CN107278903B - The quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn - Google Patents
The quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn Download PDFInfo
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- CN107278903B CN107278903B CN201710661074.3A CN201710661074A CN107278903B CN 107278903 B CN107278903 B CN 107278903B CN 201710661074 A CN201710661074 A CN 201710661074A CN 107278903 B CN107278903 B CN 107278903B
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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 quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, the following steps are included: 1) using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, it is inoculated in breeding rejuvenation integration culture medium, it is placed in culture 15d in culturing room, indoor conditions is 22-26 DEG C, 1400lux, 8h/d, then it is transferred to 25-28 DEG C, continues to cultivate 15d under the conditions of the natural lighting of 3500-4500lux, obtains Multiple Buds；2) Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root, in indoor 22-26 DEG C, 7d is cultivated under conditions of 1500lux, 10h/d, then be transferred in greenhouse 23-28 DEG C, obscurity 20-40%, relative humidity be 60-80% natural light under cultivate 25d；3) it transplants.Method of the invention can significantly shorten the seeling industry period, reduce seeling industry cost, improve kind of shoot survival percent and seedling quality.
Description
Technical field
The present invention relates to the reproduction technique fields of Illigera trifoliata (Griff.) Dunn.It is more particularly related to a kind of Illigera trifoliata (Griff.) Dunn
Quick breeding method for tissue culture.
Background technique
Illigera trifoliata (Griff.) Dunn Illigera trifoliata (Griff.) Dunn also known as I. rhodantha, hair sinomenium acutum, Folium solani verbascifolii rattan,
For Hernandiaceae sinomenium acutum platymiscium.Illigera trifoliata (Griff.) Dunn is mainly distributed on Guangxi, Guangdong, Yunnan, Sichuan, Hunan, Fujian and Guizhou etc.
Ground is used as medicine with overground part, and there is wind-dispelling to dissipate the stasis of blood, the effect of swelling and pain relieving, is chiefly used in treating treating swelling and pain by traumatic injury, shoulder Guangxi is civil
Inflammation, treating rheumatic ostealgia, rheumatism and rheumatoid illness, infantile paralysis etc., the plant rattan are production Chinese patent drug " Huatuo's wind pains
The primary raw material medicine of treasured " and its various dosage forms of medicine.Research in recent years shows that Illigera trifoliata (Griff.) Dunn alcohol extract toxicity is smaller and there have to be significant
Anti-inflammatory effect.
Illigera trifoliata (Griff.) Dunn is born in the mountain valley of height above sea level 1100-1300m more, and Regional Distribution is scattered and reserves are rare.With people
Improvement of living standard and health care consciousness enhancing, the market demand of Illigera trifoliata (Griff.) Dunn rises year by year, wild resource by
Degree excavation, is on the verge of exhaustion.Under natural conditions, Illigera trifoliata (Griff.) Dunn relies primarily on seed and is bred, but since Illigera trifoliata (Griff.) Dunn fruit is
Mature in the annual 4-5 month, southern area is in clear and bright period plum rain season at this time, and unbroken wet weather easily causes fruit because of rainwater
Impregnate and blackening rot；The picking when fruit turns yellow green or light brown from dark green, fruit become dark brown, and seed also all becomes
Must be more shrivelled, germination percentage is relatively low, emerges irregular, brings extreme difficulties to scientific research and introducing and planting.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, can significantly shorten
The seeling industry period reduces seeling industry cost, improves kind of shoot survival percent and seedling quality.
