CN103461130A - Tissue culture method for changeable protea of clematis cultivated variety - Google Patents
Tissue culture method for changeable protea of clematis cultivated variety Download PDFInfo
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- CN103461130A CN103461130A CN2013104281298A CN201310428129A CN103461130A CN 103461130 A CN103461130 A CN 103461130A CN 2013104281298 A CN2013104281298 A CN 2013104281298A CN 201310428129 A CN201310428129 A CN 201310428129A CN 103461130 A CN103461130 A CN 103461130A
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Abstract
The invention discloses a tissue culture method for changeable protea of a clematis cultivated variety. The tissue culture method comprises the steps of (1) explant disinfection treatment; (2) bud induction and bud proliferation, comprising the following steps: (1) taking current-year young internodes and terminal buds of the clematis changeable protea; (2) washing the young internodes and the terminal buds with tap water for 1 hour and washing 4 times with sterile water in sequence; and (3) absorbing the water on the surface of an explants by using sterile filter paper, and cutting petioles and damaged parts at the two ends. The tissue culture method has the advantages that the ornamental value of a female parent can be kept well and the propagation coefficient of the clematis changeable protea can be effectively improved by using the tissue culture method; compared with a cutting propagation method, the growth period is greatly shortened, the propagation coefficient of the tissue culture method is 50 to 60 times that of the cutting propagation method; the clematis changeable protea is high in quality and high in yield; on-site popularization is facilitated.
Description
Technical field
What the present invention relates to is that (" changeable sea God " English commodity are by name: Clematis ' Proteus '), belong to field of plant tissue culture technique for a kind of clematis cultivar " changeable sea God's " method for tissue culture.
Background technology
Clematis (Clematis L.) is under the jurisdiction of Ranunculaceae (Ranunculaceae) buttercup subfamily (Subfam. Ranunculoideae) anemone family (Trib. Anemoneae) clematis subtribe (Subtrib. Clematidinae), is often perennial woody climber.The world approximately has 355 kinds, and China has 147 kinds, and wherein 93 kinds is China endemic species, and peculiar rate is 63.3%.
Its sepal petal-shaped, rich color, form is changeable, and stem is overgrow, and utilizes petiole to climb up by holding on to, and growth adaptability is strong, and horticultural use is extensive, and ornamental value is high, has critical role in vertical greening, enjoys the laudatory title of " Climbing Plant queen ".In Japan and gardens, west, clematis has occupied very consequence, and majority of plant garden, park and family garden can be seen their bright and colourful figures.In addition, the many kinds in this platymiscium as the female wither (
clematis apiifolia), the root of Chinese clematis (
c. chinensis) etc. as medicinal plant, use for a long time.
The more difficult germination of its seed, multiplex cottage propagation.(English commodity are by name: Fast-propagation Clematis ' Proteus ') to take tissue culture method to carry out clematis " changeable sea God ", set up the vegetative propagation system that Multiple Buds is taken root, be conducive to increase reproduction coefficient, move towards horticultural applications for clematis " changeable sea God " and there is stronger production application value.
Summary of the invention
What the present invention proposed is a kind of clematis cultivar " changeable sea God's " method for tissue culture, its objective is and overcome the existing existing defect of propagation technique of clematis " changeable sea God ", improve the demand that its reproduction coefficient is introduced a fine variety popularization and Landscape Application to adapt to it.
Technical solution of the present invention: the method for tissue culture of a kind of clematis cultivar " changeable sea God " comprises the steps:
1) explant is disinfected;
2) bud is induced with bud and is bred.
Described step 1) explant is disinfected, and specifically comprises:
1. get clematis " changeable sea God " the living tender stipes of children and terminal bud then;
2. rinse 1 h with running water successively, writing brush is scrubbed 20 times back and forth, on superclean bench, by mass concentration, is 75% ethanol sterilizing 20 s, and aseptic water washing 2 times, be 0.1% mercury chloride sterilizing 7 min by mass concentration afterwards, uses immediately aseptic water washing 4 times;
3. blot the explant surface moisture with sterilizing filter paper, excision petiole and injury, two ends; The pollution rate of experiment statistics is respectively 54%, 36%, and brown rate is 44%, 35%.
