CN109699497A - A kind of dendrobium candidum tissue cultural method - Google Patents
A kind of dendrobium candidum tissue cultural method Download PDFInfo
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Abstract
The present invention relates to a kind of dendrobium candidum tissue cultural methods, including Initial culture, Protocorm Multiplication culture, seedling strong seedling culture of growing directly from seeds, by introduced on the basis of MS culture medium coconut water, banana puree and dendrobium nobile leaf extract etc. provide be suitble to each cultivation stage without hormone culture-medium.The present invention is 1) without progress half-light culture in advance, the time needed for shortening entire cultivation cycle, and in the process without growth regulating is carried out using hormone, so that the growth course of dendrobium candidum accelerates reproduction speed while meeting the natural law；2) step needed for banana puree, coconut juice water and the dendrobium nobile leaf extract introduced is handled is simple, and not will increase the use cost of culture medium, is appropriate for promoting；3) survival rate is greatly improved after seedling replanting, can reach 98% or more.
Description
Technical field
The present invention relates to method for tissue culture technical field more particularly to a kind of dendrobium candidum tissue cultural methods.
Background technique
Dendrobium candidum is traditional rare traditional Chinese medicine, is first of nine big mesonas.
Its growing environment is extremely harsh: dendrobium officinale is mainly grown on the rock of overhanging cliff, in natural conditions
Lower seed germination rate is extremely low, very rare.
Modern organization culture technique is bred with enabling to the seed rapid, high volume of dendrobium candidum, enables to dendrobium candidum
Resource enriched.
The growth of seed can be generally adjusted using hormone in existing culture medium, such as: a- methyl α-naphthyl acetate, indolebutyric acid
And 6-benzyl aminopurine, etc. still, hormone being used for a long time and is adjusted and is easy to cause tissue-cultured seedling to morph, is some excellent
Shape can degenerate, so that the growth of dendrobium candidum does not meet the natural law, eventually lead to some medicinal shapes of dendrobium candidum
Shape disappears.
The dendrobium candidum seedling for more meeting the growth rhythm of dendrobium candidum using the culture medium prescription of no hormone, and being cultivated
With better survival rate, such as: 105325290 A of CN disclose a kind of stretch test without hormone method for tissue culture,
In be related to without hormone culture-medium formula.
The present invention is intended to provide under a kind of new thinking without hormone culture-medium formula, mentioned with quickly being bred to dendrobium candidum
For new selection.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of dendrobium candidum tissue cultural method,
By introducing coconut water, banana puree and dendrobium nobile leaf extract etc. on the basis of MS culture medium, provide a kind of using new nothing
The method of hormone culture-medium progress tissue cultures.
The present invention is achieved through the following technical solutions:
A kind of dendrobium candidum tissue cultural method,
S1 Initial culture:
Sterilized capsule is placed on sterile stainless steel disc, is cut off the part 0.3~0.5cm end to end and is discarded, then will
Capsule middle section is cut along the longitudinal axis, is taken out seed, is equably inoculated into Initial culture primary surface, is 27~29 DEG C in temperature, illumination
Intensity is 12~15d of culture culture under 2000~2400lx, with the naked eye can be observed have jade-green protocorm in media surface
Stem；
S2 Protocorm Multiplication culture:
After S1 is cultivated, continue temperature be 27~29 DEG C, intensity of illumination be 2000~2400lx under culture culture 20~
25d, most of protocorm have been sprouted, and the seedling that grows directly from seeds is formd, while also producing the protocorm of some new proliferation, then by this
The protocorms that are newly proliferated are transferred on proliferated culture medium a bit, continue in temperature to be 27~29 DEG C, and intensity of illumination is 2000~
20~25d of culture culture, protocorm form the new seedling that grows directly from seeds under 2400lx；
S3 grows directly from seeds seedling strong seedling culture:
The seedling that will grow directly from seeds is transferred on strong seedling culture base, is continued temperature is 26 DEG C, intensity of illumination is 2000~2500lx
45~60d is cultivated in lower culture, forms seedling；
Seedling in S3 is transferred to the greenhouse that shading rate is 75~80% and continues 15~18d of culture, then by it
It is transplanted to and continues to cultivate in matrix, can transplant after seedling grows 5~8 young leaves to crop field；
Wherein, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further including the coconut water for having 10~30%, 0.7~1%
Agar and 1~3% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 1.5~3% agar, 0.2~0.5% work
Property charcoal and 5~8% dendrobium nobile leaf extract；
Strong seedling culture base includes 1000ml MS culture medium, 1.5~3% agar, 0.3~0.6% active carbon, 5~20%
Banana puree and 3~6% dendrobium nobile leaf extract.
