CN108450328A - A kind of crocodile mouth flower quick breeding method for tissue culture - Google Patents
A kind of crocodile mouth flower quick breeding method for 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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of crocodile mouths to spend quick breeding method for tissue culture.The present invention can obtain a large amount of crocodile mouth flower cultivation seedling in a short time, and to realize effective protection and the breeding to the species, and the method for the present invention is easy to operate, and easy to implement, culture medium and reagent used easily configures, at low cost, easily a wide range of to promote.
Description
Technical field
The invention belongs to field of plant reproduction, and in particular to a kind of crocodile mouth flower quick breeding method for tissue culture.
Background technology
Crocodile mouth flower (Clinacanthus nutans) is that Acanthaceae (Acanthaceae) crocodile mouth flower belongs to tall and big herbaceous plant,
The ground such as Guangdong, Guangxi, Yunnan are distributed at present, certain region is also presented in application method, in Yunnan mainly as one kind
Traditional dai medicine is then used as a kind of wild vegetable, integration of drinking and medicinal herbs to treat liver more for treating traumatic injury in Guangdong, Guangxi province
Disease, rheumatism etc..Modern research shows that crocodile mouth flower is with good anticancer, anti-inflammatory, immunological regulation, reducing blood lipid and other effects.
It is cultivated on solid medium using plant explant, obtains callus or intact plant, as plant tissue
Culture, also referred to as cultured in vitro or explant culture.Due to different types of plant in the required condition of culture of cultured in vitro and
There are prodigious differences for method etc., according to specific plant, adjustment type of culture medium, culture illumination and temperature, different trainings
The stage of supporting culture medium growth regulator proportioning, the formation of evoking adventive bud, establishes effective bud brood body on this basis respectively
System and plant regeneration system.Carry out the protection of plant with plant tissue culture technique at present and breeds on many plant species
It succeeds.
But up to the present, the research in terms of the tissue cultures of crocodile mouth flower, domestic there is not been reported.
Invention content
The object of the present invention is to provide a kind of rapid propagation methods that can obtain a large amount of crocodile mouth flower cultivation seedlings in a short time.
The crocodile mouth of the present invention spends quick breeding method for tissue culture, includes the following steps：
The induction of S1, axillary bud：It chooses crocodile mouth scape section and is used as explant, be inoculated in inducing culture after sterilization,
Until inducing axillary bud；
The induction of S2, adventitious bud：Gained axillary bud is accessed in inducing culture and is cultivated, until inducing adventitious bud；
S3, shoot proliferation：Gained adventitious bud is accessed in proliferated culture medium to the proliferation for carrying out Multiplying culture and carrying out adventitious bud；
S4, strong seedling culture：Culture in the adventitious bud access strong seedling culture base of gained proliferation is obtained into unrooted strong sprout；
S5, culture of rootage：Gained unrooted strong sprout is accessed into culture in root media and obtains tissue-cultured seedling；
S6, gained tissue-cultured seedling is subjected to transplanting culture；
Wherein, the condition of culture in S1~S5 is 22~28 DEG C, 50~80 μm of ol m of light intensity-2s-1, illumination 14h/d；
The pH of every liter of inducing culture is 5.9, is contained：1.0mg TDZ or 1.0~2.0mg BAP, 0.1mg NAA,
Surplus is MS culture mediums；
The pH of every liter of proliferated culture medium is 5.9, is contained：1.0~3.0mg TDZ or 1.0~3.0mg BAP, 0.1mg
NAA or 0.1mg IBA, surplus are MS culture mediums；
The pH of every liter of strong seedling culture base is 5.9, is contained：Peptone 0.5g/L or coconut juice 30g/L or banana 30g/L,
Surplus is MS culture mediums；
The pH of every liter of root media is 5.9, is contained：1g activated carbons and 2.0~4.0mg IBA or 0.1~0.5mg
NAA, surplus are MS culture mediums.
The explant sterilization is will to be cleaned with tap water as the stem section of explant, first with volume fraction for 75%
Alcoholic solution surface clean disinfection 10s, then aseptic water washing 2 times, then mass-volume concentration be 0.1% mercuric chloride solution
Middle immersion 8min, then uses aseptic water washing 3 times, and stem section is finally cut into 1cm long, obtains the explant of sterilization treatment.
The transplanting culture is that tissue-cultured seedling is transplanted to peat soil：Coconut palm chaff：Perlite：River sand is with 3:2:1:1 volume ratio
In the matrix of composition, it is placed in moistening shade (relative humidity is 30~50%, light transmittance is 40~50%), 20~25 DEG C of environment
Lower culture, the composite fertilizer per week using 1wt ‰ are primary, grow learned moisture and nutrition to meet tissue-cultured seedling, are cultivated
Seedling；N in the composite fertilizer:P:The mass ratio of K is 1:1:1.
