CN105918119B - A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade - Google Patents
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade Download PDFInfo
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- CN105918119B CN105918119B CN201610262030.9A CN201610262030A CN105918119B CN 105918119 B CN105918119 B CN 105918119B CN 201610262030 A CN201610262030 A CN 201610262030A CN 105918119 B CN105918119 B CN 105918119B
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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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- 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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Abstract
The present invention discloses a kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, comprises the following steps：1) draw materials：Chu chrysanthemum blade is chosen as explant；2) induction 4 of section 3) callus) callus propagation；5) differentiation of callus；6) propagation of adventitious bud and elongation；7) take root：Elongation of adventitious bud is treated to 2~3cm, and with 2~3 full extensions leaf when carry out culture of rootage.There is the present invention convenient material drawing, material to enrich；Regeneration efficiency is high, and callus induction rate is up to 100%, and differentiation rate is up to 95%, and rooting rate is up to 100%；Reproduction speed is fast, and breeding coefficient is high, and can realize the further expanding propagation of carry out to the tests for sterility of acquisition, the advantages of providing important technical support for the quick breeding and breed improvement of excellent chu chrysanthemum strain.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade.
Background technology
Chu chrysanthemum (Dendranthema morifolium CV. ' chuju '), is the distinctive traditional product of Chuzhou City, attaches most importance to
The Chinese herbal medicine wanted and industrial crops, there is the title of Chuzhou tribute chrysanthemum.Belong to medicine, tea dual-purpose good merchantable brand, there is high medicinal and health value,
In the cultivation history of China's existing centuries.Long-term chu chrysanthemum of drinking has clearing heat and detoxicating, relaxing tendons and activating collaterals, liver protection and eyesight, enhancing people
The functions such as body immunity, at the same it is evident in efficacy to coronary heart disease, hypertension.Chu chrysanthemum rank Anhui Province " four is big " famous genunie medicinal materials it
First of first and Chinese " four is big " medicine chrysanthemum.At present, chu chrysanthemum has become Chuzhou locality pillar industry, in Chuzhou area large area kind
Plant.
But at present in production, because chu chrysanthemum is numerous using traditional vegetative propagation means progress such as cuttage and plant division for a long time
Grow so that the problems such as variety deterioration of chu chrysanthemum generally existing, pest and disease damage be serious, related breed improvement and renewal speed are slow, the big day of one's doom
Make using chu chrysanthemum as medicine, tea dual-purpose good merchantable brand carries out the application of cultivation and industrialization on a large scale.Therefore, its breed improvement is strengthened,
Cultivate high yield, high-quality and pest-resistant chu chrysanthemum improved seeds are current development chu chrysanthemum industry tasks the most urgent.Relevant chu chrysanthemum at present
The research of breeding, it is concentrated mainly on and is carried out using hybridization means, far from requirement of the adaptation chrysanthemum agriculture to variety yield and quality.
Due to substantial amounts of aseptic seedling can be obtained in a short time using tissue culture technique, the good species of chu chrysanthemum can realized
While matter resource extent metaplasia is produced so that accelerate the breed improvement process of chu chrysanthemum to be possibly realized.Relevant chu chrysanthemum is in vitro again at present
Raw research is less, wherein using chu chrysanthemum blade be explant carry out Regeneration in Vitro research it has been reported that but generally existing it is numerous
The problems such as coefficient is low, repeatable poor, the easy blackening of explant is dead and atomization vitrification phenomenon is serious is grown, is seriously limited
Application of the blade in chu chrysanthemum rapid propagation in vitro.Problems in the presence of being planted for above-mentioned chu chrysanthemum, while in order to meet to advise
Modelling is planted and breed improvement is to the demand of chu chrysanthemum high quality seedling, is badly in need of developing a kind of side of the in vitro highly efficient regeneration of chu chrysanthemum blade
Method.
The content of the invention
It is an object of the invention to plant tissue culture technique, there is provided a kind of side of the in vitro highly efficient regeneration of chu chrysanthemum blade
Method.To achieve these goals, the present invention adopts the following technical scheme that：
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, comprises the following steps：
1) draw materials：Chu chrysanthemum blade is chosen as explant；
2) cut into slices：Chu chrysanthemum blade in step 1) is cut into slices；
3) induction of callus:Section in step 2) is inoculated in progress callus group in callus inducing medium
The Fiber differentiation knitted；
4) propagation of callus：The section callus of induction in step 3) is inoculated in proliferated culture medium and is cured
The propagation of injured tissue；
5) differentiation of callus：By the callus stripping and slicing after in step 4) breeding and transfer to enter in differential medium
The differentiation of row callus；
6) propagation of adventitious bud and elongation：The callus that adventitious bud is differentiated in step 5) is transferred in adventitious bud proliferation
With the propagation and elongation culture that adventitious bud is carried out in elongation medium；
7) take root：Elongation of adventitious bud is treated to 2~3cm, and with 2~3 full extensions leaf when carry out culture of rootage.
