CN105340756A - Method for Viburnum macrocephalum Fort. f. keteleeri in vitro culture and rapid propagation - Google Patents
Method for Viburnum macrocephalum Fort. f. keteleeri in vitro culture and rapid propagation Download PDFInfo
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- CN105340756A CN105340756A CN201510930226.6A CN201510930226A CN105340756A CN 105340756 A CN105340756 A CN 105340756A CN 201510930226 A CN201510930226 A CN 201510930226A CN 105340756 A CN105340756 A CN 105340756A
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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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- 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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Abstract
The invention discloses a method for Viburnum macrocephalum Fort. f. keteleeri in vitro culture and rapid propagation. The method mainly comprises the following steps: step one, selection of a propagating material; step two, disinfection of the propagating material; step three, preparation of a culture medium and sterilization; step four, inoculation and culture, which comprise culture of a Viburnum macrocephalum Fort. f. keteleeri test-tube plantlet, culture of test-tube plantlet leaf callus and rooting culture; step five, acclimatization and transplantation. By adopting the method, the propagation coefficient and survival rate of Viburnum macrocephalum Fort. f. keteleeri can be improved, the germination cycle is reduced, the seedling juvenile phase can be avoided, not only can the influence of an external condition be eliminated, but also the method is applicable in the four seasons, the occupation land for culturing seedlings can be saved, and the production cost can be lowered; moreover a large quantity of strong seedlings can be obtained within a short period of time, large-scale and industrial production can be carried out, the supply of Viburnum macrocephalum Fort. f. keteleeri seedlings can be increased, the demand for Viburnum macrocephalum Fort. f. keteleeri nursery stock in the landscaping market can be met, the in vitro conservation of high-quality germplasm can be facilitated, and a practical technical support can be provided for the rapid propagation and wide application of the Viburnum macrocephalum Fort. f. keteleeri seedlings.
Description
Technical field
The invention belongs to method for propagation, be specially a kind of method of rare flower cultured in vitro and Fast-propagation.
Background technology
Rare flower (Viburnummacrocephalumf.keteleeri), for Adoxaceae (former Caprifoliaceae) Viburnum half evergreen shrubs, flower pattern is peculiar; fruit ruby; tree-like grace, has high ornamental value, is the distinctive woody plant of Yangzhou city flower and China.Rare flower strong adaptability, robust growth, manage more extensive, and flowers and fruits grow prosperity.Because its resistance to shade can be used as the understory shrub of Qiao, filling, grass combination; After its fruit maturation, color is red gorgeous, and nutrient component is very abundant, can be used as lark seeds; Unusual flower pattern, pure white pattern, strong fragrance, can be used as excellent ornamental tree species.Both suitable Garden virescence, beautify, sweetening treatment, also can make cut-flower material, differ from one another.Therefore, rare flower is one of desirable plants material meeting the multiple demand such as urban plant community and urban ecological system stability, landscape diversity.
But under field conditions (factors), there is reproductive disorder in rare flower, seed dormancy is serious, not easily sprout, artificial land for growing field crops cuttage survival rate is extremely low, nature and artificial propagation all extremely difficult, cause current greening seedling market rare flower provenance rare, expensive, limit rare flower popularizing in various places.
Rare flower Traditional breeding processes can be divided into plant division, cuttage, seed propagation, but plant division only just can be carried out when adult seedlings base portion sprout tillers, and the fraction of the year of 3 ~ 4 once, reproduction coefficient is very low; Take root during cottage propagation more difficult, survival rate is low, must by full light fog seedbed and HORMONE TREATMENT; Seed propagation has dual dormancy because of its seed germination, and namely needing 1 ~ 2 annual layer sand to hide could germinate, and the cycle of germinateing is long, and the seedling of the sowing virgin phase is longer, within 6 ~ 8 years, just blooms, is unfavorable for extensive fast numerous application.Fast-propagation becomes the bottleneck problem of restriction rare flower application, for this reason, sets up efficient rare flower cultured in vitro and quick propagating technology has important practical value.
Summary of the invention
Technical problem to be solved by this invention is a kind of method providing simple to operate, practical rare flower cultured in vitro and Fast-propagation.