In order to realize these purposes and other advantages according to the present invention, a kind of tissue culture rapid of Illigera trifoliata (Griff.) Dunn is provided
Fast propagation method, comprising the following steps:
1) it breeds rejuvenation integration culture: using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, being inoculated in breeding rejuvenation one
Change culture medium, be placed in culture 15d in culturing room, cultivate 22-26 DEG C of room temperature, intensity of illumination 1400lux, light application time 8h/d,
Then it is transferred to 25-28 DEG C of cultivation temperature, continues to cultivate 15d under the conditions of the natural lighting of intensity of illumination 3500-4500lux, obtain
Healthy and strong Multiple Buds breed the formula of rejuvenation integration culture medium are as follows: MS+1.0-2.0mg/L BA+0.3-0.5mg/L LFS+
0.1-0.3mg/L IBA+0.1-1.0mg/L NAA；
2) hardening of taking root synchronizing culture: the Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root
In, in 22-26 DEG C of indoor cultivation temperature, intensity of illumination 1500lux, light application time cultivates 7d under conditions of being 10h/d, then is transferred to
Cultivating 25d under the natural light that cultivation temperature is 23-28 DEG C in greenhouse, relative humidity is 60-80% and obtaining is suitable for the complete of transplanting
Plant, wherein greenhouse top covers sunshade net, obscurity 20-40%, wherein the formula of hardening of taking root synchronizing culture base
Are as follows: 1/2MS+0.1-0.3mg/L IBA+1.0-2.0mg/L NAA+0.5-1.5mg/L IAA+0.3-0.5mg/L MET；
3) it transplants: the intact plant that step 2) obtains is taken out from hardening synchronizing culture base of taking root, it is residual to clean root
Stay culture medium, transplanting to seedling bed or seedling raising greenhouse.
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the acquisition of the explant in step 1)
Method is as follows: being inoculated in culture acquisition adventitious bud on propagating culture medium after taking stem section disinfection of the Illigera trifoliata (Griff.) Dunn with axillary bud, and in nothing
Cutting obtains the stem with bud of 1.0-2.0cm long as explant, spare, the formula of the propagating culture medium under the conditions of bacterium are as follows:
MS+1.5mg/L BA+0.4mg/L LFS+0.5mg/L IBA。
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, explant described in step 1) connect
Kind method are as follows: the explant is laid in the surface of breeding rejuvenation integration culture medium, and the 1/2 explant indentation is numerous
It grows in rejuvenation integration culture medium.
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the breeding rejuvenation integration culture
The formula of base are as follows: MS+1.5mg/L BA+0.4mg/L LFS+0.2mg/L IBA+0.5mg/L NAA.
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the hardening synchronizing culture of taking root
The formula of base are as follows: 1/2MS+0.2mg/L IBA+1.5mg/L NAA+1.0mg/L IAA+0.4mg/L MET.
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, in step 2), the hardening of taking root is same
Stepization culture medium also contains 1.2mg/L nano zine oxide.
Preferably, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the middle environment transplanted of step 3) are opposite
Humidity is 80-90%, 25-28 DEG C of cultivation temperature, obscurity 60-75%.
The present invention is include at least the following beneficial effects:
1) by biotechnology to Illigera trifoliata (Griff.) Dunn carry out tissue-culturing quick-propagation, cultivate in a short time largely for
The Illigera trifoliata (Griff.) Dunn seedling of field production significantly improves the breeding coefficient and seedling quality of Illigera trifoliata (Griff.) Dunn seedling, realizes scale
Production meets the needs in production；
2) work of Illigera trifoliata (Griff.) Dunn is carried out by breeding rejuvenation integration culture, hardening of taking root synchronizing culture two-steps tissue culture method
Factory's metaplasia produces, and shortens incubation time, reduces incubation step, reduces toxigenic capacity, improve Illigera trifoliata (Griff.) Dunn tissue culture seedling
Production efficiency；
3) the sterile branch health for cultivating acquisition on breeding rejuvenation integration culture medium is sturdy, without vitrification phenomenon, exhibition
Leaf is good, and degree of lignification is higher, is inoculated on root media and takes root all right, rooting rate 92% or more, educate by transplanting
Survival rate is 91.6% or more after seedling greenhouse；
4) contain 1.2mg/L nano zine oxide in hardening synchronizing culture base of taking root of the invention, can significantly promote and take root,
Rooting rate is improved, while the resistance of plant can be enhanced, improves transplanting survival rate.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, to enable those skilled in the art's reference say
Bright book text can be implemented accordingly.
