CN110959528A - Culture medium for improving differentiation rate of protocorm-like bodies of dendrobium officinale and efficient seedling culture method - Google Patents
Culture medium for improving differentiation rate of protocorm-like bodies of dendrobium officinale and efficient seedling culture method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention provides a culture medium for improving the differentiation rate of protocorm-like bodies of dendrobium officinale and a high-efficiency seedling raising method, belonging to the technical field of plant cultivation, and the culture medium comprises: the culture medium comprises a primary induction culture medium A, an enrichment culture medium B, a differentiation culture medium C, a strong seedling and rooting culture medium D and is applied to efficient seedling culture of the dendrobium officinale. The invention can prolong the transplanting time of the tissue culture seedlings and provide technical support for industrial seedling culture. Solves the problems of higher production cost, low clone protocorm differentiation rate and the like of the current tissue culture seedlings of the clone varieties of the dendrobium officinale.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a culture medium for improving the differentiation rate of protocorm-like bodies of dendrobium officinale and an efficient seedling raising method.
Background
Dendrobium officinale, also known as Equisetum nigrum, is a rare plant with homology of medicine and food, and is classified as a rare wild medicinal plant by State administration in 1987 to be protected. The record in Ben Cao gang mu of the Ming Dynasty Li Shizhen is: herba Dendrobii has effects of eliminating arthralgia, descending qi, tonifying viscera, strengthening yin and replenishing essence. Has the efficacies of improving human immunity, resisting fatigue, helping sleep, improving gastrointestinal tract digestion and the like, and the dendrobium officinale is famous as the first of the Chinese "Jiudaoxiancao". The medicinal part of Dendrobium officinale is fresh or dry stem, and Dendrobium officinale flower.
The dendrobium officinale has low selfing and bearing rate, so that a homozygous selfing line is difficult to obtain, the separation phenomenon exists in filial combined progeny, the purity of seedlings is low, the production generally adopts mutual pollination among different individuals in populations with the same genetic background, the sterile seeding and seedling raising are carried out on the mature fructified fruits, the current dendrobium officinale No. 1, dendrobium officinale No. 2, dendrobium officinale No. 1 and senshannon No. 1 are all prepared by adopting the preferential individuals of wild populations to carry out pollination and cultivation, the agronomic characters among all varieties have great difference, the requirement on the consistency of the agronomic characters and the quality characters of fresh food varieties in production is difficult to meet, the market requirements are met for developing artificial large-scale cultivation, the selection of excellent single plants through interspecies hybridization, the asexual breeding of excellent varieties is a necessary trend, and the asexual bud cutting propagation mode is adopted in the past, the propagation efficiency is low, and the problem of low protocorm differentiation rate in the current clonal protocorm breeding method causes high seedling breeding cost, so that the development of a culture medium for improving the differentiation rate of the clonal protocorm of the dendrobium officinale and the development of an efficient seedling method have important significance.
At present, related reports on the clone tissue culture research of the dendrobium officinale have been provided, but the tissue culture cutting propagation cost is high, the problems of low differentiation rate and the like exist in the pseudoprotocorm propagation, and the tissue culture cutting propagation method is a bottleneck factor for restricting the industrialized popularization and application of excellent clone varieties of the dendrobium officinale. Therefore, a tissue culture system capable of rapidly propagating the excellent clone of dendrobium officinale is urgently needed, and a foundation is laid for the popularization of excellent clone varieties and large-scale industrialized seedling.
Disclosure of Invention
The invention aims to provide a culture medium for improving the differentiation rate of protocorm-like bodies of dendrobium officinale and a rapid seedling culture method for establishing excellent clones, which can prolong the transplanting time of tissue culture seedlings and provide technical support for industrial seedling culture. Solves the problems of higher production cost, low clone protocorm differentiation rate and the like of the current tissue culture seedlings of the clone varieties of the dendrobium officinale.
The first purpose of the invention is to provide a culture medium for improving the differentiation rate of the protocorm-like body of dendrobium officinale, wherein the culture medium comprises:
the primary induction culture medium A is used for inducing axillary buds of the dendrobium officinale stem segments to germinate and forming protocorm-like bodies on the bases of partial buds;
the proliferation culture medium B is used for transferring and culturing the protocorm-like bodies formed by the germination of the seedling base parts in the primary induction culture medium A, and enabling the protocorm-like bodies to continuously divide and proliferate to form a large number of protocorms-like bodies;
the differentiation culture medium C is used for transferring the large amount of protocorms in the proliferation culture medium B and promoting the protocorms to be differentiated to form dendrobium officinale plantlets with the height of 1-2 cm;
and a strong seedling and rooting culture medium D for transferring the dendrobium officinale plantlets into the medium D and rooting the strong seedlings to form big seedlings with the height of 3-4 cm.
As a further technical scheme of the culture medium, the formula and the content of the induction culture medium A in 1L of culture medium are as follows: KNO3:1900mg、NH4NO3:1650mg、KH2PO4:170mg、MgSO4·7H2O:370mg、CaCl2:440mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2mg of naphthylacetic acid, 1-2 mg of 6-benzylaminopurine, 0g of banana, 4.4g of agar and 20g of white granulated sugar.
