CN105993633A - Xinhui mandarin orange virus-free test tube micro-grafting method - Google Patents
Xinhui mandarin orange virus-free test tube micro-grafting method Download PDFInfo
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- CN105993633A CN105993633A CN201610579304.7A CN201610579304A CN105993633A CN 105993633 A CN105993633 A CN 105993633A CN 201610579304 A CN201610579304 A CN 201610579304A CN 105993633 A CN105993633 A CN 105993633A
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- Prior art keywords
- bright red
- culture medium
- newly
- chachiensis hort
- citrus chachiensis
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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
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
-
- 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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
-
- 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
Abstract
The invention relates to the technical field of Xinhui mandarin orange seedling culture propagation and particularly relates to a Xinhui mandarin orange virus-free test tube micro-grafting method. The micro-grafting method mainly comprises the following steps: fetching 1-2mm stem tip of a target plant, and pre-culturing with an improved MT culture medium; adding liquid nitrogen to remove virus; inoculating into a subculture medium for culture; disinfecting, sterilizing and germinating the rootstock seed; grafting the cluster buds with sound development of axillary buds of Xinhui mandarin orange to the rootstock; and performing liquid culture of seedlings in the test tube. In the culture method, the improved MT culture medium is adopted for pre-culture; after the improvement, the subculture propagation coefficient is greater than 4, the vitrification phenomenon rarely happens, and the variation is greatly reduced; and with the culture method, the root system of the plant is stronger, the resistance against adverse environment is strong, the plant adapts to the damp and rainy weather environment of the south more easily, and the survival rate is also greatly increased.
Description
Technical field
The present invention relates to Citrus seedling propagating technology field, be specifically related to a kind of newly bright red Citrus chachiensis Hort. detoxification test tube plantlet of meeting
Micro-grafting method.
Background technology
Xinhui tangerine peel be newly can the dry fruit skin of bright red Citrus chachiensis Hort., because its quality is unique, as far back as bright clear before the most rough sound
Far and near, and be listed in " tribute ", marketing is both at home and abroad.
Citrus chachiensis Hort. C. Suavissima Tanaka is the most long more good with the time of storage, therefore claims " Pericarpium Citri Reticulatae ".Pericarpium Citri Reticulatae is produced with Guangdong and is preferred.History
Special title " Pericarpium citri reticuatae chachiensis " in trade, not produced in other provinces.
Newly the peel of the bright red Citrus chachiensis Hort. of meeting is exactly the famous special product Pericarpium Citri Reticulatae of newly meeting.Owing to it has the highest medicinal valency
Value, is again traditional spice and seasoning good merchantable brand, so always enjoying high reputation.North and south has the most been become as far back as the Song dynasty
One of " Guangdong products " of trade, the on sale throughout the country and area such as Nan Yang, America.And " Pericarpium citri reticuatae chachiensis " is with new meeting
Pericarpium Citri Reticulatae is top grade, therefore the Chinese medicine " ERCHEN TANG " that the Qing Dynasty's big doctor YE Tian shi is opened, write " Pericarpium Citri tangerinae " especially exactly.
Its drug effect produced because of Bu Shixin club is the most inferior, and weary fragrance and numbness mouth (i.e. bitter pungent), so Xinhui tangerine peel
Price is higher, and skin is more expensive than meat.
On the basis of Citrus genetic transformation is built upon receptor system development, there is good tissue culture's regeneration
Low the making of the key that system succeeds often, the varietY specificity of receptor system and regeneration efficiency newly can be bright red
Citrus chachiensis Hort. genetic transformation is extremely limited.Mostly traditional Citrus grafting is to use heat treatment detoxification, then in field
Shoot-tip Grafting, cuts and exposes in atmosphere, and antibacterial or virus infect easily by cutting or hide, and become
Motility rate is relatively low, is easier to the problems referred to above occur in the moist rainy climatic environment in south.The bright red Citrus chachiensis Hort. of new meeting
Root system is undeveloped, is afraid of arid, is afraid of again flood, how to make the pericarp development newly bright red Citrus chachiensis Hort. more preferable, and carries
Its color and luster high and quality, have important Research Significance.
