CN106718926A - A kind of leaf of plum Chinese ilex quick breeding method for tissue culture - Google Patents
A kind of leaf of plum Chinese ilex quick breeding method for tissue culture 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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of leaf of plum Chinese ilex quick breeding method for tissue culture.The present invention realizes wild leaf of plum Chinese ilex tissue-culturing quick-propagation, is that wild leaf of plum Chinese ilex tissue culture technique opens up new road.The method of the present invention is simple to operate, it is easy to implement, and can disposably cultivate substantial amounts of leaf of plum Chinese ilex growth seedling.The present invention can effectively shorten seedling raise period, and culture medium and reagent used by the present invention is easily configured, and low cost is easily promoted on a large scale.
Description
Technical field：
The invention belongs to field of plant reproduction, and in particular to a kind of leaf of plum Chinese ilex quick breeding method for tissue culture.
Background technology：
Leaf of plum Chinese ilex (Ilex asprella Champ.ex Benth) belongs to Aquifoliaceae Ilex, machaka, plant height
Up to 3 meters.It is distributed mainly on the ground such as China Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Guangdong, Guangxi, Hong Kong；In Philippine group
Island is also distributed.It is born in height above sea level 400-1000 meters of mountain region sparse woods or in the shrubbery of roadside more.Its root, stem, leaf can be used as medicine, taste
It is bitter, sweet, cool in nature, it is clearing heat and detoxicating, promote the production of body fluid to quench thirst.Be the Lingnan area conventional Chinese medicine such as Guangdong, Guangxi, for catching a cold, hyperpyrexia polydipsia,
Tonsillitis, sphagitis, tracheitis, pertussis etc..Leaf of plum Chinese ilex root is the primary raw material of WANGLAOJI LIANGCHA, husky small stream herbal tea etc.,
The among the people also extensive use leaf of plum Chinese ilex self-control herbal tea in the south of the Five Ridges.Blade according to research leaf of plum Chinese ilex contains ursolic acid, with calmness, disappears
The effects such as inflammation, anti-diabetic, coronary heart disease and angina pectoris.As modern medicine grinds to leaf of plum Chinese ilex active component and pharmaceutical component
Study carefully deeply, its clinical practice expands year by year, and leaf of plum Chinese ilex has wide market prospects and economic benefit.
At present, leaf of plum Ilex material is mainly derived from wild resource, and with the expanded demand in market, leaf of plum Chinese ilex provides naturally
Source increasingly depleted, it is impossible to meet the current market demand.Artificial cultivation is later important development direction.Common leaf of plum Chinese ilex
Reproduction technique is mainly seed generative propagation and branch cutting breeding.Leaf of plum Chinese ilex seed has deep-sleep, next year germinates
Characteristic, its germination breeding is significantly affected by season.In addition, leaf of plum Chinese ilex seed is small, its time-consuming effort of collection, and be difficult
Storage.Leaf of plum Chinese ilex cutting propagation, need to gather a large amount of lignifying branches raw then, there is the growth of leaf of plum Chinese ilex compared with havoc,
And in large-scale plantation, it is vulnerable to the limitation of quantity of material.
The content of the invention：
Do not influenceed by time and raw material quantity it is an object of the invention to provide a kind of nursery breeding, breeding coefficient is high,
The cultivation time is short, to cultivate the leaf of plum Chinese ilex tissue-culturing quick-propagation side that leaf of plum Chinese ilex seedling provides technical guarantee on a large scale
Method.
