CN110679484A - Dendrobium huoshanense seedling cultivation technology - Google Patents
Dendrobium huoshanense seedling cultivation technology 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
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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 discloses a dendrobium huoshanense seedling cultivation technology, which comprises the steps of clamping a disinfected capsule by using tweezers under an aseptic condition, cutting the top end of the capsule by using a scalpel, slightly shaking the capsule in a culture bottle, uniformly sowing a layer of seeds on the surface of an MS culture medium, immediately sealing the bottle opening, culturing the sowed culture bottle in a clean dark environment at the temperature of (25 +/-2) DEG C for 1 week at the pH of 5.8, culturing in an induction culture medium A, and culturing under the illumination intensity of 1500 mu mol.m‑2·s‑1～2000μmol·m‑2·s‑1And under the condition that the illumination duration is 10-12 h/d, the seeds germinate for 10-20 d, and the protocorm is formed after the seeds are continuously cultured for 55-60 d. The invention achieves the purpose of increasing the differentiation of protocorms by accurately controlling the concentration of various nutrients in a culture medium in dendrobium huoshanense, adding phytohormone to induce the growth and proliferation of the protocorms, using the agonist to induce the differentiation and growth of cells and controlling the expression of plant genes.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a dendrobium huoshanense seedling cultivation technology.
Background
The dendrobium huoshanense is a unique and rare traditional Chinese medicine with unique Anhui emblem, contains various active substances such as dendrobine, polysaccharide, flavone and the like, has the effects of replenishing vital essence and strengthening yin, promoting the production of body fluid to quench thirst, tonifying deficiency and removing deficient fire in stomach, is the most extensive and rare traditional Chinese medicine, and the market demand for the dendrobium huoshanense is gradually increased.
However, the dendrobium huoshanense is a special dendrobium variety in the dendrobium huoshanense, and is a more precious variety in the dendrobium huoshanense, the dendrobium huoshanense is difficult to plant due to the fact that the growing environment is special and distinctive, cultivated dendrobium huoshanense plants are delicate and small, the derived dendrobium huoshanense plants are small in number, good plants cannot be planted, and the original bulbodium and cluster buds of the dendrobium huoshanense are not uniformly differentiated and are small in number by using a traditional planting method.
Disclosure of Invention
The invention aims to solve the defects in the prior art, such as: by using the traditional planting method, the protocorms and cluster buds of dendrobium huoshanense have uneven differentiation and less quantity, and the dendrobium huoshanense seedling cultivation technology is provided.
In order to achieve the purpose, the invention adopts the following technical scheme:
the dendrobium huoshanense seedling breeding technology comprises the following operation steps:
s1: aseptic seeding, namely clamping the disinfected capsule by using tweezers under an aseptic condition, cutting the top end of the capsule by using a scalpel, slightly shaking the capsule in a culture bottle, uniformly sowing a layer of seeds on the surface of an MS culture medium, and immediately sealing a bottle opening;
s2: inducing protocorm, culturing in a culture flask at 25 + -2 deg.C in clean dark environment at pH5.8 for 1 week, culturing in induction medium A under illumination intensity of 1500 μmol/m-2·s-1～2000μmol·m-2·s-1Under the condition that the illumination duration is 10-12 h/d, seeds germinate for 10-20 d, and are continuously cultured for 55-60 d to form protocorms;
s3: adding the protocorm value, selecting healthy, full and pollution-free protocorm to shake and culture on a growth medium A, wherein the shaking frequency is 120 r.min-1Adding the protocorm value; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Subculturing for 55 d-65 d under the condition that the illumination duration is 10 h/d-12 h/d;
s4: inducing cluster buds, namely selecting healthy and pollution-free protocorms on an induction culture medium B to induce the cluster buds; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Subculturing for 40-55 d under the condition that the illumination duration is 10-12 h/d;
s5: the cluster buds are proliferated, and healthy and pollution-free cluster buds are selected to be placed on a growth medium B for proliferation; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Culturing for 50-70 days under the condition that the illumination time is 10-12 h/d.