Preferably, the chu chrysanthemum blade of the step 1) be field chu chrysanthemum elite plant strain then give birth to edible tender branch blade or from
Body regenerates the blade of obtained chu chrysanthemum aseptic seedling full extension.
Preferably, the method for the in vitro highly efficient regeneration of chu chrysanthemum blade also includes surface sterilization；Surface sterilization：By step 2)
In have drawn from field then give birth to edible tender branch vanes 75% absolute ethyl alcohol wipe one time after, nothing is used in aseptic working platform
Bacterium water rinses 2~3 times；Then after sterilizing 10~25s with 75% absolute ethyl alcohol, with sterile water wash 2~3 times；Then use
0.1%HgCl2 1~3min of solution disinfection, aseptic water washing 5~6 times.
Preferably, in the step 3) callus inducing medium include MS+0.1~3.0mg/L TDZ+0.01~
0.05mg/L 6-BA+30g/L sucrose+7.0g/L agar, pH=5.8.
Preferably, after 14~16d of illumination cultivation, the Callus of Leaf of induction in step 3) is inoculated in proliferated culture medium
The middle propagation for carrying out callus；Wherein callus proliferation medium be MS+0.2~2.0mg/L TDZ+30g/L sucrose+
7.0g/L agar, pH=5.8.
Preferably, after 10~14d of illumination cultivation, by the callus after breeding in step 4) be cut into (0.4~0.5) ×
(0.4~0.5)) cm2The stripping and slicing of size and transfer in differential medium carry out callus differentiation；Wherein callus
Differential medium is MS+0.2~1.0mg/L TDZ+0.05~0.2mg/L 6-BA+0.1~1.0mg/L NAA+30g/L sucrose
+ 7.0g/L agar, pH=5.8.
Preferably, after 21~28d of illumination cultivation, the callus that adventitious bud is differentiated in step 5) is transferred in adventitious bud
Propagation and the elongation culture of adventitious bud are carried out in propagation and elongation medium；Wherein adventitious bud proliferation and elongation medium are MS+
0.5~2.0mg/L 6-BA+0.05~0.2mg/L TDZ+0.2~2.0mg/L NAA+30g/L sucrose+7.0g/L agar, pH
=5.8.
Preferably, the root media in the step 7) is 1/2MS+0.05~0.3mg/L IBA+0.05~0.5mg/
L IAA+20g/L sucrose+7.0g/L agar, pH=5.8.
The advantage of the invention is that：First, convenient material drawing, material enrich；Second, regeneration efficiency is high, callus induction
Rate can be up to 100%, and differentiation rate can be up to 95%, and rooting rate can be up to 100%；Third, reproduction speed is fast, breeding coefficient is high,
And the further expanding propagation of carry out to the tests for sterility of acquisition can be realized, it is quick breeding and the product of excellent chu chrysanthemum strain
Kind improvement provides important technical support.Therefore, the method for a kind of in vitro highly efficient regeneration of chu chrysanthemum blade provided by the present invention,
The quick breeding and large-scale production for not being only chu chrysanthemum elite plant strain provide technical support, while are also the kind of later stage chu chrysanthemum
Improvement is laid a good foundation.
Brief description of the drawings
Fig. 1 is the induction of chu chrysanthemum Callus of Leaf
Fig. 2 is the propagation of callus
Fig. 3 is the differentiation of callus
Fig. 4 is propagation and the elongation of adventitious bud
Fig. 5 is taking root for adventitious bud
Embodiment
The present invention is described in detail below in conjunction with accompanying drawing.
Embodiment 1
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, concrete operations are as follows：
1st, well-grown perennial chu chrysanthemum elite plant strain is chosen from crop field, chooses chu chrysanthemum and give birth to newborn edible tender branch then
Blade is explant.
2nd, after 75% absolute ethyl alcohol wipes 1 time, in aseptic operating platform, with aseptic water washing 3 times, then with 75% wine
Essence sterilization 10s, aseptic water washing 3 times, sterilizes 1min, finally with aseptic water washing 6 times with 0.1% mercuric chloride afterwards.After sterilization
Blade is cut into 0.3 × 0.3cm2The stripping and slicing of size is standby.
