CN106069774B - A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant - Google Patents
A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant Download PDFInfo
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- CN106069774B CN106069774B CN201610564784.XA CN201610564784A CN106069774B CN 106069774 B CN106069774 B CN 106069774B CN 201610564784 A CN201610564784 A CN 201610564784A CN 106069774 B CN106069774 B CN 106069774B
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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
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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 invention discloses a kind of sinocalamus latiflorus stem end evoked callus and the methods that obtain regeneration plant, belong to forest numerous field soon.This method include the selection of explant, the Fiber differentiation and Multiplying culture of callus, the induction of embryoid and sprouting, take root, strong sprout, hardening and transplanting are buried.The culture medium and root media that inducing culture provided by the invention, proliferated culture medium, induction are sprouted, so that sinocalamus latiflorus differentiation efficiency is high, growth coefficient is high, rooting rate is high, the demand of plant growth time is short, it is adaptable after cultivation, nursery stock stalwartness is tall and straight, grows fine, neat and consistent, nursery stock incubation is substantially reduced, has saved great amount of cost.The present invention is established by the callus induction system that the nutritive issue of sinocalamus latiflorus is starting, and successfully obtains regeneration plant, for sinocalamus latiflorus seedling it is fast it is numerous provide a kind of technological means, the sinocalamus latiflorus transformation system for future, which is established, provides platform.
Description
Technical field
The invention belongs to forest numerous fields soon, and in particular to a kind of sinocalamus latiflorus stem end evoked callus simultaneously obtains regeneration plant
Method.
Background technology
Sinocalamus latiflorus is grass family Bambusoideae Dendrocalamus sinocalamus latiflorus subgenus, is divided naturally in portions such as Fujian-Taiwan Guangdong, Guangxi Guizhou Yunnan
Cloth is one of important large-scale sympodial bamboo kind of double purposes of In South China and the southeastern coastal areas, has high economic valency
Value and social value.The relevant research of sinocalamus latiflorus tissue cultures is fewer, at present mainly using anther and embryo as main explant.
1987, Yeh etc. was material by the embryo of sinocalamus latiflorus, successfully induces the callus of sinocalamus latiflorus and obtains regeneration plant, but by
It is very long in the period of becoming civilized of sinocalamus latiflorus, therefore seed is not easy to obtain, and leads to the limitation in terms of the materials of this method.Nineteen ninety Tsay etc.
Regeneration plant is obtained after inducing callus using the anther of sinocalamus latiflorus, but is being transplanted by the haplobiont that this method obtains
I.e. death in later six months, this illustrates that feasibility of this method in production is smaller；Meanwhile the system obtains the explant of bamboo class
There is a significant limitation, for example the blooming cycle of sinocalamus latiflorus is very long, and seldom blooms, therefore be not readily available anther；It is trained by anther
The regeneration plant that raises causes unpredictable shadow theoretically speaking become monoploid by diploid to the later stage development of plant
It rings.Therefore the problem that explant is difficult to obtain can be cracked by establishing the regenerating system using sinocalamus latiflorus nutritive issue as explant, be existing
New approach is opened up in sinocalamus latiflorus breeding for biotechnology applications.
