CN106942051B - A kind of culture medium and propagation method of the tissue-culturing quick-propagation of alum root blade - Google Patents
A kind of culture medium and propagation method of the tissue-culturing quick-propagation of alum root blade Download PDFInfo
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- CN106942051B CN106942051B CN201710118920.7A CN201710118920A CN106942051B CN 106942051 B CN106942051 B CN 106942051B CN 201710118920 A CN201710118920 A CN 201710118920A CN 106942051 B CN106942051 B CN 106942051B
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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 the culture mediums and propagation method of a kind of tissue-culturing quick-propagation of alum root blade.By the hormone combinations of explant selection, the selection of minimal medium and suitable dose, the highly efficient regeneration of plant in alum Root tissue culture is realized, operation of the present invention is simple, pollution rate is low, plant regeneration success rate is high, the seedling production cycle is shorter.
Description
Technical field
The present invention relates to field of plant tissue culture technique more particularly to a kind of leaf tissue culture of alum root quickly to breed
Culture medium and propagation method.
Background technique
Alum root (Heuchera micrantha) also known as coral bell, Saxifragaceae alum root category, happiness sun is shade tolerant, extremely cold-resistant, energy
The normal growth under subzero 30 DEG C of low temperature, leaf color is abundant, leaf changeable, is excellent color leaf shade ground cover plants.Ornamental value
Height, it is best in quality, there is very high economic benefit, exploitation prospect is wide, and market prospects are extremely good.
The Sterile culture method of alum root is division propagation, and breeding amount is limited, and by seasonal effect, and not being able to satisfy market needs
It asks.
Report about alum Root tissue culture is more, and but there are the following problems: 1) explant selection is single and limits to, related
Report explant all select " tender stem segments with terminal bud or axillary bud " (Sun Guofeng, 2007；Zhang Zhihong, 2011；Chen Hong, 2011；It is high
Swallow, 2015), also have been reported that " blade cannot induce budding " (Wang Jing, 2012).Explant selects the stem with terminal bud or axillary bud
Section, this destructive sampling not only obtains explant negligible amounts, but also the entire plant of parent is caused to be destroyed, and causes material unrestrained
Take；2) pertinent literature minimal medium selects MS, and hormone selects the different ratio of 6-BA and NAA, the inductivities of Multiple Buds compared with
Low and be easy to appear vitrifying, the increment multiple for being proliferated growth is generally 8-9, and the production cycle is longer.
Summary of the invention
There are aiming at the problem that, the present invention provides culture medium and the breeding side of a kind of tissue-culturing quick-propagation of alum root
Method realizes plant in alum Root tissue culture by explant, the hormone combinations of the selection of minimal medium and suitable dose
Highly efficient regeneration.This method is easy to operate, pollution rate is low, plant regeneration success rate is high, and the seeling industry period is shorter.
A kind of culture medium of the tissue-culturing quick-propagation of alum root, it includes
1) bud inducement cultivation base: 1.0 ~ 3.0 mg of LS+6-BA/0.1 ~ 0.3mg/L+ of L+ NAA
2) proliferated culture medium: 1.0 ~ 3.0 mg of LS+6-BA/0.1 ~ 0.3mg/L of L+ NAA；
3) root media: 1/2LS+ NAA0.5 ~ 1.5mg/L；
In LS and 1/2LS, 20~30g/L of sucrose, 5~8 g/L of agar are added, adjusts pH5.6~5.8.
A kind of quick breeding method for tissue culture of alum root blade, comprising the following steps:
(1) preparation of culture medium
1) bud inducement cultivation base: 1.0 ~ 3.0 mg of LS+6-BA/0.1 ~ 0.3mg/L of L+ NAA；
2) proliferated culture medium: LS+6-BA0.5 ~ 1.0 mg/0.05 ~ 0.1mg/L of L+ NAA；
3) root media: 1/2LS+ NAA0.5 ~ 1.5mg/L；
In LS and 1/2LS, 20~30g/L of sucrose, 5~8 g/L of agar are added, adjusts pH5.6~5.8.
(2) culture of alum root tissue-cultured seedling
1) explant selects: selecting explant of the alum root 1-2 leave piece of robust growth disease-free spot as tissue cultures
Body, flowing water rinses 1 ~ 2h, then clean with aseptic water washing on superclean bench, with 70% alcohol disinfecting 20-40s, 0.1% liter
Mercury solution immersion carries out sterilizing 6-8min, then multiple with aseptic water washing, spare；
2) explant prepared in step 1) the Fiber differentiation of Multiple Buds: is cut into 1 square centimeter on superclean bench
Fritter, be inoculated into bud inducement cultivation base and cultivated；Condition of culture: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is
1500Lx, light application time 12h/d；
3) Multiple Buds of the inductive formation in step 2 are inoculated on proliferated culture medium cultivate and grow seedling；Training
The condition of supporting: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；
4) when the seedling in step 3) grow when growing to 3. 0 ~ 5.0cm high, be inoculated into root media；Training
The condition of supporting: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；
5) when the seedling in step 4) grows to high 4 ~ 8cm and is longer than the root of 2cm at least 5, obtaining can
The seedling of bottle outlet plantation.