Leaf of plum Chinese ilex quick breeding method for tissue culture of the invention, it is characterised in that comprise the following steps：
A, the leaf of plum Chinese ilex of the healthy no disease and pests harm 8-12cm of selection newly take out band bud branch then, remove the leaf of branch, use wine
Smart cotton swab is removed the dust on branch surface and is cut into 3-5 sections, and every section carries the 1-2 branch section of resting bud point as explant；
B, explant is sterilized, culture induces axillary bud during the explant insertion primary after sterilizing then is lured into bud culture medium
Or terminal bud, condition of culture is：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, culture
20-30 days, into axillary-bud or top-bud, germination rate can reach more than 80%, and described primary lures bud to train for the resting bud hair tonic of explant
Every liter of base is supported to contain：1mg ZT (zeatin), 0.05mg NAA (methyl α-naphthyl acetate), 6.2g agar, 0.1g inositols, 30g sucrose, surplus
It is MS culture mediums, pH 5.8-6.0；
C, axillary-bud or top-bud is cut, to access and carry out Multiplying culture in adventitious bud proliferation culture medium and obtain adventitious bud, culture
Condition is：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, described adventitious bud proliferation
Every liter of culture medium contains：1mg 6-BA (6- benzyl amino bases purine), 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sucrose,
Balance of MS culture mediums, pH 5.8-6.0；The Multiplying culture cycle is 35-45 days, and growth coefficient can reach 5；
D, adventitious bud access strong seedling culture base is carried out strong seedling culture and obtains unrooted strong sprout, condition of culture is：1500-
2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, every liter described of strong seedling culture base contains：
0.1mg 6-BA, 0.1mg NAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS culture mediums, pH 5.8-6.0；
E, the 2-3cm unrooted strong sprouts with 3-5 blade high are accessed into rooting induction culture medium in culture obtain tissue-cultured seedling,
Condition of culture is：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, described taking root is lured
Every liter of culture medium is led to contain：1mg IBA (heteroauxin), 0.02g AC (activated carbon), 6.2g agar, 0.1g inositols, 20g sugarcanes
Sugar, balance of 1/2MS culture mediums, pH 5.8-6.0；Culture 30-45 days, rooting rate can reach more than 85%；
F, tissue-cultured seedling is carried out into transplanting culture；
Described step b's is preferably on aseptic operating platform explant sterilizing, and explant is placed in into volume fraction
30s is soaked in 75% alcoholic solution；Move on on aseptic working platform, the explant of alcohol disinfecting is put into conical flask, add matter
The mercuric chloride aqueous solution of fraction 0.1% is measured, 1 is added dropwise and is dripped tween, soak 10-15min, centre is rocked for several times, finally rushed with sterilized water
Wash 3 times, obtain the explant of sterilization treatment.
Described transplanting culture is to treat tissue culture seedling rooting 5-10 bars, during root 1-2cm long, by tissue-cultured seedling move on to temperature humidity with
In extraneous consistent culturing room, bottle cap is opened after 4 days, the tissue-cultured seedling for bearing root is kept contact higher with the external world, after 7 days
Added water taking-up rooted seedling, cleans culture medium, and 1-3min is soaked with the potassium permanganate solution of mass fraction 0.1%, moves into perlite：
Vermiculite volume ratio=3:In 2 matrix.Transplanting survival rate can reach more than 95%.
MS culture mediums in the present invention refer to the fluid nutrient medium for not adding agar, and its formula belongs to the known normal of this area
Know, described 1/2MS culture mediums are that a great number of elements halves in MS culture mediums, and other constant culture mediums.
Compared with prior art, it is of the invention with advantages below：
The present invention realizes wild leaf of plum Chinese ilex tissue-culturing quick-propagation, is that wild leaf of plum Chinese ilex tissue culture technique is opened
Ward off new road.The method of the present invention is simple to operate, it is easy to implement, and can disposably cultivate substantial amounts of leaf of plum Chinese ilex growth seedling.
The present invention can effectively shorten seedling raise period, and culture medium and reagent used by the present invention is easily configured, low cost, easily on a large scale
Promote.
Specific embodiment：
Following examples are further illustrated to of the invention, rather than limitation of the present invention.