As a further technical scheme of the culture medium, the formula and the content of the proliferation culture medium B in 1L of culture medium are as follows:
KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.5 mg of 6-benzylaminopurine, 100g of banana, 4.4g of agar and 30g of white granulated sugar.
As a further technical scheme of the seedling culture method, the formula and the content of the differentiation medium C in 1L of culture medium are as follows:
KNO3:1250mg、NH4NO3:225mg、KH2PO4:175mg、MgSO4·7H2O:325mg、CaCl2:165mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.5 mg of naphthylacetic acid, 0mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar and 20g of white granulated sugar.
As a further technical scheme of the seedling culture method, the formula and the content of the strong seedling and rooting culture medium D in 1L culture medium are as follows: KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.1 mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar, 30g of white granulated sugar and 0.5g/L of activated carbon.
As a further technical scheme of the seedling culture method, the preparation process of each component of the culture medium in 1L of culture medium according to the formula comprises the following steps:
(1) measuring about 500ml of water, putting the water into a hot water pot, and heating;
(2) weighing 4.4g of agar powder;
(3) adding agar powder when the water temperature reaches 80 ℃, stirring uniformly, and heating to boil;
(4) adding medicines required by each culture medium, wherein the medicines comprise pre-prepared element mother liquor and trace element mother liquor;
(5) weighing 70-100g of banana when preparing the culture medium B, C, D, cutting into small pieces, and scattering and juicing by using a juicer;
(6) adding the squeezed banana juice into a hot water pot;
(7) adding 0.5g of activated carbon when preparing a culture medium D;
(8) adding 20-30g of white granulated sugar;
(9) adding water to a constant volume of 1L after the medicine and the white granulated sugar are completely dissolved, and uniformly stirring;
(10) adjusting the pH value to 5.8-6.2 by using pH test paper;
(11) subpackaging, namely pouring the prepared culture medium into a tissue culture bottle, wherein the pouring amount is determined according to the volume of the tissue culture bottle and is generally about 1/5 of a container;
(12) sterilizing with high pressure steam at 121 deg.C under 1.5Mpa for 21 min;
(13) and (4) placing the sterilized culture medium into an inoculation chamber for standing, and cooling and solidifying the culture medium.
The second purpose of the invention is to provide a high-efficiency seedling raising method for improving the differentiation rate of the protocorm-like body of dendrobium officinale, wherein the high-efficiency seedling raising method uses the culture medium, and the high-efficiency seedling raising method further comprises the following steps:
(1) stem induction culture of excellent single dendrobium officinale plant
Cutting stem segments of fresh dendrobium officinale strips into one segment with each section by using a scalpel, carrying an axillary bud, clamping the cut segments by using forceps, horizontally placing the cut segments in a culture medium A for culturing, promoting the axillary bud to germinate, and forming a pseudobulbus on the base of a seedling partially.
(2) Proliferation of Dendrobium officinale protocorm
And uniformly transferring the pseudoprotocorms formed by the germination of the seedling bases onto a culture medium B by using tweezers on an ultra-clean workbench, and culturing and promoting the pseudoprotocorms to continuously divide and proliferate to form a large number of pseudocorms.
(3) Differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultraclean workbench by using a spoon, uniformly transferring the protocorms to a culture medium C, and culturing and differentiating the protocorms to form dendrobium officinale plantlets with the height of about 1-2 cm.
(4) Strong seedling of dendrobium officinale plantlet
Carefully clamping the seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, inserting the seedlings into a strong seedling and rooting culture medium D, and forming big seedlings with the uniformity and the height of about 3-4cm through strong seedling.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a superclean bench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out the plantlets one by one, and inserting the plantlets into a strong seedling and rooting culture medium D for culturing for 60-90 days.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
Placing the tissue culture bottles on an off-ground seedling bed of the multi-span greenhouse in batches, hardening the seedlings under natural light for three weeks, taking out the bottle seedlings, cleaning a root culture medium, and transplanting the bottle seedlings into a loose-scale matrix.
As a further technical scheme of the seedling raising method, the method specifically comprises the following steps:
(1) stem induction culture of excellent single dendrobium officinale plant
Washing fresh dendrobium officinale strips with tap water, putting the washed fresh dendrobium officinale strips into a super-clean workbench, sequentially adding the fresh dendrobium officinale strips into 75% alcohol solution to soak for 2min under the aseptic condition of the super-clean workbench, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in 0.1% arsenic-mercury solution for 10min, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in 1% sodium hypochlorite solution for 10min, and rinsing the fresh dendrobium officinale strips with sterile water for 6 times. The treated stem segments were cut into one segment with an axillary bud by each section using a scalpel, and the cut segments were placed flat in medium A by being held with forceps. Placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and ultraviolet sterilizing the culture environment every day for 30 min. In about 30 days or so, the axillary buds germinate, forming partially pseudobulbus at the base of the seedling.
(2) Proliferation of Dendrobium officinale protocorm
The pseudoprotocorm formed by the germination of the seedling base part is evenly transferred to a culture medium B by tweezers on an ultra-clean workbench. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms.