The 3rd, leafcutting proximal ends are inoculated in the induction that callus is carried out in callus inducing medium, warp upwards
15d illumination cultivation is crossed, substantial amounts of light green callus is induced at paddle cutout, and the inductivity of callus is
83.7%；Wherein the inducing culture of callus is MS+0.1mg/L TDZ+0.01mg/L 6-BA+30g/L sucrose+7.0g/
L agar, pH=5.8；
4th, the blade for inducing callus is transferred and the Multiplying culture of callus, illumination is carried out in proliferated culture medium
The substantial amounts of open-textured callus of green is obtained after cultivating 12d；The proliferated culture medium of callus is MS+0.2mg/L
TDZ+30g/L sucrose+7.0g/L agar, pH=5.8；
5th, by the callus after propagation, it is cut into 0.4 × 0.4cm2The stripping and slicing of size simultaneously transfers to enter in differential medium
The differentiation culture of row callus；The substantial amounts of indefinite bud point of green, and callus are differentiated after illumination cultivation 24d, on callus
The differentiation rate of tissue is 89.4%；Wherein the differential medium of callus is MS+0.2mg/L TDZ+0.05mg/L 6-BA+
0.1mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；
6th, the callus of adventitious bud clump will be differentiated, transferring, it is indefinite to be carried out in adventitious bud proliferation and the culture medium of elongation
The propagation of bud and elongation；The adventitious bud largely extended, wherein adventitious bud proliferation and elongation medium are obtained after illumination cultivation 16d
For MS+0.5mg/L 6-BA+0.05mg/L TDZ+0.2mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；
7th, treat Elongation of adventitious bud to 2cm, and with 2 full extensions leaf when, be transferred in root media and given birth to
Root culture；After illumination cultivation 16d, the base portion of adventitious bud grows a large amount of elongated adventitious roots, and the inductivity of adventitious root is up to
100%；Wherein root media is 1/2MS+0.05mg/L IBA+0.05mg/LIAA+20g/L sucrose+7.0g/L agar, pH
=5.8.
Embodiment 2
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, concrete operations are as follows：
1st, well-grown perennial chu chrysanthemum elite plant strain is chosen from crop field, chooses chu chrysanthemum and give birth to newborn edible tender branch then
Blade is explant.
2nd, after 75% absolute ethyl alcohol wipes 1 time, in aseptic operating platform, with aseptic water washing 3 times, then with 75% wine
Essence sterilization 15s, aseptic water washing 2 times, sterilizes 2min, finally with aseptic water washing 6 times with 0.1% mercuric chloride afterwards.After sterilization
Blade is cut into 0.3 × 0.5cm2The stripping and slicing of size is standby.
The 3rd, leafcutting proximal ends are inoculated in the induction that callus is carried out in callus inducing medium, warp upwards
14d illumination cultivation is crossed, substantial amounts of light green callus is induced at paddle cutout, and the inductivity of callus is
100% (Fig. 1)；Wherein the inducing culture of callus be MS+1.5mg/L TDZ+0.03mg/L 6-BA+30g/L sucrose+
7.0g/L agar, pH=5.8；
4th, the blade for inducing callus is transferred and the Multiplying culture of callus, illumination is carried out in proliferated culture medium
The substantial amounts of open-textured callus (Fig. 2) of green is obtained after cultivating 10d；Wherein the proliferated culture medium of callus is MS+
1.0mg/L TDZ+30g/L sucrose+7.0g/L agar, pH=5.8；
5th, by the callus after propagation, it is cut into 0.4 × 0.5cm2The stripping and slicing of size simultaneously transfers to enter in differential medium
The differentiation of row callus；After illumination cultivation 21d, the substantial amounts of indefinite bud point (Fig. 3) of green is differentiated in callus stripping and slicing, and
The differentiation rate of callus is 97.4%；The differential medium of callus is MS+0.5mg/L TDZ+0.1mg/L 6-BA+
0.5mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；
6th, by the callus after differentiation, transfer and the propagation of adventitious bud is carried out in adventitious bud proliferation and the culture medium of elongation
And elongation, the adventitious bud (Fig. 4) largely extended is obtained after illumination cultivation 14d；Wherein adventitious bud proliferation and elongation medium be
MS+1.0mg/L 6-BA+0.1mg/L TDZ+0.5mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；
7th, treat Elongation of adventitious bud to 2cm, and with 2 full extensions leaf when, be transferred in root media and given birth to
Root culture；After illumination cultivation 14d, the base portion of adventitious bud grows a large amount of sturdy adventitious roots (Fig. 5), and the inductivity of adventitious root is high
Up to 100%；Wherein root media is 1/2MS+0.1mg/L IBA+0.1mg/L IAA+20g/L sucrose+7.0g/L agar, pH
=5.8.