MS culture mediums in the present invention are international culture medium, ingredient and preparation method can referring to Tan Wencheng, wear
Plan is just edited,《Ornamental plant tissue culture technique》, Beijing：China Forestry Publishing House, 1991.
Since crocodile mouth scape section differentiation capability is poor, the ability of evoking adventive bud is poor.Therefore the present invention will be with site
Material of the axillary bud that stem section induces as evoking adventive bud, effect are very ideal.Stem section with site is inoculated in induction training
It supports in base, general 10d can induce axillary bud.Axillary bud is inoculated in inducing culture, general 30d can be induced not
Normal bud is transferred to shoot proliferation, general one month can be primary with subculture, and the adventitious bud of proliferation carries out strong sprout training in strong seedling culture base
It supports, is transferred in root media after 20d, 20d can take root for forming tissue-cultured seedling, general one month in tissue culture transplantation of seedlings to matrix
After can grow to 5~6cm high, obtain cultivation seedling.
Due to the tissue-cultured seedling very little of crocodile mouth flower, it is usually no more than 3cm, dehydration is easy to after being transplanted to outdoor or nutrient lacks
It is weary and dead.Therefore tissue-cultured seedling is migrated to peat soil by the present invention：Coconut palm chaff：Perlite：River sand volume ratio is 3:2:1:1 mixing
It in matrix, is placed in the environment of moistening, concealment and cultivates, water, fertilising is to meet the moisture and nutrition need needed for tissue-cultured seedling growth
It wants, ensures that tissue-cultured seedling has higher survival rate.
Compared with prior art, of the invention to has the following advantages：
The present invention from explant be induced to obtain cultivation seedling only need 140d, greatly accelerate crocodile mouth flower breeding speed
Degree, by shoot proliferation, can be proliferated 6.07 times or so every month, therefore can obtain a large amount of tissue-cultured seedling in the short term,
For tissue-cultured seedling after transplanting, survival rate is up to 97% or more, the Chinese herbal medicine so as to make crocodile mouth spend this traditional and novel
Vegetables are able to preserve and develop, further to be laid a good foundation from now on using the species.
The method of the present invention is easy to operate, easy to implement, and culture medium used and reagent easily configure, at low cost, easily a wide range of
It promotes.
Description of the drawings
Fig. 1 is the rapid propagation in vitro photo of crocodile mouth scape section in embodiment 1.Wherein, A：Crocodile mouth scape section is in MS+1.0mg/L
20d or so is cultivated on TDZ culture mediums；B：Axillary bud cultivates the bud clump after 30d on MS+1.0mg/L TDZ culture mediums；D：Axillary bud exists
The bud clump formed after 30d is cultivated on organic additive peptone and MS culture mediums；E：Axillary bud is in MS+1.0mg/L BAP+0.1mg/
It is proliferated on L NAA culture mediums；G：On improvement MS+3.0mg/L IBA culture mediums after culture 30d, root system is formed sprout；H：Crocodile mouth
(peat soil in flower seedling replanting to mixed-matrix：Coconut palm chaff：Perlite：River sand 3:2:2:1) in；I：Crocodile after field management 120d
Mouth spends plant.
Specific implementation mode
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1：
Spend the stem section of plant as explant using the crocodile mouth acquired from South China Botanical Garden Chinese Academy of Sciences, by stem section explant
After being cleaned up with tap water, is first cleaned for 75% alcohol water blend surface with volume fraction and sterilize 10s, sterile water elution 2 times,
8min is impregnated in quality concentration of volume percent is 0.1% mercuric chloride solution, and with sterile water washing 3 times, finally by stem later
Section is cut into 1cm sizes, is inoculated on inducing culture and cultivates, and condition of culture is 22~28 DEG C, and light intensity is 50 μm of ol m-2s-1, light
According to axillary bud is induced after cultivating 10d under conditions of 14h/d, axillary bud, which is then gone to inducing culture, continues to cultivate, and is induced after 30d
Go out adventitious bud；Then this adventitious bud is transferred to the proliferation that adventitious bud is carried out in propagating culture medium, condition of culture is 22~28 DEG C, light
It is 50 μm of ol m by force-2s-1, illumination 14h/d, one month is a subculture cycle, and each subculture cycle is proliferated 6.07 times.It will proliferation
Adventitious bud be transferred in strong seedling culture base and cultivate, condition of culture is 22~28 DEG C, and light intensity is 50 μm of ol m-2s-1, illumination 14h/d,
It is transferred in root media after 20d, it is made to take root, test tube seedling is grown up to after 20d.The test tube seedling is transplanted to containing peat soil：Coconut palm
Chaff：Perlite：River sand volume ratio is 3:2:2:In 1 mixed-matrix, it is placed in the environment of moistening, shade and cultivates, outdoor temperature
Between 20~25 DEG C, through watering, fertilising, the composite fertilizer (N of application 1 ‰ per week：P：K is 1：1：1) once, to meet test tube seedling
Moisture and nutritional need needed for growth, through routine culture, test tube seedling grows to 4~5cm high after one month, and survival rate is up to
97%, this test tube seedling is planted as cultivation seedling cultivate (Fig. 1) in using fiery ash as the basin of matrix at this time.