(3) Differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultra-clean workbench by using a spoon and uniformly transferring the protocorms to a culture medium C. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm-like bodies in the tissue culture bottle can be observed to form seedlings with the height of about 1-2cm after differentiation for about 60-90 days.
(4) Strong seedling of dendrobium officinale plantlet
Carefully clamping the seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, and inserting the seedlings into a strong seedling and rooting culture medium D. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The seedlings in the tissue culture bottle can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm after the seedlings are strengthened for about 60-90 days.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a superclean bench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out the plantlets one by one, and inserting the plantlets into a strong seedling and rooting culture medium D. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The big seedlings in the tissue culture bottle have good growth vigor and equal uniformity after about 60 to 90 days, the leaves are large and thick, the leaves are oblong, the color of the leaves is dark green, each seedling has at least 3 to 4 developed and robust root systems, and the plant height reaches 6 to 8 cm.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the middle ten days of the first half year to the late 6 months, a layer of shading net with shading rate of 50% is arranged on each layer of the culture bottle cover, and the seedling culture bottles are placed on the multi-span greenhouse off-ground seedbed in the morning of 9% on sunny days: 00 hours to 16 pm: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off for three weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 95%;
the tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the beginning of 9 months to the beginning of 11 months in the next half year, a layer of shading net with shading rate of 50% is arranged on the culture bottle cover, and the culture bottle cover is arranged on the multi-span greenhouse off-ground seedbed in the morning of 10% in sunny days: 00 hours to afternoon 15: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off the seedlings for two weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 90%.
As a further technical scheme of the seedling raising method, the step (2) of the method can be repeatedly carried out.
As a further technical scheme of the seedling culture method, the protocorm can continuously divide and proliferate to generate new protocorms in the process of subculture, but the number of times of grafting is not more than 10.
Compared with the prior art, the culture medium for improving the differentiation rate of the protocorm-like bodies of the dendrobium officinale and the high-efficiency seedling raising method provided by the invention have the following beneficial effects:
the culture medium improves the induction, proliferation and differentiation rates of the clone protocorm of the dendrobium officinale, and promotes the survival rate of strong seedlings, rooting and transplanting.
The cultivation method of the invention not only accelerates the cultivation of the dendrobium officinale clone seedlings, but also has simple operation, good seedling consistency and transplanting survival rate, overcomes the problems of large individual character difference, inconsistent growth and unstable quality of the sterile sowed seeds, and has great significance for the development of the dendrobium officinale industry.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments.
Example 1
The first purpose of the invention is to provide a culture medium for improving the differentiation rate of the protocorm-like body of dendrobium officinale, wherein the culture medium comprises:
the primary induction culture medium A is used for inducing axillary buds of the dendrobium officinale stem segments to germinate and forming protocorm-like bodies on the bases of partial buds;
the proliferation culture medium B is used for transferring and culturing the protocorm-like bodies formed by the germination of the seedling base parts in the primary induction culture medium A, and enabling the protocorm-like bodies to continuously divide and proliferate to form a large number of protocorms-like bodies;
the differentiation culture medium C is used for transferring the large amount of protocorms in the proliferation culture medium B and promoting the protocorms to be differentiated to form dendrobium officinale plantlets with the height of 1-2 cm;
and a strong seedling and rooting culture medium D for transferring the dendrobium officinale plantlets into the medium D and rooting the strong seedlings to form big seedlings with the height of 3-4 cm.
As a further technical scheme of the culture medium, the formula and the content of the induction culture medium A in 1L of culture medium are as follows: KNO3:1900mg、NH4NO3:1650mg、KH2PO4:170mg、MgSO4·7H2O:370mg、CaCl2:440mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2mg of naphthylacetic acid, 1-2 mg of 6-benzylaminopurine, 0g of banana, 4.4g of agar and 20g of white granulated sugar.
As a further technical scheme of the culture medium, the formula and the content of the proliferation culture medium B in 1L of culture medium are as follows:
KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.5 mg of 6-benzylaminopurine, 100g of banana, 4.4g of agar and 30g of white granulated sugar.
As a further technical scheme of the seedling culture method, the formula and the content of the differentiation medium C in 1L of culture medium are as follows:
KNO3:1250mg、NH4NO3:225mg、KH2PO4:175mg、MgSO4·7H2O:325mg、CaCl2:165mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.5 mg of naphthylacetic acid, 0mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar and 20g of white granulated sugar.
As a further technical scheme of the seedling culture method, the formula and the content of the strong seedling and rooting culture medium D in 1L culture medium are as follows: KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.1 mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar, 30g of white granulated sugar and 0.5g/L of activated carbon.
