CN106417013A - Tissue culture method of ceratostigma plumbaginoides - Google Patents
Tissue culture method of ceratostigma plumbaginoides Download PDFInfo
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- CN106417013A CN106417013A CN201610799972.0A CN201610799972A CN106417013A CN 106417013 A CN106417013 A CN 106417013A CN 201610799972 A CN201610799972 A CN 201610799972A CN 106417013 A CN106417013 A CN 106417013A
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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 tissue culture method of ceratostigma plumbaginoides. The tissue culture method comprises following steps: step one, a ceratostigma plumbaginoides plant growing strongly is selected, the upper half part of a branch is picked up and processed into small sections of 3-5 cm, 1-2 leaf buds are reserved at each small section, the small section is soaked with 75% ethanol, is rinsed with sterile water 2-3 times and then is placed in an active carbon nanoparticle aqueous solution with the massic volume fraction being 0.1-0.3 to be soaked for 8-10 min, the small section is washed with the sterile water 3-5 times, surface moisture is removed, and a ceratostigma plumbaginoides explant is obtained; step two, differentiation culture is performed; step three, is performed; step four, rooting culture is performed, wherein each of a differentiation culture medium, a hardening-off medium and a rooting culture medium comprises components as follows: 30-50 g/L of a banana fermentation product, 20-30 g/L of mashed potatoes, 3-5 g/L of peptone, 2-3 g/L of trypsin and 5-10 g/L of yeast extract, the rooting culture medium further contains 10%-20% (v/v) of scindapsus aureus leaf extract, and the banana fermentation product is a substance prepared from bananas through fermentation for 3-5 d under the oxygen-free condition at the temperature of 25-28 DEG C.
Description
Technical field
The present invention relates to a kind of method for tissue culture of ceratostigma plumbaginoides Bunge.
Background technology
Ceratostigma plumbaginoides Bunge (Classification system：Ceratostigma plumbaginoides) have another name called mountain ash bavin, false indigo (Henan), angle
Post flower etc., perennial vertical herbage for many years, is Plumbaginaceae, ceratostigma plumbaginoides Bunge platymiscium.Ceratostigma plumbaginoides Bunge is rich in plumbagin, and rheumatism is closed
Section pain, traumatic injury, pyogenic infections dislike sore and tinea has therapeutic action.Meanwhile, ceratostigma plumbaginoides Bunge also has high ornamental value.It is
A kind of integrate medicinal with ornamental value plant.
However, ceratostigma plumbaginoides Bunge seed propagation, its seed Natural seed setting rate is low, is difficult to obtain seed, domestic introducing and planting is most
Number is using import seeds cultivation, expensive.And utilize cutting propagation rooting rate relatively low, be difficult to obtain a large amount of seedlings.Closely
Nian Lai, gradually adopts method for tissue culture that ceratostigma plumbaginoides Bunge is cultivated, but due to using MS culture medium etc. in incubation
The overgenerous culture medium of nutrition, easily causes a large amount of pollutions of ceratostigma plumbaginoides Bunge, leads to its survival rate relatively low, and also results in resource wave
Take and environmental pollution.
Content of the invention
It is an object of the invention to solving at least the above and/or defect, and provide at least will be described later excellent
Point.
It is a still further object of the present invention to provide a kind of method for tissue culture of ceratostigma plumbaginoides Bunge.
The present invention provide technical scheme be：
.
The present invention includes at least following advantage：
The present invention is carried out disinfection to segment using active carbon nanoparticles solution, and active carbon nanoparticles particle can adsorb to fall on segment
Various bacteriums, to bacterium, there is killing action simultaneously, ensure that the thorough disinfection to ceratostigma plumbaginoides Bunge explant, after will not polluting
The operation of phase.