Summary of the invention
In order to solve above-mentioned technical problem, it is an object of the invention to provide that a kind of anti-adverse environment is strong, become
Motility rate is high, be beneficial to improve the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting of pericarp development and quality.
For realizing above-mentioned technical purpose, the present invention uses following technical scheme:
Newly the bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting, mainly comprises the steps:
Step one, detoxification newly can the acquisitions of bright red Citrus chachiensis Hort. Multiple Buds:
The bright red Citrus chachiensis Hort. mother's branch of newly meeting of sterilization, sterilization treatment is cut stem apex 1-2mm on superclean bench, turns
Move on to improve preculture in MT culture medium, after carrying out preculture 3-5 days under 4 DEG C of dark conditions, by stem apex
Tissue transfers to protect frost in culture medium to adapt to 1h, after be transferred into cryovial and put into rapidly liquid nitrogen
In, make stem-tip tissue fast cooling to-196 DEG C lasting 1h, be quickly transferred in 45 DEG C of water solution the most again
Freezing recovery, be then seeded in subculture medium, 25 DEG C of light culture carried out conventional illumination cultivation after one week, obtained
Obtaining detoxification newly can bright red Citrus chachiensis Hort. Multiple Buds；
Step 2, Root-stock selection:
Fructus Citri Limoniae or fragrant citrus seed are inoculated into 1/2MS culture medium at superclean bench after ethanol, mercuric chloride sterilization,
After seed germination, pick out seedling sturdy, that growth conditions is good, treat that growth of seedling to second true leaf is formed
After, treat micrografting as inoculation stock；
Step 3, micrografting:
Superclean bench is placed inoculating groove, in inoculating groove, then places the wafer of sterilizing and by Oryza glutinosa
Paper is fixed in inoculating groove, is blocked by the inoculation stock of step 2, be placed on glutinous at distance root 4-5cm
On rice paper；The detoxification again step one subculture multiplication obtained newly can bright red Citrus chachiensis Hort. Multiple Buds cambium layer alignment stock
And insert in stock otch, wafer doubling newly can be fixed after bright red Citrus chachiensis Hort. Multiple Buds by compacting stock and detoxification,
Obtain micrografting Seedling；
Step 4, cultivation are transplanted:
In test tube, inject the liquid medium of 1/4 volume, and annular puts into 2cm filter paper as fixing, warp
After sterilizing, the micrografting Seedling that step 3 is obtained, transfer in liquid medium, be placed in temperature 25 DEG C, light
Cultivate 30 days according under the isoperibol that intensity is 2500-3000lux, treat that stock root starts growth, grow thickly
After bud-leaf sheet normally unfolds growth, it is placed in seedling exercising in nature light and, after 30 days, transplantation of seedlings can be washed, transplant into
Motility rate is close to 100%.
Wherein, in step one, described improvement MT culture medium includes the component of following content: NH4NO3
1700-1800mg/L；KNO31800-2000mg/L；CaCl2·2H2O 8300-8500mg/L；MgSO4·7H2O
7300-7500mg/L；KH2PO43150-3200mg/L；KI 0.80-0.90mg/L；H3BO3 6.0-6.5
mg/L；MnSO4·4H2O 22.0-23.0mg/L；Na2MoO4·2H2O 0.2-0.3mg/L；CuSO4·5H2O
0.02-0.03mg/L；CoCl2·6H2O 0.02-0.03mg/L；FeSO4·7H2O 27.0-28.0mg/L；
Na2-EDTA 37.0-38.0mg/L；Nicotinic acid 4-6mg/L；Pyridoxine hydrochloride 19-21mg/L；Hydrochloric acid sulfur
Amine element 0.3-0.5mg/L；Glycine 1.5-2.5mg/L.
