CN112400698A - Seedling detoxification cultivation method - Google Patents
Seedling detoxification cultivation method Download PDFInfo
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- CN112400698A CN112400698A CN202011626956.4A CN202011626956A CN112400698A CN 112400698 A CN112400698 A CN 112400698A CN 202011626956 A CN202011626956 A CN 202011626956A CN 112400698 A CN112400698 A CN 112400698A
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- 238000001784 detoxification Methods 0.000 title claims abstract description 61
- 238000012364 cultivation method Methods 0.000 title claims abstract description 18
- 230000005684 electric field Effects 0.000 claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000003206 sterilizing agent Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 11
- 230000004083 survival effect Effects 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
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- 238000004140 cleaning Methods 0.000 claims description 7
- 230000003203 everyday effect Effects 0.000 claims description 7
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 238000000338 in vitro Methods 0.000 claims description 3
- 239000003973 paint Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 4
- 239000002574 poison Substances 0.000 abstract description 2
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- 241000196324 Embryophyta Species 0.000 description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 239000008223 sterile water Substances 0.000 description 12
- 238000005286 illumination Methods 0.000 description 10
- 238000005406 washing Methods 0.000 description 6
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 235000011430 Malus pumila Nutrition 0.000 description 4
- 235000015103 Malus silvestris Nutrition 0.000 description 4
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- 235000014443 Pyrus communis Nutrition 0.000 description 4
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- 231100000252 nontoxic Toxicity 0.000 description 4
- 230000003000 nontoxic effect Effects 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
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- 241000220225 Malus Species 0.000 description 3
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- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
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- 102000004877 Insulin Human genes 0.000 description 1
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- 244000141359 Malus pumila Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A method for culturing the seedlings without poison features that the inoculated bud leaves are put in high-voltage electric field for high-voltage removing treatment. The cultivation method utilizes the high-voltage electric field to detoxify the nursery stock, and has the advantages of high detoxification rate, simple detoxification steps, convenient operation, easy establishment of conditions required by detoxification, low cost, short detoxification treatment period and good detoxification effect.
Description
Technical Field
The invention relates to the field of nontoxic seedling cultivation, in particular to a seedling detoxification cultivation method for detoxification by using a high-voltage electric field.
Background
According to long-term research by scientists, the cause of crop quality deterioration is mainly infection by plant viruses. In order to obtain high yield and improve the growth performance of plants, a large amount of plant viruses are accumulated in plant cells in asexual propagation for a long time, so that the life span, the properties, the yield and the like of the plants are influenced to a certain extent, and the plants are easy to suffer from diseases. Viruses are a special organism without cellular structure, their structure is very simple and consists of a protein coat and internal genetic material. Viruses cannot survive independently and must live inside the cells of other organisms, leaving the living cell shell without showing any signs of life activity. Asexual reproduction plants need to be frequently detoxified to strengthen the characteristics of the plants and prevent and control some diseases. In the production process, virus-free seedlings, namely virus-free seedlings, must be cultured.
The cultivation of non-toxic plants is an urgent need of society. The traditional detoxification methods comprise a heat treatment detoxification method, a stem tip culture detoxification method, a callus detoxification method and the like, but the long-term practice shows that the detoxification methods have more or less defects: for example, the heat treatment detoxification method has the problems of low success rate of detoxification, not wide application range, plant death caused by detoxification temperature and the like; the stem tip culture detoxification method has the problems of high stem tip stripping technology, capability of improving detoxification rate by combining with other detoxification modes and the like. The callus detoxification method has the problems of unstable genetic property and incapability of developing into a complete plant.
Therefore, the existing plant detoxification method has many defects, so that the requirements of production, experimental research and other plant detoxification can not be met.
Disclosure of Invention
The invention provides a seedling detoxification cultivation method for detoxification by using a high-voltage electric field, aiming at various technical problems in the background art. Compared with the prior detoxification method for removing plant viruses, the method provided by the invention has the difference that a series of problems of low detoxification rate, great difficulty in detoxification technology, complex detoxification steps, high conditions required for detoxification, long detoxification treatment period and the like can be effectively avoided.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for culturing the seedlings without poison features that the inoculated bud leaves are put in high-voltage electric field for high-voltage removing treatment.
