CN105123517B - The culture of Chinese herbaceous peony leaf tissue is directly into the culture medium and cultural method of bud - Google Patents
The culture of Chinese herbaceous peony leaf tissue is directly into the culture medium and cultural method of bud Download PDFInfo
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- CN105123517B CN105123517B CN201510525936.0A CN201510525936A CN105123517B CN 105123517 B CN105123517 B CN 105123517B CN 201510525936 A CN201510525936 A CN 201510525936A CN 105123517 B CN105123517 B CN 105123517B
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- herbaceous peony
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Abstract
The invention belongs to the field of tissue culture of plant, and in particular to a kind of Chinese herbaceous peony leaf tissue culture is directly into the culture medium and cultural method of bud.This method is to choose tender Chinese herbaceous peony blade in late March；25 ± 2 DEG C of temperature on culture medium are inoculated in after sterilizing, light culture 2 months obtains adventitious bud；The culture medium is improvement MS minimal mediums, 0.1~3.0mgL of addition TDZ‑1+ coconut juice 5~15%, additional saccharose 30gL‑1, caseinhydrolysate 600mgL‑1, agar 6.4gL‑1, pH is 5.8.Using this method Chinese herbaceous peony blade bud ratio can be made to reach as high as 85%, efficiently solve problem, saved human and material resources, shorten the time of Chinese herbaceous peony regeneration, be the fast numerous offer technical support of Chinese herbaceous peony improved seeds, wide market.
Description
Technical field
The invention belongs to the field of tissue culture of plant, and in particular to a kind of Chinese herbaceous peony leaf tissue culture is directly into the training of bud
Support base and cultural method.
Background technology
Chinese herbaceous peony (Paeonia lactiflora Pall.) is traditional famous flower of China, ranks " flower phase ", is only second to " flower
King " tree peony, in existing more than the 4000 years cultivation history of China.The flower of Chinese herbaceous peony is very large, and pattern is gorgeous, and flower pattern is attractive in appearance, fragrance of a flower fragrance
It is strongly fragrant, it is deep to be liked by broad masses of the people, and some have the kind of novel character, market prospects are particularly wide.Such as common 3
~5 years raw Chinese herbaceous peony cultivar prices are generally 20~30 yuan, and the more kind of novelty such as ' gold wheel ' price is about common
10 times of kind, and demand is huge.And how fast and efficiently to carry out these novel kinds expanding numerous, it is to put in grower
Problem in the urgent need to address in front.
Current the most frequently used propagation method of Chinese herbaceous peony is division propagation and seed propagation, although the former can keep the property of original kind
Shape, but need plant strain growth to divide once for 3~5 years, and the cycle is longer, breeding coefficient is relatively low, the maternal plant next year after plant division
Ornamental value is decreased obviously；The latter can neither keep the character of original kind, need just bloom by 4~5 juvenile phase again, because
This, both of which is not suitable for being commercially produced on a large scale.Plant Tissue Breeding is that one kind of micropropagation of plants has the most
The method of effect, compared with Traditional breeding processes, tissue cultures can keep original kind and merit, expand its line of breeding
Number, and do not influenceed by season, it can be operated with the anniversary, the efficiency of breeding has been significantly increased.CN102428872B is disclosed
A kind of method that complete Chinese herbaceous peony plant is obtained by Seed embryo culture, but because embryo can not keep maternal character completely, because
The expansion that this cannot be used for improved seeds is numerous.Beautiful et al. paper ' Herbaceous peony ' big rich and honour ' inducing clumping bud and the lifes delivered of Wu Hong
Root technology ' (Wu Hongjuan, Shen Miaomiao, it is big in dawn south Herbaceous peonies ' big rich and honour ' inducing clumping bud and rooting technique Northeast forestries
Learn journal, 2011,39 (9):20-22.), ' Chinese herbaceous peony different cultivars Primary culture compares and ' cinnabar is sentenced ' inducing clumping bud is ground
