CN108308039A - Tissue culture medium (TCM) and its preparation method and application for dendrobium candidum tissue culture - Google Patents
Tissue culture medium (TCM) and its preparation method and application for dendrobium candidum 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 tissue culture medium (TCM) and its preparation method and application for dendrobium candidum tissue culture, which includes separate inducing culture, proliferated culture medium, root media；Wherein, inducing culture includes B5 medium, 6 BA, IAA, adenine, folic acid, vitamin B2；Proliferated culture medium includes B5 medium, 6 BA, IBA, streptomysin, mashed potatoes, coconut juice；Root media includes 1/2 B5 medium, CPA, GA3, NAA, lactoalbumin hydrolysate, beef broth, activated carbon.The tissue culture medium (TCM) fast and efficiently can obtain dendrobium candidum by tissue cultures, while the preparation method process is simple and raw material is easy to get.
Description
Technical field
The present invention relates to tissue culture medium (TCM)s, and in particular, to a kind of tissue culture medium (TCM) for dendrobium candidum tissue culture
And its preparation method and application.
Background technology
Dendrobium candidum is a kind of herbaceos perennial that happiness is shady and cool, happiness it is warm, moist, with 1000 millimeters of annual rainfall
Above, the environment of half cloudy half light, 1 monthly mean temperature is grown in the subtropical zone remote, thickly forested mountains higher than 8 DEG C to be preferred, suitable growth temperature
For 15 to 28 degree, suitable growth air humidity is 60% or more, not very stringent to clay fertilizer requirement, wild mostly in loose and thick tree
It is grown on skin or trunk, some is also grown in crack of stone.Dendrobium candidum category aerial root system, major requirement root permeability is good, adopts
Matrix preferably ventilated drainage, under suitable temperature humidity, the speed of growth is fast, and survival ability is very strong.Every year
The end of spring and the beginning of summer, biennial stem top, which is saved, extracts inflorescence out, and Post flowering grows sprouting from stem foot and develops into stem, and autumn and winter, which enters, stops
The dormancy phase.
The average Natural seed setting rate of dendrobium candidum is only 0.31%, wherein when not being fully deployed also with same day sepal of blooming
The spontaneous pollination success rate of artificial pollination setting percentage highest, dendrobium candidum is only 30% or so, while the wild seed of dendrobium candidum
Survival rate it is very low；To sum up several factors and then results in dendrobium candidum there is larger difficulties in reproductive process.
Invention content
The object of the present invention is to provide a kind of tissue culture medium (TCM) for dendrobium candidum tissue culture and preparation method thereof and
Using the tissue culture medium (TCM) fast and efficiently can obtain dendrobium candidum, while the preparation method process letter by tissue cultures
Single and raw material is easy to get.
To achieve the goals above, the present invention provides a kind of tissue culture medium (TCM)s for dendrobium candidum tissue culture, should
Tissue culture medium (TCM) includes separate inducing culture, proliferated culture medium, root media；
Wherein, inducing culture include B5 medium, 0.8-1.2mg/L 6-BA, 0.1-0.3mg/L IAA, 0.01-
The vitamin B2 of the adenine of 0.05mg/L, the folic acid of 0.01-0.03mg/L, 0.04-0.06mg/L；
Proliferated culture medium include B5 medium, 0.2-0.5mg/L 6-BA, 0.05-0.1mg/L IBA, 0.05-0.08mg/L
Streptomysin, the mashed potatoes of 15-20g/L, 4-8g/L coconut juice；
Root media include 1/2 B5 medium, 0.4-0.8mg/L CPA, 0.02-0.06mg/L GA3、0.01-
The activated carbon of the lactoalbumin hydrolysate of NAA, 0.2-0.4mg/L of 0.05mg/L, the beef broth of 2-6g/L, 7-10g/L.
The present invention also provides the preparation method of the above-mentioned tissue culture medium (TCM) for dendrobium candidum tissue culture, the preparations
Method is to mix each component in inducing culture, proliferated culture medium, root media.
Invention further provides a kind of as the above-mentioned tissue culture medium (TCM) for dendrobium candidum tissue culture is iron
Application in skin stem of noble dendrobium tissue cultures.