Realizing technical scheme of the present invention is:
(1) selection of propagating materials
To choose on rare flower adult plants axillalry bud as propagating materials.
(2) sterilization of propagating materials
Get the material sheared, after running water running water 2h, with hairbrush, washing powder, surface clean is clean, with the alcohol surface sterilization 30s of 70% (volumetric concentration), 0.1% (mass concentration) tween soaks vibration flushing 20 ~ 30min, 0.3% (mass concentration) javelle water sterilization 15min, finally uses aseptic water washing 5 ~ 7 times.
(3) medium preparation, sterilizing
Medium is prepared:
The best initial medium of rare flower stem apex Primary culture is: MS+BA1.0+ZT8.0+NAA0.2;
(configure the stem apex Primary culture base of a liter---MS (medium): I 50ml, II 5ml, III 5ml, IV 5ml; BA (6-benzyl aminopurine): 10ml; ZT (zeatin): 80ml; NAA (methyl α-naphthyl acetate): 2ml; Be settled to 1L) rare flower differentiation cultivate optimal medium be: MS+BA1.0+ZT15;
(configure the differentiation medium of a liter---MS (medium): I 50ml, II 5ml, III 5ml, IV 5ml; BA (6-benzyl aminopurine): 10ml; ZT (zeatin): 150ml; Be settled to 1L)
Rare flower Callus of Leaf optimal medium is: MS+BA0.5+NAA2.0+2,4-D0.5 ~ 1.0;
(configure the Callus of Leaf medium of a liter---MS (medium): I 50ml, II 5ml, III 5ml, IV 5ml; BA (6-benzyl aminopurine): 5ml; NAA (methyl α-naphthyl acetate): 20ml; 2,4-D (2,4-dichlorphenoxyacetic acid): 5 ~ 10ml; Be settled to 1L)
Rare flower rooting of vitro seedling induction optimal medium is: MS+NAA8.0;
(configure the root induction medium of a liter---MS (medium): I 50ml, II 5ml, III 5ml, IV 5ml; NAA (methyl α-naphthyl acetate): 80ml; Be settled to 1L)
Often kind of medium adds 20g/L sucrose, agar powder 6.8g/L, pH6.0 ~ 6.2,121 DEG C of autoclave sterilization 18min, and medium temperature 25 DEG C ± 2 DEG C, illumination 12h/d, illuminance is about 2000lx.
(4) inoculate, cultivate, comprise the cultivation of rare flower test-tube plantlet, test-tube plantlet blade callus tissue culture and culture of rootage;
By axillalry bud kind on Primary culture base MS, in seeded process, the incision of rare flower explant generally all can brown stain rapidly, thus pollution medium, cut by explant is immersed sterile water, in Primary culture base, increase 0.5%Vc, the number of times increasing switching medium can greatly reduce brown stain and pollute.
Through 40d growth after, crowd shoots is cut into band joint seedling, transfer into differentiation cultivate medium carry out shoot proliferation cultivation.
In rare flower bud Subculture, unnecessary blade is cut into about 1cm × 0.5cm pane, is inoculated in rare flower Callus of Leaf medium.
Treat that Calli Differentiation becomes bud, move into root media and carry out root induction.
(5) rooting culture
The rooting tube plantlet reaching transplanting standard is moved into greenhouse, open bottleneck hardening 2 ~ 3d, take out and clean medium, transplant in the matrix that humus soil+silica (volume ratio 2:1) is housed, first 2 ~ 3 week every day sprayed water 3 ~ 4 times, water in right amount afterwards, cultivate 50 ~ 60d and can be transplanted into land for growing field crops.
Compared with prior art, its remarkable advantage is in the present invention:
1, the present invention uses the axillalry bud of rare flower as material, and draw materials conveniently, quantity is sufficient, uses the technology of tissue cultures, and axillalry bud is inoculated in medium after sterilization, and the short period forms callus until formation seedling plants of taking root;
2, the present invention has mainly screened the optimal medium that applicable rare flower stem apex startup, differentiation, callus induction and rooting of vitro seedling are induced, improvement has been done to rare flower isolated culture condition, the survival rate that effective raising rare flower is bred in vitro, improves the reproduction coefficient of rare flower seedling;
3, the method is that the in-vitro propagate nursery of rare flower provides technical support, plants matter preferably and Fast-propagation provides practical scheme for rare flower retains.