Study carefully ' (Wu Hongjuan, in dawn south Chinese herbaceous peony different cultivars Primary cultures compare and ' cinnabar is sentenced ' inducing clumping bud research Inner Mongol agricultures
Industry college journal, 2010,31 (4):29-33.) report the method that Multiple Buds are induced by explant of underground resting bud, the party
Method is numerous not enough in the presence of 3 points for the expansion of improved seeds：1. the underground resting bud limited amount that a plant is included, is outer using it
The reproductive efficiency of implant is not high；2. every plant of tissue-cultured seedling can only produce 3~5 Multiple Buds, and breeding coefficient is not high；3. whole process must
The process of a underground bud Primary culture must be undergone, substantial amounts of human and material resources are expended, cost is improved.What Hu Yingquan et al. was delivered
Paper ' inductive technology of Chinese herbaceous peony adventitious bud ' (Hu Yingquan, Feng Haihua, when the precious inductive technology for insulting Chinese herbaceous peony adventitious buds, Shanxi Forestry
Science and technology, 2003, supplementary issue:23-24,33.) tissue cultures are carried out to Chinese herbaceous peony tender stem, in M S+BA 3.5mg/L+ sucrose 30g/L+ fine jades
On fat 6g/L culture mediums can a step into bud, shorten into the bud time, but the paper data are more ambiguous, the time of statistics is not also made
Explanation, the inductivity highest of final bud only has 74%.' 5 Cultivars of Chinese Herbaceous Peony callus inductions of the paper that Wang Ji phoenix et al. is delivered
And differentiation research ' (Wang Jifeng, Li Qing, Meng understand .5 Cultivars of Chinese Herbaceous Peony callus induction and differentiation research Beijing Forestry University and learned
Report, 2011,32 (3):Successfully induce callus in 213-216.), and differentiate adventitious bud, but its callus browning it is serious,
Vitrifying, frangible, inductivity is low, and only 7.95%.Paper that Sun Xiao plums et al. are delivered ' Chinese herbaceous peony callus induction and indefinite
The research of bud differentiation ' (Sun Xiaomei, Cheng Wan, Liu Ping, Zhou Wenqiang, wangdan, Yang Hong light Chinese herbaceous peonies callus induction and adventitious bud point
The research Agricultural University Of Shenyang journal of change, 2014-02,45 (1):24-27.) Chinese herbaceous peony cotyledon, stem, blade are cultivated, with
Base portion stem is optimal adventitious buds differentiation explant, but differentiation rate only has 35%, and need to pass through callus induction period.To sum up institute
State, it is preceding successfully to differentiate adventitious bud per capita in Chinese herbaceous peony tissue culture procedures, but they are more with embryo, underground resting bud, stem
Sections etc. are explant, and not only differentiation rate is low, and subculture number is more (implementing in two steps), and experimentation is cumbersome, and the cycle is longer, and
Preferable effect can not be obtained.
The content of the invention
The present invention is in order to solve the key technical problem during Chinese herbaceous peony tissue culture there is provided one kind using Chinese herbaceous peony blade as explant
The step of body one, into the culture medium and cultural method of bud, is the fast numerous offer technical support of Chinese herbaceous peony improved seeds.
The Chinese herbaceous peony leaf tissue culture that the present invention is used is directly into the culture medium of bud, it is characterised in that improvement MS is trained substantially
Support base, 0.1~3.0mgL of addition TDZ-1+ coconut juice 5~15%, additional saccharose 30gL-1, caseinhydrolysate 600mgL-1,
Agar 6.4gL-1, pH is 5.8.
The invention also discloses a kind of Chinese herbaceous peony leaf tissue culture directly into the cultural method of bud, it is characterised in that including
Following steps：Late March chooses tender Chinese herbaceous peony blade；25 ± 2 DEG C of temperature on culture medium, light culture 2 are inoculated in after sterilizing
Month, obtain adventitious bud；The culture medium is improvement MS minimal mediums, 0.1~3.0mgL of addition TDZ-1+ coconut juice 5~
15%, additional saccharose 30gL-1, caseinhydrolysate 600mgL-1, agar 6.4gL-1, pH is 5.8.