The cultivation process is as follows:
s1, picking the bud and leaf parts of the plants with viruses;
s2: performing surface cleaning and surface sterilization treatment on the bud leaves picked in the step S1;
s3: inoculating the bud leaves processed in the step S2 into a culture medium, and placing the bud leaves in an environment suitable for survival for later use;
s4: placing the inoculated bud leaves in the step S3 in a high-voltage electric field device, and carrying out high-voltage treatment for 25-50 minutes every day for 7-15 days;
s5: taking out the bud leaves subjected to the electric field treatment in the step S4, and then carrying out tissue culture to form callus and proliferate new parts;
s6: and (4) selecting the newly-bred parts formed in the step (S5) for culturing, and growing complete detoxified plants.
The bud part selected in step S1 is a tender leaf bud.
The size of the bud leaf part selected in the step S1 is judged by the survival in vitro, and the length is 1-3 cm.
The surface sterilization in step S2 is to soak the leaves with a sterilizing agent for 20-40S.
The sterilizing agent is one of ethanol solution, sodium hypochlorite and hydrogen peroxide.
The culture medium in the step S3 is MS +6-BA0.5mg/L + NAA0.1mg/L, MS +6-BA2.0mg/L + NAA0.1mg/L + GA30.3mg/L、-MS+BA0.7mg/L+IBA0.3 mg/L.
In step S3, the environment suitable for survival is at a temperature of 20-26 deg.C and a light period of 12-16 h, and the light intensity is 1500-3000 lx.
The high-voltage electric field in the high-voltage electric field device in the step S4 is 10-15 kv/cm.
The high-voltage electric field device in the step S4 includes an upper electrode plate, a lower electrode plate, and an electric field space; the electric field space is formed by enclosing a PE film and is arranged between the upper electrode plate and the lower electrode plate; an air inlet and an air outlet are respectively arranged on two sides of the electric field space; and insulating paint is uniformly coated on the surfaces of the upper electrode plate and the lower electrode plate of the high-voltage electric field device.
Compared with the prior art, the cultivation method disclosed by the invention utilizes the high-voltage electric field to detoxify the nursery stock, and has the advantages of high detoxification rate, simple detoxification steps, convenience in operation, easiness in establishment of conditions required for detoxification, low cost, short detoxification treatment period and good detoxification effect.
Drawings
Fig. 1 is a schematic structural diagram of a high-voltage electric field device according to the present invention.
FIG. 2 is a table comparing the ozone content in the conventional detoxification method and the high-voltage electric field detoxification method of the present invention.
In fig. 1: an upper electrode plate 1, a PE film 2, an air inlet 3, an air outlet 4 and a lower electrode plate 5.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The invention discloses a cultivation method of nontoxic seedlings, which combines a high-voltage electric field with cultivation of nontoxic seedlings in plant breeding, has simple processing steps, only places plants in the high-voltage electric field, has simple and easy-to-use processing process, is a physical process in the whole processing process, has the function of detoxifying viruses in plants by using the effect of field intensity, does not need to worry about the problem of chemical dose of the plants, and only needs to ensure the survival of the plants in the whole process.
Unlike sterilization and disinfection in the conventional manner, disinfection generally means "a method of killing pathogenic microorganisms but not necessarily bacterial spores", and sterilization means "a method of killing all microorganisms (including bacterial spores) on an object". The detoxification in the invention refers to the removal of RNA virus parasitizing in plant cells, and avoids the character of influencing plant growth. The cultivation of the plant virus-free seedlings has the advantages of strong growth potential of fruit trees, high yield, good quality, good commodity performance and objective economic benefit. Besides, the method has important significance in plant pathology, and enriches the content of plant pathology.
According to the seedling detoxification cultivation method disclosed by the invention, in the cultivation process of seedlings, inoculated bud leaves are placed in a high-voltage electric field for high-voltage detoxification treatment.