Described step 2) bud is induced and bud propagation, specifically comprises:
1. induce cultivation
After flushing, explant is placed in to culture dish, with behind scissors, tweezers excision petiole, injury, two ends, bud is inoculated in to the sterilized inducing culture 1/2 MS+ growth regulatory substance NAA 0.5mg/L+ growth regulatory substance TDZ agar 7g/L that sucrose 20 ~ 30 g/L that 1.0 mg/L+mass concentration is 3%+mass concentration is 0.7%, the pH value is 5.8, cultivate 4 weeks, 25 ℃ of cultivation temperature, illumination 16 h/d, illumination 2400 1x;
2. adventitious bud proliferation is cultivated
The Multiple Buds of inducing is divided into to simple bud, then simple bud is accessed to the agar 7g/L that sucrose 20 ~ 30 g/L that 1/2 MS+growth regulatory substance 6-BA 1.0 mg/L+growth regulatory substance KT 0.5mg/L+growth regulatory substance NAA 0.5 mg/L+mass concentration is 3%+mass concentration is 0.7%, medium, the pH value is 5.8, cultivate temperature, the same 1. inducing clumping bud of illumination condition 4 weeks;
3. strong seedling culture
Proliferated culture medium middle period look indefinite bud dark green, that length is 1 ~ 2 cm is cut one by one, in access MS minimal medium, cultivated for 4 weeks, the same 1. inducing clumping bud of illumination condition;
4. culture of rootage
By growing way after strong seedling culture, vigorous bud grafting enters in root media 1/2 MS+ growth regulatory substance IBA 0.3 mg/L, cultivates 40 days.Temperature, the same 1. inducing clumping bud of illumination condition;
5. hardening and transplanting
The blake bottle bottle cap of clematis " changeable sea God " plant that takes root is opened, after room temperature lower refining seedling 6 d, take out test-tube plantlet, clean, be transplanted to ready perlite, vermiculite, peat perlite, its weight ratio is perlite: in the nutritive cube of vermiculite: peat=1:1:1, keep humidity 85% ~ 95%, temperature, the same 2. inducing clumping bud of illumination condition, survival rate reaches 95%.
Advantage of the present invention: the application method for tissue culture, can keep preferably maternal ornamental value, effectively improve clematis " changeable sea God's " reproduction coefficient.With cottage propagation, compare, growth cycle significantly shortens, the reproduction coefficient of tissue culture method is cottage propagation 50 ~ 60 times.The kind high-quality, output is considerable and be convenient to promote on the spot.
Embodiment
Embodiment
The method for tissue culture of a kind of clematis cultivar " changeable sea God ", its incubation step is as follows:
Step 1, explant are disinfected
Choose and give birth to then young tender stipes and terminal bud, rinse successively 1.5 h, be 75% ethanol sterilizing 20 s by mass concentration on superclean bench, uses aseptic water washing 2 times, by mass concentration, is 0.1% mercuric chloride sterilizing 7 min afterwards, uses immediately aseptic water washing 3-4 time.
Step 2, bud are induced with bud and are bred
1. induce cultivation
After flushing, explant is placed in to culture dish, with behind scissors, tweezers excision petiole, injury, two ends, bud is inoculated in to upper 5 weeks of sterilized inducing culture 1/2 MS+NAA 0.5 mg/L+TDZ 1.0 mg/L, the agar 7g/L that sucrose 20 ~ 30 g/L that mass concentration is 3%+mass concentration is 0.7%, medium, pH 5.7,25 ℃ of cultivation temperature; Illumination condition: illumination/dark, illumination 16 h/d wherein, illuminance is 2400 1x.
2. adventitious bud proliferation is cultivated
The Multiple Buds induced in inducing culture is divided into to simple bud, accesses in 1/2 MS+ growth regulatory substance 6-BA 1.0 mg/L+ growth regulatory substance KT 0.5 mg/L+ growth regulatory substance NAA 0.5 mg/L proliferated culture mediums and cultivate 4 weeks illumination conditions with 1. inducing cultivation.
3. strong seedling culture
The indefinite bud that proliferated culture medium middle period look is dark green, length is 2 cm left and right cuts one by one, in access MS minimal medium, cultivates 25 days, and illumination condition is with 1. inducing cultivation.
4. culture of rootage
By growing way after strong seedling culture, vigorous bud accesses root media 1/2 MS+ growth regulatory substance IBA 0.3 mg/L after cutting the basal part of stem brown material, cultivates after 6 weeks and takes root, and illumination condition is with 1. inducing cultivation.
5. hardening and transplanting
The blake bottle bottle cap of plant of taking root is opened, and after room temperature lower refining seedling 6 d, takes out test-tube plantlet, clean, be transplanted to ready perlite: vermiculite: on peat (1:1:1) seedbed, keep 20 ~ 25 ℃ of temperature, humidity 85% ~ 95%, suitably shade, and survival rate reaches 90%.
Described MS minimal medium is comprised of macroelement, trace element, molysite and organic principle.