And, the culture medium of the present invention is provided without traditional MS culture medium, and banana fermentate and mashed potatoes etc. is adopted to become
Divide culture ceratostigma plumbaginoides Bunge, because ceratostigma plumbaginoides Bunge is not high to managerial demand, during culture, both can guarantee that the normal of ceratostigma plumbaginoides Bunge
Growth, simultaneously and not waste of resource, save production cost, and be difficult microbiological contamination in incubation.The culture of rootage of the present invention
Epipremnum aureum leaf extract is contained, the material of taking root wherein containing can effective stimulus ceratostigma plumbaginoides Bunge plant be taken root, and safety non-toxic in base,
Raw material is easy to get.The composition such as the licorice containing and N methyl taurine can effectively stimulate plant to take root so as to get
The radical mesh of ceratostigma plumbaginoides Bunge plant is more, and length is also longer, improves the survival rate after transplanting.
Contain substantial amounts of coconut milk in the culture medium of the present invention, contain in coconut milk protein, fat, vitamin C and calcium,
The mineral matters such as phosphorus, iron, potassium, magnesium, sodium, contain more moisture and anthelmintic substance so that the nutrition of culture medium is abundanter simultaneously,
And the growth of ceratostigma plumbaginoides Bunge explant can be promoted, beneficial to rooting of ceratostigma plumbaginoides Bunge explant.
The present invention carries out tissue-culturing quick-propagation by tissue culture technique to ceratostigma plumbaginoides Bunge, cultivates big at short notice
Amount is available for the ceratostigma plumbaginoides Bunge seedling of field production, improves breeding coefficient and the seedling quality of ceratostigma plumbaginoides Bunge seedling, realizes scale metaplasia
Produce, meet the needs on producing.
Using the method for the present invention, the plantlet in vitro rooting rate of acquisition more than 95%, every plant of average band 5-10 root, root is long
For 4-8 centimetre, realize the large-scale production of ceratostigma plumbaginoides Bunge tissue culture seedling.
Specific embodiment
The present invention is described in further detail below, with make those skilled in the art with reference to specification word being capable of evidence
To implement.
The present invention provides a kind of method for tissue culture of ceratostigma plumbaginoides Bunge, including：
Step one, the ceratostigma plumbaginoides Bunge plant of selection robust growth, pick the top half of branch, at this top half branch
Manage the segment for 3～5cm, each segment retains 1～2 leaf bud, infiltrates this segment using 75% ethanol, afterwards with aseptic
Water rinses 2～3 times, then is placed in immersion 8～10min in the active carbon nanoparticles aqueous solution that quality volume fraction is 0.1～0.3, it
Use aseptic water washing 3-5 time afterwards, removing surface moisture obtains ceratostigma plumbaginoides Bunge explant；
Step 2, differentiation culture：The ceratostigma plumbaginoides Bunge obtaining in step one explant is inoculated in differential medium and cultivates 25
～35 days until the lateral bud redifferentiation of ceratostigma plumbaginoides Bunge explant grows Multiple Buds；
Step 3, strong seedling culture：To train in strong seedling culture base through the ceratostigma plumbaginoides Bunge explant of differentiation culture in step 2
Support and obtain healthy and strong plant in 35～45 days；
Step 4, culture of rootage：Ceratostigma plumbaginoides Bunge stalwartness plant through obtaining in step 3 is placed in culture in root media
Obtain the sapling with root within 45～55 days；
Wherein, the condition of culture of described differentiation culture, strong seedling culture and culture of rootage is：Cultivation temperature is 23-27 DEG C, light
It is 2000-2500lux according to intensity, periodicity of illumination is 10-15 hour/sky；
Described differential medium, described strong seedling culture base and described root media all comprise：Banana fermentate 30～
50g/L, mashed potatoes 20～30g/L, peptone 3～5g/L, trypsase 2～3g/L and yeast extract 5～10g/L,
Described root media also comprises：The epipremnum aureum leaf extract of 10～20% (v/v), epipremnum aureum leaf extract is by epipremnum aureum
After the particle that it is 200 mesh for granularity that leaf is pulverized, cross the liquid that 300 mesh sieves obtain；
Banana fermentate is the material that the oxygen free condition bottom fermentation that banana is placed in 25～28 DEG C obtains for 3～5 days.