Wherein, inducing culture is to contain N- phenyl-N ' -1,2,3- thiadiazoles -5- ureas (TDZ) in every liter of culture medium
1.0mg and α-naphthylacetic acid (NAA) 0.1mg, remaining composition are MS；Proliferated culture medium is fast containing 6- benzyl amino in every liter of culture medium
Purine (BAP) 1.0mg, α-naphthylacetic acid (NAA) 0.1mg, remaining composition are MS；Strong seedling culture base is to contain albumen in every liter of culture medium
Peptone 0.5g L-1, remaining composition is MS；Root media is to contain activated carbon 1g and indole -3-butyric acid (IBA) in every liter of culture medium
3.0mg, remaining composition are MS.
Embodiment 2
Spend the stem section of plant as explant using the crocodile mouth acquired from South China Botanical Garden Chinese Academy of Sciences, by stem section explant
After being cleaned up with tap water, is first cleaned for 75% alcohol water blend surface with volume fraction and sterilize 10s, sterile water elution 2 times,
8min is impregnated in quality concentration of volume percent is 0.1% mercuric chloride solution, and with sterile water washing 3 times, finally by stem later
Section is cut into 1cm sizes, is inoculated on inducing culture and cultivates, and condition of culture is 22~28 DEG C, and light intensity is 45 μm of ol m-2s-1, light
According to axillary bud is induced after cultivating 14d under conditions of 14h/d, axillary bud, which is then gone to inducing culture, continues to cultivate, and is induced after 30d
Go out adventitious bud；Then this adventitious bud is transferred to the proliferation that adventitious bud is carried out in propagating culture medium, condition of culture is 22~28 DEG C, light
It is 50 μm of ol m by force-2s-1, illumination 14h/d, one month is a subculture cycle, and each subculture cycle is proliferated 6.07 times.It will proliferation
Adventitious bud be transferred in strong seedling culture base and cultivate, condition of culture is 22~28 DEG C, and light intensity is 50 μm of ol m-2s-1, illumination 14h/d,
It is transferred in root media after 20d, it is made to take root, test tube seedling is grown up to after 20d.The test tube seedling is transplanted to containing coconut palm chaff：Pearl
Rock volume ratio is 2:In 1 mixed-matrix, it is placed in the environment of moistening, shade and cultivates, outdoor temperature is between 20~25 DEG C, through pouring
Water, fertilising, the composite fertilizer (N of application 1 ‰ per week：P：K is 1：1：1) once, with meet test tube seedling growth needed for moisture and
Nutritional need, through routine culture, test tube seedling grows to 4~5cm high after one month, and survival rate is up to 96%, at this time by this test tube seedling
It plants in using fiery ash as the basin of matrix and cultivates as cultivation seedling.