In the above-mentioned technical solutions, the present invention is by adjusting in each inducing culture, proliferated culture medium, root media
Component and content simultaneously should so that the tissue culture medium (TCM) fast and efficiently can obtain dendrobium candidum by tissue cultures
Preparation method process is simple and raw material is easy to get.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific implementation mode
The specific implementation mode of the present invention is described in detail below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of tissue culture medium (TCM) for dendrobium candidum tissue culture, which includes respective
Independent inducing culture, proliferated culture medium, root media；
Wherein, inducing culture includes the 6-BA of B5 medium, 0.8-1.2mg/L（6- benzyl aminoadenines）、0.1-0.3mg/L
IAA（Heteroauxin）, the adenine of 0.01-0.05mg/L, the folic acid of 0.01-0.03mg/L, 0.04-0.06mg/L dimension life
Plain B2；
Proliferated culture medium include B5 medium, 0.2-0.5mg/L 6-BA, 0.05-0.1mg/L IBA（Indolebutyric acid）、
The coconut juice of the streptomysin of 0.05-0.08mg/L, the mashed potatoes of 15-20g/L, 4-8g/L；
Root media includes the CPA of 1/2 B5 medium, 0.4-0.8mg/L（Cyproterone acetate）, 0.02-0.06mg/L
GA3（Gibberellin）, 0.01-0.05mg/L NAA（Methyl α-naphthyl acetate）, the lactoalbumin hydrolysate of 0.2-0.4mg/L, 2-6g/L beef
The activated carbon of juice, 7-10g/L.
In above-mentioned each culture medium, the range of pH can select in a wide range, but in order to enable the culture medium
Promote the tissue cultures of dendrobium candidum, it is preferable that inducing culture, proliferated culture medium, root media are satisfied by the following conditions：
PH is 5.3-5.5.
Wherein, the mode of the adjusting of pH can also select in a wide range, but consider from the stability of culture medium,
Preferably, the adjusting of pH of inducing culture, proliferated culture medium, root media is carried out by the way of buffer solution.
In the medium of the present invention, in order to preferably for it is each tissue excellent nutrient and suitable culture item are provided
Part, it is preferable that inducing culture, proliferated culture medium and root media agar and 25-30g/L containing 6.5-7.0g/L
Sucrose.
In the present invention, in order to enable inducing culture can advantageously promote the tissue cultures of dendrobium candidum, it is preferable that
The use condition of inducing culture is：It is carried out in the case of alternation of light and darkness, illumination is existed simultaneously using incandescent lamp and blue-ray light
Lower progress, alternation of light and darkness condition are：Light application time is 12-14h/ days, and interlunation is 10-12h/ days, intensity of illumination 1700-
1900lux, cultivation temperature are 21-23 DEG C, relative humidity 50-60%.
In the present invention, in order to enable proliferated culture medium can advantageously promote the tissue cultures of dendrobium candidum, it is preferable that
Preferably, the use condition of proliferated culture medium is：It is carried out in the case of alternation of light and darkness, illumination is same using incandescent lamp and ultraviolet light
When in the presence of carry out, alternation of light and darkness condition is：Light application time is 12-14h/ days, and interlunation is 10-12h/ days, intensity of illumination
For 2000-2100lux, cultivation temperature is 21-23 DEG C, relative humidity 50-60%.
In the present invention, in order to enable root media can advantageously promote the tissue cultures of dendrobium candidum, it is preferable that
The use condition of root media is：It is carried out in the case of alternation of light and darkness, illumination is using natural light, alternation of light and darkness condition：
Light application time is 12-14h/ days, and interlunation is 10-12h/ days, intensity of illumination 2000-2100lux, cultivation temperature 21-
23 DEG C, relative humidity 50-60%.
The present invention also provides the preparation method of the above-mentioned tissue culture medium (TCM) for dendrobium candidum tissue culture, the preparations
Method is to mix each component in inducing culture, proliferated culture medium, root media.
Invention further provides a kind of as the above-mentioned tissue culture medium (TCM) for dendrobium candidum tissue culture is iron
Application in skin stem of noble dendrobium tissue cultures.
The present invention will be described in detail by way of examples below.