Described macroelement is: potassium nitrate KNO
3, 1900 mgL
-1, ammonium nitrate NH
4nO
3, 1650 mgL
-1, potassium dihydrogen phosphate KH
2pO
4, 170 mgL
-1, magnesium sulfate MgSO
47 H
2o, 370 mgL
-1, calcium chloride CaCl
24 H
2o, 440 mgL
-1.
Described trace element is: potassium iodide KI, 0.83 mgL
-1, boric acid H
3bO
3, 6.2 mgL
-1, or manganese sulphate MnSO44 H
2o, 22.3 mgL
-1or zinc sulphate ZnSO
47 H
2o, 8.6 mgL
-1or sodium molybdate Na
2moO
42 H
2o, 0.25 mgL
-1, copper sulphate CuSO
45 H
2o, 0.025 mgL
-1, cobalt chloride CoCl
26 H
2o, 0.025 mgL
-1.
Described molysite is: EDTA-Na2 Na
2eDTA, 37.3 mgL
-1, ferrous sulfate FeSO
47 H
2o, 27.8 mgL
-1.
Described organic principle is: inositol C
6h
12o
62H
2o, 100 mgL
-1, glycine NH
2cH
2cOOH, 2 mgL
-1, thiamine hydrochloride C
12h
17c
lOs2HCl, 0.1 mgL
-1, puridoxine hydrochloride C
8h
11o
3nHCl, 0.5 mgL
-1, nicotinic acid NC
5h
4cOOH, 0.5 mgL
-1.
The composition of described 1/2 MS minimal medium is: macroelement is half of MS minimal medium, all the other composition trace elements, molysite, and organic principle is identical with MS minimal medium organic principle.
Described growth regulatory substance 6-BA is 6-benzyladenine; NAA is methyl α-naphthyl acetate; KT is kinetin; IBA is indolebutyric acid, and TDZ is thidiazuron.
Claims (7)
1. the method for tissue culture of a clematis cultivar " changeable sea God ", is characterized in that the method comprises the steps:
1) explant is disinfected;
2) bud is induced with bud and is bred.
2. the method for tissue culture of a kind of clematis cultivar according to claim 1 " changeable sea God ", is characterized in that described step 1) explant disinfects, and specifically comprises:
1. get clematis " changeable sea God " the living tender stipes of children and terminal bud then;
2. rinse 1 h with running water successively, writing brush is scrubbed 20 times back and forth, on superclean bench, by mass concentration, is 75% ethanol sterilizing 20 s, and aseptic water washing 2 times, be 0.1% mercury chloride sterilizing 7 min by mass concentration afterwards, uses immediately aseptic water washing 4 times;
3. blot the explant surface moisture with sterilizing filter paper, excision petiole and injury, two ends; The pollution rate of experiment statistics is respectively 54%, 36%, and brown rate is 44%, 35%.
3. the method for tissue culture of clematis cultivar according to claim 1 " changeable sea God ", is characterized in that described step 2) bud is induced and bud is bred, specifically comprise:
1. induce cultivation
After flushing, explant is placed in to culture dish, with behind scissors, tweezers excision petiole, injury, two ends, bud is inoculated in to sterilized inducing culture 1/2 MS+ growth regulatory substance NAA 0.5mg/L+ growth regulatory substance TDZ agar 7 g/L that sucrose 20 ~ 30 g/L+mass concentration is 0.7% that 1.0 mg/L+mass concentration is 3%, the pH value is 5.8, cultivate 4 weeks, 25 ℃ of cultivation temperature, illumination 16 h/d, illumination 2400 1x;
2. adventitious bud proliferation is cultivated
The Multiple Buds of inducing is divided into to simple bud, then simple bud is accessed to the medium of agar 7 g/L that sucrose 20 ~ 30 g/L+mass concentration is 0.7% that 1/2 MS+growth regulatory substance 6-BA 1.0 mg/L+growth regulatory substance KT 0.5mg/L+growth regulatory substance NAA 0.5 mg/L+mass concentration is 3%, the pH value is 5.8, cultivate temperature, the same 1. inducing clumping bud of illumination condition 4 weeks;
3. strong seedling culture
Proliferated culture medium middle period look indefinite bud dark green, that length is 1 ~ 2 cm is cut one by one, in access MS minimal medium, cultivated for 4 weeks, the same 1. inducing clumping bud of illumination condition;
4. culture of rootage
By growing way after strong seedling culture, vigorous bud grafting enters in root media 1/2 MS+ growth regulatory substance IBA 0.3 mg/L, cultivates 40 days; Temperature, the same 1. inducing clumping bud of illumination condition;
5. hardening and transplanting
The blake bottle bottle cap of clematis " changeable sea God " plant that takes root is opened, after room temperature lower refining seedling 6 d, take out test-tube plantlet, clean, be transplanted in the ready nutritive cube be comprised of perlite, vermiculite, peat, the weight ratio between perlite, vermiculite and peat is 1:1:1, keeps humidity 85% ~ 95%, temperature, illumination condition are with (2) inducing clumping bud, and survival rate reaches 95%.