In one of embodiment of the present invention, preferably, in described step 2, described differential medium also wraps
Contain：Coconut milk 30～40mg/L, 0.3～1.2mg/L 6-BA, 0.3-0.9mg/L NAA and 4.8-5.8g/L agar, and adjust
Initial pH value is 5.8.
In one of embodiment of the present invention, preferably, in described step 3, strong seedling culture base comprises：Coconut
Newborn 40～50mg/L, 0.4-1.2mg/L 6-BA, 0.3-0.9mg/L IAA and 3.8-4.8g/L agar, and adjust initial pH value
For 5.8.
In one of embodiment of the present invention, preferably, in described step 4, root media also comprises：Coconut palm
Son breast 50～60mg/L, 1.0-2.0mg/L NAA, 0.1～0.3mg/L licorice, 1.5-2.5mg/L N- methyl ox sulphur
Acid and 3.8-4.8g/L agar, and adjust initial pH value for 5.8.
In one of embodiment of the present invention, preferably, also carrying out step 5 after described step 4, described
Step 5 includes：Hardening and transplanting：First the plant with root will be obtained in step 4 in 25 DEG C of lower refining seedlings of temperature 3-7 days, afterwards
It is transplanted in sandy soil earth again and grows 40～50 days, then transplant to grown in field, wherein, the growth conditions in land for growing field crops is again：Temperature
For 25-30 DEG C, relative humidity is 75-80%, and sunshade rate is 70%.
In one of embodiment of the present invention, preferably, described coconut milk be coconut through 100 mesh filtered through gauze it
The liquid obtaining afterwards.
Embodiment 1
A kind of method for tissue culture of ceratostigma plumbaginoides Bunge, comprises the steps：
Step one, the ceratostigma plumbaginoides Bunge plant of selection robust growth, pick the top half of branch, at this top half branch
Manage the segment for 3cm, each segment retains 1 leaf bud, infiltrates this segment using 75% ethanol, use rinsed with sterile water afterwards
2 times, then it is placed in immersion 8min in the active carbon nanoparticles aqueous solution that quality volume fraction is 0.1%, use aseptic water washing 3 afterwards
Secondary, remove surface moisture and obtain ceratostigma plumbaginoides Bunge explant；
Step 2, differentiation culture：The ceratostigma plumbaginoides Bunge obtaining in step one explant is inoculated in differential medium and cultivates 25
It is until the lateral bud redifferentiation of ceratostigma plumbaginoides Bunge explant grows Multiple Buds；
Step 3, strong seedling culture：To train in strong seedling culture base through the ceratostigma plumbaginoides Bunge explant of differentiation culture in step 2
Support and obtain healthy and strong plant in 35 days；
Step 4, culture of rootage：Ceratostigma plumbaginoides Bunge stalwartness plant through obtaining in step 3 is placed in culture in root media
Obtain the sapling with root within 45 days；
Wherein, the condition of culture of described differentiation culture, strong seedling culture and culture of rootage is：Cultivation temperature is 23 DEG C, illumination
Intensity is 2000lux, and periodicity of illumination is 10 hours/day；
Described differential medium, described strong seedling culture base and described root media all comprise：Banana fermentate 30g/L,
Mashed potatoes 20g/L, peptone 3g/L, trypsase 2g/L and yeast extract 5g/L,
In described step 2, differential medium also comprises：Coconut milk 30mg/L, 0.3mg/L 6-BA, 0.3mg/L NAA and
4.8g/L agar, and adjust initial pH value for 5.8
Described root media also comprises：The epipremnum aureum leaf extract of 10% (v/v), epipremnum aureum leaf extract is by epipremnum aureum leaf powder
Broken be 200 mesh for granularity particle after, cross the liquid that obtains of 300 mesh sieves；Coconut milk 50mg/L, 1.0mg/L NAA, 0.1mg/L
Licorice, 1.5mg/L N methyl taurine and 3.8g/L agar, and adjust initial pH value for 5.8.