It should be noted that experimental method described in following embodiments is unless otherwise specified conventional method, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
Embodiment 1:
A kind of quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, comprising the following steps:
1) it breeds rejuvenation integration culture: using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, being inoculated in breeding rejuvenation one
Change culture medium, be placed in culture 15d in culturing room, cultivate 22-26 DEG C of room temperature, intensity of illumination 1400lux, light application time 8h/d,
Then it is transferred to 25-28 DEG C of cultivation temperature, continues to cultivate 15d under the conditions of the natural lighting of intensity of illumination 3500lux, obtains stalwartness
Multiple Buds breed the formula of rejuvenation integration culture medium are as follows: MS+1.0mg/L BA+0.3mg/L LFS+0.1mg/L IBA+
0.1mg/L NAA；
2) hardening of taking root synchronizing culture: the Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root
In, in 22-26 DEG C of indoor cultivation temperature, intensity of illumination 1500lux, light application time cultivates 7d under conditions of being 10h/d, then is transferred to
Cultivating 25d under the natural light that cultivation temperature is 23-28 DEG C in greenhouse, relative humidity is 60-80% and obtaining is suitable for the complete of transplanting
Plant, wherein greenhouse top covers sunshade net, obscurity 20%, wherein the formula of hardening of taking root synchronizing culture base are as follows: 1/
2MS+0.1mg/L IBA+1.0mg/L NAA+0.5mg/L IAA+0.3mg/L MET；
3) it transplants: the intact plant that step 2) obtains is taken out from hardening synchronizing culture base of taking root, it is residual to clean root
Stay culture medium, transplanting to seedling bed or seedling raising greenhouse, the envionmental humidity of transplanting is 80-90%, 25-28 DEG C of cultivation temperature,
Obscurity is 60%.
Wherein, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the inoculation side of explant described in step 1)
Method are as follows: the explant is laid in the surface of breeding rejuvenation integration culture medium, and the 1/2 explant indentation breeding is multiple
In strong integration culture medium.
Wherein, MS is MS minimal medium, and BA is 6-benzyladenine, and LFS is spirit hair element, and IBA is indolebutyric acid, NAA
For methyl α-naphthyl acetate, IAA is heteroauxin, and MET is paclobutrazol.
Embodiment 2:
A kind of quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, comprising the following steps:
1) it breeds rejuvenation integration culture: using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, being inoculated in breeding rejuvenation one
Change culture medium, be placed in culture 15d in culturing room, cultivate 22-26 DEG C of room temperature, intensity of illumination 1400lux, light application time 8h/d,
Then it is transferred to 25-28 DEG C of cultivation temperature, continues to cultivate 15d under the conditions of the natural lighting of intensity of illumination 4500lux, obtains stalwartness
Multiple Buds breed the formula of rejuvenation integration culture medium are as follows: MS+2.0mg/L BA+0.5mg/L LFS+0.3mg/L IBA+
1.0mg/L NAA；
2) hardening of taking root synchronizing culture: the Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root
In, in 22-26 DEG C of indoor cultivation temperature, intensity of illumination 1500lux, light application time cultivates 7d under conditions of being 10h/d, then is transferred to
Cultivating 25d under the natural light that cultivation temperature is 23-28 DEG C in greenhouse, relative humidity is 60-80% and obtaining is suitable for the complete of transplanting
Plant, wherein greenhouse top covers sunshade net, obscurity 40%, wherein the formula of hardening of taking root synchronizing culture base are as follows: 1/
2MS+0.3mg/L IBA+2.0mg/L NAA+1.5mg/L IAA+0.5mg/L MET；
3) it transplants: the intact plant that step 2) obtains is taken out from hardening synchronizing culture base of taking root, it is residual to clean root
Stay culture medium, transplanting to seedling bed or seedling raising greenhouse, the envionmental humidity of transplanting is 80-90%, 25-28 DEG C of cultivation temperature,
Obscurity is 75%.
Wherein, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the inoculation side of explant described in step 1)
Method are as follows: the explant is laid in the surface of breeding rejuvenation integration culture medium, and the 1/2 explant indentation breeding is multiple
In strong integration culture medium.
Wherein, MS is MS minimal medium, and BA is 6-benzyladenine, and LFS is spirit hair element, and IBA is indolebutyric acid, NAA
For methyl α-naphthyl acetate, IAA is heteroauxin, and MET is paclobutrazol.