Embodiment 1
1）By the stem eye of dendrobium candidum（Length is 0.5cm）It carries out disinfection（The stem eye is first placed in 65 weight % alcohol water blends
Middle immersion 40s then places it in 4 weight % aqueous sodium hypochlorite solutions and impregnates 3min, cleaning 4 is carried out finally by distilled water
It is secondary）To obtain explant；
2）Explant is placed in inducing culture（IAA, 0.03mg/L including B5 medium, 6-BA, 0.2mg/L of 1mg/L
The sucrose of adenine, the folic acid of 0.02mg/L, the vitamin B2 of 0.05mg/L, the agar of 6.8g/L and 28g/L, pH 5.4）In
Carry out Fiber differentiation（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and blue-ray light, cultivates
Time is 13 days；Alternation of light and darkness condition is：Light application time is 13h/ days, and interlunation is 11h/ days, and intensity of illumination is
1800lux, cultivation temperature are 22 DEG C, relative humidity 55%）To obtain protocorm；
3）Protocorm is placed in proliferated culture medium（IBA, 0.07mg/ including B5 medium, 6-BA, 0.08mg/L of 0.4mg/L
The sucrose of the streptomysin of L, the mashed potatoes of 18g/L, the coconut juice of 6g/L, the agar of 6.8g/L and 28g/L, pH 5.4）In increased
Grow culture（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and ultraviolet light, and incubation time is
18 days；Alternation of light and darkness condition is：Light application time is 13h/ days, and interlunation is 11h/ days, intensity of illumination 2050lux, culture
Temperature is 22 DEG C, relative humidity 55%）To obtain proliferation seedling；
4）Proliferation seedling is placed in root media（GA including 1/2 B5 medium, CPA, 0.04mg/L of 0.6mg/L3、
The lactoalbumin hydrolysate of NAA, 0.3mg/L of 0.03mg/L, the beef broth of 4g/L, the activated carbon of 8g/L, 6.8g/L agar and
The sucrose of 28g/L, pH 5.4）Middle carry out culture of rootage（It is carried out in the case of alternation of light and darkness, illumination uses natural light, culture
Time is 8 days；Alternation of light and darkness condition is：Light application time is 13h/ days, and interlunation is 11h/ days, intensity of illumination 2050lux,
Cultivation temperature is 22 DEG C, relative humidity 55%）To obtain the seedling of dendrobium candidum.
Embodiment 2
1）By the stem eye of dendrobium candidum（Length is 0.4cm）It carries out disinfection（The stem eye is first placed in 60 weight % alcohol water blends
Middle immersion 30s then places it in 3 weight % aqueous sodium hypochlorite solutions and impregnates 3min, cleaning 3 is carried out finally by distilled water
It is secondary）To obtain explant；
2）Explant is placed in inducing culture（IAA, 0.01mg/L including B5 medium, 6-BA, 0.1mg/L of 0.8mg/L
Adenine, the folic acid of 0.01mg/L, the vitamin B2 of 0.04mg/L, the agar of 6.5g/L and the sucrose of 25g/L, pH 5.3）
Middle carry out Fiber differentiation（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and blue-ray light, trains
It is 10 days to support the time；Alternation of light and darkness condition is：Light application time is 12h/ days, and interlunation is 10h/ days, and intensity of illumination is
1700lux, cultivation temperature are 21 DEG C, relative humidity 50%）To obtain protocorm；
3）Protocorm is placed in proliferated culture medium（IBA, 0.05mg/ including B5 medium, 6-BA, 0.05mg/L of 0.2mg/L
The sucrose of the streptomysin of L, the mashed potatoes of 15g/L, the coconut juice of 4g/L, the agar of 6.5g/L and 25g/L, pH 5.3）In increased
Grow culture（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and ultraviolet light, and incubation time is
15 days；Alternation of light and darkness condition is：Light application time is 12h/ days, and interlunation is 10h/ days, intensity of illumination 2000lux, culture
Temperature is 21 DEG C, relative humidity 50%）To obtain proliferation seedling；
4）Proliferation seedling is placed in root media（GA including 1/2 B5 medium, CPA, 0.02mg/L of 0.4mg/L3、
The lactoalbumin hydrolysate of NAA, 0.2mg/L of 0.01mg/L, the beef broth of 2g/L, the activated carbon of 7g/L, 6.5g/L agar and
The sucrose of 25g/L, pH 5.3）Middle carry out culture of rootage（It is carried out in the case of alternation of light and darkness, illumination uses natural light, culture
Time is 7 days；Alternation of light and darkness condition is：Light application time is 12h/ days, and interlunation is 10h/ days, intensity of illumination 2000lux,
Cultivation temperature is 21 DEG C, relative humidity 50%）To obtain the seedling of dendrobium candidum.