As a further technical scheme of the seedling culture method, the preparation process of each component of the culture medium in 1L of culture medium according to the formula comprises the following steps:
(1) measuring about 500ml of water, putting the water into a hot water pot, and heating;
(2) weighing 4.4g of agar powder;
(3) adding agar powder when the water temperature reaches 80 ℃, stirring uniformly, and heating to boil;
(4) adding medicines required by each culture medium, wherein the medicines comprise pre-prepared element mother liquor and trace element mother liquor;
(5) weighing 70-100g of banana when preparing the culture medium B, C, D, cutting into small pieces, and scattering and juicing by using a juicer;
(6) adding the squeezed banana juice into a hot water pot;
(7) adding 0.5g of activated carbon when preparing a culture medium D;
(8) adding 20-30g of white granulated sugar;
(9) adding water to a constant volume of 1L after the medicine and the white granulated sugar are completely dissolved, and uniformly stirring;
(10) adjusting the pH value to 5.8-6.2 by using pH test paper;
(11) subpackaging, namely pouring the prepared culture medium into a tissue culture bottle, wherein the pouring amount is determined according to the volume of the tissue culture bottle and is generally about 1/5 of a container;
(12) sterilizing with high pressure steam at 121 deg.C under 1.5Mpa for 21 min;
(13) and (4) placing the sterilized culture medium into an inoculation chamber for standing, and cooling and solidifying the culture medium.
The second purpose of the invention is to provide a high-efficiency seedling raising method for improving the differentiation rate of the protocorm-like body of dendrobium officinale, wherein the high-efficiency seedling raising method uses the culture medium, and the high-efficiency seedling raising method further comprises the following steps:
(1) stem induction culture of excellent single dendrobium officinale plant
Cutting stem segments of fresh dendrobium officinale strips into one segment with each section by using a scalpel, carrying an axillary bud, clamping the cut segments by using forceps, horizontally placing the cut segments in a culture medium A for culturing, promoting the axillary bud to germinate, and forming a pseudobulbus on the base of a seedling partially.
(2) Proliferation of Dendrobium officinale protocorm
And uniformly transferring the pseudoprotocorms formed by the germination of the seedling bases onto a culture medium B by using tweezers on an ultra-clean workbench, and culturing and promoting the pseudoprotocorms to continuously divide and proliferate to form a large number of pseudocorms.
(3) Differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultraclean workbench by using a spoon, uniformly transferring the protocorms to a culture medium C, and culturing and differentiating the protocorms to form dendrobium officinale plantlets with the height of about 1-2 cm.
(4) Strong seedling of dendrobium officinale plantlet
Carefully clamping the seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, inserting the seedlings into a strong seedling and rooting culture medium D, and forming big seedlings with the uniformity and the height of about 3-4cm through strong seedling.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a superclean bench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out the plantlets one by one, and inserting the plantlets into a strong seedling and rooting culture medium D for culturing for 60-90 days.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
Placing the tissue culture bottles on an off-ground seedling bed of the multi-span greenhouse in batches, hardening the seedlings under natural light for three weeks, taking out the bottle seedlings, cleaning a root culture medium, and transplanting the bottle seedlings into a loose-scale matrix.
As a further technical scheme of the seedling raising method, the method specifically comprises the following steps:
(1) stem induction culture of excellent single dendrobium officinale plant
Washing fresh dendrobium officinale strips with tap water, putting the washed fresh dendrobium officinale strips into a super-clean workbench, sequentially adding the fresh dendrobium officinale strips into 75% alcohol solution to soak for 2min under the aseptic condition of the super-clean workbench, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in 0.1% arsenic-mercury solution for 10min, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in 1% sodium hypochlorite solution for 10min, and rinsing the fresh dendrobium officinale strips with sterile water for 6 times. The treated stem segments were cut into one segment with an axillary bud by each section using a scalpel, and the cut segments were placed flat in medium A by being held with forceps. Placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and ultraviolet sterilizing the culture environment every day for 30 min. In about 30 days or so, the axillary buds germinate, forming partially pseudobulbus at the base of the seedling.
(2) Proliferation of Dendrobium officinale protocorm
The pseudoprotocorm formed by the germination of the seedling base part is evenly transferred to a culture medium B by tweezers on an ultra-clean workbench. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms.
(3) Differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultra-clean workbench by using a spoon and uniformly transferring the protocorms to a culture medium C. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm-like bodies in the tissue culture bottle can be observed to form seedlings with the height of about 1-2cm after differentiation for about 60-90 days.
(4) Strong seedling of dendrobium officinale plantlet
Carefully clamping the seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, and inserting the seedlings into a strong seedling and rooting culture medium D. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The seedlings in the tissue culture bottle can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm after the seedlings are strengthened for about 60-90 days.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a superclean bench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out the plantlets one by one, and inserting the plantlets into a strong seedling and rooting culture medium D. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The big seedlings in the tissue culture bottle have good growth vigor and equal uniformity after about 60 to 90 days, the leaves are large and thick, the leaves are oblong, the color of the leaves is dark green, each seedling has at least 3 to 4 developed and robust root systems, and the plant height reaches 6 to 8 cm.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the middle ten days of the first half year to the late 6 months, a layer of shading net with shading rate of 50% is arranged on each layer of the culture bottle cover, and the seedling culture bottles are placed on the multi-span greenhouse off-ground seedbed in the morning of 9% on sunny days: 00 hours to 16 pm: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off for three weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 95%;
the tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the beginning of 9 months to the beginning of 11 months in the next half year, a layer of shading net with shading rate of 50% is arranged on the culture bottle cover, and the culture bottle cover is arranged on the multi-span greenhouse off-ground seedbed in the morning of 10% in sunny days: 00 hours to afternoon 15: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off the seedlings for two weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 90%.