S6: rooting and strengthening seedling, selecting healthy and pollution-free cluster buds with the bud height of 1.5-2.5 cm on 1/2MS culture medium, rooting and strengthening seedling(ii) a The illumination intensity is 1500 mu mol.m-2·s-1～ 3000μmol·m-2·s-1Culturing for 65d to 75d under the condition that the illumination duration is 10h/d to 12 h/d;
s7: the subculture times of protocorm and cluster buds are controlled within 4 generations.
Preferably, the capsule needs to be pretreated, the surface of the capsule is fully cleaned by clear water, residual moisture is dried in the shade, then the capsule is wrapped by clean paper and stored in a refrigerator at 4 ℃ for a week, the pretreated capsule is firstly soaked in 75% ethanol for 15min to 20min and then is burnt on an alcohol lamp for 1s to 2s for later use.
Preferably, the induction culture medium A is based on MS liquid culture medium, and 0.05-0.2 mg-L of 2, 4-dichlorophenoxyacetic acid is added-10.05 to 0.1 mg.L of Kinetin (KT)-1。
Preferably, the growth medium A is based on MS solid medium, and 100-200 g/L of potato juice and 0.5-1.0 mg/L of naphthylacetic acid (NAA) are added-10.5-1.0 mg-L of 6-benzylamino adenine (6-BA)-1In the medium of (1).
Preferably, the induction culture medium B is based on an MS solid culture medium, and 0.1-0.3 mg.L of Kinetin (KT) is added-10.2 to 0.4 mg/L of naphthylacetic acid (NAA)-10.3-0.5 mg-L of 6-benzylamino adenine (6-BA)-1。
Preferably, the induction culture medium B is based on an MS solid culture medium, and 0.1-0.2 mg-L Kinetin (KT) is added-10.1 to 0.2 mg/L of naphthylacetic acid (NAA)-10.3-0.4 mg-L of 6-benzylamino adenine (6-BA)-1。
Preferably, 0.8% agar and 3.0% sucrose are added to the induction medium A, the induction medium B, the growth medium A and the growth medium B.
Compared with the prior art, the invention has the beneficial effects that:
1. the method comprises the steps of accurately controlling the concentration of various nutrients in a culture medium in dendrobium huoshanense, adding plant hormone to induce the growth and proliferation of protocorms, using the agonist to induce the differentiation and growth of cells, and controlling the expression of plant genes to achieve the purpose of increasing the differentiation of the protocorms;
2. through comparing the experiment, can draw the in-process of artificial cultivation Huoshan rice stem of noble dendrobium, Kinetin (KT) influences great at the in-process of extension, and kinetin's concentration is crossed lowly, and then embryo growing situation is not good, and if use concentration crosses lowly, has the effect of restraineing again to the growth of plant, so the ratio of kinetin is very important, and better growth effect is guaranteed to accurate control content.
