CN113796315B - Quick propagation method of endangered wild plant cymbidium floribundum - Google Patents
Quick propagation method of endangered wild plant cymbidium floribundum Download PDFInfo
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- CN113796315B CN113796315B CN202111191192.5A CN202111191192A CN113796315B CN 113796315 B CN113796315 B CN 113796315B CN 202111191192 A CN202111191192 A CN 202111191192A CN 113796315 B CN113796315 B CN 113796315B
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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
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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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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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention relates to the technical field of plant tissue culture, and particularly discloses a rapid propagation method of endangered wild plant cymbidium floribundum. The method comprises the steps of aseptic germination of seeds, protocorm proliferation, protocorm differentiation and sprouting, bud rooting and tissue culture seedling transplantation. The time for the seeds to germinate into protocorms in an aseptic mode is 90 days, the time for the protocorms to proliferate is 20 days, the proliferation efficiency can reach 5 protocorms, the time for the protocorms to differentiate 2 cm-sized buds is 30 days, the time for the buds to root is 10 days, and the survival rate of the tissue culture seedlings transplanted into a substrate after the tissue culture seedlings are cultured for 30 days to be rooted is 90%. The method of the invention has the culture time of 60 days from the proliferation, differentiation and sprouting of the protocorm to the rooting of the buds to the tissue culture seedling of the florists orchid, greatly shortens the time compared with other propagation modes such as the plant division propagation and the like, and can quickly provide a large amount of raw materials for the application of the florists orchid with high quality.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation method of endangered wild plant cymbidium floribundum.
Background
Cymbidium floribundum Lindl is an epiphytic plant of Cymbidium in the orchid family, is a member of two species in the appendix of international trade convention for endangered wild animal and plant species, and is a national grade I protective wild plant. The Dolichos floribunda is distributed in Asian countries such as India of China and is distributed in provinces such as Guangdong, Zhejiang, Yunnan, Hainan and Hunan. The leaves of the orchids are fleshy, are pleased with yin and direct sunlight, are pleased with dampness and dry, can resist high temperature and low temperature, have gorgeous and various colors, have long flowering phase, are easy to cultivate, have higher ornamental value, and are one of more ideal potted flowers; the roots of the cymbidium floribundum can be used as a medicine for nourishing yin, clearing away the lung-heat, eliminating phlegm and relieving cough; the florets have abundant variation in plant type, flower number, flower color and the like, have wide adaptability and have important application value in breeding new varieties.
However, on the one hand, due to the deterioration or loss of habitat, cutting or direct harvesting for horticultural appreciation, etc., the actual level of development has reduced the population by more than 30% over the last 10 years; on the other hand, the orchids are mainly propagated by plants, but the propagation speed is slow; the field germination rate of seeds is low; the literature on the cymbidium floribundum tissue culture technology is not reported a lot, and in addition to a plant growth regulator, a moringa leaf juice, a medlar leaf and other nutritional agents are added in the existing cymbidium floribundum tissue culture medium, so that the propagation cost is increased.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for rapidly propagating endangered wild orchids. The invention provides the following technical scheme:
a method for rapidly propagating endangered wild plant cymbidium floribundum comprises the following specific steps:
1) sterile germination of seeds: sterilizing mature capsules picked in the field, cutting capsules, taking out seeds, inoculating the seeds into a germination culture medium, carrying out dark culture for 30 days, then transferring to illumination culture, and carrying out aseptic germination until the seeds germinate to form protocorms; the method for disinfecting the seeds comprises the steps of soaking the seeds in 75% ethanol for 3 minutes, soaking the seeds in 2% sodium hypochlorite for 14 minutes, soaking the seeds in sterile water and shaking the seeds for 6 times, wherein the shaking time is one minute to two minutes each time; the germination culture medium of the seeds is a culture medium containing 3g/L Huabao I, 4g/L peptone and 0.1g/L active carbon;
2) proliferation of protocorm: transferring the protocorm formed in the step 1) into a culture medium for inducing the proliferation of the protocorm, and conventionally culturing until more protocorms are differentiated; the culture medium for proliferation of protocorm is 1/2MS culture medium containing 1.0mg/L naphthylacetic acid, 2.0 mg/L6-benzylaminopurine and 2.0g/L hydrolyzed milk protein;
3) protocorm differentiation and budding: transferring the protocorm formed in the step 1) or the step 2) into a culture medium for differentiating buds, and conventionally culturing until adventitious buds are differentiated; the culture medium for differentiation and budding is 1/2MS culture medium containing 0.5mg/L naphthylacetic acid and 1.0 mg/L6-benzylaminopurine;
4) and (3) bud rooting: cutting off buds with the size of about 3-5 cm formed in the step 3), transferring the buds into a rooting induction culture medium, and conventionally culturing until roots are formed to obtain a cymbidium floribundum tissue culture seedling; the rooting induction culture medium is 1/2MS culture medium containing 2mg/L naphthylacetic acid;
5) transplanting tissue culture seedlings: transplanting the tissue culture seedlings subjected to rooting culture for 30 days in the step 4) into different matrixes, and culturing until the tissue culture seedlings survive; the matrix is a humus soil and vesuvianite mixed matrix with the volume ratio of 1: 1.