Embodiment 1：
The wild leaf of plum Chinese ilex of a, the healthy no disease and pests harm 8-12cm of selection or so newly takes out band bud branch；Remove the miscellaneous of branch
Leaf, wipes the dust on branch surface away and is cut into 3-5 sections using the alcohol swab of volume fraction 75%, every section of 3cm or so, with 1-2
Individual resting bud point, using this stem section as explant；
B, explant sterilizing：On aseptic operating platform, the stem section (explant) that above-mentioned preliminarily pasteurized is obtained is placed in volume
30s is soaked in the alcoholic solution of fraction 75%；Move on on aseptic working platform, the stem section of alcohol disinfecting is put into conical flask, add
The mercuric chloride aqueous solution of mass fraction 0.1%, is added dropwise 1 and drips tween, soaks 10-15min, and centre is rocked for several times, finally uses sterilized water
Rinse 3 times, obtain the explant of sterilization treatment；
C, explant Fiber differentiation：Cultivated during the explant insertion primary of sterilized treatment is lured into bud culture medium, cultivate bar
Part is：1500Lx, 24 DEG C -28 DEG C, light application time 14h, interlunation 10h are cultivated 20-30 days, the resting bud hair tonic of explant
Into axillary-bud or top-bud, germination rate can reach more than 80%.Described primary adventitious bud induction culture base is：MS+1mg/L ZT+
The sucrose of 0.05mg/L NAA+6.2g/L agar powder+0.1g/L inositols+3%, pH 5.8-6.0, i.e., described primary lures bud culture
Every liter of base contains：1mg ZT, 0.05mg NAA, 6.2g agar, 0.1g inositols, 30g sucrose, balance of MS culture mediums, pH 5.8-
6.0, its every liter compound method is that 1mg ZT, 0.05mg NAA, 6.2g agar, 0.1g inositols, 30g sucrose are added into a small amount of liquid
In body MS culture mediums, pH value to 5.8-6.0 then is adjusted with liquid MS medium constant volume to 1L again, sterilized standby.
D, adventitious bud proliferation culture：The axillary-bud or top-bud of above-mentioned resulting a length of 1cm or so is cut, adventitious bud is accessed
Cultivated in proliferated culture medium and obtain adventitious bud, condition of culture is：1500Lx, 24 DEG C -28 DEG C, light application time 14h, interlunation
10h, culture is a cycle in 35-45 days, and growth coefficient can reach 5.Described adventitious bud proliferation Media Components are：MS+
The sucrose of 1mg/L6-BA+0.05mg/LNAA+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0, i.e., described is indefinite
Every liter of bud proliferated culture medium contains：1mg 6-BA, 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS trainings
Foster base, pH 5.8-6.0, its every liter compound method is by 1mg 6-BA, 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sugarcanes
Sugar is added in a small amount of liquid MS medium, then with liquid MS medium constant volume to 1L, adjusts pH value to 5.8-6.0, is sterilized standby
With.
E, strong seedling culture：Culture during the adventitious bud for obtaining is put into strong seedling culture base will be bred and obtains within 30-45 days unrooted strong sprout,
Condition of culture is：1500Lx, 24 DEG C -28 DEG C, light application time 14h, interlunation 10h.Described strong seedling culture base composition is：MS
The sucrose of+0.1mg/L6-BA+0.1mg/LNAA+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0, i.e., described is strong
Every liter of seedling culture medium contains：0.1mg 6-BA, 0.1mg NAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS cultures
Base, pH 5.8-6.0, its every liter compound method is by 0.1mg 6-BA, 0.1mg NAA, 6.2g agar, 0.1g inositols, 20g sugarcanes
Sugar is added in a small amount of liquid MS medium, then with liquid MS medium constant volume to 1L, adjusts pH value to 5.8-6.0, is sterilized standby
With.