4. the method for tissue culture of a kind of clematis cultivar according to claim 3 " changeable sea God ", is characterized in that described MS minimal medium is comprised of macroelement, trace element, molysite and organic principle.
5. the method for tissue culture of a kind of clematis cultivar according to claim 4 " changeable sea God ", is characterized in that described macroelement is: potassium nitrate KNO
3, 1900 mgL
-1, ammonium nitrate NH
4nO
3, 1650 mgL
-1, potassium dihydrogen phosphate KH
2pO
4, 170 mgL
-1, magnesium sulfate MgSO
47 H
2o, 370 mgL
-1, calcium chloride CaCl
24 H
2o, 440 mgL
-1;
Described trace element is: potassium iodide KI, 0.83 mgL
-1, boric acid H
3bO
3, 6.2 mgL
-1, or manganese sulphate MnSO44 H
2o, 22.3 mgL
-1or zinc sulphate ZnSO
47 H
2o, 8.6 mgL
-1or sodium molybdate Na
2moO
42 H
2o, 0.25 mgL
-1, copper sulphate CuSO
45 H
2o, 0.025 mgL
-1, cobalt chloride CoCl
26 H
2o, 0.025 mgL
-1;
Described molysite is: EDTA-Na2 Na
2eDTA, 37.3 mgL
-1, ferrous sulfate FeSO
47 H
2o, 27.8 mgL
-1;
Described organic principle is: inositol C
6h
12o
62H
2o, 100 mgL
-1, glycine NH
2cH
2cOOH, 2 mgL
-1, thiamine hydrochloride C
12h
17c
lOs2HCl, 0.1 mgL
-1, puridoxine hydrochloride C
8h
11o
3nHCl, 0.5 mgL
-1, nicotinic acid NC
5h
4cOOH, 0.5 mgL
-1.
6. the method for tissue culture of a kind of clematis cultivar according to claim 3 " changeable sea God ", the composition that it is characterized in that described 1/2 MS minimal medium is: macroelement is half of MS minimal medium, all the other composition trace elements, molysite, organic principle is identical with MS minimal medium organic principle.
7. the method for tissue culture of a kind of clematis cultivar according to claim 3 " changeable sea God ", is characterized in that described growth regulatory substance 6-BA is 6-benzyladenine; NAA is methyl α-naphthyl acetate; KT is kinetin; IBA is indolebutyric acid, and TDZ is thidiazuron.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104206281A (en) * | 2014-09-29 | 2014-12-17 | 江苏农林职业技术学院 | Tissue cultivation method for elegant-purple clematis |
| CN107041307A (en) * | 2017-04-26 | 2017-08-15 | 江苏农林职业技术学院 | Clematis Henry method for tissue culture |
| CN110547202A (en) * | 2019-10-09 | 2019-12-10 | 江苏农林职业技术学院 | clematis chinensis proliferation tissue culture method |
Citations (2)
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| CN102265787A (en) * | 2010-06-04 | 2011-12-07 | 上海上房园艺有限公司 | Tissue culture method of president clematis |
| CN103202229A (en) * | 2013-04-11 | 2013-07-17 | 福建农林大学 | Tissue culturing and rapid propagating method for chloranthy florida var. plena |
-
2013
- 2013-09-22 CN CN201310428129.8A patent/CN103461130B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102265787A (en) * | 2010-06-04 | 2011-12-07 | 上海上房园艺有限公司 | Tissue culture method of president clematis |
| CN103202229A (en) * | 2013-04-11 | 2013-07-17 | 福建农林大学 | Tissue culturing and rapid propagating method for chloranthy florida var. plena |
Non-Patent Citations (2)
| Title |
|---|
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| 张启香等: "铁线莲品种‘Multi-Blue’不定根的诱导培养及其发生过程的解剖学观察", 《植物资源与环境学报》, vol. 19, no. 1, 31 December 2010 (2010-12-31), pages 80 - 85 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104206281A (en) * | 2014-09-29 | 2014-12-17 | 江苏农林职业技术学院 | Tissue cultivation method for elegant-purple clematis |
| CN107041307A (en) * | 2017-04-26 | 2017-08-15 | 江苏农林职业技术学院 | Clematis Henry method for tissue culture |
| CN110547202A (en) * | 2019-10-09 | 2019-12-10 | 江苏农林职业技术学院 | clematis chinensis proliferation tissue culture method |
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| CN103461130B (en) | 2015-05-20 |
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