Embodiment 3
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, concrete operations are as follows：
1st, well-grown perennial chu chrysanthemum elite plant strain is chosen from crop field, chooses chu chrysanthemum and give birth to the young tender of newborn branch then
Blade is explant.
2nd, after 75% absolute ethyl alcohol wipes 1 time, in aseptic operating platform, with aseptic water washing 2 times, then with 75% alcohol
25s is sterilized, aseptic water washing 3 times, 3min is sterilized with 0.1% mercuric chloride afterwards, finally with aseptic water washing 5 times.Will after sterilization
Blade is cut into 0.5 × 0.5cm2The stripping and slicing of size is standby.
The 3rd, leafcutting proximal ends are inoculated in the induction that callus is carried out in callus inducing medium, warp upwards
16d illumination cultivation is crossed, substantial amounts of callus is induced at paddle cutout, and the inductivity of callus is 100%；Callus
The inducing culture of tissue is MS+3.0mg/L TDZ+0.05mg/L 6-BA+30g/L sucrose+7.0g/L agar, pH=5.8；
4th, the blade for inducing callus is transferred and the Multiplying culture of callus, illumination is carried out in proliferated culture medium
The substantial amounts of green close callus of quality is obtained after cultivating 14d；The proliferated culture medium of callus is MS+2.0mg/L
TDZ+30g/L sucrose+7.0g/L agar, pH=5.8；
5th, by the callus after propagation, it is cut into 0.5 × 0.5cm2The stripping and slicing of size simultaneously transfers to enter in differential medium
The differentiation of row callus；Substantial amounts of indefinite bud point, the differentiation of callus are differentiated after illumination cultivation 28d, on callus
Rate is 79.4%；The differential medium of callus is MS+1.0mg/L TDZ+0.2mg/L 6-BA+1.0mg/L NAA+30g/
L sucrose+7.0g/L agar, pH=5.8；
6th, by the callus after differentiation, transfer and the propagation of adventitious bud is carried out in adventitious bud proliferation and the culture medium of elongation
And elongation, after illumination 21d, obtain the adventitious bud largely extended；Adventitious bud proliferation and elongation medium are MS+2.0mg/L 6-
BA+0.2mg/L TDZ+2.0mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；
7th, treat Elongation of adventitious bud to 3cm, and with 3 full extensions leaf when, be transferred in root media and given birth to
Root culture, after illumination cultivation 21d, the base portion of adventitious bud grows a large amount of short and thick adventitious roots, and the inductivity of adventitious root is up to
97.6%；Wherein root media is 1/2MS+0.3mg/L IBA+0.5mg/LIAA+20g/L sucrose+7.0g/L agar, pH=
5.8。
Embodiment 4
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, concrete operations are as follows：
Test influence of the blade inoculation mode to chu chrysanthemum blade in-vitro regeneration efficiency：Only change the inoculation side of leafcutting
Formula, by the leafcutting after sterilization respectively in a manner of proximal ends and distal shaft end are upward, it is inoculated in callus inducing medium
In, cultivated under light.Remaining step is the same as embodiment 2.After illumination cultivation 14-16d, the induction situation of callus is counted.As a result
Show that induction of the vaccination ways of (table 1) blade to callus plays an important role.The upward inoculation side of blade proximal ends
Formula is more conducive to the induction of callus and the differentiation in later stage.
Influence of the vaccination ways of table 1 to chu chrysanthemum blade Regeneration in Vitro
Note：Data are average ± standard error, and each processing includes 30 explants, and each processing is repeated 3 times.
Embodiment 5
A kind of method of the in vitro highly efficient regeneration of chu chrysanthemum blade, concrete operations are as follows：
Test influence of the source of blade to chu chrysanthemum in-vitro regeneration efficiency：Only change the source of leafcutting, in the future
The leafcutting after the elite plant strain sterilization of field and the chu chrysanthemum tests for sterility of fast numerous acquisition are come from, with the upward side of proximal ends
Formula, it is inoculated in callus inducing medium, is cultivated under light respectively.Remaining step is the same as embodiment 2.Illumination cultivation 14-16d
Afterwards, the induction situation of callus is counted.As a result induction no significant difference of the source of (table 2) blade to callus is shown.
Influence of the blade source of table 2 to chu chrysanthemum blade Regeneration in Vitro
Note：Data are average ± standard error, and each processing includes 30 explants, and each processing is repeated 3 times.