As a further technical scheme of the seedling raising method, the step (2) of the method can be repeatedly carried out.
As a further technical scheme of the seedling culture method, the protocorm can continuously divide and proliferate to generate new protocorms in the process of subculture, but the number of times of grafting is not more than 10.
Example 2
The invention is further illustrated below with reference to example 1 and the relevant experimental results of the invention are published. Experiment one:
1. the culture medium A, B, C, D was prepared.
2. A method of culturing.
(1) Stem induction culture of excellent single dendrobium officinale plant
Washing mature fresh dendrobium officinale strips in late 12 months with tap water, putting the strips into a clean bench, sequentially adding the strips into 75% alcohol solution to soak for 2min under the aseptic condition of the clean bench, rinsing the strips with sterile water for 6 times, soaking the strips in 0.1% arsenic-mercury solution for 10min, rinsing the strips with sterile water for 6 times, soaking the strips in 1% sodium hypochlorite solution for 10min, and rinsing the strips with sterile water for 6 times. The treated stem segments were cut into one segment with one axillary bud per node with a scalpel, and the cut segments were kept flat in medium A with 1mg of 6-benzylaminopurine held by forceps. Placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and ultraviolet sterilizing the culture environment every day for 30 min. In about 30 days or so, the axillary buds germinate, forming partially pseudobulbus at the base of the seedling.
(2) Proliferation of Dendrobium officinale protocorm
The pseudobulb formed by the germination of the seedling base is evenly transferred to a culture medium B on an ultra-clean workbench by using tweezers, wherein the naphthalene acetic acid is 0.2mg, and the 6-benzylaminopurine is 0.5 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms.
(3) Differentiation of Dendrobium officinale protocorm
The protocorm-like bodies were picked up on a clean bench with a spoon and transferred evenly onto medium C, where naphthylacetic acid was 0.2 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm-like bodies in the tissue culture bottle can be observed to form seedlings with the height of about 1-2cm after differentiation for about 60-90 days.
(4) Strong seedling of dendrobium officinale plantlet
The seedlings formed by differentiating the protocorm-like bodies are carefully clamped by tweezers on a clean bench and placed on a stainless steel inoculation culture disc disinfected in advance to separate each seedling, and the seedlings are inserted into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg, and the 6-benzylaminopurine is 0 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The seedlings in the tissue culture bottle can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm after the seedlings are strengthened for about 60-90 days.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a super-clean workbench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out one plantlet by one, and inserting the plantlets into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg and the 6-benzylaminopurine is 0 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The big seedlings in the tissue culture bottle have good growth vigor and equal uniformity after about 60 to 90 days, the leaves are large and thick, the leaves are oblong, the color of the leaves is dark green, each seedling has at least 3 to 4 developed and robust root systems, and the plant height reaches 6 to 8 cm.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the middle ten days of the first half year to the late 6 months, a layer of shading net with shading rate of 50% is arranged on each layer of the culture bottle cover, and the seedling culture bottles are placed on the multi-span greenhouse off-ground seedbed in the morning of 9% on sunny days: 00 hours to 16 pm: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off for three weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 95%.
Experiment two:
1. the culture medium A, B, C, D was prepared.
2. A method of culturing.
(1) Stem induction culture of excellent single dendrobium officinale plant
Washing mature fresh dendrobium officinale strips in late 3 months with tap water, putting the strips into a clean bench, sequentially adding the strips into 75% alcohol solution to soak for 2min under the aseptic condition of the clean bench, rinsing the strips with sterile water for 6 times, soaking the strips in 0.1% arsenic-mercury solution for 10min, rinsing the strips with sterile water for 6 times, soaking the strips in 1% sodium hypochlorite solution for 10min, and rinsing the strips with sterile water for 6 times. The treated stem segments were cut into one segment with one axillary bud per node with a scalpel, and the cut segments were kept flat in medium A with 2mg of 6-benzylaminopurine held by forceps. Placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and ultraviolet sterilizing the culture environment every day for 30 min. In about 30 days or so, the axillary buds germinate, forming partially pseudobulbus at the base of the seedling.
(2) Proliferation of Dendrobium officinale protocorm
The pseudobulb formed by the germination of the seedling base is transferred evenly onto the culture medium B by tweezers on an ultra-clean workbench, wherein the naphthalene acetic acid is 0.2mg, and the 6-benzylaminopurine is 0.5 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms.
(3) Differentiation of Dendrobium officinale protocorm
The protocorm-like bodies were picked up on a clean bench with a spoon and transferred evenly onto medium C, where naphthylacetic acid was 0.2 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm-like bodies in the tissue culture bottle can be observed to form seedlings with the height of about 1-2cm after differentiation for about 60-90 days.