(3) hardening and transplanting of tissue-cultured seedling
Seedling band bottle described in step 5) is moved into greenhouse together, lid is first gone to place 2-5 days, bottle outlet simultaneously cleans root
Culture medium is transplanted into peat: in mixed-matrix of the perlite by volume 3:1, managing to finished product seedling, is out of the garden.
Beneficial effects of the present invention
One, operation of the present invention step is simple, and explant selects the raw healthy young leaves of 1-2, and explant source is wide, sampling
Irreversible injury will not be caused to maternal plant, guaranteed that maternal plant growth is unaffected to greatest extent, improved growth efficiency,
Pollution rate is reduced, provides healthy and strong seedling for subsequent proliferation；
Two, the present invention adjusts in conjunction with different hormone combinations and concentration by using LS culture medium prescription, improves and grow thickly
Bud induced efficiency (each explant 8-10), improves proliferation multiplying power (growth coefficient reaches 15-20 times), shortens growth week
Phase (inducing clumping bud 30-40 days, proliferation growth 15-20 days is taken root and grown 15-20 days)；Also the matter of seedling is further improved
Amount, makes transplanting survival rate be up to 98% or more.
Three, it using method of the invention, generally only needs seedling can be obtained within 60-80 days, is one kind not by factors such as seasons
It influences, the method for high-quality alum root seedling efficiently, is quickly provided, improved variety popularization speed can be accelerated, improve the plantation of field kind and produce
Amount.
Four, the alum Root tissue culture system that the present invention establishes will provide theoretical foundation and technical support for industrial seedling rearing,
Method is easy to operate, pollution rate is low, plant regeneration success rate is high.
Specific embodiment
By following embodiment, the present invention is described in further detail, but the contents of the present invention are not limited thereto.
Embodiment 1
A kind of method of the tissue-culturing quick-propagation of alum root blade, successively carries out step:
(1) preparation of culture medium, each component including minimal medium and each stage culture medium of tissue culture with every liter contained by weight
Amount are as follows:
1) minimal medium: inducing clumping bud culture, proliferation, strong sprout culture medium be all made of LS minimal medium, take root
Culture uses 1/2LS.
2) inducing clumping bud culture medium are as follows: 2.0 0.2 mg/L of mg/L+NAA of LS+6-BA+sucrose 20g/L+ fine jade
Rouge 8g/L, pH5.6~5.8
3) proliferation, strong seedling culture base are as follows: 1.0 mg/L+NAA of LS+6-BA, 0.1 mg/L+ sucrose 25g/L+ agar
5g/L, pH5.6~5.8.
4) root media are as follows: 1/2LS+NAA0.5mg/L+sucrose 30g/L+ agar 5g/L, pH5.6~5.8；
(2) culture of alum root tissue-cultured seedling
1) explant selects: selecting explant of the alum root 1-2 leave piece of robust growth disease-free spot as tissue cultures
Body, flowing water rinses 1 ~ 2h, then clean with aseptic water washing on superclean bench, with 70% alcohol disinfecting 20-40s, 0.1% mercuric chloride
Solution immersion carries out sterilizing 6-8min, then multiple with aseptic water washing, spare；
2) explant prepared in step 1) the Fiber differentiation of Multiple Buds: is cut into 1 square centimeter on superclean bench
The fritter of left and right, is inoculated into bud inducement cultivation base and is cultivated；Condition of culture: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is
1500Lx, light application time 12h/d；By 30-40d, the explant of an inoculation averagely can induce 8-10 Multiple Buds；
3) Multiple Buds of the inductive formation in step 2 are inoculated on proliferated culture medium cultivate and grow seedling；Training
The condition of supporting: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；Incubation time: 15-20d, proliferation
Multiplying power 15-20；
4) when the seedling in step 3) grow grow to 3. 0-5.0cm it is high when, be inoculated into root media；Culture
Condition: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；Incubation time: 15-20d, rooting rate
100%；
5) it when the seedling in step 4) grows to high 4- 8cm and is longer than the root of 2cm at least 5, obtains to go out
The seedling of bottle plantation.
(3) hardening and transplanting of tissue-cultured seedling
Seedling band bottle described in step 5) is moved into greenhouse together, lid is first gone to place 2-5 days, bottle outlet simultaneously cleans root
Culture medium is transplanted into peat: in mixed-matrix of the perlite by volume 3:1, managing to finished product seedling, is out of the garden.
Embodiment 2
In this example, the preparation of step (1) culture medium, each component including minimal medium and each stage culture medium of tissue culture
With every liter contained by weight are as follows:
2) inducing clumping bud culture medium are as follows: 3.0 0.2 mg/L of mg/L+NAA of LS+6-BA+sucrose 20g/L+ fine jade
Rouge 8g/L, pH5.6~5.8
3) proliferation, strong seedling culture base are as follows: 0.5 mg/L+NAA of LS+6-BA, 0.1 mg/L+ sucrose 25g/L+ agar
5g/L, pH5.6~5.8.