Embodiment 3
1）By the stem eye of dendrobium candidum（Length is 0.6cm）It carries out disinfection（The stem eye is first placed in 70 weight % alcohol water blends
Middle immersion 60s then places it in 5 weight % aqueous sodium hypochlorite solutions and impregnates 4min, cleaning 5 is carried out finally by distilled water
It is secondary）To obtain explant；
2）Explant is placed in inducing culture（IAA, 0.05mg/L including B5 medium, 6-BA, 0.3mg/L of 1.2mg/L
Adenine, the folic acid of 0.03mg/L, the vitamin B2 of 0.06mg/L, the agar of 7.0g/L and the sucrose of 30g/L, pH 5.5）
Middle carry out Fiber differentiation（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and blue-ray light, trains
It is 15 days to support the time；Alternation of light and darkness condition is：Light application time is 14h/ days, and interlunation is 12h/ days, and intensity of illumination is
1900lux, cultivation temperature are 23 DEG C, relative humidity 60%）To obtain protocorm；
3）Protocorm is placed in proliferated culture medium（IBA, 0.08mg/L including B5 medium, 6-BA, 0.1mg/L of 0.5mg/L
Streptomysin, the mashed potatoes of 20g/L, the coconut juice of 8g/L, the agar of 7.0g/L and the sucrose of 30g/L, pH 5.5）In increased
Grow culture（It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress using incandescent lamp and ultraviolet light, and incubation time is
20 days；Alternation of light and darkness condition is：Light application time is 14h/ days, and interlunation is 12h/ days, intensity of illumination 2100lux, culture
Temperature is 23 DEG C, relative humidity 60%）To obtain proliferation seedling；
4）Proliferation seedling is placed in root media（GA including 1/2 B5 medium, CPA, 0.06mg/L of 0.8mg/L3、
The lactoalbumin hydrolysate of NAA, 0.4mg/L of 0.05mg/L, the beef broth of 6g/L, the activated carbon of 10g/L, 7.0g/L agar and
The sucrose of 30g/L, pH 5.5）Middle carry out culture of rootage（It is carried out in the case of alternation of light and darkness, illumination uses natural light, culture
Time is 10 days；Alternation of light and darkness condition is：Light application time is 14h/ days, and interlunation is 12h/ days, and intensity of illumination is
2100lux, cultivation temperature are 23 DEG C, relative humidity 60%）To obtain the seedling of dendrobium candidum.
Comparative example 1
It is carried out according to the method for embodiment 1, except that inducing culture is：Including B5 medium, 0.6mg/L 6-BA,
The agar of the adenine of IAA, 0.008mg/L of 0.08mg/L, the folic acid of 0.008mg/L, the vitamin B2 of 0.03mg/L, 6g/L
With the sucrose of 20g/L.
Comparative example 2
It is carried out according to the method for embodiment 1, except that inducing culture is：Including B5 medium, 1.3mg/L 6-BA,
The agar of the adenine of IAA, 0.06mg/L of 0.4mg/L, the folic acid of 0.05mg/L, the vitamin B2 of 0.07mg/L, 7.2g/L
With the sucrose of 32g/L.
Comparative example 3
It is carried out according to the method for embodiment 1, except that root media is：Including 1/2 B5 medium, 0.3mg/L
The GA of CPA, 0.01mg/L3, the lactoalbumin hydrolysate of NAA, 0.1mg/L of 0.008mg/L, the beef broth of 1g/L, 5g/L activity
The sucrose of charcoal, the agar of 6g/L and 20g/L.
Comparative example 4
It is carried out according to the method for embodiment 1, except that root media is：Including 1/2 B5 medium, 1mg/L CPA,
The GA of 0.08mg/L3, the lactoalbumin hydrolysate of NAA, 0.5mg/L of 0.08mg/L, the beef broth of 7g/L, 11g/L activated carbon,
The agar of 8g/L and the sucrose of 28g/L.
As a result it counts：
It is tested respectively in above-described embodiment and comparative example with the dendrobium candidum stem with bud of 20cm length, then calculates step
Rapid 3）In proliferation rate and step 4）In survival rate, concrete outcome is shown in Table 1.