Embodiment
In order to understand the present invention better, illustrate technical scheme of the present invention below by specific embodiment.
Embodiment 1
1. the selection of propagating materials
Axillalry bud on Agricultural College Affiliated to Yangzhou Univ.'s rare flower adult plants taken from by examination material.
2. the sterilization of propagating materials
After running water running water 2h, with hairbrush, washing powder, surface clean is clean, with the alcohol surface sterilization 30s of 70%, 0.1% tween soaks the flushing 20 ~ 30min that vibrates, and 0.3% hypochlorite disinfectant 15min, finally uses aseptic water washing 5 ~ 7 times.
3. medium preparation, sterilizing
Rare flower stem apex Primary culture initial medium is: MS+BA1.0+ZT8.0+NAA0.2;
The medium that rare flower differentiation is cultivated is: MS+BA1.0+ZT15;
Rare flower Callus of Leaf medium is: MS+BA0.5+NAA2.0+2,4-D0.5 ~ 1.0;
Rare flower rooting of vitro seedling inducing culture is: MS+NAA8.0.
Different culture media adds 20g/L sucrose, agar powder 6.8g/L, pH6.0 ~ 6.2,121 DEG C of autoclave sterilization 18min, and medium temperature 25 DEG C ± 2 DEG C, illumination 12h/d, illuminance is about 2000lx.
4. inoculate, cultivate
Axillalry bud is inoculated on Primary culture base, in seeded process, the incision of rare flower explant generally all can brown stain rapidly, thus pollution medium, cut by explant is immersed sterile water, increase 0.5%Vc in the medium, the number of times increasing switching medium can greatly reduce brown stain and pollute.
Through 40d growth after, crowd shoots is cut into band joint seedling, transfer into differentiation cultivate medium carry out shoot proliferation cultivation.
In rare flower bud Subculture, unnecessary blade is cut into about 1cm × 0.5cm pane, is inoculated in rare flower Callus of Leaf medium.
Treat that Calli Differentiation becomes bud, move into root media and carry out root induction.
5. rooting culture
The rooting tube plantlet reaching transplanting standard is moved into greenhouse, open bottleneck hardening 2 ~ 3d, take out and clean medium, transplant in the matrix that humus soil+silica (volume ratio 2:1) is housed, first 2 ~ 3 week every day sprayed water 3 ~ 4 times, water in right amount afterwards, cultivate 50 ~ 60d and can be transplanted into land for growing field crops.
Effect illustrates:
Rare flower axillalry bud is inoculated on Primary culture base, and through 40d growth, bud germination rate is 86.7%, and growing way is outstanding, and leaf look is green; In squamous subculture, Bud polarization is effective, and rapidly, Multiple Buds quantity is many, and inductivity is high, and clump Shoot propagation multiple reaches 10.57 in growth.Get blade after 10d cultivated by callus tissue culture base, the edge callus growth that blade contacts with medium is rapid, and 30d rear blade disappears, and form bulk band white and absinthe-green numerous microspike, callus induction rate reaches 94.3%.Be inoculated into root media, after 30d, seedling starts to take root successively, and rooting rate reaches 83.3%.After domestication, transplanting survival rate can reach more than 70%.
The present invention preferentially solves a difficult problem for a large amount of Fast-propagation rare flower, and has done detailed introduction by example, but will be appreciated that above-mentioned description should not be considered to restriction of the present invention.After those skilled in the art have read foregoing, for multiple amendment of the present invention and substitute will be all apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (6)
1. a method for rare flower cultured in vitro and Fast-propagation, is characterized in that comprising the following steps:
Step one, selects the axillalry bud of rare flower as propagating materials;
Step 2, the sterilization of propagating materials;
Step 3, medium preparation, sterilizing;
Described medium concrete configuration is determined through comparative trial, finds best medium under kinds of schemes;
The best initial medium of rare flower stem apex Primary culture is: MS+BA1.0+ZT8.0+NAA0.2;
The optimal medium that rare flower differentiation is cultivated is: MS+BA1.0+ZT15;
Rare flower Callus of Leaf optimal medium is: MS+BA0.5+NAA2.0+2,4-D0.5 ~ 1.0;
Rare flower rooting of vitro seedling induction optimal medium is: MS+NAA8.0;
Step 4, inoculation, cultivation, comprise the cultivation of rare flower test-tube plantlet, test-tube plantlet blade callus tissue culture and culture of rootage;
Step 5, rooting culture.