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow be familiar with this art
Personage can understand present invention and be carried out, and it is not intended to limit the scope of the present invention.It is all according to the present invention
The equivalent change or modification that Spirit Essence is made, it should all cover within the scope of the present invention.
Claims (6)
- A kind of 1. method of the in vitro highly efficient regeneration of chu chrysanthemum blade, it is characterised in that：Comprise the following steps：1) draw materials：Chu chrysanthemum blade is chosen as explant；2) cut into slices：Chu chrysanthemum blade in step 1) is cut into slices；3) induction of callus:Section in step 2) is inoculated in callus inducing medium and carries out callus Fiber differentiation；4) propagation of callus：The section callus of induction in step 3) is inoculated in progress callus group in proliferated culture medium The propagation knitted；5) differentiation of callus：Callus stripping and slicing after breeding in step 4) and transferring is cured in differential medium The differentiation of injured tissue；6) propagation of adventitious bud and elongation：The callus that adventitious bud is differentiated in step 5) is transferred in adventitious bud proliferation and stretched Propagation and the elongation culture of adventitious bud are carried out in long culture medium；7) take root：Elongation of adventitious bud is treated to 2~3cm, and with 2~3 full extensions leaf when carry out culture of rootage；Wherein, callus inducing medium includes MS+0.1~3.0mg/L TDZ+0.01~0.05mg/L in the step 3) 6-BA+30g/L sucrose+7.0g/L agar, pH=5.8；Callus proliferation medium is MS+0.2~2.0mg/L TDZ+30g/L sucrose+7.0g/L agar in the step 4), PH=5.8；The differential medium of callus is MS+0.2~1.0mg/L TDZ+0.05~0.2mg/L 6-BA+ in the step 5) 0.1~1.0mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；Adventitious bud proliferation and elongation medium are MS+0.5~2.0mg/L 6-BA+0.05~0.2mg/L in the step 6) TDZ+0.2~2.0mg/L NAA+30g/L sucrose+7.0g/L agar, pH=5.8；Root media in the step 7) is 1/2MS+0.05~0.3mg/L IBA+0.05~0.5mg/L IAA+20g/L Sucrose+7.0g/L agar, pH=5.8.
- A kind of 2. method of in vitro highly efficient regeneration of chu chrysanthemum blade according to claim 1, it is characterised in that：The step 1) Chu chrysanthemum blade give birth to the blade or the chu chrysanthemum aseptic seedling that obtains of Regeneration in Vitro of edible tender branch then by field chu chrysanthemum elite plant strain The blade of full extension.
- A kind of 3. method of in vitro highly efficient regeneration of chu chrysanthemum blade according to claim 2, it is characterised in that：The chu chrysanthemum leaf The method of the in vitro highly efficient regeneration of piece also includes surface sterilization；Surface sterilization：The children raw then in the field that had drawn from step 1) is tender Aseptic water washing is used after the absolute ethyl alcohol of the vanes 75% of branch wipes one time, in aseptic working platform 2~3 times；Then use After 75% absolute ethyl alcohol sterilizes 10~25s, with sterile water wash 2~3 times；Then with 0.1%HgCl2 solution disinfections 1~ 3min, aseptic water washing 5~6 times.
- A kind of 4. method of in vitro highly efficient regeneration of chu chrysanthemum blade according to claim 1, it is characterised in that：Illumination cultivation 14 After~16d, the Callus of Leaf of induction in step 3) is inoculated in the propagation that callus is carried out in proliferated culture medium.
- A kind of 5. method of in vitro highly efficient regeneration of chu chrysanthemum blade according to claim 1, it is characterised in that：Illumination cultivation 10 After~14d, the callus after breeding in step 4) is cut into (0.4~0.5) × (0.4~0.5) cm2The stripping and slicing of size simultaneously turns It is connected to the differentiation that callus is carried out in differential medium.
- A kind of 6. method of in vitro highly efficient regeneration of chu chrysanthemum blade according to claim 1, it is characterised in that：Illumination cultivation 21 After~28d, the callus that adventitious bud is differentiated in step 5) is transferred and carried out not in adventitious bud proliferation and elongation medium The propagation of normal bud and elongation culture.
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| CN108901850A (en) * | 2018-07-26 | 2018-11-30 | 中国科学院合肥物质科学研究院 | A kind of method of spun gold emperor chrysanthemum stem apex Regeneration in Vitro |
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| CN101965799B (en) * | 2010-11-12 | 2012-10-17 | 郑州大学 | Tissue culture quick propagation method for Opisthopappus Shih |
| CN102090342B (en) * | 2011-01-10 | 2013-01-16 | 南京农业大学 | Method for establishing high-efficient regeneration system of dendranthema zawadskii |
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