In described step 3, strong seedling culture base comprises：Coconut milk 40mg/L, 0.4mg/L 6-BA, 0.3mg/L IAA and
3.8g/L agar, and adjust initial pH value for 5.8.
Banana fermentate is the material that the oxygen free condition bottom fermentation that banana is placed in 25 DEG C obtains for 3 days.
Step 5, hardening and transplanting：First the plant with root will be obtained in step 4 in 25 DEG C of lower refining seedlings of temperature 3 days, it
It is transplanted to afterwards in sandy soil earth again and grows 40 days, then transplant to grown in field, wherein, the growth conditions in land for growing field crops is again：Temperature is
25 DEG C, relative humidity is 75%, and sunshade rate is 70%.
Described coconut milk is the liquid through obtaining after 100 mesh filtered through gauze for the coconut.
Embodiment 2
A kind of method for tissue culture of ceratostigma plumbaginoides Bunge, comprises the steps：
Step one, the ceratostigma plumbaginoides Bunge plant of selection robust growth, pick the top half of branch, at this top half branch
Manage the segment for 5cm, each segment retains 2 leaf buds, infiltrates this segment using 75% ethanol, use rinsed with sterile water afterwards
3 times, then it is placed in immersion 10min in the active carbon nanoparticles aqueous solution that quality volume fraction is 0.3%, use aseptic water washing 5 afterwards
Secondary, remove surface moisture and obtain ceratostigma plumbaginoides Bunge explant；
Step 2, differentiation culture：The ceratostigma plumbaginoides Bunge obtaining in step one explant is inoculated in differential medium and cultivates 35
It is until the lateral bud redifferentiation of ceratostigma plumbaginoides Bunge explant grows Multiple Buds；
Step 3, strong seedling culture：To train in strong seedling culture base through the ceratostigma plumbaginoides Bunge explant of differentiation culture in step 2
Support and obtain healthy and strong plant in 45 days；
Step 4, culture of rootage：Ceratostigma plumbaginoides Bunge stalwartness plant through obtaining in step 3 is placed in culture in root media
Obtain the sapling with root within 55 days；
Wherein, the condition of culture of described differentiation culture, strong seedling culture and culture of rootage is：Cultivation temperature is 27 DEG C, illumination
Intensity is 2500lux, and periodicity of illumination is 15 hours/day；
Described differential medium, described strong seedling culture base and described root media all comprise：Banana fermentate 50g/L,
Mashed potatoes 30g/L, peptone 5g/L, trypsase 3g/L and yeast extract 10g/L,
In described step 2, differential medium also comprises：Coconut milk 40mg/L, 1.2mg/L 6-BA, 0.9mg/L NAA and
5.8g/L agar, and adjust initial pH value for 5.8
Described root media also comprises：The epipremnum aureum leaf extract of 20% (v/v), epipremnum aureum leaf extract is by epipremnum aureum leaf powder
Broken be 200 mesh for granularity particle after, cross the liquid that obtains of 300 mesh sieves；Coconut milk 60mg/L, 2.0mg/L NAA, 0.3mg/L
Licorice, 2.5mg/L N methyl taurine and 4.8g/L agar, and adjust initial pH value for 5.8.
In described step 3, strong seedling culture base comprises：Coconut milk 50mg/L, 1.2mg/L 6-BA, 0.9mg/L IAA and
4.8g/L agar, and adjust initial pH value for 5.8.
Banana fermentate is the material that the oxygen free condition bottom fermentation that banana is placed in 28 DEG C obtains for 5 days.
Step 5, hardening and transplanting：First the plant with root will be obtained in step 4 in 25 DEG C of lower refining seedlings of temperature 3-7 days,
It is transplanted to afterwards in sandy soil earth again and grows 50 days, then transplant to grown in field, wherein, the growth conditions in land for growing field crops is again：Temperature
For 30 DEG C, relative humidity is 80%, and sunshade rate is 70%.
Described coconut milk is the liquid through obtaining after 100 mesh filtered through gauze for the coconut.