3. 6-benzylamino adenine is added in the growth and development process to accelerate the growth of plants, after cells are differentiated, 6-benzylamino adenine is added to accelerate the healing of the division part in the differentiation process of the plants and help the stems and leaves of plant tissues to differentiate and heal the wounds, so that the growth of protocorms and cluster buds is improved;
4. change the cultivation mode, through the shake culture solution for when stem on the hillside stem on the earth embryo bud can be abundant nutrient solution contact, also make the plant embryo can be abundant contact with the air, avoid under the environment of stewing solid-state and liquid culture medium environment, the partial plant embryo that appears is not abundant with nutrient solution, air contact, and the growth that leads to is bad.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the illustrated orientations or positional relationships for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
The dendrobium huoshanense seedling cultivation technology comprises the following specific operation steps:
s1: aseptic seeding, namely clamping the disinfected capsule by using tweezers under an aseptic condition, cutting the top end of the capsule by using a scalpel, slightly shaking the capsule in a culture bottle, uniformly spreading a layer of seeds on the surface of an MS culture medium, immediately sealing a bottle opening, pretreating the capsule, fully cleaning the surface by using clear water, drying residual moisture in the shade, wrapping the capsule by using clean paper, storing the capsule in a refrigerator at 4 ℃ for a week, soaking the pretreated capsule in 75% ethanol for 15min, and then firing the capsule on an alcohol lamp for 1s for later use;
s2: inducing protocorm, culturing in 24 deg.C clean dark environment at pH5.8, culturing in inducing culture medium A containing MS liquid culture medium and 2, 4-dichlorophenoxyacetic acid 0.05-0.2 mg/L-10.05 to 0.1 mg.L of Kinetin (KT)-1The light intensity is 1500 mu mol · m-2·s-1～2000μmol·m-2·s-1Under the condition that the illumination duration is 11h/d, seeds germinate for 10d, and are continuously cultured for 55d to form protocorms;
table 1: the influence (%) of the induction culture medium A with different hormone ratios on the induction of dendrobium huoshanense protocorm under the same environment is as follows:
according to the table, the MS +2, 4-D0.1 mg.L can be selected-1+KT 0.05mg·L-1The induction rate of the culture medium is maximized, and the induction rate is reduced by continuously adding the culture medium on the basis of the concentration, so that the induction culture medium is most suitable for induction of protocorms, and the most suitable induction concentration is obtained through stepwise gradual comparison.
The proportion of different hormones has great influence on the induction of dendrobium huoshanense protocorms, and along with the increase of the culture time, KT does not have great improvement effect along with the increase of the concentration, but has a descending trend, so that the situation that only the original corms of dendrobium huoshanense are induced is shownThe better induction culture effect can be obtained only within a more proper KT concentration range, namely 0.05 mg.L-1The method is most suitable, wherein 2,4-D is also suitable for inducing protocorms only under the condition of certain concentration, when the concentration is insufficient, the best effect cannot be achieved, and the concentration of 2,4-D is required to reach 0.1 mg.L-1Can be more suitable for induction of protocorms.
The hormone is used for inducing a large amount of dendrobium huoshanense embryo tissues in a plant tissue culture mode, the induction of dendrobium huoshanense protocorms is influenced in many aspects, the plant hormone can influence the differential expression of plant somatic embryo genes, and the development of cells is influenced by changing a gene expression system in the cells.
S3: adding the value of protocorm, selecting healthy, full and pollution-free protocorm to shake culture on a growth medium A, wherein the growth medium A is based on an MS solid culture medium, and adding 100-200 g/L of potato juice and 0.5-1.0 mg.L of naphthylacetic acid (NAA)-10.5-1.0 mg-L of 6-benzylamino adenine (6-BA)-1Oscillation frequency of 120 r.min-1Adding the protocorm value; the illumination intensity is 1500 mu mol.m-2·s-1Subculturing for 55d under the condition that the illumination time is 10 h/d;
table 2: the influence (%) of growth medium A with different hormone ratios on the proliferation of dendrobium huoshanense protocorm under the same environment is as follows:
| protocorm increment rate (%) | Potato juice (g/L) | NAA(mg·L-1) | 6-BA(mg·L-1) |
| 8.5 | 100 | 0.5 | 0.5 |
| 10.6 | 100 | 1.0 | 1.0 |
| 7.1 | 200 | 0.5 | 0.5 |
| 9.3 | 200 | 1.0 | 1.0 |
According to the table, the MS +2, 4-D0.1 mg.L can be selected-1+KT 0.05mg·L-1The culture medium has maximized proliferation rate, and is most suitable for protocorm proliferation through the cooperation of NAA and potato juice.