The cultivation process is as follows:
s1, picking the bud and leaf parts of the plants with viruses;
s2: performing surface cleaning and surface sterilization treatment on the bud leaves picked in the step S1;
s3: inoculating the bud leaves processed in the step S2 into a culture medium, and placing the bud leaves in an environment suitable for survival for later use;
s4: placing the inoculated bud leaves in the step S3 in a high-voltage electric field device, and carrying out high-voltage treatment for 25-50 minutes every day for 7-15 days;
s5: taking out the bud leaves subjected to the electric field treatment in the step S4, and then carrying out tissue culture to form callus and proliferate new parts;
s6: and (4) selecting the newly-bred parts formed in the step (S5) for culturing, and growing complete detoxified plants.
As a preferable technical scheme, the bud leaf part selected in the step S1 is a tender leaf and a tender bud, and the size of the selected bud leaf part is judged by easy in vitro survival, and the length is 1-3 cm.
Preferably, the surface sterilization in step S2 is performed by soaking and sterilizing the bud leaves with a sterilizing agent for 20 to 40 seconds. The sterilizing agent is one of ethanol solution, sodium hypochlorite and hydrogen peroxide, and the preferable scheme is as follows: 70-75% of ethanol solution, 5.5-6.5% of sodium hypochlorite and 2.7-3.3% of hydrogen peroxide
The culture medium disclosed in step S3 is MS +6-BA0.5mg/L + NAA0.1mg/L or MS +6-BA2.0mg/L + NAA0.1mg/L + GA30.3mg/L or-MS + BA0.7mg/L + IBA0.3mg/L. When the inoculation culture is carried out in the culture medium, the environment suitable for survival is at the temperature of 20-25 ℃, the illumination period of 12-16 h and the illumination intensity of 1500-.
As a preferred example, the high voltage electric field in the high voltage electric field apparatus disclosed in the step S4 is 10-15 kv/cm. As shown in fig. 1, the high-voltage electric field device comprises an upper electrode plate 1, a lower electrode plate 5 and an electric field space. The electric field space is formed by enclosing the PE film, and the electric field space in the embodiment is a rectangular frame body formed by enclosing the PE film and is arranged between the upper electrode plate and the lower electrode plate; an air inlet 3 and an air outlet 4 are respectively arranged on two sides of the electric field space, and insulating paint is uniformly coated on the surfaces of the upper electrode plate and the lower electrode plate. The electric field space in the whole high-voltage electric field device forms a device capable of eliminating the interference of ozone and ions.
And (4) placing the inoculated bud leaves in the electric field space for high-voltage electric field detoxification. Under the action of a high-voltage electric field, the hydrophobic groups of the protein expose and limit the proximity of a substrate and an active site to inhibit the activity of an insulin B chain, and can change the secondary structures of papain, pepsin, polyphenol oxidase and peroxidase to ensure that protein molecules are aggregated. The inactivation of the virus by the high-voltage electric field may depend on the aggregation of the protein by the high-voltage electric field, the exposure of hydrophobic groups and sulfydryl is generated by the coat protein under the action of the high-voltage electric field, and partial disulfide bonds are crosslinked to form protein aggregation, so that the coat protein of the virus is denatured and loses the ability of infecting cells.
FIG. 2 is a comparison of ozone generation capacity of a conventional detoxification device and a high-voltage electric field detoxification device according to the present invention under a high-voltage environment of 200 kv/m. It can be seen from the figure that the concentration value (0.6 mg ^) of the ozone function is exceeded by the traditional detoxification device when the device is electrified for about 50min
) The high voltage electric field in this embodiment can beThe effect of ozone was removed within 2 hours.
The invention is described in detail below with reference to examples, and any modifications made based on the invention are within the scope of the invention.
Example 1
A high-voltage electric field detoxification method for apple seedlings comprises the following specific steps: selecting tender shoot leaves with the length of 1-1.5cm on apple seedlings, cleaning up the obvious dirt on the surfaces of the tender shoot leaves under running water by using a soft brush, then placing the tender shoot leaves in a sterilized bottle, adding a sodium hypochlorite solution into the bottle, wherein the concentration of the sodium hypochlorite solution is preferably 5.5% -6.5%, soaking for 20-30s, pouring out a sterilizing agent, adding a sterile water washing sterilizing agent into the bottle, pouring out the sterile water, repeating the sterile water washing process for 3-5 times, and ensuring that the sterilizing agent on the surfaces of the tender shoot leaves is washed clean.