Further, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further including the coconut water for having 15~25%, 0.8~1%
Agar and 2~3% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 2~3% agar, 0.3~0.5% activity
Charcoal and 6~8% dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, wherein further include 2~3% agar, 0.4~0.6% activity
Charcoal, 10~15% banana puree and 4~5% dendrobium nobile leaf extract.
Further, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further include have 20% coconut water, 0.9% agar and
2.5% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further include 2.5% agar, 0.4% active carbon and
6% dendrobium nobile leaf extract；
Strong seedling culture base includes 1000ml MS culture medium, wherein further including 2.5% agar, 0.5% active carbon, 12%
Banana puree and 4.5% dendrobium nobile leaf extract.
Further, the preceding two panels young leaves by seedling since root is won, and uses group after adding 20ml water by 10g young leaves
It knits and filters to obtain dendrobium nobile leaf extract after bruisher is smashed to pieces.
Compared with prior art, the present invention has following usefulness:
1) this method is without progress half-light culture in advance, the time needed for shortening entire cultivation cycle, and in the process
Growth regulating (hormone needed for growth course is provided by coconut water, banana puree and dendrobium nobile leaf extract) is carried out without use hormone,
So that the growth course of dendrobium candidum accelerates reproduction speed while meeting the natural law；
2) step needed for banana puree, coconut juice water and the dendrobium nobile leaf extract introduced is handled is simple, and not will increase culture
The use cost of base is appropriate for promoting；
3) survival rate is greatly improved after seedling replanting, can reach 98% or more.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
A kind of dendrobium candidum tissue cultural method, comprising the following steps:
S1 Initial culture:
Sterilized capsule is placed on sterile stainless steel disc, is cut off the part 0.3cm end to end and is discarded, then will be in capsule
Section is cut along the longitudinal axis, is taken out seed, is equably inoculated into Initial culture primary surface, is 27 DEG C in temperature, intensity of illumination is
Culture culture 12d, with the naked eye can be observed have jade-green protocorm in media surface under 2000lx；
S2 Protocorm Multiplication culture:
After S1 is cultivated, continue in temperature to be 27 DEG C, intensity of illumination is culture culture 20d, most of protocorm under 2000lx
Stem has been sprouted, and the seedling that grows directly from seeds is formd, while also producing the protocorm of some new proliferation, then by the protocorm of these new proliferation
Stem is transferred on proliferated culture medium, continues in temperature to be 27 DEG C, and intensity of illumination is culture culture 20d, protocorm shape under 2000lx
The seedling that grows directly from seeds of Cheng Xin；
S3 grows directly from seeds seedling strong seedling culture:
The seedling that will grow directly from seeds is transferred on strong seedling culture base, and continuation is cultivated in the case where temperature is 26 DEG C, intensity of illumination is 2000lx
45d is cultivated, seedling is formed；
Wherein, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further include have 10% coconut water, 0.7% agar and
1% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further include 1.5% agar, 0.2% active carbon and
5% dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, 1.5% agar, 0.3% active carbon, 5% banana puree and
3% dendrobium nobile leaf extract；
Seedling in S3 is transferred to the greenhouse that shading rate is 75% to continue to cultivate 15d, is then transplanted to base
Continue to cultivate in matter, can transplant after seedling grows 5 young leaves to crop field.