Embodiment 3:
A kind of quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, comprising the following steps:
1) it breeds rejuvenation integration culture: using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, being inoculated in breeding rejuvenation one
Change culture medium, be placed in culture 15d in culturing room, cultivate 22-26 DEG C of room temperature, intensity of illumination 1400lux, light application time 8h/d,
Then it is transferred to 25-28 DEG C of cultivation temperature, continues to cultivate 15d under the conditions of the natural lighting of intensity of illumination 4000lux, obtains stalwartness
Multiple Buds breed the formula of rejuvenation integration culture medium are as follows: MS+1.5mg/L BA+0.4mg/L LFS+0.2mg/L IBA+
0.5mg/L NAA；
2) hardening of taking root synchronizing culture: the Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root
In, in 22-26 DEG C of indoor cultivation temperature, intensity of illumination 1500lux, light application time cultivates 7d under conditions of being 10h/d, then is transferred to
Cultivating 25d under the natural light that cultivation temperature is 23-28 DEG C in greenhouse, relative humidity is 60-80% and obtaining is suitable for the complete of transplanting
Plant, wherein greenhouse top covers sunshade net, obscurity 30%, wherein the formula of hardening of taking root synchronizing culture base are as follows: 1/
2MS+0.2mg/L IBA+1.5mg/L NAA+1.0mg/L IAA+0.4mg/L MET；
3) it transplants: the intact plant that step 2) obtains is taken out from hardening synchronizing culture base of taking root, it is residual to clean root
Stay culture medium, transplanting to seedling bed or seedling raising greenhouse, the envionmental humidity of transplanting is 80-90%, 25-28 DEG C of cultivation temperature,
Obscurity is 65%.
Wherein, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the inoculation side of explant described in step 1)
Method are as follows: the explant is laid in the surface of breeding rejuvenation integration culture medium, and the 1/2 explant indentation breeding is multiple
In strong integration culture medium.
Wherein, MS is MS minimal medium, and BA is 6-benzyladenine, and LFS is spirit hair element, and IBA is indolebutyric acid, NAA
For methyl α-naphthyl acetate, IAA is heteroauxin, and MET is paclobutrazol.
Wherein, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn, the acquisition methods of the explant in step 1)
It is as follows: to be inoculated in culture acquisition adventitious bud on propagating culture medium after taking stem section disinfection of the Illigera trifoliata (Griff.) Dunn with axillary bud, and in sterile item
Cutting obtains the stem with bud of 1.0-2.0cm long as explant, the formula of the propagating culture medium are as follows: MS+1.5mg/L under part
BA+0.4mg/L LFS+0.5mg/L IBA。
Embodiment 4:
On the basis of embodiment 3, the quick breeding method for tissue culture of the Illigera trifoliata (Griff.) Dunn is described in step 2)
Hardening of taking root synchronizing culture base also contains 1.2mg/L nano zine oxide.
In order to illustrate the effect of the quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn of the invention, the present inventor's needle
Comparative study has been carried out to above-described embodiment 1-4,5 bottles of samples are prepared with every group of embodiment, 24 plants every bottle, totally 100 plants, are united respectively
Plant is survived after take root plant and the rooting rate that cleaning root remaining medium obtains before meter transplanting, and transplanting culture 30 days
Several and survival rate, as a result see the table below 1.As shown in Table 1, the rooting rate and survival rate for the Illigera trifoliata (Griff.) Dunn that method of the invention obtains are equal
It is higher.
The rooting rate and survival rate of the different embodiments of table 1
| It takes root plant number/strain | Rooting rate % | Survive plant number/strain | Survival rate % | |
| Embodiment 1 | 95 | 95 | 87 | 91.6 |
| Embodiment 2 | 92 | 92 | 85 | 92.4 |
| Embodiment 3 | 93 | 93 | 90 | 96.8 |
| Embodiment 4 | 99 | 99 | 97 | 98.0 |
The present inventor is object with embodiment 4, studies the hardening of taking root of the nano zine oxide containing various concentration
Influence of the synchronizing culture base to the rooting rate during Illigera trifoliata (Griff.) Dunn tissue culture and the survival rate of plant after transplanting culture 30 days, sample
This quantity is 5 bottles, 24 plants every bottle, totally 100 plants, the results are shown in Table 2.