F, differentiation culture of rootage：Above-mentioned resulting 2-3cm is high with the 3-5 unrooted strong sprout access rooting induction of blade
Culture obtains tissue-cultured seedling for 30-45 days in culture medium, and rooting rate can reach more than 85%.Condition of culture is：1500Lx, 24 DEG C -28
DEG C, light application time 14h, interlunation 10h.Described rooting induction Media Components are：1/2MS+1mg/L IBA+0.02g/L
The sucrose of AC+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0, i.e., every liter described of rooting induction culture medium contains：
1mg IBA, 0.02gAC, 6.2g agar, 0.1g inositols, 20g sucrose, balance of 1/2MS culture mediums, pH 5.8-6.0, its every liter
Compound method is that 1mg IBA, 0.02gAC, 6.2g agar, 0.1g inositols, 20g sucrose are added in liquid 1/2MS culture mediums,
Then with liquid MS medium constant volume to 1L, pH value to 5.8-6.0 is adjusted, is sterilized standby.
G, transplanting culture：Treat tissue culture seedling rooting 5-10 bars, during root 1-2cm long, by tissue-cultured seedling move on to temperature humidity with it is extraneous
In consistent culturing room, bottle cap is opened after 4 days, make the tissue-cultured seedling contact higher with external world's holding of taking root, add water taking-up after 7 days
Rooted seedling, cleans culture medium；1-3min is soaked with the potassium permanganate solution of mass fraction 0.1%, perlite is moved into：Vermiculite volume
Than being 3:Cultivated in 2 matrix, be derived from leaf of plum Chinese ilex seedling.
Embodiment 2：
The wild leaf of plum Chinese ilex of a, the healthy no disease and pests harm 8-12cm of selection or so newly takes out band bud branch；Remove the miscellaneous of branch
Leaf, wipes the dust on branch surface away and is cut into 3-5 sections using the alcohol swab of volume fraction 75%, every section of 3cm or so, with 1-2
Individual resting bud point, the branch processed using this is used as explant；
B, explant sterilizing：On aseptic operating platform, the stem section (explant) that above-mentioned preliminarily pasteurized is obtained is placed in volume
30s is soaked in the alcoholic solution of fraction 75%；Move on on aseptic working platform, the stem section of alcohol disinfecting is put into conical flask, add
The mercuric chloride aqueous solution of mass fraction 0.1%, is added dropwise 1 and drips tween, soaks 10-15min, and centre is rocked for several times, finally uses sterilized water
Rinse 3 times, obtain the explant of sterilization treatment；
C, explant Fiber differentiation：Cultivated during the explant insertion primary of sterilized treatment is lured into bud culture medium, cultivate bar
Part is：2000Lx, 24 DEG C -28 DEG C, light application time 12hh, interlunation 12h are cultivated 20-30 days, the resting bud hair tonic of explant
Into axillary-bud or top-bud, germination rate can reach more than 80%.Described primary adventitious bud induction culture base is：MS+1mg/L ZT+
The sucrose of 0.05mg/L NAA+6.2g/L agar powder+0.1g/L inositols+3%, pH 5.8-6.0, i.e., described primary lures bud culture
Every liter of base contains：1mg ZT, 0.05mg NAA, 6.2g agar, 0.1g inositols, 30g sucrose, balance of MS culture mediums, pH 5.8-
6.0, its every liter compound method is that 1mg ZT, 0.05mg NAA, 6.2g agar, 0.1g inositols, 30g sucrose are added into a small amount of liquid
In body MS culture mediums, pH value to 5.8-6.0 then is adjusted with liquid MS medium constant volume to 1L again, sterilized standby.
D, adventitious bud proliferation culture：The axillary-bud or top-bud of above-mentioned resulting a length of 1cm or so is cut, adventitious bud is accessed
Cultivated in proliferated culture medium and obtain adventitious bud, condition of culture is：2000Lx, 24 DEG C -28 DEG C, light application time 12hh, interlunation
12h, culture is a cycle in 35-45 days, and growth coefficient can reach 5.Described adventitious bud proliferation Media Components are：MS+
The sucrose of 1mg/L6-BA+0.05mg/LNAA+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0, i.e., described is indefinite
Every liter of bud proliferated culture medium contains：1mg 6-BA, 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS trainings
Foster base, pH 5.8-6.0, its every liter compound method is by 1mg 6-BA, 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sugarcanes
Sugar is added in a small amount of liquid MS medium, then with liquid MS medium constant volume to 1L, adjusts pH value to 5.8-6.0, is sterilized standby
With.