Embodiment 2
A kind of dendrobium candidum tissue cultural method, comprising the following steps:
S1 Initial culture:
Sterilized capsule is placed on sterile stainless steel disc, is cut off the part 0.4cm end to end and is discarded, then will be in capsule
Section is cut along the longitudinal axis, is taken out seed, is equably inoculated into Initial culture primary surface, is 28 DEG C in temperature, intensity of illumination is
Culture culture 13d, with the naked eye can be observed have jade-green protocorm in media surface under 2200lx；
S2 Protocorm Multiplication culture:
After S1 is cultivated, continue in temperature to be 28 DEG C, intensity of illumination is culture culture 23d, most of protocorm under 2200lx
Stem has been sprouted, and the seedling that grows directly from seeds is formd, while also producing the protocorm of some new proliferation, then by the protocorm of these new proliferation
Stem is transferred on proliferated culture medium, continues in temperature to be 28 DEG C, and intensity of illumination is culture culture 23d, protocorm shape under 2200lx
The seedling that grows directly from seeds of Cheng Xin；
S3 grows directly from seeds seedling strong seedling culture:
The seedling that will grow directly from seeds is transferred on strong seedling culture base, and continuation is cultivated in the case where temperature is 26 DEG C, intensity of illumination is 2300lx
50d is cultivated, seedling is formed；
Wherein, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further include have 15% coconut water, 0.8% agar and
2% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 2% agar, 0.3% active carbon and 6%
Dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, wherein further include 2% agar, 0.4% active carbon, 10%
Banana puree and 4% dendrobium nobile leaf extract；
Seedling in S3 is transferred to the greenhouse that shading rate is 78% to continue to cultivate 16d, is then transplanted to base
Continue to cultivate in matter, can transplant after seedling grows 7 young leaves to crop field.
Embodiment 3
A kind of dendrobium candidum tissue cultural method, comprising the following steps:
S1 Initial culture:
Sterilized capsule is placed on sterile stainless steel disc, is cut off the part 0.5cm end to end and is discarded, then will be in capsule
Section is cut along the longitudinal axis, is taken out seed, is equably inoculated into Initial culture primary surface, is 29 DEG C in temperature, intensity of illumination is
Culture culture 15d, with the naked eye can be observed have jade-green protocorm in media surface under 2400lx；
S2 Protocorm Multiplication culture:
After S1 is cultivated, continue in temperature to be 29 DEG C, intensity of illumination is culture culture 25d, most of protocorm under 2400lx
Stem has been sprouted, and the seedling that grows directly from seeds is formd, while also producing the protocorm of some new proliferation, then by the protocorm of these new proliferation
Stem is transferred on proliferated culture medium, continues in temperature to be 29 DEG C, and intensity of illumination is culture culture 25d, protocorm shape under 2400lx
The seedling that grows directly from seeds of Cheng Xin；
S3 grows directly from seeds seedling strong seedling culture:
The seedling that will grow directly from seeds is transferred on strong seedling culture base, and continuation is cultivated in the case where temperature is 26 DEG C, intensity of illumination is 2500lx
60d is cultivated, seedling is formed；
Wherein, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further include have 20% coconut water, 0.9% agar and
2.5% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further include 2.5% agar, 0.4% active carbon and
6% dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, 2.5% agar, 0.5% active carbon, 12% banana puree and
4.5% dendrobium nobile leaf extract；
Seedling in S3 is transferred to the greenhouse that shading rate is 80% to continue to cultivate 18d, is then transplanted to base
Continue to cultivate in matter, can transplant after seedling grows 8 young leaves to crop field.