Table 3: the influence (%) of the culture environment on the proliferation of dendrobium huoshanense protocorms:
according to the table, the better granular protocorm can be dispersed when the culture is carried out in the shaking liquid culture medium, through vibrating the culture medium, nutrient substances and gas are continuously exchanged in the process of continuously vibrating the dendrobium, the metabolism of cells is facilitated, the growth of protocorm is prevented from being inhibited, the pure solid culture medium is used, some germs can not well contact the nutrient substances in the culture medium, the pure liquid culture medium is used, the germs are too immersed in the culture solution, the contact area with air is limited, the limited ability to carbon dioxide will also inhibit proliferation of protocorms, while in a shaking liquid medium environment, the plants are alternately contacted with the nutrient solution and the air, and the dendrobium can be intermittently, alternately and simultaneously contacted with the nutrient solution and the air, so that the protocorm can be well added.
S4: inducing cluster buds by selecting healthy and pollution-free protocorms on an induction medium B, wherein the induction medium B is based on an MS solid medium and is added with 0.1-0.3 mg.L of Kinetin (KT)-10.2 to 0.4 mg/L of naphthylacetic acid (NAA)-10.3-0.5 mg-L of 6-benzylamino adenine (6-BA)-1(ii) a The illumination intensity is 1500 mu mol.m-2·s-1Subculturing for 40d under the condition that the illumination time is 10 h/d;
table 4: the influence (%) of the induction medium B with different hormone ratios on the induction of the dendrobium huoshanense cluster buds under the same environment is as follows:
as is clear from the above table, 0.2 mg. multidot.L of Kinetin (KT) was added to 1/2MS medium-10.2 mg. L of Naphthylacetic acid (NAA)-16-benzylaminopurine (6-BA)0.3 mg. L-1The experiment group adopts an orthogonal experiment method, nine experiment groups are designed, the optimal concentration can be obtained, the result is obtained through variance analysis, the intuition and the variance analysis are combined with each other, the KT is the factor with the largest influence in the inducing process of the dendrobium huoshanense cluster buds, the added concentration directly influences the growth condition of the cluster buds, and when the concentration is too high, the inhibiting phenomenon can occur, so that the concentration ratio of the KT is very important.
S5: proliferating cluster buds, selecting healthy and pollution-free cluster buds on a growth culture medium B for proliferating cluster buds, wherein the induction culture medium B is based on an MS liquid culture medium and added with 1.0-1.5 mg.L of naphthylacetic acid (NAA)-10.5-1.5 mg.L of 6-benzylamino adenine (6-BA)-16-benzyl1.0-1.5 mg/L of amino adenine (6-BA)-1In the medium of (1); the illumination intensity is 1500 mu mol.m-2·s-1Culturing for 50 days under the condition that the illumination time is 10 h/d.
Table 5: the influence (%) of growth medium B with different hormone ratios on the induction of dendrobium huoshanense cluster buds under the same environment is as follows:
as is clear from the above table, 1.5 mg. L of Kinetin (KT) was added to the MS medium-11.0 mg. L of Naphthylacetic acid (NAA)-16-benzylaminopurine (6-BA)1.5 mg. L-1The average increment rate of each cluster bud is 3.4, the most suitable growth rate of the cluster buds of dendrobium huoshanense is obtained through a comparison experiment, and the KT can be obviously influenced greatly on the elongation of the plant through the comparison experiment in the early experimental experience, so that the most suitable KT concentration is obtained through the comparison emphatically.