Embodiment 3
A kind of method for tissue culture of ceratostigma plumbaginoides Bunge, comprises the steps：
Step one, the ceratostigma plumbaginoides Bunge plant of selection robust growth, pick the top half of branch, at this top half branch
Manage the segment for 4cm, each segment retains 2 leaf buds, infiltrates this segment using 75% ethanol, use rinsed with sterile water afterwards
3 times, then it is placed in immersion 9min in the active carbon nanoparticles aqueous solution that quality volume fraction is 0.2%, use aseptic water washing 4 afterwards
Secondary, remove surface moisture and obtain ceratostigma plumbaginoides Bunge explant；
Step 2, differentiation culture：The ceratostigma plumbaginoides Bunge obtaining in step one explant is inoculated in differential medium and cultivates 30
It is until the lateral bud redifferentiation of ceratostigma plumbaginoides Bunge explant grows Multiple Buds；
Step 3, strong seedling culture：To train in strong seedling culture base through the ceratostigma plumbaginoides Bunge explant of differentiation culture in step 2
Support and obtain healthy and strong plant in 40 days；
Step 4, culture of rootage：Ceratostigma plumbaginoides Bunge stalwartness plant through obtaining in step 3 is placed in culture in root media
Obtain the sapling with root within 50 days；
Wherein, the condition of culture of described differentiation culture, strong seedling culture and culture of rootage is：Cultivation temperature is 25 DEG C, illumination
Intensity is 2250lux, and periodicity of illumination is 12.5 hours/day；
Described differential medium, described strong seedling culture base and described root media all comprise：Banana fermentate 40g/L,
Mashed potatoes 25g/L, peptone 4g/L, trypsase 2.5g/L and yeast extract 7.5g/L,
In described step 2, differential medium also comprises：Coconut milk 35mg/L, 0.75mg/L 6-BA, 0.6mg/L NAA
With 5.3g/L agar, and adjust initial pH value be 5.8.
Described root media also comprises：The epipremnum aureum leaf extract of 15% (v/v), epipremnum aureum leaf extract is by epipremnum aureum leaf powder
Broken be 200 mesh for granularity particle after, cross the liquid that obtains of 300 mesh sieves；Coconut milk 55mg/L, 1.5mg/L NAA, 0.2mg/L
Licorice, 2.0mg/L N methyl taurine and 5.3g/L agar, and adjust initial pH value for 5.8.
In described step 3, strong seedling culture base comprises：Coconut milk 45mg/L, 0.8mg/L 6-BA, 0.6mg/L IAA and
5.3g/L agar, and adjust initial pH value for 5.8.
Banana fermentate is the material that the oxygen free condition bottom fermentation that banana is placed in 27 DEG C obtains for 4 days.
Step 5, hardening and transplanting：First the plant with root will be obtained in step 4 in 25 DEG C of lower refining seedlings of temperature 5 days, it
It is transplanted to afterwards in sandy soil earth again and grows 45 days, then transplant to grown in field, wherein, the growth conditions in land for growing field crops is again：Temperature is
28 DEG C, relative humidity is 77%, and sunshade rate is 70%.
Described coconut milk is the liquid through obtaining after 100 mesh filtered through gauze for the coconut.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and embodiment
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Realize other modification, therefore under the universal being limited without departing substantially from claim and equivalency range, the present invention does not limit
In specific details and shown here embodiment.