Embodiment 4
A kind of dendrobium candidum tissue cultural method, except the following contents is, remaining content is same as Example 1, specific different
Are as follows:
Initial culture base includes 1000ml MS culture medium, wherein further including having 25% coconut water, 1% agar and 3%
Dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 3% agar, 0.5% active carbon and 8%
Dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, wherein further include 3% agar, 0.6% active carbon, 15%
Banana puree and 5% dendrobium nobile leaf extract.
In Examples 1 to 4, preceding two panels young leaves of the seedling since root is won, after adding 20ml water by 10g young leaves
Filtered after being smashed to pieces with tissue mashing machine dendrobium nobile leaf extract for configure culture medium；Banana puree is using well-done but unputrefied perfume (or spice)
Any of several broadleaf plants, gross weight mashed into paste are spare (can provide a greater amount of natural growth hormone in this way)；Coconut water is broken using mature coconut fruit
It is spare to open collection.
In above-described embodiment 1~4, culture medium with a thickness of 3cm in when Initial culture each culture dish；Protocorm Multiplication training
Support and be transferred in culture bottle and carry out, and in culture bottle culture medium with a thickness of 7cm, 3 plants every bottle；Seedling strong seedling culture of growing directly from seeds changes bottle
Carry out, and in culture bottle culture medium with a thickness of 8cm, 1 plant every bottle.
In order to reduce workload, the error caused by repeatedly weighing is reduced, common drug is made into higher than required concentration
10-100 times of mother liquor.MS culture medium difference mother liquor method is as shown in the table:
(1) a great number of elements mother liquor
A great number of elements is weighed according to 10 times of numerical value high when using, and after respectively weighing various compounds, removes CaCl2·
2H2Outside O is individually prepared, remaining compound is blended in 500mL beaker plus the dissolution of appropriate distilled water, stirs dissolution with glass bar,
It pours into 1000mL volumetric flask and is settled to scale with distilled water, set in bottle and save, labeled indicating compound name (or
Number), cycles of concentration prepares date and makers-up's name, CaCl2·2H2O, which is prepared, to be ibid placed in another bottle.
(2) preparation of microelement mother liquor
100 times of numerical value is concentrated as required to weigh, various compounds is weighed remove molysite (FeSO respectively4·7H2O and Na2-
EDTA.2H2O) individually prepared as one group it is outer, remaining compound can mix be placed in beaker plus the dissolution of a small amount of distilled water after, it is fixed
Hold in 1000mL volumetric flask, sets in bottle and save, it is labelled.
(3) molysite is prepared
By FeSO4·7H2O and Na2-EDTA.2H2O is dissolved in respectively in 450mL distilled water, and heating is stirred continuously, after dissolution
Mixing is placed in water-bath and heats (50 DEG C) and be stirred continuously to solution in golden yellow (about 20~30min of heating), adds water constant volume
To 1000mL, pH value is adjusted to be placed in bottle to 5.5, it is labelled be placed at room temperature for it is cooling after, then refrigerate.
(4) organic matter mother liquor
50 times, after remaining is weighed respectively are concentrated by mother liquor requirement, dissolution, constant volume is placed in bottle in 500mL volumetric flask
Middle preservation, it is labelled.
Mother liquor is stored in 2~4 DEG C of refrigerator, especially organic species, unsuitable too long, the inorganic salts mother liquor of period of storage
It is preferably finished in one month, if discovery has mould and precipitating to generate, cannot reuse.