The blade sterilization treatment is combined with the sterilizing methods that mercuric chloride is combined using alcohol using alcohol with mercuric chloride
Sterilizing methods, are comprised the following steps that：
1. young leaflet tablet is first rinsed into 10min in flowing water, removes exterior earth, then cleaned with liquid detergent, flowing water
Rinse well；
2. young leaflet tablet is placed on superclean bench, be positioned in beaker, with 75% alcohol disinfecting 30s, sterilized water is anti-
Rinse 5~6 times again；
3. 3min is sterilized with 0.1% mercuric chloride again, sterilized water is rinsed 5~6 times repeatedly；
4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, is cut into 1cm × 1cm
Size be inoculated on inducing culture, to avoid cross-infection, 3 blades of every bottle of inoculation.
Beneficial effects of the present invention are embodied in：
(1) Chinese herbaceous peony blade bud ratio reaches as high as 85% in the present invention, overcomes Chinese herbaceous peony blade and breaks up by callus
The difficult technology barrier of adventitious bud.
(2) in the present invention culture of Chinese herbaceous peony leaf tissue without the direct step of callus phase into bud, it is to avoid because many
Secondary subculture and the problem of labor intensive, material resources, the time of Chinese herbaceous peony regeneration is shortened, while reducing the pollution danger in switching process
Evil.
(3) blade of every inoculation can form 3-8 adventitious bud in the present invention, improve breeding coefficient.
Brief description of the drawings
Fig. 1 is the Chinese herbaceous peony blade being inoculated on culture medium.
Fig. 2 is adventitious bud formation process.
Embodiment
Embodiment 1
It is ' purple to transplant the Chinese herbaceous peony support osmanthus type kind in Yangzhou University's gardening and plant protection institute Chinese herbaceous peony Germplasm Resources
Phoenix plumage ' it is examination material to carry out this research work：
(1) draw materials the time：On March 28th, 2014；
(2) selection：Choose tender blade；
(3) sterilizing and inoculation of blade：
After tender leaf is adopted, the sterilizing methods being combined using alcohol with mercuric chloride are comprised the following steps that：
1. young leaflet tablet is first rinsed into 10min in flowing water, removes exterior earth, then with liquid detergent (Shanghai and yellowish-white cat
Co., Ltd) clean, flowing water is rinsed well；
2. young leaflet tablet is placed on superclean bench, be positioned in beaker, with 75% alcohol disinfecting 30s, sterilized water is anti-
Rinse 5~6 times again；
3. 3min is sterilized with 0.1% mercuric chloride (Shanghai Chinese and Western chemical plant) again, sterilized water is rinsed 5~6 times repeatedly；
4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, is cut into 1cm × 1cm
Size be inoculated on inducing culture, to avoid cross-infection, 3 blades of every bottle of inoculation are inoculated with 200 bottles altogether.
(4) culture medium prescription and cultural method：
1. culture medium prescription：Improve MS minimal mediums, addition TDZ 1.0mgL-1+ coconut juice 5%, additional saccharose 30g
L-1, hydrolysis network protein 60 0mgL-1, agar 6.4gL-1, pH is 5.8.TDZ is purchased from Beijing Suo Laibao Science and Technology Ltd, sugarcane
Sugar is purchased from Shanghai Experimental Reagent Co., Ltd., caseinhydrolysate and is purchased from Sangon Biotech (Shanghai) Co., Ltd., agar
It is purchased from Beijing Suo Laibao Science and Technology Ltd, coconut juice and is purchased from Shanghai Jia Guo Food Co., Ltd.