Invention content
It is an object of the invention in view of the shortcomings of the prior art, providing a kind of sinocalamus latiflorus stem end evoked callus and obtaining again
The method of raw plant.It is matched by adjusting specific plant culture and different plant hormones, optimum culture condition induction
The callus at sinocalamus latiflorus stem end, and obtain regeneration plant after induction is broken up.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant, specifically include following steps：
（1）The selection of explant：Perennial sinocalamus latiflorus edible tender branch is taken to be rinsed 2-3 hours through flowing water for explant,
The ethyl alcohol surface sterilization of 70wt% 45 seconds, aseptic water washing 3-5 times is rear to be sterilized with the mercuric chloride concussion of 0.1wt%, and the time of disinfection is
8 minutes, aseptic water washing 5-10 times；Surface moisture is blotted with aseptic paper afterwards, it is spare as explant；
（2）The Fiber differentiation and Multiplying culture of callus：By step（1）Obtained aseptic explant is cut into 0.5-1 cm
Segment, be placed on the inducing culture of callus, callus initially forms after being cultivated 30-40 days under dark condition；It lures
Various states are presented in derived callus, and the faint yellow and fine and close embryo callus of separation is inoculated on proliferated culture medium,
It is cultivated in the dark state, replaces a subculture every two weeks, the Multiplying culture time is 3-4 weeks；
（3）The induction and sprouting of embryoid：Separation step（2）It is proliferated good callus and is inoculated into the training that induction is sprouted
It supports on base, is cultivated 30-50 days under illumination condition, embryoid gradually sprout by induction；Inducing the condition of culture sprouted is：Illumination 16
H, 2000 lux of intensity of illumination；The efficiency of differentiation is in 50 %；
（4）It takes root：By step（3）In the Multiple Buds that induce detached from callus, be that a unit is put per 3-5 plant
It is placed in root media；It can see sprouting for root within 15 days or so, root is gradually sturdy after 30-40 days, and the efficiency taken root reaches
90%；
（5）Strong sprout, hardening and transplanting are buried：Step（4）Tissue-cultured seedling in middle root media carries out while taking root
The proliferation of bud is put into tap water after tissue culture seedling rooting 3-4 roots in half-open ampuliform state hardening 2-3 days, after by plant from culture
It takes out in base, after cleaning culture medium, then places 1 day；It transplants after burying, hot-house culture 1-2 weeks, hot-house culture condition is：Illumination
16 h, 2000 lux of intensity, 21-26 DEG C of temperature；Shade astigmatism culture is then carried out, is sprayed water daily, humidity is maintained at 40wt%-
60wt%；Survival rate 90%.
Step（2）The inducing culture of the callus：MS+BAP 0.3 mg/L+2, 4-D 4 mg/L+IBA
0.2 mg/L+ 2-（N- morpholinoes）0.5 g/L+ sucrose of ethanesulfonic acid, 50 mg/L+ agar powders 8 g/L, PH 5.0.
Step（2）The proliferated culture medium：50 mg/L+PVP 0.5g/L+2,4-D of MS+ sucrose, 4 mg/L+ agar powders
8 g/L, PH 5.8.
Step（3）The culture medium that the induction is sprouted：NB culture mediums+30 g/L+ 6-BA of sucrose, 3 mg/L+1.5
0.1 mg/L+250 mg/L PVP+ agar powders of mg/L KT+NAA 8 g/L, PH 5.8.
Step（4）Described in root media：0.2 mg/L+ agar powders 8g/L, PH 6.0 of MS+NAA.
The beneficial effects of the present invention are：
（1）It establishes and is the callus induction system of starting by the nutritive issue of sinocalamus latiflorus, and successfully obtain regeneration plant, for fiber crops
The fast of bamboo seedling numerous provides a kind of technological means；
（2）Sinocalamus latiflorus transformation system foundation for future provides platform.
Description of the drawings
Fig. 1 is the whole process that induction sinocalamus latiflorus regenerating system is originated by stem end：Figure 1A is induces on the inducing culture of stem end
Go out callus；Figure 1B is callus squamous subculture；Fig. 1 C are initial stage form of the callus on inducing culture；Fig. 1 D
For regeneration plant induced synthesis；Fig. 1 E are grown for regeneration plant fasciation；Fig. 1 F are regeneration plant root induction；Fig. 1 G are planted for regeneration
Strain is cultivated in soil to be survived.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited only to these embodiments.