2. the method for rare flower cultured in vitro according to claim 1 and Fast-propagation, is characterized in that, the sterilization method of propagating materials described in step 2, through plurality of reagents process, comprises running water, alcohol, tween, clorox and sterile water.
3. the method for rare flower cultured in vitro according to claim 1 and Fast-propagation, it is characterized in that, in step 3, each medium adds 20g/L sucrose, agar powder 6.8g/L, pH6.0 ~ 6.2,121 DEG C of autoclave sterilization 18min, medium temperature 25 DEG C ± 2 DEG C, illumination 12h/d, illuminance is about 2000lx.
4. the method for rare flower cultured in vitro according to claim 1 and Fast-propagation, it is characterized in that, explant is specifically immersed sterile water and cuts by inoculation described in step 4, cultivation, 0.5%Vc is increased in Primary culture base, prevent brownization, first cultivate into test-tube plantlet with axillalry bud, the blade of recycling test-tube plantlet carries out tissue cultures.
5. the method for rare flower cultured in vitro according to claim 1 and Fast-propagation, it is characterized in that, rooting culture described in step 5 is that the rooting tube plantlet reaching transplanting standard is moved into greenhouse, open bottleneck hardening 2 ~ 3d, transplant cultivation 50 ~ 60d in the matrix that humus soil+silica (volume ratio 2:1) is housed and can be transplanted into land for growing field crops.
6. the method for rare flower cultured in vitro according to claim 5 and Fast-propagation, is characterized in that, transplanting survival rate can reach more than 70%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108040872A (en) * | 2017-11-29 | 2018-05-18 | 云南省农业科学院花卉研究所 | A kind of Vitro Quick Reproduction cultural method of feverfew |
| CN109156361A (en) * | 2018-10-25 | 2019-01-08 | 吉林农业大学 | A kind of in vitro quick breeding by group culture method of pruning Jia Opulus |
| CN109964814A (en) * | 2018-10-10 | 2019-07-05 | 北华大学 | A kind of rapid propagation in vitro method of warm batten Jia Opulus |
| CN114568065A (en) * | 2022-03-10 | 2022-06-03 | 陕西省西安植物园 | Method for quickly raising seedlings by seeding agar flowers |
| CN116472963A (en) * | 2023-04-27 | 2023-07-25 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108040872A (en) * | 2017-11-29 | 2018-05-18 | 云南省农业科学院花卉研究所 | A kind of Vitro Quick Reproduction cultural method of feverfew |
| CN108040872B (en) * | 2017-11-29 | 2021-03-30 | 云南省农业科学院花卉研究所 | In-vitro rapid propagation culture method for white chrysanthemum |
| CN109964814A (en) * | 2018-10-10 | 2019-07-05 | 北华大学 | A kind of rapid propagation in vitro method of warm batten Jia Opulus |
| CN109964814B (en) * | 2018-10-10 | 2022-06-24 | 北华大学 | In-vitro rapid propagation method of viburnum sargentii |
| CN109156361A (en) * | 2018-10-25 | 2019-01-08 | 吉林农业大学 | A kind of in vitro quick breeding by group culture method of pruning Jia Opulus |
| CN114568065A (en) * | 2022-03-10 | 2022-06-03 | 陕西省西安植物园 | Method for quickly raising seedlings by seeding agar flowers |
| CN116472963A (en) * | 2023-04-27 | 2023-07-25 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
| CN116472963B (en) * | 2023-04-27 | 2024-02-09 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
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Application publication date: 20160224 |