Influence of the nano zine oxide of 2 various concentration of table to Illigera trifoliata (Griff.) Dunn tissue culture rooting rate and survival rate
As can be seen from Table 2, nano zine oxide takes root with facilitation to Illigera trifoliata (Griff.) Dunn, and with taking root, hardening is synchronized
The nano oxidized zinc concentration that culture medium contains increases, and the rooting rate of Illigera trifoliata (Griff.) Dunn is in the trend of reduction after first increasing, and survival rate
It is stable compared to relatively, but ratio is not added in the embodiment of nano zine oxide, and survival rate significantly improves, and illustrates that nano zine oxide has
The effect for promoting plant to survive.Wherein, when being 1.2mg/L with nano oxidized Zn content, rooting rate that Illigera trifoliata (Griff.) Dunn tissue culture obtains
Highest.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (4)
1. a kind of quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn, which comprises the following steps:
1) it breeds rejuvenation integration culture: using the stem with bud of Illigera trifoliata (Griff.) Dunn as explant, being inoculated in breeding rejuvenation integration training
Base is supported, culture 15d in culturing room is placed in, cultivates 22-26 DEG C of room temperature, intensity of illumination 1400lux, light application time 8h/d, then
It is transferred to 25-28 DEG C of cultivation temperature, continues to cultivate 15d under the conditions of the natural lighting of intensity of illumination 3500-4500lux, is obtained healthy and strong
Multiple Buds, breed rejuvenation integration culture medium formula are as follows: MS+1.0-2.0mg/L BA+0.3-0.5mg/L LFS+0.1-
0.3mg/L IBA+0.1-1.0mg/L NAA；
2) hardening of taking root synchronizing culture: the Multiple Buds that step 1) culture obtains are inoculated into hardening synchronizing culture base of taking root,
In 22-26 DEG C of indoor cultivation temperature, intensity of illumination 1500lux, light application time cultivates 7d under conditions of being 10h/d, then is transferred to big
Cultivating 25d under the natural light that cultivation temperature is 23-28 DEG C in canopy, relative humidity is 60-80% and obtaining is suitable for the complete plant transplanted
Strain, wherein greenhouse top covers sunshade net, obscurity 20-40%, wherein the formula of hardening of taking root synchronizing culture base are as follows:
1/2MS+0.1-0.3mg/L IBA+1.0-2.0mg/L NAA+0.5-1.5mg/L IAA+0.3-0.5mg/L MET；
3) it transplants: the intact plant that step 2) obtains is taken out from hardening synchronizing culture base of taking root, clean root residual training
Support base, transplanting to seedling bed or seedling raising greenhouse；
The acquisition methods of explant in step 1) are as follows: being inoculated in breeding culture after taking stem section disinfection of the Illigera trifoliata (Griff.) Dunn with axillary bud
Culture obtains adventitious bud on base, and aseptically cutting obtains the stem with bud of 1.0-2.0cm long as explant, standby
With the formula of the propagating culture medium are as follows: MS+1.5mg/L BA+0.4mg/L LFS+0.5mg/L IBA；
The formula of the hardening synchronizing culture base of taking root are as follows: 1/2MS+0.2mg/L IBA+1.5mg/L NAA+1.0mg/L
IAA+0.4mg/L MET；
In step 2), the hardening synchronizing culture base of taking root also contains 1.2mg/L nano zine oxide.
2. the quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn as described in claim 1, which is characterized in that described in step 1)
The inoculation method of explant are as follows: the explant is laid in the surface of breeding rejuvenation integration culture medium, and described outer by 1/2
In implant indentation breeding rejuvenation integration culture medium.
3. the quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn as described in claim 1, which is characterized in that the breeding rejuvenation
The formula of integrated culture medium are as follows: MS+1.5mg/L BA+0.4mg/L LFS+0.2mg/L IBA+0.5mg/L NAA.
4. the quick breeding method for tissue culture of Illigera trifoliata (Griff.) Dunn as described in claim 1, which is characterized in that transplanting in step 3)
Envionmental humidity be 80-90%, 25-28 DEG C of cultivation temperature, obscurity 60-75%.
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