MS minimal medium formulas are improved in the present invention is：
A great number of elements
Potassium nitrate KNO3 250mg/L
Ammonium nitrate NH4NO3 500mg/L
Potassium dihydrogen phosphate KH2PO4 550mg/L
Magnesium sulfate MgSO4·7H2O 250mg/L
Calcium nitrate Ca (NO3)2·4H2O 500mg/L
Trace element
KI KI 0.83mg/L
Boric acid H3BO3 6.2mg/L
Manganese sulfate MnSO4·4H2O 22.3mg/L
Zinc sulfate ZnSO4·7H2O 8.6mg/L
Sodium molybdate Na2MoO4·2H2O 0.25mg/L
Copper sulphate CuSO4·5H2O 0.025mg/L
Cobalt chloride CoCl2·6H2O 0.025mg/L
Molysite
Disodium ethylene diamine tetraacetate Na2·EDTA 37.3mg/L
Ferrous sulfate FeSO4·7H2O 27.8mg/L
Organic principle
Inositol 100mg/L
Glycine 2mg/L
Thiamine hydrochloride VB10.1mg/L
Puridoxine hydrochloride VB60.5mg/L
Nicotinic acid VB50.5mg/L
Sucrose 30g/L
Agar 6.4g/L.
2. condition of culture：25 ± 2 DEG C of temperature, light culture 2 months.
(5) statistical indicator and method：Explant starts statistics after tissue culture room culture 60d and sprouted and browning situation.
Tissue number/inoculation blade amt × 100% of bud ratio=budding；
Tissue number/inoculation blade amt × 100% of melting brown rate=browning.
(6) result：The blade inoculation of 1cm × 1cm sizes as shown in Figure 1 is subjected to light culture in bud differential medium,
1.5~2 months it is observed that at the beginning of the adventitious bud formed as shown in Figure 2, blade bud ratio is up to 84.60%, and every blade can
3-8 adventitious bud is formed, melting brown rate is only 1.93%.
Claims (3)
1. a kind of Chinese herbaceous peony leaf tissue culture is directly into the culture medium of bud, it is characterised in that the culture medium is：Improve MS basic
Culture medium, 0.1~3.0mgL of addition TDZ-1+ coconut juice 5~15%, additional saccharose 30gL-1, caseinhydrolysate 600mgL-1, agar 6.4gL-1, pH is 5.8；The improvement MS minimal mediums formula is as follows：
A great number of elements：Potassium nitrate KNO3250mg/L, ammonium nitrate NH4NO3500mg/L, potassium dihydrogen phosphate KH2PO4550mg/L,
Magnesium sulfate MgSO4·7H2O 250mg/L, calcium nitrate Ca (NO3)2·4H2O 500mg/L；
Trace element：KI KI 0.83mg/L, boric acid H3BO36.2mg/L, manganese sulfate MnSO4·4H2O 22.3mg/L, sulphur
Sour zinc ZnSO4·7H2O 8.6mg/L, sodium molybdate Na2MoO4·2H2O 0.25mg/L, copper sulphate CuSO4·5H2O 0.025mg/
L, cobalt chloride CoCl2·6H2O 0.025mg/L；
Molysite：Disodium ethylene diamine tetraacetate Na2EDTA 37.3mg/L, ferrous sulfate FeSO4·7H2O 27.8mg/L；
Organic principle：Inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB6
0.5mg/L, nicotinic acid VB50.5mg/L, sucrose 30g/L, agar 6.4g/L.