It is 100% that seedling, which is transferred to greenhouse to continue the survival rate of culture, in Examples 1 to 4, continues Statistics Implementation
In example 1~4 seedling from greenhouse be transplanted to crop field after 2nd month, the 6th month and 12nd month when dendrobium candidum growth shape
Condition see the table below:
| 2nd month | 6th month | 12nd month | |
| Embodiment 1 | Survival rate 98.2%, 10~12cm of plant height | 15~18cm of plant height | Plant height 25cm or more |
| Embodiment 2 | Survival rate 98.4%, 11~14cm of plant height | 17~20cm of plant height | Plant height 25cm or more |
| Embodiment 3 | Survival rate 98.9%, 11~13cm of plant height | 15~19cm of plant height | Plant height 25cm or more |
| Embodiment 4 | Survival rate 98.7%, 10~15cm of plant height | 15~20cm of plant height | Plant height 25cm or more |
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (5)
1. a kind of dendrobium candidum tissue cultural method, which comprises the following steps:
S1 Initial culture:
Sterilized capsule is placed on sterile stainless steel disc, is cut off the part 0.3~0.5cm end to end and is discarded, then by capsule
Middle section is cut along the longitudinal axis, is taken out seed, is equably inoculated into Initial culture primary surface, is 27~29 DEG C in temperature, intensity of illumination
For 12~15d of culture culture under 2000~2400lx, it with the naked eye can be observed have jade-green protocorm in media surface；
S2 Protocorm Multiplication culture:
After S1 is cultivated, continuing in temperature to be 27~29 DEG C, intensity of illumination is 20~25d of culture culture under 2000~2400lx,
Most of protocorm has been sprouted, and the seedling that grows directly from seeds is formd, while also producing the protocorm of some new proliferation, then that these are new
The protocorm of proliferation is transferred on proliferated culture medium, continues in temperature to be 27~29 DEG C, intensity of illumination is under 2000~2400lx
Culture 20~25d of culture, protocorm form the new seedling that grows directly from seeds；
S3 grows directly from seeds seedling strong seedling culture:
The seedling that will grow directly from seeds is transferred on strong seedling culture base, and continuation is trained in the case where temperature is 26 DEG C, intensity of illumination is 2000~2500lx
45~60d of culture is supported, seedling is formed；
Wherein, by weight,
Initial culture base includes 1000ml MS culture medium, wherein further including the agar of the coconut water for having 10~30%, 0.7~1%
With 1~3% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 1.5~3% agar, 0.2~0.5% active carbon
With 5~8% dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, 1.5~3% agar, 0.3~0.6% active carbon, 5~20% perfume (or spice)
Any of several broadleaf plants mud and 3~6% dendrobium nobile leaf extract.
2. a kind of dendrobium candidum tissue cultural method according to claim 1, which is characterized in that by weight,
Initial culture base includes 1000ml MS culture medium, wherein further including the agar of the coconut water for having 15~25%, 0.8~1%
With 2~3% dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further include 2~3% agar, 0.3~0.5% active carbon and
6~8% dendrobium nobile leaf extract；
Strong seedling culture base include 1000ml MS culture medium, wherein further include 2~3% agar, 0.4~0.6% active carbon, 10
~15% banana puree and 4~5% dendrobium nobile leaf extract.
3. a kind of dendrobium candidum tissue cultural method according to claim 2, which is characterized in that by weight,
Initial culture base includes 1000ml MS culture medium, wherein further including having 20% coconut water, 0.9% agar and 2.5%
Dendrobium nobile leaf extract；
Proliferated culture medium includes 1000ml MS culture medium, wherein further including 2.5% agar, 0.4% active carbon and 6%
Dendrobium nobile leaf extract；
Strong seedling culture base includes 1000ml MS culture medium, wherein further include 2.5% agar, 0.5% active carbon, 12% perfume (or spice)
Any of several broadleaf plants mud and 4.5% dendrobium nobile leaf extract.
4. any a kind of dendrobium candidum tissue cultural method according to claim 1~3, which is characterized in that will be in S3
Seedling be transferred to shading rate be 75~80% greenhouse continue 15~18d of culture, be then transplanted in matrix after
Continuous culture, can transplant after seedling grows 5~8 young leaves to crop field.
5. a kind of dendrobium candidum tissue cultural method according to claim 4, which is characterized in that by seedling since root
Preceding two panels young leaves win, add by 10g young leaves and filter to obtain dendrobium nobile leaf extract after being smashed to pieces after 20ml water with tissue mashing machine.
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