As preferably, in step one, described improvement MT culture medium includes the component of following content: NH4NO3
1750mg/L；KNO31900mg/L；CaCl2·2H2O 8400mg/L；MgSO4·7H2O 7400mg/L；
KH2PO43180mg/L；KI 0.83mg/L；H3BO36.3mg/L；MnSO4·4H2O 22.6mg/L；
Na2MoO4·2H2O 0.25mg/L；CuSO4·5H2O 0.025mg/L；CoCl2·6H2O 0.025mg/L；
FeSO4·7H2O 27.8mg/L；Na2-EDTA 37.3mg/L；Nicotinic acid 5mg/L；Pyridoxine hydrochloride 20mg/L；
Thiamine hydrochloride 0.4mg/L；Glycine 2mg/L.
As preferably, in step one, the formula of described protection culture medium is: improvement MT culture medium+30 (wt) %
Glycerol+15 (wt) % ethylene glycol+15 (wt) % dimethyl sulfoxide+sucrose 50g/L.
As preferably, in step one, the formula of described subculture medium is: improvement MT culture medium+BA
2mg/L+NAA 0.05mg/L+ agar 6.5g/L+ sucrose 50g/L.
As preferably, in step 4, the formula of described liquid medium is: improvement MT culture medium+IBA 0.5
mg/L。
The present invention has the following beneficial effect:
(1) this culture method will newly can bright red Citrus chachiensis Hort. mother's branch by improvement MT culture medium preculture, through liquid nitrogen take off
Poison, recovery of thawing, utilize Fructus Citri Limoniae/fragrant citrus as stock, substantially increase anti-adverse environment, improve
Result pericarp development in period and quality, it is to avoid " mostly traditional newly can bright red Citrus chachiensis Hort. grafting be to use heat treatment detoxification,
Then at field Shoot-tip Grafting, cut and expose in atmosphere, antibacterial or virus infect easily by cutting or
Person hides " problem；
(2) this culture method uses improvement MT culture medium preculture, and after improvement, subculture value-added coefficient is more than 4,
The most do not occur that vitrification phenomenon, variation also greatly reduce, and allow and newly understand the pericarp development of bright red Citrus chachiensis Hort. more
Good, color and luster and quality are more excellent；Solve in prior art " with general MS culture medium culturing, axillalry bud
Blade easily comes off, vitrification or variation；Although having by tradition MT culture medium culturing vitrification degree
Reduced, but and growth regulator arrange in pairs or groups after excessive growth easily occurs, mutation probability reduces inconspicuous " problem.
(3) the whole process of this operation is all carried out in gnotobasis, it is ensured that seedling grafting process is not by pathogen
Invasion, without potential danger.Make root system of plant flourishing by water planting, it is easier to adapt to south humidity many
The climatic environment of rain, survival rate is greatly improved.
Detailed description of the invention
In the following detailed description, by the way of explanation, only describe some exemplary enforcement of the present invention
Example.Describe the most illustrative rather than be used for limiting scope of the claims.
Embodiment 1
Newly the bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting, mainly comprises the steps:
Step one, detoxification newly can the acquisitions of bright red Citrus chachiensis Hort. Multiple Buds:
The bright red Citrus chachiensis Hort. mother's branch of newly meeting of sterilization, sterilization treatment is cut stem apex 2mm on superclean bench, transfer
Preculture in improvement MT culture medium, carries out preculture after 3 days, by stem-tip tissue under 4 DEG C of dark conditions
Transfer to protect frost in culture medium to adapt to 1h, after be transferred into cryovial and rapidly in input liquid nitrogen, make
Stem-tip tissue fast cooling to-196 DEG C persistently 1h, be quickly transferred in 45 DEG C of water defrosting the most again and recover,
Being then seeded in subculture medium, 25 DEG C of light culture carried out conventional illumination cultivation after one week, it is thus achieved that detoxification is new
Can bright red Citrus chachiensis Hort. Multiple Buds；The above-mentioned condition of culture of strict control, is beneficial to improve the anti-adverse environment of later stage Seedling
And result fruit-setting rate in period and quality；
Described improvement MT culture medium includes the component of following content: NH4NO31700mg/L；KNO3 1800