2. a kind of Chinese herbaceous peony leaf tissue culture is directly into the cultural method of bud, it is characterised in that comprise the following steps：Late March
Choose tender Chinese herbaceous peony blade；It is inoculated in after sterilizing on culture medium, 25 ± 2 DEG C of temperature, light culture 2 months, obtains adventitious bud；Institute
It is improvement MS minimal mediums, 0.1~3.0mgL of addition TDZ to state culture medium-1+ coconut juice 5~15%, additional saccharose 30gL-1, caseinhydrolysate 600mgL-1, agar 6.4gL-1, pH is 5.8；Wherein, the improvement MS minimal mediums are formulated such as
Under：
A great number of elements：Potassium nitrate KNO3250mg/L, ammonium nitrate NH4NO3500mg/L, potassium dihydrogen phosphate KH2PO4550mg/L,
Magnesium sulfate MgSO4·7H2O 250mg/L, calcium nitrate Ca (NO3)2·4H2O 500mg/L；
Trace element：KI KI 0.83mg/L, boric acid H3BO36.2mg/L, manganese sulfate MnSO4·4H2O 22.3mg/L, sulphur
Sour zinc ZnSO4·7H2O 8.6mg/L, sodium molybdate Na2MoO4·2H2O 0.25mg/L, copper sulphate CuSO4·5H2O 0.025mg/
L, cobalt chloride CoCl2·6H2O 0.025mg/L；
Molysite：Disodium ethylene diamine tetraacetate Na2EDTA 37.3mg/L, ferrous sulfate FeSO4·7H2O 27.8mg/L；
Organic principle：Inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB6
0.5mg/L, nicotinic acid VB50.5mg/L, sucrose 30g/L, agar 6.4g/L.
3. method according to claim 2, it is characterised in that blade sterilization treatment is using alcohol with going out that mercuric chloride is combined
Bacterium method, is comprised the following steps that：
1. young leaflet tablet is first rinsed into 10min in flowing water, removes exterior earth, then cleaned with liquid detergent, flowing water is rinsed
Totally；
2. young leaflet tablet is placed on superclean bench, be positioned in beaker, with 75% alcohol disinfecting 30s, sterilized water is rushed repeatedly
Wash 5~6 times；
3. 3min is sterilized with 0.1% mercuric chloride again, sterilized water is rinsed 5~6 times repeatedly；
4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, is cut into the big of 1cm × 1cm
It is small to be inoculated on culture medium, to avoid cross-infection, 3 blades of every bottle of inoculation.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428872A (en) * | 2011-10-14 | 2012-05-02 | 扬州大学 | Culture medium and culture method for cultivating immature embryo of double-petal paeonia lactiflora |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0523071A (en) * | 1991-07-22 | 1993-02-02 | Mitsubishi Agricult Mach Co Ltd | Method for proliferating adventive embryo of paeonia by tissue culture |
| JPH06217659A (en) * | 1993-01-27 | 1994-08-09 | Mitsubishi Agricult Mach Co Ltd | Method of observing chromosome of peony |
| JP2009232691A (en) * | 2008-03-26 | 2009-10-15 | Naraken Chusho Kigyo Sien Center | Method for tissue culture of paeonia lactiflora pallas and browning suppressing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428872A (en) * | 2011-10-14 | 2012-05-02 | 扬州大学 | Culture medium and culture method for cultivating immature embryo of double-petal paeonia lactiflora |
Non-Patent Citations (6)
| Title |
|---|
| TDZ 和暗培养对蝴蝶兰叶片不定芽发生的影响;陆顺教等;《热带农业科学》;20150715;第35卷(第7期);第22-26，35页 * |
| TDZ 高效诱导蝴蝶兰叶片不定芽及植株再生;程强强等;《植物科学学报》;20111231;第29卷(第4期);第524-530页 * |
| 一种快速有效的741杨离体叶片再生芽方法;吴双秀等;《植物研究》;20040731;第24卷(第3期);第347-350页 * |
| 光皮桦叶片再生体系的建立;孙晓敏等;《西北农林科技大学学报（自然科学版）》;20120430;第40卷(第4期);第61-67页 * |
| 杜仲叶片为外植体的植株再生;向阳等;《分子植物育种》;20031231;第1卷(第5/6期);第825-826页 * |
| 草莓高效离体叶片再生体系的建立;王翡等;《西北植物学报》;20101231;第30卷(第5期);第1045-1049页 * |
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