Wherein, inducing culture is to contain 6-benzyl aminopurine (BAP) 1.0mg and α-naphthylacetic acid in every liter of culture medium
(NAA) 0.1mg, remaining composition are MS；Proliferated culture medium is to contain 6-benzyl aminopurine (BAP) 2.0mg, α-in every liter of culture medium
Methyl α-naphthyl acetate (NAA) 0.1mg, remaining composition are MS；Strong seedling culture base is to contain coconut juice 30g L in every liter of culture medium-1, remaining composition
For MS；Root media is in every liter of culture medium
MS。
Embodiment 3
Spend the stem section of plant as explant using the crocodile mouth acquired from South China Botanical Garden Chinese Academy of Sciences, by stem section explant
After being cleaned up with tap water, is first cleaned for 75% alcohol water blend surface with volume fraction and sterilize 10s, sterile water elution 2 times,
8min is impregnated in quality concentration of volume percent is 0.1% mercuric chloride solution, and with sterile water washing 3 times, finally by stem later
Section is cut into 1cm sizes, is inoculated on inducing culture and cultivates, and condition of culture is 22~28 DEG C, and light intensity is 45 μm of ol m-2s-1, light
According to axillary bud is induced after cultivating 14d under conditions of 14h/d, axillary bud, which is then gone to inducing culture, continues to cultivate, and is induced after 30d
Go out adventitious bud；Then this adventitious bud is transferred to the proliferation that adventitious bud is carried out in propagating culture medium, condition of culture is 22~28 DEG C, light
It is 50 μm of ol m by force-2s-1, illumination 14h/d, one month is a subculture cycle, and each subculture cycle is proliferated 6.07 times.It will proliferation
Adventitious bud be transferred in strong seedling culture base and cultivate, condition of culture is 22~28 DEG C, and light intensity is 50 μm of ol m-2s-1, illumination 14h/d,
It is transferred in root media after 20d, it is made to take root, test tube seedling is grown up to after 20d.The test tube seedling is transplanted to containing peat soil：River
Husky volume ratio is 2:In 1 mixed-matrix, it is placed in the environment of moistening, shade and cultivates, outdoor temperature is between 20~25 DEG C, through pouring
Water, fertilising, the composite fertilizer (N of application 1 ‰ per week：P：K is 1：1：1) once, with meet test tube seedling growth needed for moisture and
Nutritional need, through routine culture, test tube seedling grows to 4~5cm high after one month, and survival rate is up to 95%, at this time by this test tube seedling
It plants in using fiery ash as the basin of matrix and cultivates as cultivation seedling.
Wherein, inducing culture is to contain 6-benzyl aminopurine (BAP) 2.0mg and α-naphthylacetic acid in every liter of culture medium
(NAA) 0.1mg, remaining composition are MS；Proliferated culture medium is to contain N- phenyl-N ' -1,2,3- thiadiazoles -5- in every liter of culture medium
Urea (TDZ) 2.0mg, α-naphthylacetic acid (NAA) 0.1mg, remaining composition are MS；Strong seedling culture base is to contain banana in every liter of culture medium
Juice 30g L-1, remaining composition is MS；Root media is to contain activated carbon 1g and indole -3-butyric acid (IBA) in every liter of culture medium
3.0mg and α-naphthylacetic acid (NAA) 0.1mg, remaining composition are MS.
Embodiment 4
Spend the stem section of plant as explant using the crocodile mouth acquired from South China Botanical Garden Chinese Academy of Sciences, by stem section explant
After being cleaned up with tap water, is first cleaned for 75% alcohol water blend surface with volume fraction and sterilize 10s, sterile water elution 2 times,
8min is impregnated in quality concentration of volume percent is 0.1% mercuric chloride solution, and with sterile water washing 3 times, finally by stem later
Section is cut into 1cm sizes, is inoculated on inducing culture and cultivates, and condition of culture is 22~28 DEG C, and light intensity is 45 μm of ol m-2s-1, light
According to axillary bud is induced after cultivating 14d under conditions of 14h/d, axillary bud, which is then gone to inducing culture, continues to cultivate, and is induced after 30d
Go out adventitious bud；Then this adventitious bud is transferred to the proliferation that adventitious bud is carried out in propagating culture medium, condition of culture is 22~28 DEG C, light
It is 50 μm of ol m by force-2s-1, illumination 14h/d, one month is a subculture cycle, and each subculture cycle is proliferated 6.07 times.It will proliferation
Adventitious bud be transferred in strong seedling culture base and cultivate, condition of culture is 22~28 DEG C, and light intensity is 50 μm of ol m-2s-1, illumination 14h/d,
It is transferred in root media after 20d, it is made to take root, test tube seedling is grown up to after 20d.The test tube seedling is transplanted to containing peat soil：It is precious
Pearl rock volume ratio is 2:It in 1 mixed-matrix, is placed in the environment of moistening, shade and cultivates, outdoor temperature is between 20~25 DEG C, warp
Watering, fertilising, the composite fertilizer (N of application 1 ‰ per week：P：K is 1：1：1) once, to meet the moisture needed for test tube seedling growth
And nutritional need, through routine culture, test tube seedling grows to 4~5cm high after one month, and survival rate is up to 98%, at this time by this test tube
Seedling is planted in using fiery ash as the basin of matrix as cultivation seedling and is cultivated.