E, strong seedling culture：Culture during the adventitious bud for obtaining is put into strong seedling culture base will be bred and obtains within 30-45 days unrooted strong sprout,
Condition of culture is：2000Lx, 24 DEG C -28 DEG C, light application time 12hh, interlunation 12h.Described strong seedling culture base composition is：
The sucrose of MS+0.1mg/L6-BA+0.1mg/LNAA+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0 are that is, described
Every liter of strong seedling culture base contains：0.1mg 6-BA, 0.1mg NAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS trainings
Foster base, pH 5.8-6.0, its every liter compound method is by 0.1mg 6-BA, 0.1mg NAA, 6.2g agar, 0.1g inositols, 20g
Sucrose is added in a small amount of liquid MS medium, then with liquid MS medium constant volume to 1L, adjusts pH value to 5.8-6.0, sterilizing
It is standby.
F, differentiation culture of rootage：Above-mentioned resulting 2-3cm is high with the 3-5 unrooted strong sprout access rooting induction of blade
Culture obtains tissue-cultured seedling for 30-45 days in culture medium, and rooting rate can reach more than 85%.Condition of culture is：2000Lx, 24 DEG C -28
DEG C, light application time 12hh, interlunation 12h.Described rooting induction Media Components are：1/2MS+1mg/L IBA+0.02g/
The sucrose of L AC+6.2g/L agar powder+0.1g/L inositols+2%, pH 5.8-6.0, i.e., every liter described of rooting induction culture medium contains
Have：1mg IBA, 0.02gAC, 6.2g agar, 0.1g inositols, 20g sucrose, balance of 1/2MS culture mediums, pH 5.8-6.0, its
Every liter of compound method is that 1mg IBA, 0.02gAC, 6.2g agar, 0.1g inositols, 20g sucrose are added into liquid 1/2MS cultures
In base, then with liquid MS medium constant volume to 1L, pH value to 5.8-6.0 is adjusted, sterilized standby.
G, transplanting culture：Treat tissue culture seedling rooting 5-10 bars, during root 1-2cm long, by tissue-cultured seedling move on to temperature humidity with it is extraneous
In consistent culturing room, bottle cap is opened after 4 days, make the tissue-cultured seedling contact higher with external world's holding of taking root, add water taking-up after 7 days
Rooted seedling, cleans culture medium；1-3min is soaked with the potassium permanganate solution of mass fraction 0.1%, perlite is moved into：Vermiculite is 3:
In 2 matrix, leaf of plum Chinese ilex seedling is derived from.
Claims (3)
1. a kind of leaf of plum Chinese ilex quick breeding method for tissue culture, it is characterised in that comprise the following steps：
A, the leaf of plum Chinese ilex of the healthy no disease and pests harm 8-12cm of selection newly take out band bud branch then, remove the leaf of branch, use alcohol swab
Wipe the dust on branch surface away and be cut into 3-5 sections, every section carries the 1-2 branch section of resting bud point as explant；
B, explant is sterilized, culture induces axillary bud or top during the explant insertion primary after sterilizing then is lured into bud culture medium
Bud, condition of culture is：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, described is first
In generation, lures every liter of bud culture medium to contain：1mg ZT, 0.05mg NAA, 6.2g agar, 0.1g inositols, 30g sucrose, balance of MS cultures
Base, pH 5.8-6.0；
C, axillary-bud or top-bud is cut, to access and carry out Multiplying culture in adventitious bud proliferation culture medium and obtain adventitious bud, condition of culture
For：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, described adventitious bud proliferation culture
Every liter of base contains：1mg 6-BA, 0.05mgNAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS culture mediums, pH
5.8-6.0；
D, adventitious bud access strong seedling culture base is carried out strong seedling culture and obtains unrooted strong sprout, condition of culture is：1500-2000Lx,
24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, every liter described of strong seedling culture base contains：0.1mg6-BA、
0.1mg NAA, 6.2g agar, 0.1g inositols, 20g sucrose, balance of MS culture mediums, pH 5.8-6.0；
E, the 2-3cm unrooted strong sprouts with 3-5 blade high are accessed into rooting induction culture medium in culture obtain tissue-cultured seedling, cultivate
Condition is：1500-2000Lx, 24 DEG C -28 DEG C, light application time 12h-14h, interlunation 12h-10h, described rooting induction training
Every liter of base is supported to contain：1mg IBA, 0.02g AC, 6.2g agar, 0.1g inositols, 20g sucrose, balance of 1/2MS culture mediums, pH
5.8-6.0；
F, tissue-cultured seedling is carried out into transplanting culture.