mg/L；CaCl2·2H2O 8300mg/L；MgSO4·7H2O 7300mg/L；KH2PO43150mg/L；
KI 0.80mg/L；H3BO36.0mg/L；MnSO4·4H2O 22.0mg/L；Na2MoO4·2H2O 0.2mg/L；
CuSO4·5H2O 0.02mg/L；CoCl2·6H2O 0.02mg/L；FeSO4·7H2O 27.0mg/L；Na2-EDTA
37.0mg/L；Nicotinic acid 4mg/L；Pyridoxine hydrochloride 19mg/L；Thiamine hydrochloride 0.3mg/L；Glycine
1.5mg/L；
The formula of described protection culture medium is: improvement MT culture medium+30 (wt) % glycerol+15 (wt) %
Ethylene glycol+15 (wt) % dimethyl sulfoxide+sucrose 50g/L；
The formula of described subculture medium is: improvement MT culture medium+BA 2mg/L+NAA 0.05mg/L+ agar
6.5g/L+ sucrose 50g/L；
Step 2, Root-stock selection:
Fructus Citri Limoniae or fragrant citrus seed are inoculated into 1/2MS culture medium at superclean bench after ethanol, mercuric chloride sterilization,
After seed germination, pick out seedling sturdy, that growth conditions is good, treat that growth of seedling to second true leaf is formed
After, treat micrografting as inoculation stock；
Step 3, micrografting:
Superclean bench is placed inoculating groove, in inoculating groove, then places the wafer of sterilizing and by Oryza glutinosa
Paper is fixed in inoculating groove, is blocked by the inoculation stock of step 2, be placed on glutinous at distance root 4-5cm
On rice paper；The detoxification again step one subculture multiplication obtained newly can bright red Citrus chachiensis Hort. Multiple Buds cambium layer alignment stock
And insert in stock otch, wafer doubling newly can be fixed after bright red Citrus chachiensis Hort. Multiple Buds by compacting stock and detoxification,
Obtain micrografting Seedling；
Step 4, cultivation are transplanted:
In test tube, inject the liquid medium of 1/4 volume, and annular puts into 2cm filter paper as fixing, warp
After sterilizing, the micrografting Seedling that step 3 is obtained, transfer in liquid medium, be placed in temperature 25 DEG C, light
Cultivate 30 days according under the isoperibol that intensity is 2500-3000lux, treat that stock root starts growth, grow thickly
After bud-leaf sheet normally unfolds growth, it is placed in seedling exercising in nature light and, after 30 days, transplantation of seedlings can be washed, transplant into
Motility rate 99%.
Wherein, the formula of described liquid medium is: improvement MT culture medium+IBA 0.5mg/L.
Embodiment 2
Newly the bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting, mainly comprises the steps:
Step one, detoxification newly can the acquisitions of bright red Citrus chachiensis Hort. Multiple Buds:
The bright red Citrus chachiensis Hort. mother's branch of newly meeting of sterilization, sterilization treatment is cut stem apex 1mm on superclean bench, transfer
Preculture in improvement MT culture medium, carries out preculture after 5 days, by stem-tip tissue under 4 DEG C of dark conditions
Transfer to protect frost in culture medium to adapt to 1h, after be transferred into cryovial and rapidly in input liquid nitrogen, make
Stem-tip tissue fast cooling to-196 DEG C persistently 1h, be quickly transferred in 45 DEG C of water defrosting the most again and recover,
Being then seeded in subculture medium, 25 DEG C of light culture carried out conventional illumination cultivation after one week, it is thus achieved that detoxification is new
Can bright red Citrus chachiensis Hort. Multiple Buds；
Described improvement MT culture medium includes the component of following content: NH4NO31800mg/L；KNO3 2000
mg/L；CaCl2·2H2O 8500mg/L；MgSO4·7H2O 7500mg/L；KH2PO43200mg/L；
KI 0.90mg/L；H3BO36.5mg/L；MnSO4·4H2O 23.0mg/L；Na2MoO4·2H2O 0.3mg/L；
CuSO4·5H2O 0.03mg/L；CoCl2·6H2O 0.03mg/L；FeSO4·7H2O 28.0mg/L；Na2-EDTA
38.0mg/L；Nicotinic acid 6mg/L；Pyridoxine hydrochloride 21mg/L；Thiamine hydrochloride 0.5mg/L；Sweet ammonia
Acid 2.5mg/L；
The formula of described protection culture medium is: improvement MT culture medium+30 (wt) % glycerol+15 (wt) %
Ethylene glycol+15 (wt) % dimethyl sulfoxide+sucrose 50g/L；
The formula of described subculture medium is: improvement MT culture medium+BA 2mg/L+NAA 0.05mg/L+ agar
6.5g/L+ sucrose 50g/L；
Step 2, Root-stock selection:
Fructus Citri Limoniae or fragrant citrus seed are inoculated into 1/2MS culture medium at superclean bench after ethanol, mercuric chloride sterilization,