Embodiment 1
A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant, specifically include following steps：
（1）The selection of explant：Perennial sinocalamus latiflorus edible tender branch is taken to be rinsed 3 hours through flowing water, 70wt% for explant
Ethyl alcohol surface sterilization 45 seconds, aseptic water washing 4 times, the rear mercuric chloride concussion disinfection with 0.1wt%, the time of disinfection is 8 minutes,
Aseptic water washing 8 times；Surface moisture is blotted with aseptic paper afterwards, it is spare as explant；
（2）The Fiber differentiation and Multiplying culture of callus：By step（1）Obtained aseptic explant is cut into 0.5 cm's
Segment is placed on the inducing culture of callus, and callus initially forms after being cultivated 30 days under dark condition；It induces
Various states are presented in callus, and the faint yellow and fine and close embryo callus of separation is inoculated on proliferated culture medium, in dark
It is cultivated under state, replaces a subculture every two weeks, the Multiplying culture time is 3 weeks；
（3）The induction and sprouting of embryoid：Separation step（2）It is proliferated good callus and is inoculated into the training that induction is sprouted
It supports on base, is cultivated 30 days under illumination condition, embryoid gradually sprout by induction；Inducing the condition of culture sprouted is：16 h of illumination, light
According to 2000 lux of intensity；The efficiency of differentiation is in 50 %；
（4）It takes root：By step（3）In the Multiple Buds that induce detached from callus, every 3 plants are placed for a unit
Enter in root media；The efficiency taken root reaches 90%；
（5）Strong sprout, hardening and transplanting are buried：Step（4）Tissue-cultured seedling in middle root media carries out while taking root
The proliferation of bud is put into tap water after tissue culture seedling rooting 3 in half-open ampuliform state hardening 2 days, after by plant from culture medium
It takes out, after cleaning culture medium, then places 1 day；It transplants after burying, hot-house culture 1 week, hot-house culture condition is：16 h of illumination, by force
Spend 2000 lux, 25 DEG C of temperature；Shade astigmatism culture is then carried out, is sprayed water daily, humidity is maintained at 40wt%；Survival rate 90%.
Step（2）The inducing culture of the callus：MS+BAP 0.3 mg/L+2, 4-D 4 mg/L+IBA
0.2 mg/L+ 2-（N- morpholinoes）0.5 g/L+ sucrose of ethanesulfonic acid, 50 mg/L+ agar powders 8 g/L, PH 5.0.
Step（2）The proliferated culture medium：50 mg/L+PVP 0.5g/L+2,4-D of MS+ sucrose, 4 mg/L+ agar powders
8 g/L, PH 5.8.
Step（3）The culture medium that the induction is sprouted：NB culture mediums+30 g/L+ 6-BA of sucrose, 3 mg/L+1.5
0.1 mg/L+250 mg/L PVP+ agar powders of mg/L KT+NAA 8 g/L, PH 5.8.
Step（4）Described in root media：0.2 mg/L+ agar powders 8g/L, PH 6.0 of MS+NAA.
Embodiment 2
A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant, specifically include following steps：
（1）The selection of explant：Perennial sinocalamus latiflorus edible tender branch is taken to be rinsed 2 hours through flowing water, 70wt% for explant
Ethyl alcohol surface sterilization 45 seconds, aseptic water washing 5 times, the rear mercuric chloride concussion disinfection with 0.1wt%, the time of disinfection is 8 minutes,
Aseptic water washing 10 times；Surface moisture is blotted with aseptic paper afterwards, it is spare as explant；
（2）The Fiber differentiation and Multiplying culture of callus：By step（1）Obtained aseptic explant is cut into the small of 1 cm
Section, is placed on the inducing culture of callus, callus initially forms after being cultivated 40 days under dark condition；What is induced is cured
Various states are presented in injured tissue, and the faint yellow and fine and close embryo callus of separation is inoculated on proliferated culture medium, in dark shape
It is cultivated under state, replaces a subculture every two weeks, the Multiplying culture time is 4 weeks；
（3）The induction and sprouting of embryoid：Separation step（2）It is proliferated good callus and is inoculated into the training that induction is sprouted
It supports on base, is cultivated 50 days under illumination condition, embryoid gradually sprout by induction；Inducing the condition of culture sprouted is：16 h of illumination, light
According to 2000 lux of intensity；The efficiency of differentiation is in 50 %；
（4）It takes root：By step（3）In the Multiple Buds that induce detached from callus, every 5 plants are placed for a unit
Enter in root media；The efficiency taken root reaches 90%；
（5）Strong sprout, hardening and transplanting are buried：Step（4）Tissue-cultured seedling in middle root media carries out while taking root
The proliferation of bud is put into tap water after tissue culture seedling rooting 4 in half-open ampuliform state hardening 3 days, after by plant from culture medium
It takes out, after cleaning culture medium, then places 1 day；It transplants after burying, hot-house culture 2 weeks, hot-house culture condition is：16 h of illumination, by force
Spend 2000 lux, 26 DEG C of temperature；Shade astigmatism culture is then carried out, is sprayed water daily, humidity is maintained at 60wt%；Survival rate 90%.