Wherein, inducing culture is to contain 6-benzyl aminopurine (BAP) 1.0mg and α-naphthylacetic acid in every liter of culture medium
(NAA) 0.1mg, remaining composition are MS；Proliferated culture medium is to contain N- phenyl-N ' -1,2,3- thiadiazoles -5- in every liter of culture medium
Urea (TDZ) 1.0mg, indole -3-butyric acid (IBA) 0.1mg, remaining composition are MS；Strong seedling culture base is to contain in every liter of culture medium
Bananas juice 30g L-1, remaining composition is MS；Root media is to contain activated carbon 1g and indole -3-butyric acid in every liter of culture medium
(IBA) 2.0mg and α-naphthylacetic acid (NAA) 0.4mg, remaining composition are MS.
Claims (3)
1. a kind of crocodile mouth spends quick breeding method for tissue culture, which is characterized in that include the following steps：
The induction of S1, axillary bud：It chooses crocodile mouth scape section and is used as explant, be inoculated in inducing culture after sterilization, until
Induce axillary bud；
The induction of S2, adventitious bud：Gained axillary bud is accessed in inducing culture and is cultivated, until inducing adventitious bud；
S3, shoot proliferation：Gained adventitious bud is accessed in proliferated culture medium to the proliferation for carrying out Multiplying culture and carrying out adventitious bud；
S4, strong seedling culture：Culture in the adventitious bud access strong seedling culture base of gained proliferation is obtained into unrooted strong sprout；
S5, culture of rootage：Gained unrooted strong sprout is accessed into culture in root media and obtains tissue-cultured seedling；
S6, gained tissue-cultured seedling is subjected to transplanting culture；
Wherein, the condition of culture in S1~S5 is 22~28 DEG C, 50~80 μm of ol m of light intensity-2s-1, illumination 14h/d；
The pH of every liter of inducing culture is 5.9, is contained：1.0mg TDZ or 1.0~2.0mg BAP, 0.1mg NAA, surplus
For MS culture mediums；
The pH of every liter of proliferated culture medium is 5.9, is contained：1.0~3.0mg TDZ or 1.0~3.0mg BAP, 0.1mg NAA
Or 0.1mg IBA, surplus are MS culture mediums；
The pH of every liter of strong seedling culture base is 5.9, is contained：Peptone 0.5g/L or coconut juice 30g/L or banana 30g/L, surplus
For MS culture mediums；
The pH of every liter of root media is 5.9, is contained：1g activated carbons and 2.0~4.0mg IBA or 0.1~0.5mg
NAA, surplus are MS culture mediums.
2. crocodile mouth according to claim 1 spends quick breeding method for tissue culture, which is characterized in that the explant sterilizing
Disinfection is will to be cleaned with tap water as the stem section of explant, and disinfection is cleaned on the alcoholic solution surface for being first 75% with volume fraction
10s, then aseptic water washing 2 times, then 8min is impregnated in the mercuric chloride solution that mass-volume concentration is 0.1%, then with sterile
Water rinses 3 times, and stem section is finally cut into 1cm long, obtains the explant of sterilization treatment.
3. crocodile mouth according to claim 1 spends quick breeding method for tissue culture, which is characterized in that the transplanting, which is cultivated, is
Tissue-cultured seedling is transplanted to peat soil：Coconut palm chaff：Perlite：River sand is with 3:2:1:In the matrix of 1 volume ratio composition, it is placed in relatively wet
Degree is 30~50%, light transmittance is cultivated in the environment of being 40~50%, 20~25 DEG C, the composite fertilizer one per week using 1wt ‰
It is secondary, obtain cultivation seedling；N in the composite fertilizer:P:The mass ratio of K is 1:1:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113940203A (en) * | 2021-11-18 | 2022-01-18 | 中国热带农业科学院热带作物品种资源研究所 | Single-node cutting rapid propagation method for red bud flowers |
| CN114617062A (en) * | 2022-01-30 | 2022-06-14 | 山东农业大学 | Tissue culture and rapid propagation method for crocodile flower |
-
2018
- 2018-01-24 CN CN201810069097.XA patent/CN108450328A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| BIHUA CHEN等: "The Rapid Propagation Technique of the Medicinal Plant Clinacanthus nutans by Tissue Culture", 《NEW YORK SCIENCE JOURNAL》 * |
| 王大平: "《园林苗圃学》", 31 January 2014 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113940203A (en) * | 2021-11-18 | 2022-01-18 | 中国热带农业科学院热带作物品种资源研究所 | Single-node cutting rapid propagation method for red bud flowers |
| CN114617062A (en) * | 2022-01-30 | 2022-06-14 | 山东农业大学 | Tissue culture and rapid propagation method for crocodile flower |
| CN114617062B (en) * | 2022-01-30 | 2022-11-01 | 山东农业大学 | Tissue culture and rapid propagation method for crocodile flower |
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