After seed germination, pick out seedling sturdy, that growth conditions is good, treat that growth of seedling to second true leaf is formed
After, treat micrografting as inoculation stock；
Step 3, micrografting:
Superclean bench is placed inoculating groove, in inoculating groove, then places the wafer of sterilizing and by Oryza glutinosa
Paper is fixed in inoculating groove, is blocked by the inoculation stock of step 2, be placed on glutinous at distance root 4-5cm
On rice paper；The detoxification again step one subculture multiplication obtained newly can bright red Citrus chachiensis Hort. Multiple Buds cambium layer alignment stock
And insert in stock otch, wafer doubling newly can be fixed after bright red Citrus chachiensis Hort. Multiple Buds by compacting stock and detoxification,
Obtain micrografting Seedling；
Step 4, cultivation are transplanted:
In test tube, inject the liquid medium of 1/4 volume, and annular puts into 2cm filter paper as fixing, warp
After sterilizing, the micrografting Seedling that step 3 is obtained, transfer in liquid medium, be placed in temperature 25 DEG C, light
Cultivate 30 days according under the isoperibol that intensity is 2500-3000lux, treat that stock root starts growth, grow thickly
After bud-leaf sheet normally unfolds growth, it is placed in seedling exercising in nature light and, after 30 days, transplantation of seedlings can be washed, transplant into
Motility rate 98%.
Wherein, the formula of described liquid medium is: improvement MT culture medium+IBA 0.5mg/L.
Embodiment 3
Newly the bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting, mainly comprises the steps:
Step one, detoxification newly can the acquisitions of bright red Citrus chachiensis Hort. Multiple Buds:
The bright red Citrus chachiensis Hort. mother's branch of newly meeting of sterilization, sterilization treatment is cut stem apex 2mm on superclean bench, transfer
Preculture in improvement MT culture medium, carries out preculture after 5 days, by stem-tip tissue under 4 DEG C of dark conditions
Transfer to protect frost in culture medium to adapt to 1h, after be transferred into cryovial and rapidly in input liquid nitrogen, make
Stem-tip tissue fast cooling to-196 DEG C persistently 1h, be quickly transferred in 45 DEG C of water defrosting the most again and recover,
Being then seeded in subculture medium, 25 DEG C of light culture carried out conventional illumination cultivation after one week, it is thus achieved that detoxification is new
Can bright red Citrus chachiensis Hort. Multiple Buds；
Described improvement MT culture medium includes the component of following content: NH4NO31750mg/L；KNO3 1900
mg/L；CaCl2·2H2O 8400mg/L；MgSO4·7H2O 7400mg/L；KH2PO43180mg/L；
KI 0.83mg/L；H3BO36.3mg/L；MnSO4·4H2O 22.6mg/L；Na2MoO4·2H2O 0.25mg/L；
CuSO4·5H2O 0.025mg/L；CoCl2·6H2O 0.025mg/L；FeSO4·7H2O 27.8mg/L；
Na2-EDTA 37.3mg/L；Nicotinic acid 5mg/L；Pyridoxine hydrochloride 20mg/L；Thiamine hydrochloride 0.4mg/L；
Glycine 2mg/L；
The formula of described protection culture medium is: improvement MT culture medium+30 (wt) % glycerol+15 (wt) %
Ethylene glycol+15 (wt) % dimethyl sulfoxide+sucrose 50g/L；
The formula of described subculture medium is: improvement MT culture medium+BA 2mg/L+NAA 0.05mg/L+ agar
6.5g/L+ sucrose 50g/L；
Step 2, Root-stock selection:
Fructus Citri Limoniae or fragrant citrus seed are inoculated into 1/2MS culture medium at superclean bench after ethanol, mercuric chloride sterilization,
After seed germination, pick out seedling sturdy, that growth conditions is good, treat that growth of seedling to second true leaf is formed
After, treat micrografting as inoculation stock；
Step 3, micrografting:
Superclean bench is placed inoculating groove, in inoculating groove, then places the wafer of sterilizing and by Oryza glutinosa
Paper is fixed in inoculating groove, is blocked by the inoculation stock of step 2, be placed on glutinous at distance root 4-5cm
On rice paper；The detoxification again step one subculture multiplication obtained newly can bright red Citrus chachiensis Hort. Multiple Buds cambium layer alignment stock
And insert in stock otch, wafer doubling newly can be fixed after bright red Citrus chachiensis Hort. Multiple Buds by compacting stock and detoxification,
Obtain micrografting Seedling；