(4) Strong seedling of dendrobium officinale plantlet
The seedlings formed by differentiating the protocorm-like bodies are carefully clamped by tweezers on a clean bench and placed on a stainless steel inoculation culture disc disinfected in advance to separate each seedling, and the seedlings are inserted into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg, and the 6-benzylaminopurine is 0 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The seedlings in the tissue culture bottle can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm after the seedlings are strengthened for about 60-90 days.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a super-clean workbench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out one plantlet by one, and inserting the plantlets into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg and the 6-benzylaminopurine is 0.1 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The big seedlings in the tissue culture bottle have good growth vigor and equal uniformity after about 60 to 90 days, the leaves are large and thick, the leaves are oblong, the color of the leaves is dark green, each seedling has at least 3 to 4 developed and robust root systems, and the plant height reaches 6 to 8 cm.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the middle ten days of the first half year to the late 6 months, a layer of shading net with shading rate of 50% is arranged on each layer of the culture bottle cover, and the seedling culture bottles are placed on the multi-span greenhouse off-ground seedbed in the morning of 9% on sunny days: 00 hours to 16 pm: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off for three weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 95%.
Experiment one:
1. the culture medium A, B, C, D was prepared.
2. A method of culturing.
(1) Stem induction culture of excellent single dendrobium officinale plant
Washing mature fresh dendrobium officinale strips in late 12 months with tap water, putting the strips into a clean bench, sequentially adding the strips into 75% alcohol solution to soak for 2min under the aseptic condition of the clean bench, rinsing the strips with sterile water for 6 times, soaking the strips in 0.1% arsenic-mercury solution for 10min, rinsing the strips with sterile water for 6 times, soaking the strips in 1% sodium hypochlorite solution for 10min, and rinsing the strips with sterile water for 6 times. The treated stem segments were cut into one segment with one axillary bud per node with a scalpel, and the cut segments were kept flat in medium A with 2mg of 6-benzylaminopurine held by forceps. Placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and ultraviolet sterilizing the culture environment every day for 30 min. In about 30 days or so, the axillary buds germinate, forming partially pseudobulbus at the base of the seedling.
(2) Proliferation of Dendrobium officinale protocorm
The pseudobulb formed by the germination of the seedling base is evenly transferred to a culture medium B on an ultra-clean workbench by using tweezers, wherein the naphthalene acetic acid is 0.2mg, and the 6-benzylaminopurine is 0 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms.
(3) Differentiation of Dendrobium officinale protocorm
The protocorm-like bodies were picked up on a clean bench with a spoon and transferred evenly onto medium C, where naphthylacetic acid was 0.2 mg. Culturing the tissue culture bottle in an environment with a temperature of 25 deg.C, illumination of 2000lx and illumination of 12h per day, and ultraviolet sterilizing the culture environment for 30min per day. The protocorm-like bodies in the tissue culture bottle can be observed to form seedlings with the height of about 1-2cm after differentiation for about 60-90 days.
(4) Strong seedling of dendrobium officinale plantlet
The seedlings formed by differentiating the protocorm-like bodies carefully clamped by tweezers on a clean bench are placed on a stainless steel inoculation culture disc disinfected in advance to separate each seedling, and the seedlings are inserted into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg, and the 6-benzylaminopurine is 0.1 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The seedlings in the tissue culture bottle can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm after the seedlings are strengthened for about 60-90 days.
(5) Rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a super-clean workbench by using forceps, separating each plantlet on a stainless steel inoculation culture disc disinfected in advance, picking out one plantlet by one, and inserting the plantlets into a strong seedling and rooting culture medium D, wherein the naphthylacetic acid is 0.2mg and the 6-benzylaminopurine is 0.1 mg. Placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx, illumination of 12h every day for culture, and ultraviolet sterilizing the culture environment every day for 30 min. The big seedlings in the tissue culture bottle have good growth vigor and equal uniformity after about 60 to 90 days, the leaves are large and thick, the leaves are oblong, the color of the leaves is dark green, each seedling has at least 3 to 4 developed and robust root systems, and the plant height reaches 6 to 8 cm.
(6) Hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the beginning of 9 months to the beginning of 11 months in the next half year, a layer of shading net with shading rate of 50% is arranged on the culture bottle cover, and the culture bottle cover is arranged on the multi-span greenhouse off-ground seedbed in the morning of 10% in sunny days: 00 hours to afternoon 15: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off the seedlings for two weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 90%.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The culture medium for improving the differentiation rate of the protocorm-like body of the dendrobium officinale is characterized by comprising:
the primary induction culture medium A is used for inducing axillary buds of the dendrobium officinale stem segments to germinate and forming protocorm-like bodies on the bases of partial buds;
the proliferation culture medium B is used for transferring and culturing the protocorm-like bodies formed by the germination of the seedling base parts in the primary induction culture medium A, and enabling the protocorm-like bodies to continuously divide and proliferate to form a large number of protocorms-like bodies;
the differentiation culture medium C is used for transferring the large amount of protocorms in the proliferation culture medium B and promoting the protocorms to be differentiated to form dendrobium officinale plantlets with the height of 1-2 cm;
and a strong seedling and rooting culture medium D for transferring the dendrobium officinale plantlets into the medium D and rooting the strong seedlings to form big seedlings with the height of 3-4 cm.
2. The culture medium for increasing the differentiation rate of the protocorm-like body of dendrobium officinale according to claim 1, wherein the formula and the content of the induction culture medium A in 1L of culture medium are as follows: KNO3:1900mg、NH4NO3:1650mg、KH2PO4:170mg、MgSO4·7H2O:370mg、CaCl2:440mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2mg of naphthylacetic acid, 1-2 mg of 6-benzylaminopurine, 0g of banana, 4.4g of agar and 20g of white granulated sugar.