Wherein the time for the seeds to aseptically germinate to form protocorms in the step 1) is 90 days.
Wherein, the proliferation efficiency of the protocorm in the step 2) for 20 days can reach 5 protocorms.
Wherein the cultivation time for differentiating 2 cm buds from the protocorm in the step 3) is 30 days.
Wherein, the rooting time of the buds in the step 4) is 10 days.
Wherein, the survival rate of the tissue culture seedlings in the step 5) reaches 90 percent after 30 days of transplanting culture, and the plants grow obviously.
Wherein the culture medium in the steps 1), 2), 3) and 4) contains 30g/L of sucrose and 5g/L of agar powder, and the pH value is 5.4.
Wherein, the seed germination culture conditions in the step 1) are firstly to culture in dark light for 30 days, then to transfer to illumination culture, the illumination time is 14h/d, the illumination intensity is 2000lx, and the temperature is 25 +/-1 ℃.
Wherein, the culture conditions of the conventional culture in the steps 2), 3) and 4) are illumination intensity 2000lx and illumination time 14h/d, and the temperature of the constant temperature incubator is 25 +/-1 ℃.
Compared with the prior art, the invention has the following beneficial effects:
1) the method of the invention forms protocorm after seeds germinate for 90 days, the culture time from the propagation, differentiation and sprouting of the protocorm to the rooting of the bud to the acquisition of the tissue culture seedling is 60 days, and a large amount of excellent raw materials can be rapidly, massively and excellently provided for the application of the cymbidium.
2) The method has important significance in the aspects of protection and preservation of wild orchids, requirements of artificial cultivation on seedlings, cultivation of new varieties and the like.
Drawings
FIG. 1: is the mature capsule of wild orchis polycarpa (field picking place: Hunan mulberry planting) used in the examples of the invention.
FIG. 2: is protocorm formed by aseptic germination of seeds after 90 days of culture in the embodiment of the invention.
FIG. 3: is the proliferation of protocorms in the examples of the invention.
FIG. 4: is the case of protocorm sprouting in the examples of the present invention.
FIG. 5: is the rooting condition of the buds in the embodiment of the invention.
FIG. 6: is the survival condition of the tissue culture seedling in the embodiment of the invention.
Detailed Description
In order to express the present invention more clearly, preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The medium formulations used in the examples below are different from the medium used in the reported case for the tissue culture of orchids.
Example 1
A method for rapidly propagating endangered wild plant orchida florida comprises the following steps:
1) sterile germination of seeds: sterilizing mature capsules picked in the field, cutting capsules, taking out seeds, inoculating the seeds in a germination culture medium, performing dark culture for 30 days, then transferring to illumination culture, wherein the illumination time is 14h/d, the illumination intensity is 2000lx, the temperature is 25 +/-1 ℃, and performing aseptic germination until the seeds germinate to form protocorms; the seed disinfection method comprises the steps of soaking the seeds in 75% ethanol for 3 minutes, soaking the seeds in 2% sodium hypochlorite for 14 minutes, soaking the seeds in sterile water and shaking the seeds for 6 times, wherein the shaking time is one minute to two minutes each time; the seed germination culture medium is a culture medium containing 3g/L Huabao I, 4g/L peptone and 0.1g/L active carbon, and the time for the seeds to germinate aseptically to form protocorms is 90 days.