4) root media are as follows: 1/2LS+NAA1.0mg/L+sucrose 30g/L+ agar 5g/L, pH5.6~5.8；
Remaining step, technique are the same as embodiment 1.
Embodiment 3
In this example, the preparation of step (1) culture medium, each component including minimal medium and each stage culture medium of tissue culture
With every liter contained by weight are as follows:
2) inducing clumping bud culture medium are as follows: 2.0 0.1 mg/L of mg/L+NAA of LS+6-BA+sucrose 20g/L+ fine jade
Rouge 8g/L, pH5.6~5.8
3) proliferation, strong seedling culture base are as follows: 1.0 mg/L+NAA of LS+6-BA, 0.2 mg/L+ sucrose 25g/L+ agar
5g/L, pH5.6~5.8.
4) root media are as follows: 1/2LS+NAA1.5mg/L+sucrose 30g/L+ agar 5g/L, pH5.6~5.8；
Remaining step, technique are the same as embodiment 1.
Claims (2)
1. a kind of quick breeding method for tissue culture of alum root blade, which comprises the following steps:
(1) preparation of culture medium
1) bud inducement cultivation base: 2.0 ~ 3.0 mg of LS+6-BA/0.1 ~ 0.2 mg/L of L+ NAA；
2) proliferated culture medium: LS+6-BA0.5 ~ 1.0 mg/0.1 ~ 0.2 mg/L of L+NAA；
3) root media: 0.5 ~ 1.5mg/L of 1/2LS+ NAA；
In LS and 1/2LS, 20~30g/L of sucrose, 5~8 g/L of agar are added, adjusts pH5.6~5.8；
(2) culture of alum root tissue-cultured seedling
1) explant selects: selecting explant of the alum root 1-2 leave piece of robust growth disease-free spot as tissue cultures, flows
Water rinses 1 ~ 2h, then clean with aseptic water washing on superclean bench, with 70% alcohol disinfecting 20-40s, 0.1% mercuric chloride solution
Immersion carries out sterilizing 6-8min, then multiple with aseptic water washing, spare；
2) explant prepared in step 1) the Fiber differentiation of Multiple Buds: is cut into 1 square centimeter small on superclean bench
Block is inoculated into bud inducement cultivation base and is cultivated；Condition of culture: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx,
Light application time 12h/d；
3) Multiple Buds of the inductive formation in step 2 are inoculated on proliferated culture medium cultivate and grow seedling；Cultivate item
Part: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；
4) it when growing to a height of 3. 0 ~ 5.0cm when cultivate the seedling grown in step 3), is inoculated into root media；Training
The condition of supporting: cultivation temperature is 25 ± 2 DEG C, and illumination light intensity is 1500Lx, light application time 12h/d；
5) when the seedling in step 4) grows to a height of 4 ~ 8cm and is longer than the root of 2cm at least 5, bottle outlet is obtained
The seedling of plantation.
2. the method according to claim 1, wherein further including following steps:
(3) hardening and transplanting of tissue-cultured seedling
Seedling band bottle described in step 5) is moved into greenhouse together, lid is first gone to place 2-5 days, bottle outlet simultaneously cleans root culture
Base is transplanted into peat: in mixed-matrix of the perlite by volume 3:1, managing to finished product seedling, is out of the garden.
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| CN107683770B (en) * | 2017-10-25 | 2019-11-26 | 上海市农业科学院 | A kind of breeding method of alum root ' smearing tea ' seedling |
| CN110122327B (en) * | 2018-02-02 | 2021-05-18 | 江苏省中国科学院植物研究所 | Method for establishing alum root rachis in-vitro regeneration system |
| CN108496798A (en) * | 2018-03-23 | 2018-09-07 | 贵州思源农旅综合开发有限公司 | A kind of tissue culture propagation method of alum root " kimonos " |
| CN108513911A (en) * | 2018-07-02 | 2018-09-11 | 杭州市园林绿化股份有限公司 | A kind of regenerated tissue cultures abductive approach of alum root petiole adventitious bud high frequency |
| CN109122315B (en) * | 2018-08-24 | 2021-06-29 | 上海市农业科学院 | Method for cultivating seedlings by using alum root and petioles |
| CN109430056B (en) * | 2018-11-19 | 2021-11-09 | 上海市农业科学院 | Method for inducing regeneration of adventitious buds of alum roots |
| CN112136609A (en) * | 2019-06-27 | 2020-12-29 | 贵州思源农旅综合开发有限公司 | Facility cultivation method of alum root tissue culture seedlings |
| CN110199885A (en) * | 2019-07-16 | 2019-09-06 | 上海市农业科学院 | A method of separation alum root secondary color leaf obtains pure color aseptic seedling |
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