2. leaf of plum Chinese ilex quick breeding method for tissue culture according to claim 1, it is characterised in that described step b
By explant sterilizing be on aseptic operating platform, explant to be placed in the alcoholic solution of volume fraction 75% and soaks 30s；Move on to
On aseptic working platform, the explant of alcohol disinfecting is put into conical flask, adds the mercuric chloride aqueous solution of mass fraction 0.1%, drop
Drop tween of plus 1, soaks 10-15min, and centre rocks for several times, finally with aseptic water washing 3 times, obtains the explant of sterilization treatment.
3. leaf of plum Chinese ilex quick breeding method for tissue culture according to claim 1, it is characterised in that described step f
Transplanting culture be to treat tissue culture seedling rooting 5-10 bars, during root 1-2cm long, tissue-cultured seedling is moved on into temperature humidity consistent with the external world
In culturing room, bottle cap is opened after 4 days, make the tissue-cultured seedling for bearing root keep contact higher, the taking-up that added water after 7 days to take root with the external world
Seedling, cleans matrix, and 1-3min is soaked with the potassium permanganate solution of mass fraction 0.1%, moves into perlite：Vermiculite volume ratio=3:
In 2 culture medium.
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Cited By (5)
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| CN108029559A (en) * | 2017-12-29 | 2018-05-15 | 南京林业大学 | A kind of method of quickly breeding bearberry tissue-cultured seedling |
| CN108450334A (en) * | 2018-05-02 | 2018-08-28 | 四川七彩林业开发有限公司 | A kind of narrow hat Chinese ilex method for tissue culture of sunlight |
| CN110476808A (en) * | 2019-08-01 | 2019-11-22 | 广州中医药大学(广州中医药研究院) | A kind of abductive approach of Flos Ilicis Asprellae callus |
| CN110604053A (en) * | 2019-07-25 | 2019-12-24 | 广东省农业科学院作物研究所 | In-vitro culture rapid propagation method of holly |
| CN113767765A (en) * | 2021-11-01 | 2021-12-10 | 广东兴源农林有限公司 | Method for raising seedlings of roughhaired holly by cutting |
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| 蔡时可等: "岗梅的组织培养和快速繁殖", 《植物生理学通讯》 * |
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| CN108029559A (en) * | 2017-12-29 | 2018-05-15 | 南京林业大学 | A kind of method of quickly breeding bearberry tissue-cultured seedling |
| CN108450334A (en) * | 2018-05-02 | 2018-08-28 | 四川七彩林业开发有限公司 | A kind of narrow hat Chinese ilex method for tissue culture of sunlight |
| CN108450334B (en) * | 2018-05-02 | 2020-04-24 | 四川七彩林科股份有限公司 | Tissue culture method for ilex strictus |
| CN110604053A (en) * | 2019-07-25 | 2019-12-24 | 广东省农业科学院作物研究所 | In-vitro culture rapid propagation method of holly |
| CN110476808A (en) * | 2019-08-01 | 2019-11-22 | 广州中医药大学(广州中医药研究院) | A kind of abductive approach of Flos Ilicis Asprellae callus |
| CN113767765A (en) * | 2021-11-01 | 2021-12-10 | 广东兴源农林有限公司 | Method for raising seedlings of roughhaired holly by cutting |
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