Step 4, cultivation are transplanted:
In test tube, inject the liquid medium of 1/4 volume, and annular puts into 2cm filter paper as fixing, warp
After sterilizing, the micrografting Seedling that step 3 is obtained, transfer in liquid medium, be placed in temperature 25 DEG C, light
Cultivate 30 days according under the isoperibol that intensity is 2500-3000lux, treat that stock root starts growth, grow thickly
After bud-leaf sheet normally unfolds growth, it is placed in seedling exercising in nature light and, after 30 days, transplantation of seedlings can be washed, transplant into
Motility rate 98%.
Wherein, the formula of described liquid medium is: improvement MT culture medium+IBA 0.5mg/L.
It is demonstrated experimentally that this culture method uses improvement MT culture medium preculture, subculture value-added coefficient after improvement
More than 4, the most do not occur that vitrification phenomenon, variation also greatly reduce；This culture method makes newly can be bright red
The pericarp development of Citrus chachiensis Hort. is more preferable, and color and luster and quality are more excellent, it is easier to adapt to the moist rainy climatic environment in south,
Survival rate is also greatly improved.
The foregoing is only the schematic detailed description of the invention of the present invention, be not limited to the model of the present invention
Enclose.Any those skilled in the art, done on the premise of without departing from the design of the present invention and principle
Equivalent variations and amendment, all should belong to the scope of protection of the invention.
Claims (6)
1. the bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of new meeting, it is characterised in that mainly comprise the steps:
Step one, detoxification newly can the acquisitions of bright red Citrus chachiensis Hort. Multiple Buds:
The bright red Citrus chachiensis Hort. mother's branch of newly meeting of sterilization, sterilization treatment is cut stem apex 1-2mm on superclean bench, transfer
Preculture in improvement MT culture medium, after carrying out preculture 3-5 days under 4 DEG C of dark conditions, by stem apex group
Knit and transfer to protect frost in culture medium to adapt to 1h, after be transferred into cryovial and rapidly in input liquid nitrogen,
Make stem-tip tissue fast cooling to-196 DEG C lasting 1h, being quickly transferred in 45 DEG C of water defrosting the most again and recovering,
Being then seeded in subculture medium, 25 DEG C of light culture carried out conventional illumination cultivation after one week, it is thus achieved that detoxification is new
Can bright red Citrus chachiensis Hort. Multiple Buds；
Step 2, Root-stock selection:
Fructus Citri Limoniae or fragrant citrus seed are inoculated into 1/2MS culture medium at superclean bench after ethanol, mercuric chloride sterilization,
After seed germination, pick out seedling sturdy, that growth conditions is good, treat that growth of seedling to second true leaf is formed
After, treat micrografting as inoculation stock；
Step 3, micrografting:
Superclean bench is placed inoculating groove, in inoculating groove, then places the wafer of sterilizing and by Oryza glutinosa
Paper is fixed in inoculating groove, is blocked by the inoculation stock of step 2, be placed on glutinous at distance root 4-5cm
On rice paper；The detoxification again step one subculture multiplication obtained newly can bright red Citrus chachiensis Hort. Multiple Buds cambium layer alignment stock
And insert in stock otch, wafer doubling newly can be fixed after bright red Citrus chachiensis Hort. Multiple Buds by compacting stock and detoxification,
Obtain micrografting Seedling；
Step 4, cultivation are transplanted:
In test tube, inject the liquid medium of 1/4 volume, and annular puts into 2cm filter paper as fixing, warp
After sterilizing, the micrografting Seedling that step 3 is obtained, transfer in liquid medium, be placed in temperature 25 DEG C, light
Cultivate 30 days according under the isoperibol that intensity is 2500-3000lux, treat that stock root starts growth, grow thickly
After bud-leaf sheet normally unfolds growth, it is placed in seedling exercising in nature light and, after 30 days, transplantation of seedlings can be washed.