2) Proliferation of protocorm: transferring the protocorm formed in the step 1) into a culture medium for inducing the proliferation of the protocorm, and conventionally culturing until more protocorms are differentiated; the culture medium for protocorm proliferation is 1/2MS culture medium containing 1.0mg/L naphthylacetic acid, 2.0 mg/L6-benzylaminopurine and 2.0g/L hydrolyzed milk protein, and the proliferation efficiency of the protocorm proliferation culture for 20 days can reach 5 protocorms.
3) Protocorm differentiation and budding: transferring the protocorm formed in the step 1) or the step 2) into a culture medium for differentiating buds, and conventionally culturing until adventitious buds are differentiated; the culture medium for protocorm budding is 1/2MS culture medium containing 0.5mg/L naphthylacetic acid and 1.0 mg/L6-benzylaminopurine; the cultivation time for the protocorm to differentiate 2 cm buds was 30 days.
4) And (3) bud rooting: cutting off buds with the size of about 3-5 cm formed in the step 3), transferring the buds into a rooting induction culture medium, and conventionally culturing until roots are formed to obtain a cymbidium floribundum tissue culture seedling; the culture medium for rooting the buds is 1/2MS culture medium containing 2mg/L naphthylacetic acid, and the time for rooting the buds is 10 days.
5) Transplanting tissue culture seedlings: transplanting the tissue culture seedlings subjected to rooting culture for 30 days in the step 4) into different matrixes, and culturing until the tissue culture seedlings survive; the matrix for transplanting is a humus soil and vesuvianite mixed matrix with the volume ratio of 1: 1. The survival rate of the tissue culture seedlings reaches 90 percent after 30 days of transplanting culture, and the plants grow obviously.
The culture medium in the steps 1), 2), 3) and 4) contains 30g/L of sucrose and 5g/L of agar powder, the pH value is 5.4, the culture conditions of conventional culture are that the illumination intensity is 2000lx, the illumination time is 14h/d, and the temperature of a constant-temperature incubator is 26 +/-1 ℃.
The result shows that the method can form protocorms after seeds germinate for 90 days, the whole process of forming tissue culture seedlings from the protocorms from proliferation, differentiation and sprouting to the roots of the sprouts only needs 60 days, a large amount of excellent raw materials can be rapidly provided for the application of the cymbidium floribundum in a large amount and high quality, the method has important significance on the aspects of protection and preservation of wild cymbidium floribundum resources, requirements of artificial cultivation on seedlings, cultivation of new varieties and the like, and the market application prospect is wide.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for rapidly propagating endangered wild plant orchida florida is characterized by comprising the following steps:
1) sterile germination of seeds: sterilizing mature capsules picked in the field, cutting capsules, taking out seeds, inoculating the seeds into a germination culture medium, carrying out dark culture for 30 days, then transferring to illumination culture, and carrying out aseptic germination until the seeds germinate to form protocorms; the method for disinfecting the seeds comprises the steps of soaking the seeds in 75% ethanol for 3 minutes, soaking the seeds in 2% sodium hypochlorite for 14 minutes, soaking the seeds in sterile water and shaking the seeds for 6 times, wherein the shaking time is one minute to two minutes each time; the germination culture medium of the seeds consists of a culture medium of 3g/L Huabao I, 4g/L peptone, 0.1g/L active carbon, 30g/L sucrose and 5g/L agar powder, and the pH value is 5.4;
2) proliferation of protocorm: transferring the protocorm formed in the step 1) into a culture medium for inducing the proliferation of the protocorm, and conventionally culturing until more protocorms are differentiated; the culture medium for proliferation of protocorm is 1/2MS culture medium containing 1.0mg/L naphthylacetic acid, 2.0 mg/L6-benzylaminopurine, 2.0g/L lactoprotein hydrolysate, 30g/L sucrose and 5g/L agar powder, and the pH value is 5.4;
3) protocorm differentiation and budding: transferring the protocorm formed in the step 1) or the step 2) into a culture medium for differentiating buds, and conventionally culturing until adventitious buds are differentiated; the culture medium for differentiation and budding is 1/2MS culture medium of 0.5mg/L naphthylacetic acid, 1.0 mg/L6-benzylaminopurine, 30g/L sucrose and 5g/L agar powder, and the pH value is 5.4;
4) and (3) bud rooting: cutting off buds with the size of 3-5 cm formed in the step 3), transferring the buds into a rooting induction culture medium, and conventionally culturing until roots are formed to obtain a cymbidium floribundum tissue culture seedling; the rooting induction culture medium is 1/2MS culture medium containing 2mg/L naphthylacetic acid, 30g/L sucrose and 5g/L agar powder, and the pH value is 5.4;
5) transplanting tissue culture seedlings: transplanting the tissue culture seedlings subjected to rooting culture for 30 days in the step 4) into different matrixes, and culturing until the tissue culture seedlings survive; the matrix is a humus soil and vesuvianite mixed matrix with the volume ratio of 1: 1.