3. The culture medium for increasing the differentiation rate of the protocorm-like body of dendrobium officinale according to claim 1, wherein the formula and the content of the proliferation culture medium B in 1L of culture medium are as follows:
KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.5 mg of 6-benzylaminopurine, 100g of banana, 4.4g of agar and 30g of white granulated sugar.
4. The culture medium for increasing the differentiation rate of the protocorm-like body of dendrobium officinale according to claim 1, wherein the formula and the content of the differentiation medium C in 1L culture medium are as follows:
KNO3:1250mg、NH4NO3:225mg、KH2PO4:175mg、MgSO4·7H2O:325mg、CaCl2:165mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.5 mg of naphthylacetic acid, 0mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar and 20g of white granulated sugar.
5. The culture medium for increasing the differentiation rate of the protocorm-like body of dendrobium officinale according to claim 1, wherein the formula and the content of the strong seedling and rooting culture medium D in 1L culture medium are as follows: KNO3:1267mg、NH4NO3:1100mg、KH2PO4:113mg、MgSO4·7H2O:247mg、CaCl2:293mg、KI:0.83mg、H3BO3:6.2mg、MnSO4·4H2O:22.3mg、ZnSO4·7H2O:8.6mg、Na2-EDTA:37.3mg、FeSO427.8mg of 7H2O, 0.5mg of nicotinic acid, 0.5mg of pyridoxine hydrochloride, 0.1mg of thiamine hydrochloride, 2.0mg of glycine, 0.2-0.3 mg of naphthylacetic acid, 0-0.1 mg of 6-benzylaminopurine, 70g of banana, 4.4g of agar, 30g of white granulated sugar and 0.5g/L of activated carbon.
6. The culture medium for increasing the differentiation rate of Dendrobium candidum protocorm according to claim 2, 3, 4 or 5, wherein the formulation of each component of the culture medium in 1L of culture medium comprises the following steps:
(1) measuring about 500ml of water, putting the water into a hot water pot, and heating;
(2) weighing 4.4g of agar powder;
(3) adding agar powder when the water temperature reaches 80 ℃, stirring uniformly, and heating to boil;
(4) adding medicines required by each culture medium, wherein the medicines comprise pre-prepared element mother liquor and trace element mother liquor;
(5) weighing 70-100g of banana when preparing the culture medium B, C, D, cutting into small pieces, and scattering and juicing by using a juicer;
(6) adding the squeezed banana juice into a hot water pot;
(7) adding 0.5g of activated carbon when preparing a culture medium D;
(8) adding 20-30g of white granulated sugar;
(9) adding water to a constant volume of 1L after the medicine and the white granulated sugar are completely dissolved, and uniformly stirring;
(10) adjusting the pH value to 5.8-6.2 by using pH test paper;
(11) subpackaging, namely pouring the prepared culture medium into a tissue culture bottle, wherein the pouring amount is determined according to the volume of the tissue culture bottle and is generally about 1/5 of a container;
(12) sterilizing with high pressure steam at 121 deg.C under 1.5Mpa for 21 min;
(13) and (4) placing the sterilized culture medium into an inoculation chamber for standing, and cooling and solidifying the culture medium.
7. An efficient seedling raising method for improving the differentiation rate of the protocorm-like bodies of the dendrobium officinale, which is characterized by using the culture medium of claim 6 and further comprising the following steps:
(1) stem induction culture of excellent single dendrobium officinale plant
Cutting stem segments of fresh stems of mature dendrobium officinale into one segment with each section by a scalpel, carrying an axillary bud, clamping the cut segments by a pair of tweezers, horizontally placing the cut segments in a culture medium A for culturing, promoting the axillary bud to germinate, and forming a pseudobulbus on the base of a seedling;
(2) proliferation of Dendrobium officinale protocorm
Uniformly transferring the pseudoprotocorms formed by the germination of the seedling bases onto a culture medium B by using tweezers on an ultra-clean workbench, and culturing and promoting the pseudoprotocorms to continuously divide and proliferate to form a large number of pseudocorms;
(3) differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultraclean workbench by using a spoon, uniformly transferring the protocorms to a culture medium C, and culturing and differentiating the protocorms to form dendrobium officinale plantlets with the height of about 1-2 cm;
(4) strong seedling of dendrobium officinale plantlet
Carefully clamping out seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, inserting the seedlings into a strong seedling and rooting culture medium D, and forming big seedlings with basically consistent uniformity and height of about 3-4cm through strong seedlings;
(5) rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a super-clean workbench by using forceps, placing the dendrobium officinale plantlets on a stainless steel inoculation culture disc which is disinfected in advance, separating each plantlet, picking out one plantlet by one plantlet, and inserting the plantlets into a strong seedling and rooting culture medium D for culturing for 60-90 days;
(6) hardening and transplanting of dendrobium officinale tissue culture seedlings
Placing the tissue culture bottles on an off-ground seedling bed of the multi-span greenhouse in batches, hardening the seedlings under natural light for three weeks, taking out the bottle seedlings, cleaning a root culture medium, and transplanting the bottle seedlings into a loose-scale matrix.