Claims (6)
1. a kind of method for tissue culture of ceratostigma plumbaginoides Bunge is it is characterised in that comprise the steps：
Step one, the ceratostigma plumbaginoides Bunge plant of selection robust growth, pick the top half of branch, this top half branch are processed as
The segment of 3～5cm, each segment retains 1～2 leaf bud, infiltrates this segment using 75% ethanol, floated with sterilized water afterwards
Wash 2～3 times, then be placed in immersion 8～10min in the active carbon nanoparticles aqueous solution that quality volume fraction is 0.1～0.3, use afterwards
Aseptic water washing 3-5 time, removes surface moisture and obtains ceratostigma plumbaginoides Bunge explant；
Step 2, differentiation culture：The ceratostigma plumbaginoides Bunge obtaining in step one explant is inoculated in differential medium and cultivates 25～35
It is until the lateral bud redifferentiation of ceratostigma plumbaginoides Bunge explant grows Multiple Buds；
Step 3, strong seedling culture：35 will be cultivated through the ceratostigma plumbaginoides Bunge explant of differentiation culture in strong seedling culture base in step 2
Obtain healthy and strong plant within～45 days；
Step 4, culture of rootage：By the ceratostigma plumbaginoides Bunge stalwartness plant through obtaining in step 3 be placed in root media culture 45～
Obtain the sapling with root within 55 days；
Wherein, the condition of culture of described differentiation culture, strong seedling culture and culture of rootage is：Cultivation temperature is 23-27 DEG C, and illumination is strong
Spend for 2000-2500lux, periodicity of illumination is 10-15 hour/sky；
Described differential medium, described strong seedling culture base and described root media all comprise：Banana fermentate 30～50g/L,
Mashed potatoes 20～30g/L, peptone 3～5g/L, trypsase 2～3g/L and yeast extract 5～10g/L,
Described root media also comprises：The epipremnum aureum leaf extract of 10～20% (v/v), epipremnum aureum leaf extract is by epipremnum aureum leaf powder
Broken be 200 mesh for granularity particle after, cross the liquid that obtains of 300 mesh sieves；
Banana fermentate is the material that the oxygen free condition bottom fermentation that banana is placed in 25～28 DEG C obtains for 3～5 days.
2. the method for tissue culture of ceratostigma plumbaginoides Bunge as claimed in claim 1 is it is characterised in that in described step 2, described differentiation
Culture medium also comprises：Coconut milk 30～40mg/L, 0.3～1.2mg/L 6-BA, 0.3-0.9mg/L NAA and 4.8-5.8g/L fine jade
Fat, and adjust initial pH value for 5.8.
3. the method for tissue culture of ceratostigma plumbaginoides Bunge as claimed in claim 1 is it is characterised in that in described step 3, strong seedling culture
Base comprises：Coconut milk 40～50mg/L, 0.4-1.2mg/L 6-BA, 0.3-0.9mg/LIAA and 3.8-4.8g/L agar, and adjust
Section initial pH value is 5.8.
4. the method for tissue culture of ceratostigma plumbaginoides Bunge as claimed in claim 1 is it is characterised in that in described step 4, culture of rootage
Base also comprises：Coconut milk 50～60mg/L, 1.0-2.0mg/L NAA, 0.1～0.3mg/L licorice, 1.5-2.5mg/L
N methyl taurine and 3.8-4.8g/L agar, and adjust initial pH value for 5.8.
5. the method for tissue culture of ceratostigma plumbaginoides Bunge as claimed in claim 1 is it is characterised in that also carry out after described step 4
Step 5, described step 5 includes：Hardening and transplanting：First the plant with root will be obtained in step 4 in 25 DEG C of lower refining seedlings of temperature
3-7 days, it is transplanted to afterwards in sandy soil earth again and grows 40～50 days, then transplant to grown in field again, wherein, the growth bar in land for growing field crops
Part is：Temperature is 25-30 DEG C, and relative humidity is 75-80%, and sunshade rate is 70%.
6. the method for tissue culture of described ceratostigma plumbaginoides Bunge as arbitrary in claim 2～4 is it is characterised in that described coconut milk is coconut palm
The liquid through obtaining after 100 mesh filtered through gauze for the son.
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| CN107691222A (en) * | 2017-11-10 | 2018-02-16 | 四川农业大学 | A kind of method that indigo plant spends red efficiently cuttage and quick-propagation |
| CN112715366A (en) * | 2021-01-29 | 2021-04-30 | 四川农业大学 | Method for efficiently and quickly rooting and strengthening tissue culture seedlings of Minjiang snowflake |
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