2. the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting as claimed in claim 1, it is characterised in that:
In step one, described improvement MT culture medium includes the component of following content: NH4NO3
1700-1800mg/L；KNO31800-2000mg/L；CaCl2·2H2O 8300-8500mg/L；MgSO4·7H2O
7300-7500mg/L；KH2PO43150-3200mg/L；KI 0.80-0.90mg/L；H3BO3 6.0-6.5
mg/L；MnSO4·4H2O 22.0-23.0mg/L；Na2MoO4·2H2O 0.2-0.3mg/L；CuSO4·5H2O
0.02-0.03mg/L；CoCl2·6H2O 0.02-0.03mg/L；FeSO4·7H2O 27.0-28.0mg/L；
Na2-EDTA 37.0-38.0mg/L；Nicotinic acid 4-6mg/L；Pyridoxine hydrochloride 19-21mg/L；Thiamine hydrochloride
Element 0.3-0.5mg/L；Glycine 1.5-2.5mg/L.
3. the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting as claimed in claim 2, it is characterised in that
In step one, described improvement MT culture medium includes the component of following content: NH4NO31750mg/L；
KNO31900mg/L；CaCl2·2H2O 8400mg/L；MgSO4·7H2O 7400mg/L；KH2PO4 3180
mg/L；KI 0.83mg/L；H3BO36.3mg/L；MnSO4·4H2O 22.6mg/L；Na2MoO4·2H2O 0.25
mg/L；CuSO4·5H2O 0.025mg/L；CoCl2·6H2O 0.025mg/L；FeSO4·7H2O 27.8mg/L；
Na2-EDTA 37.3mg/L；Nicotinic acid 5mg/L；Pyridoxine hydrochloride 20mg/L；Thiamine hydrochloride 0.4mg/L；
Glycine 2mg/L.
4. the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting as claimed in claim 3, it is characterised in that
In step one, the formula of described protection culture medium is: improvement MT culture medium+30 (wt) % glycerol+15
(wt) % ethylene glycol+15 (wt) % dimethyl sulfoxide+sucrose 50g/L.
5. the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting as claimed in claim 3, it is characterised in that
In step one, the formula of described subculture medium is: improvement MT culture medium+BA 2mg/L+NAA
0.05mg/L+ agar 6.5g/L+ sucrose 50g/L.