The treated tender bud leaves are inoculated to a culture medium of MS +6-BA0.5mg/L + NAA0.1mg/L to ensure that the bud leaves grow and survive. The culture conditions are that the temperature is 20-25 ℃, the illumination period is 15-16 h, and the illumination intensity is 2000-3000 lx.
The culture medium is placed in an electric field with intensity of 15kv/cm for 25-30min every day for 15 days. And performing tissue culture on the explant treated by the electric field to form callus, and proliferating new plant bodies. Taking the newly proliferated part for culturing and rooting, and then transferring the newly proliferated part into soil to complete the culture of the virus-free plant body.
Example 2
A high-voltage electric field detoxification method for pear tree detoxification comprises the following specific steps: selecting 1.5-3cm long tender bud leaves on a pear tree, cleaning up the obvious dirt on the surfaces of the tender bud leaves under running water by using a soft brush, then placing the tender bud leaves in a sterilized bottle, adding 70% alcohol into the bottle, soaking for 30-40s, pouring out a sterilizing agent, adding sterile water into the bottle to wash the sterilizing agent, pouring out the sterile water, and repeating the sterile water washing process for 3-5 times to ensure that the sterilizing agent on the surfaces of the tender bud leaves is washed clean.
Inoculating the treated tender bud to MS +6-BA2.0mg/L + NAA0.1mg/L + GA3The growth was allowed to survive on 0.3mg/L medium. The culture conditions are 24-26 ℃, the illumination period is 12-13 h, and the illumination intensity is 1500-.
The culture medium is placed in an electric field with intensity of 10kv/cm for 40-50min every day for 12 days. And performing tissue culture on the explant treated by the electric field to form callus, and proliferating new plant bodies. Taking the newly proliferated part for culturing and rooting, and then transferring the newly proliferated part into soil to complete the culture of the virus-free plant body.
Example 3
A high-voltage electric field detoxification method for apple saplings comprises the following specific steps: selecting tender bud leaves with the length of 2-3cm on an apple tree, cleaning up the obvious dirt on the surfaces of the tender bud leaves under running water by using a soft brush, then placing the tender bud leaves in a sterilized bottle, adding hydrogen peroxide with the concentration of 2.7% -3.3% into the bottle, soaking for 35-40s, pouring out a sterilizing agent, adding a sterile water washing sterilizing agent into the bottle, pouring out the sterile water, repeating the sterile water washing process for 3-5 times, and ensuring that the sterilizing agent on the surfaces of the tender bud leaves is washed clean.
Inoculating the treated tender bud to MS +6-BA2.0mg/L + NAA0.1mg/L + GA3The growth was allowed to survive on 0.3mg/L medium. The culture conditions are that the temperature is 23-27 ℃, the illumination period is 12-13 h, and the illumination intensity is 2000-3000 lx.
The culture medium is placed in an electric field with intensity of 15kv/cm for 30-45min every day for 7 days. And performing tissue culture on the explant treated by the electric field to form callus, and proliferating new plant bodies. Taking the newly proliferated part for culturing and rooting, and then transferring the newly proliferated part into soil to complete the culture of the virus-free plant body.
Example 4
A high-voltage electric field detoxification method for pear saplings comprises the following specific steps: selecting tender shoot leaves with the length of 3cm on a pear tree, cleaning up the obvious dirt on the surfaces of the tender shoot leaves under running water by using a soft brush, then placing the tender shoot leaves in a sterilized bottle, adding a hydrogen peroxide solution with the concentration of 3-3.3% into the bottle, soaking for 35-40s, pouring out a sterilizing agent, adding sterile water into the bottle, pouring out the sterile water, and repeating the sterile water washing process for 3-5 times to ensure that the sterilizing agent on the surfaces of the tender shoot leaves is completely washed out.
The treated tender bud and leaf are inoculated to a culture medium of-MS + BA0.7mg/L + IBA0.3mg/L to ensure that the bud and leaf grow and survive. The culture conditions are that the temperature is 23-26 ℃, the illumination period is 15-16 h, and the illumination intensity is 1800-.