2. The method for the rapid propagation of the endangered wild plant orchids according to claim 1, wherein the time for the seeds to germinate aseptically to form protocorms in step 1) is 90 days.
3. The method for rapid propagation of the endangered wild plant, orchidaceae, according to claim 1, wherein the propagation efficiency of the protocorm propagation culture in step 2) for 20 days is up to 5 protocorms.
4. The method for the rapid propagation of the endangered wild plant orchids according to claim 1, wherein the cultivation time for differentiating 2 cm buds from the protocorm in the step 3) is 30 days.
5. The method for the rapid propagation of the endangered wild plant orchidaceae hynchoides as claimed in claim 1, wherein the time for the shoots to root in the step 4) is 10 days.
6. The method for rapidly propagating orchidaceae, which is an endangered wild plant, according to claim 1, wherein the survival rate of the tissue culture seedlings in the step 5) after 30 days of transplanting culture reaches 90%, and the plants grow obviously.
7. The method for rapid propagation of an endangered wild plant, cymbidium floribundum as claimed in claim 1, wherein the seed germination culture conditions in step 1) are that after culturing in the dark for 30 days, the seeds are transferred to illumination culture, the illumination time is 14h/d, the illumination intensity is 2000lx, and the temperature is 25 ± 1 ℃.
8. The method for rapidly propagating orchidaceae, which is an endangered wild plant, according to claim 1, wherein the cultivation conditions of the conventional cultivation in the steps 2), 3) and 4) are that the illumination intensity is 2000lx, the illumination time is 14h/d, and the temperature of the constant-temperature incubator is 25 +/-1 ℃.
9. Use of the method according to any one of claims 1 to 8 for the rapid propagation of an endangered wild plant, orchidaceae.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577973A (en) * | 2012-03-12 | 2012-07-18 | 云南山里红生物科技有限公司 | Tissue culture method for Cymbidium floribundum |
| CN107691224A (en) * | 2017-11-13 | 2018-02-16 | 秦素梅 | A kind of seed propagation method of floribunda orchid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577973A (en) * | 2012-03-12 | 2012-07-18 | 云南山里红生物科技有限公司 | Tissue culture method for Cymbidium floribundum |
| CN107691224A (en) * | 2017-11-13 | 2018-02-16 | 秦素梅 | A kind of seed propagation method of floribunda orchid |
Non-Patent Citations (5)
| Title |
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| 多花兰的离体萌发和试管成苗技术研究;丁长春等;《安徽农业科学》;20110301(第07期);全文 * |
| 多花兰种子无菌萌发及离体快繁研究;唐凤鸾等;《广西植物》;20121130;第32卷(第6期);全文 * |
| 多花兰种子繁育技术研究;王郑昊等;《山东林业科技》;20130215(第01期);全文 * |
| 蜜蜂兰组培快繁技术体系的建立;饶宝蓉等;《福建农业科技》;20210328;第52卷(第3期);全文 * |
| 野生兰6个珍贵品种组织培养技术研究初探;周双清等;《热带林业》;20131215(第04期);全文 * |
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