate and seedling survival rate were tested using the preparation methods of the embodiments and comparative examples, and the results are shown in Table 1 below.

[0027] Table 1 Performance test results

[0028] From the above performance test results, it can be seen that Examples 1-3 have higher root induction rates and seedling survival rates; in particular, the comprehensive performance of Example 3 is the most outstanding. However, since Comparative Examples 1-6 do not adopt the necessary technical solutions, their corresponding performance tests are significantly worse than those of the Examples. The above experimental results further prove the importance of the technical solutions defined in the present invention for its technical effects.

[0029] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for improving the rapid propagation rate of bifolia tissue culture, characterized in that: The steps include: S1: Preparation and disinfection of explants: Using a two-leaf Gesneriaceae plant as the mother plant, taking young and tender new leaves as explants, washing them clean and moving them into a clean bench, soaking them in alcohol and washing them with sterile water; then dripping disinfectant, shaking them constantly to make the explants fully contact with the disinfectant, washing them with sterile water for 3-6 times, and then placing the explants on sterile filter paper to absorb moisture to obtain sterilized explants; S2: Adventitious bud induction culture: Cut the explant leaves disinfected in step S1, place the leaves facing upward on the induction medium for induction culture, and transfer the leaves to the proliferation medium after adventitious buds grow out of the cut positions of the leaves; S3: Proliferation culture of adventitious buds: cutting the adventitious buds from the leaves in step S2, inoculating them into a proliferation medium for proliferation and expansion culture, so as to achieve rapid proliferation of the buds, form robust bud seedlings, and obtain adventitious bud tissues; S4: Seedling rooting culture: The seedlings are cut from the adventitious bud tissue obtained in step S3, and inoculated into a seedling rooting medium for rooting culture to promote root growth, thereby obtaining rooted seedlings with a high rooting rate and a well-developed root system. After the roots grow to 2-3 cm, the seedlings can be taken out of the bottle for planting; S5: Transplanting of tissue culture seedlings: Take out the seedlings in step S4 from the culture bottle, wash off the culture medium with clean water, transfer to the water curtain greenhouse, and plant them in pots; after cultivation, place the leaves of the seedlings parallel to the diagonal of the pot; sprinkle with 2000 times zinc after transplanting, the humidity of the planting environment is 75-90%, the temperature is 20-30℃, the planting substrate is imported peat soil, and after the roots grow out, start to fertilize and water to complete the transplanting.

2. The method for improving the proliferation rate of rapid propagation of Scutellaria baicalensis tissue culture according to claim 1, characterized in that: Step S1 is specifically as follows: selecting a healthy, pest-free, and well-growing two-leaf Gesneriaceae plant as the mother plant, taking the young and tender new leaves of the top that have grown for 10-20 days as the explant, rinsing them with tap water, moving them into a clean bench, soaking them in 75% alcohol for 30-60 seconds, washing them twice with sterile water, and then treating them with a 0.1% HgCl2 solution with 2 drops of TWeen-20 for 5-8 minutes, shaking them continuously during this period to allow the explant to fully contact with the disinfectant, and washing them with sterile water 3-6 times; placing the explant on sterile filter paper to absorb moisture.

3. A method for improving the proliferation rate of rapid propagation of bifolia jasmine according to claim 1, characterized in that: The induction medium in step S2 is 1 / 3MS+6-BA 1.0-3.0mg / L+NAA 0.1-0.2mg / L; the sucrose content of the medium is 25-30g / L, and the pH is 5.8-6.

0.

4. The method for improving the proliferation rate of rapid propagation of bifolia jasmine according to claim 1, characterized in that: In step S2, the induction culture environment temperature is 24-28° C., the illumination time is 8-10 hours, the illumination intensity is 500-1000 lux, and the culture is carried out for 40-50 days.

5. The method for improving the proliferation rate of rapid propagation of Scutellaria spp. in tissue culture according to claim 1, characterized in that: The proliferation culture medium in step S3 is: 1 / 2MS + 6-BA 0.5-1.5 mg / L + NAA 0.05-0.1 mg / L, wherein the sucrose content of the culture medium is 25-30 g / L, and the pH is 5.8-6.

0.

6. The method for improving the proliferation rate of rapid propagation of bifolia jasmine according to claim 1, characterized in that: The environment temperature of the proliferation culture in step S3 is 24-28° C., the illumination time is 10-12 hours, the illumination intensity is 1500-2000 lux, and the culture is carried out for 35-40 days.

7. The method for improving the rapid propagation rate of the tissue culture of the bifolia jasmine according to claim 1, characterized in that: The rooting medium in step S4 is: 1 / 2MS+IBA 0.1-0.2mg / L+NAA0.1-0.2mg / L, the sugar content of the medium is 25-30 / L, and the pH is 5.8-6.

0.

8. The method for improving the proliferation rate of rapid propagation of Scutellaria spp. in tissue culture according to claim 1, characterized in that: The environment temperature of the rooting culture in step S4 is 24-28° C., the light duration is 8-10 hours, the light intensity is 2000-2500 lux, and the culture is 35-40 days, and the seedlings grow to 4-5 leaves.

9. The method for improving the proliferation rate of rapid tissue culture of Chrysanthemum bifolium according to claim 1, characterized in that: In step S5, after planting, 2000 times of zinc disulfide is applied once, and the humidity of the planting environment is 75-90% and the temperature is 20-30°C.

10. The method for improving the proliferation rate of rapid propagation of Lophatherum spp. in tissue culture according to claim 1, characterized in that: In step S5, the tissue culture seedling transplanting matrix is ​​an imported peat matrix.

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