8. The high-efficiency seedling raising method for improving the differentiation rate of the protocorm-like body of the dendrobium officinale according to claim 7, which is characterized by comprising the following steps of:
(1) stem induction culture of excellent single dendrobium officinale plant
Washing fresh dendrobium officinale strips with tap water, putting the washed fresh dendrobium officinale strips into a super-clean workbench, sequentially adding the fresh dendrobium officinale strips into a 75% alcohol solution to soak for 2min under the aseptic condition of the super-clean workbench, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in a 0.1% arsenic-mercury solution for 10min, rinsing the fresh dendrobium officinale strips with sterile water for 6 times, soaking the fresh dendrobium officinale strips in a 1% sodium hypochlorite solution for 10min, and rinsing the fresh dendrobium officinale strips with sterile water for 6 times;
cutting each section of the treated stem into a section with an axillary bud by using a scalpel, clamping the cut section by using a pair of forceps, and horizontally placing the cut section in a culture medium A;
placing the tissue culture bottle in a culture room at 25 deg.C, illuminating at 2000lx for 12h every day, and sterilizing the culture environment with ultraviolet rays for 30min every day;
about 30 days or so, the axillary buds germinate, and part of the axillary buds form pseudobulb at the base of the seedling;
(2) proliferation of Dendrobium officinale protocorm
Uniformly transferring the pseudoprotocorm formed by the germination of the seedling base part onto a culture medium B by using tweezers on an ultra-clean workbench;
placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx and illumination of 12h every day for culture, and sterilizing the culture environment with ultraviolet rays every day for 30 min;
the protocorm in the tissue culture bottle can be observed to continuously divide and proliferate to form a large amount of protocorms;
(3) differentiation of Dendrobium officinale protocorm
Picking out protocorms on an ultra-clean workbench by using a spoon and uniformly transferring the protocorms to a culture medium C;
placing the tissue culture bottle in an environment with the temperature of 25 deg.C, illumination of 2000lx and illumination of 12h every day for culture, and sterilizing the culture environment with ultraviolet rays every day for 30 min;
the protocorm-like bodies in the tissue culture bottle can be observed to form small seedlings with the height of about 1-2cm after differentiation for about 60-90 days;
(4) strong seedling of dendrobium officinale plantlet
Carefully clamping out seedlings formed by differentiation of protocorm-like bodies on a superclean workbench by using forceps, placing the seedlings on a stainless steel inoculation culture disc disinfected in advance, separating each seedling, and inserting the seedlings into a strong seedling and rooting culture medium D;
placing the tissue culture bottle in an environment with the temperature of 25 ℃, the illumination of 2000lx and the illumination of 12h every day for culture, and sterilizing the culture environment for 30min every day by ultraviolet rays;
after the seedlings in the tissue culture bottle are strengthened, the seedlings can be observed to form big seedlings with the uniformity basically consistent and the height of about 3-4cm in about 60-90 days;
(5) rooting of dendrobium officinale plantlets
Carefully clamping dendrobium officinale plantlets on a super-clean workbench by using forceps, placing the dendrobium officinale plantlets on a stainless steel inoculation culture disc which is disinfected in advance, separating each plantlet, picking out the plantlets one by one, and inserting the plantlets into a strong seedling and rooting culture medium D;
placing the tissue culture bottle in an environment with the temperature of 25 ℃, the illumination of 2000lx and the illumination of 12h every day for culture, and sterilizing the culture environment for 30min every day by ultraviolet rays;
good growth vigor of the big seedlings in the tissue culture bottle can be observed in about 60-90 days, the uniformity is equivalent, the leaves are large and thick, the shape of the long ellipse is long, the color of the leaves is dark green, each seedling has at least 3-4 developed and robust root systems, and the plant height reaches 6-8 cm;
(6) hardening and transplanting of dendrobium officinale tissue culture seedlings
The tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the middle ten days of the first half year to the late 6 months, a layer of shading net with shading rate of 50% is arranged on each layer of the culture bottle cover, and the seedling culture bottles are placed on the multi-span greenhouse off-ground seedbed in the morning of 9% on sunny days: 00 hours to 16 pm: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off for three weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 95%;
the tissue culture bottles can be placed on the multi-span greenhouse off-ground seedbed in batches from the beginning of 9 months to the beginning of 11 months in the next half year, a layer of shading net with shading rate of 50% is arranged on the culture bottle cover, and the culture bottle cover is arranged on the multi-span greenhouse off-ground seedbed in the morning of 10% in sunny days: 00 hours to afternoon 15: 00, opening an external shading net for shading, taking out the bottle seedlings after hardening off the seedlings for two weeks in the rainy days, cleaning a root culture medium, transplanting the bottle seedlings into a pine scale matrix, and ensuring the survival rate to be more than 90%.
9. The method for raising seedlings of dendrobium officinale Kimura et Migo with high efficiency according to claim 7 or 8, wherein the step (2) can be repeated.
10. The method for raising seedlings of dendrobium officinale Kimura et Migo with high efficiency as claimed in claim 9, wherein the protocorm can continuously divide and proliferate to generate new protocorm during the subculture process, but the number of grafting generations does not exceed 10.
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