S6: rooting and strengthening seedlings, namely selecting healthy and pollution-free cluster buds with the bud height of 2.0cm on 1/2MS culture medium to root and strengthen the seedlings; the illumination intensity is 1500 mu mol.m-2·s-1Culturing for 65d under the condition that the illumination time is 10 h/d;
the subculture frequency of protocorm and cluster bud is controlled within 4 generations, and 0.8% agar and 3.0% sucrose are added into the induction culture medium A, the induction culture medium B, the growth culture medium A and the growth culture medium B.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. The dendrobium huoshanense seedling cultivation technology is characterized by comprising the following operation steps:
s1: aseptic seeding, namely clamping the disinfected capsule by using tweezers under an aseptic condition, cutting the top end of the capsule by using a scalpel, slightly shaking the capsule in a culture bottle, uniformly sowing a layer of seeds on the surface of an MS culture medium, and immediately sealing a bottle opening;
s2: inducing protocorm, culturing in a culture flask at 25 + -2 deg.C in clean dark environment at pH5.8 for 1 week, culturing in induction medium A under illumination intensity of 1500 μmol/m-2·s-1～2000μmol·m-2·s-1Under the condition that the illumination duration is 10-12 h/d, seeds germinate for 10-20 d, and are continuously cultured for 55-60 d to form protocorms;
s3: adding the protocorm value, selecting healthy, full and pollution-free protocorm to shake and culture on a growth medium A, wherein the shaking frequency is 120 r.min-1Adding the protocorm value; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Subculturing for 55 d-65 d under the condition that the illumination duration is 10 h/d-12 h/d;
s4: inducing cluster buds, namely selecting healthy and pollution-free protocorms on an induction culture medium B to induce the cluster buds; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Subculturing for 40-55 d under the condition that the illumination duration is 10-12 h/d;
s5: the cluster buds are proliferated, and healthy and pollution-free cluster buds are selected to be placed on a growth medium B for proliferation; the illumination intensity is 1500 mu mol.m-2·s-1～2000μmol·m-2·s-1Culturing for 50-70 d under the condition that the illumination duration is 10-12 h/d;
s6: rooting and strengthening seedlings, namely selecting healthy and pollution-free cluster buds with the bud height of 1.5-2.5 cm on 1/2MS culture medium to root and strengthen the seedlings; the illumination intensity is 1500 mu mol.m-2·s-1～3000μmol·m-2·s-1Culturing for 65d to 75d under the condition that the illumination duration is 10h/d to 12 h/d;
s7: the subculture times of protocorm and cluster buds are controlled within 4 generations.
2. The dendrobe seedling cultivation technology according to claim 1, wherein capsules need to be pretreated, the surfaces of the capsules are sufficiently cleaned with clear water, residual moisture is dried in the shade, the capsules are wrapped with clean paper and stored in a refrigerator at 4 ℃ for one week, the pretreated capsules are soaked in 75% ethanol for 15-20 min and then burned on an alcohol lamp for 1-2 s.
3. The dendrobe seedling cultivation technology as claimed in claim 1, wherein the induction medium A is MS liquid medium, and 0.05-0.2 mg-L of 2, 4-dichlorophenoxyacetic acid is added-10.05 to 0.1 mg.L of Kinetin (KT)-1。
4. The dendrobe seedling cultivation technology as claimed in claim 1, wherein the growth medium A is based on MS solid medium, and 100-200 g/L of potato juice and 0.5-1.0 mg-L of naphthylacetic acid (NAA) are added-10.5-1.0 mg-L of 6-benzylamino adenine (6-BA)-1In the medium of (1).
5. The dendrobe seedling cultivation technology as claimed in claim 1, wherein the induction medium B is based on MS solid medium, and 0.1-0.3 mg-L Kinetin (KT) is added-10.2 to 0.4 mg/L of naphthylacetic acid (NAA)-10.3-0.5 mg-L of 6-benzylamino adenine (6-BA)-1。
6. The dendrobe seedling cultivation technology as claimed in claim 1, wherein the induction medium B is MS liquid medium and Naphthalene Acetic Acid (NAA) is added in an amount of 1.0-1.5 mg-L-10.5-1.5 mg-L of 6-benzylamino adenine (6-BA)-11.0-1.5 mg-L of 6-benzylamino adenine (6-BA)-1In the medium of (1).
7. The dendrobe seedling cultivation technology as claimed in claim 1, wherein 0.8% agar and 3.0% sucrose are added to each of the induction medium A, the induction medium B, the growth medium A and the growth medium B.
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| CN113229145A (en) * | 2021-04-27 | 2021-08-10 | 陈大卫 | Technology for carrying out spray breeding on dendrobium huoshanense cells |
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Application publication date: 20200114 |