Table 1
| Proliferation rate | Survival rate % | |
| Embodiment 1 | 1：45 | 95 |
| Embodiment 2 | 1：43 | 98 |
| Embodiment 3 | 1：44 | 96 |
| Comparative example 1 | 1：8 | 97 |
| Comparative example 2 | 1：16 | 95 |
| Comparative example 3 | 1：42 | 48 |
| Comparative example 4 | 1：43 | 65 |
The preferred embodiment of the present invention has been described above in detail, still, the tool during present invention is not limited to the embodiments described above
Body details can carry out a variety of simple variants, these letters to technical scheme of the present invention within the scope of the technical concept of the present invention
Monotropic type all belongs to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (9)
1. a kind of tissue culture medium (TCM) for dendrobium candidum tissue culture, which is characterized in that the tissue culture medium (TCM) includes respective
Independent inducing culture, proliferated culture medium, root media；
Wherein, the inducing culture include B5 medium, 0.8-1.2mg/L 6-BA, 0.1-0.3mg/L IAA, 0.01-
The vitamin B2 of the adenine of 0.05mg/L, the folic acid of 0.01-0.03mg/L, 0.04-0.06mg/L；
The proliferated culture medium include B5 medium, 0.2-0.5mg/L 6-BA, 0.05-0.1mg/L IBA, 0.05-
The coconut juice of the streptomysin of 0.08mg/L, the mashed potatoes of 15-20g/L, 4-8g/L；
The root media include 1/2 B5 medium, 0.4-0.8mg/L CPA, 0.02-0.06mg/L GA3、0.01-
The activated carbon of the lactoalbumin hydrolysate of NAA, 0.2-0.4mg/L of 0.05mg/L, the beef broth of 2-6g/L, 7-10g/L.
2. tissue culture medium (TCM) according to claim 1, wherein the inducing culture, proliferated culture medium, root media
It is satisfied by the following conditions：PH is 5.3-5.5.
3. tissue culture medium (TCM) according to claim 1, wherein the inducing culture, proliferated culture medium, root media
The adjusting of pH carried out by the way of buffer solution.
4. tissue culture medium (TCM) according to claim 1, wherein the inducing culture, proliferated culture medium and training of taking root
The sucrose of foster the base agar containing 6.5-7.0g/L and 25-30g/L.
5. according to the tissue culture medium (TCM) described in any one of claim 1-4, wherein the use condition of the inducing culture
For：It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress, alternation of light and darkness condition using incandescent lamp and blue-ray light
For：Light application time is 12-14h/ days, and interlunation is 10-12h/ days, intensity of illumination 1700-1900lux, and cultivation temperature is
21-23 DEG C, relative humidity 50-60%.
6. according to the tissue culture medium (TCM) described in any one of claim 1-4, wherein the use condition of the proliferated culture medium
For：It is carried out in the case of alternation of light and darkness, illumination exists simultaneously lower progress, alternation of light and darkness condition using incandescent lamp and ultraviolet light
For：Light application time is 12-14h/ days, and interlunation is 10-12h/ days, intensity of illumination 2000-2100lux, and cultivation temperature is
21-23 DEG C, relative humidity 50-60%.
7. according to the tissue culture medium (TCM) described in any one of claim 1-4, wherein the use condition of the root media
For：It is carried out in the case of alternation of light and darkness, illumination is using natural light, alternation of light and darkness condition：Light application time is 12-14h/ days,
Interlunation is 10-12h/ days, intensity of illumination 2000-2100lux, and cultivation temperature is 21-23 DEG C, relative humidity 50-
60%。
8. a kind of preparation of tissue culture medium (TCM) for dendrobium candidum tissue culture as described in any one of claim 1-7
Method, which is characterized in that the preparation method is that by each group in the inducing culture, proliferated culture medium, root media
Divide and is mixed.
9. a kind of tissue culture medium (TCM) for dendrobium candidum tissue culture as described in any one of claim 1-7 is iron
Application in skin stem of noble dendrobium tissue cultures.
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| CN111066656A (en) * | 2020-01-16 | 2020-04-28 | 贵州师范大学 | Culture medium group and method for dendrobium officinale tissue culture |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111066656A (en) * | 2020-01-16 | 2020-04-28 | 贵州师范大学 | Culture medium group and method for dendrobium officinale tissue culture |
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