The culture medium is placed in an electric field with intensity of 13kv/cm for 40-45min every day for 12 days. And performing tissue culture on the explant treated by the electric field to form callus, and proliferating new plant bodies. Taking the newly proliferated part for culturing and rooting, and then transferring the newly proliferated part into soil to complete the culture of the virus-free plant body.
Claims (10)
1. A seedling detoxification cultivation method is characterized in that in the seedling cultivation process, inoculated bud leaves are placed in a high-voltage electric field for high-voltage detoxification treatment.
2. The seedling detoxification cultivation method according to claim 1, wherein the cultivation process is as follows:
s1, picking the bud and leaf parts of the plants with viruses;
s2: performing surface cleaning and surface sterilization treatment on the bud leaves picked in the step S1;
s3: inoculating the bud leaves processed in the step S2 into a culture medium, and placing the bud leaves in an environment suitable for survival for later use;
s4: placing the inoculated bud leaves in the step S3 in a high-voltage electric field device, and carrying out high-voltage treatment for 25-50 minutes every day for 7-15 days;
s5: taking out the bud leaves subjected to the electric field treatment in the step S4, and then carrying out tissue culture to form callus and proliferate new parts;
s6: and (4) selecting the newly-bred parts formed in the step (S5) for culturing, and growing complete detoxified plants.
3. The seedling detoxification cultivation method according to claim 2, wherein the bud part selected in the step S1 is a tender leaf bud.
4. The seedling detoxification cultivation method according to claim 3, wherein the size of the bud leaf part selected in the step S1 is judged as the survival rate in vitro, and the length is 1-3 cm.
5. The seedling detoxification cultivation method as claimed in claim 2, wherein the surface sterilization in the step S2 is to soak the leaves of the buds for 20-40S with a sterilizing agent.
6. The seedling detoxification cultivation method according to claim 5, wherein the sterilizing agent is one of ethanol solution, sodium hypochlorite and hydrogen peroxide.
7. The seedling detoxification cultivation method of claim 2, wherein the culture medium in the step S3 is MS +6-BA0.5mg/L + NAA0.1mg/L, MS +6-BA2.0mg/L + NAA0.1mg/L + GA30.3mg/L, -MS + BA0.7mg/L + IBA0.3mg/L.
8. The seedling detoxification cultivation method as claimed in claim 7, wherein in the step S3, the environment suitable for survival is at a temperature of 20-26 ℃ and a light intensity of 1500-.
9. The seedling detoxification cultivation method according to claim 2, wherein the high voltage electric field in the high voltage electric field device in the step S4 is 10-15 kv/cm.
10. The seedling detoxification cultivation method according to claim 9, wherein the high voltage electric field device in step S4 comprises an upper electrode plate, a lower electrode plate, and an electric field space; the electric field space is formed by enclosing a PE film and is arranged between the upper electrode plate and the lower electrode plate; an air inlet and an air outlet are respectively arranged on two sides of the electric field space; and insulating paint is uniformly coated on the surfaces of the upper electrode plate and the lower electrode plate of the high-voltage electric field device.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1541515A (en) * | 2003-11-07 | 2004-11-03 | 山东农业大学 | Three-stage amplifying method of inventing novel germplasm of drupe fruit tree utilizing distant hybridization |
| CN1875691A (en) * | 2006-07-21 | 2006-12-13 | 吕平 | A plant quick propagation method |
| CN101912761A (en) * | 2010-07-05 | 2010-12-15 | 洪昆喨 | Dielectric discharge reactor of uniform electric field |
| CN102696487A (en) * | 2012-07-05 | 2012-10-03 | 江苏省中国科学院植物研究所 | Method for building leaf in vitro regeneration system of begonia rex |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1541515A (en) * | 2003-11-07 | 2004-11-03 | 山东农业大学 | Three-stage amplifying method of inventing novel germplasm of drupe fruit tree utilizing distant hybridization |
| CN1875691A (en) * | 2006-07-21 | 2006-12-13 | 吕平 | A plant quick propagation method |
| CN101912761A (en) * | 2010-07-05 | 2010-12-15 | 洪昆喨 | Dielectric discharge reactor of uniform electric field |
| CN102696487A (en) * | 2012-07-05 | 2012-10-03 | 江苏省中国科学院植物研究所 | Method for building leaf in vitro regeneration system of begonia rex |
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