6. the newly bright red Citrus chachiensis Hort. detoxification test tube plantlet micro-grafting method of meeting as claimed in claim 3, it is characterised in that
In step 4, the formula of described liquid medium is: improvement MT culture medium+IBA 0.5mg/L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107278629A (en) * | 2017-07-13 | 2017-10-24 | 邓国祥 | A kind of grafting implantation methods of Xinhui citrus reticulata |
| CN107750695A (en) * | 2017-11-23 | 2018-03-06 | 荔浦县万家兴果蔬专业合作社 | A kind of engrafting method of Ponkan |
| CN108739373A (en) * | 2018-04-23 | 2018-11-06 | 湖南科技学院 | A kind of method of fragrant shaddock stem apex numerous detoxification soon |
| CN108811835A (en) * | 2018-07-27 | 2018-11-16 | 中国科学院华南植物园 | A kind of rapid propagation method of citrus detoxification and gemmule grafting |
| CN108848994A (en) * | 2018-04-18 | 2018-11-23 | 宁夏林业研究院股份有限公司 | A kind of method of suitable grape micrografting seedling Liquid Culture |
| CN114793902A (en) * | 2022-05-11 | 2022-07-29 | 浙江省柑橘研究所 | Citrus hybrid seed grafted seedling cultivation method |
| CN115005103A (en) * | 2022-07-15 | 2022-09-06 | 湖北省农业科学院果树茶叶研究所 | Culture medium capable of inducing axillary buds of citrus tissues and method for propagating citrus seedlings |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418350A (en) * | 2008-10-28 | 2009-04-29 | 南京农业大学 | Method for removing strawberry light yellow edge virus by ultra low temperature technique |
| EP2288253A1 (en) * | 2008-06-13 | 2011-03-02 | Syngenta Participations AG | New method for the production of somatic embryos from euphorbia pulcherrima plant tissue |
| CN103141381A (en) * | 2013-02-24 | 2013-06-12 | 周世永 | Tissue culture method for oriental lily |
| CN103262793A (en) * | 2013-04-06 | 2013-08-28 | 上饶师范学院 | Method of removing virus for Jiangxi Qianshan red-bud taro through vitrification cryopreservation |
| CN103314863A (en) * | 2013-07-16 | 2013-09-25 | 李锋 | Efficient method for obtaining tulip callus |
-
2016
- 2016-07-21 CN CN201610579304.7A patent/CN105993633A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2288253A1 (en) * | 2008-06-13 | 2011-03-02 | Syngenta Participations AG | New method for the production of somatic embryos from euphorbia pulcherrima plant tissue |
| CN101418350A (en) * | 2008-10-28 | 2009-04-29 | 南京农业大学 | Method for removing strawberry light yellow edge virus by ultra low temperature technique |
| CN103141381A (en) * | 2013-02-24 | 2013-06-12 | 周世永 | Tissue culture method for oriental lily |
| CN103262793A (en) * | 2013-04-06 | 2013-08-28 | 上饶师范学院 | Method of removing virus for Jiangxi Qianshan red-bud taro through vitrification cryopreservation |
| CN103314863A (en) * | 2013-07-16 | 2013-09-25 | 李锋 | Efficient method for obtaining tulip callus |
Non-Patent Citations (3)
| Title |
|---|
| 丁明华等: "成年态南丰蜜橘试管嫁接育苗技术研究", 《热带亚热带植物学报》 * |
| 张胜友: "《工厂化栽培金针菇新技术》", 28 February 2010, 华中科技大学出版社 * |
| 王国平等: "《柑橘黄龙病及其防治》", 31 July 2007, 金盾出版社 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107278629A (en) * | 2017-07-13 | 2017-10-24 | 邓国祥 | A kind of grafting implantation methods of Xinhui citrus reticulata |
| CN107750695A (en) * | 2017-11-23 | 2018-03-06 | 荔浦县万家兴果蔬专业合作社 | A kind of engrafting method of Ponkan |
| CN108848994A (en) * | 2018-04-18 | 2018-11-23 | 宁夏林业研究院股份有限公司 | A kind of method of suitable grape micrografting seedling Liquid Culture |
| CN108739373A (en) * | 2018-04-23 | 2018-11-06 | 湖南科技学院 | A kind of method of fragrant shaddock stem apex numerous detoxification soon |
| CN108739373B (en) * | 2018-04-23 | 2022-03-04 | 湖南科技学院 | Method for rapid propagation and detoxification of grapefruit stem tips |
| CN108811835A (en) * | 2018-07-27 | 2018-11-16 | 中国科学院华南植物园 | A kind of rapid propagation method of citrus detoxification and gemmule grafting |
| CN114793902A (en) * | 2022-05-11 | 2022-07-29 | 浙江省柑橘研究所 | Citrus hybrid seed grafted seedling cultivation method |
| CN115005103A (en) * | 2022-07-15 | 2022-09-06 | 湖北省农业科学院果树茶叶研究所 | Culture medium capable of inducing axillary buds of citrus tissues and method for propagating citrus seedlings |
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