Step（2）The inducing culture of the callus：MS+BAP 0.3 mg/L+2, 4-D 4 mg/L+IBA
0.2 mg/L+ 2-（N- morpholinoes）0.5 g/L+ sucrose of ethanesulfonic acid, 50 mg/L+ agar powders 8 g/L, PH 5.0.
Step（2）The proliferated culture medium：50 mg/L+PVP 0.5g/L+2,4-D of MS+ sucrose, 4 mg/L+ agar powders
8 g/L, PH 5.8.
Step（3）The culture medium that the induction is sprouted：NB culture mediums+30 g/L+ 6-BA of sucrose, 3 mg/L+1.5
0.1 mg/L+250 mg/L PVP+ agar powders of mg/L KT+NAA 8 g/L, PH 5.8.
Step（4）Described in root media：0.2 mg/L+ agar powders 8g/L, PH 6.0 of MS+NAA.
Embodiment 3
A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant, specifically include following steps：
（1）The selection of explant：Perennial sinocalamus latiflorus edible tender branch is taken to be rinsed 3 hours through flowing water, 70wt% for explant
Ethyl alcohol surface sterilization 45 seconds, aseptic water washing 3 times, the rear mercuric chloride concussion disinfection with 0.1wt%, the time of disinfection is 8 minutes,
Aseptic water washing 8 times；Surface moisture is blotted with aseptic paper afterwards, it is spare as explant；
（2）The Fiber differentiation and Multiplying culture of callus：By step（1）Obtained aseptic explant is cut into 0.8 cm's
Segment is placed on the inducing culture of callus, and callus initially forms after being cultivated 35 days under dark condition；It induces
Various states are presented in callus, and the faint yellow and fine and close embryo callus of separation is inoculated on proliferated culture medium, in dark
It is cultivated under state, replaces a subculture every two weeks, the Multiplying culture time is 3 weeks；
（3）The induction and sprouting of embryoid：Separation step（2）It is proliferated good callus and is inoculated into the training that induction is sprouted
It supports on base, is cultivated 40 days under illumination condition, embryoid gradually sprout by induction；Inducing the condition of culture sprouted is：16 h of illumination, light
According to 2000 lux of intensity；The efficiency of differentiation is in 50 %；
（4）It takes root：By step（3）In the Multiple Buds that induce detached from callus, every 4 plants are placed for a unit
Enter in root media；The efficiency taken root reaches 90%；
（5）Strong sprout, hardening and transplanting are buried：Step（4）Tissue-cultured seedling in middle root media carries out while taking root
The proliferation of bud is put into tap water after tissue culture seedling rooting 3 in half-open ampuliform state hardening 2 days, after by plant from culture medium
It takes out, after cleaning culture medium, then places 1 day；It transplants after burying, hot-house culture 2 weeks, hot-house culture condition is：16 h of illumination, by force
Spend 2000 lux, 21 DEG C of temperature；Shade astigmatism culture is then carried out, is sprayed water daily, humidity is maintained at 50wt%；Survival rate 90%.
Step（2）The inducing culture of the callus：MS+BAP 0.3 mg/L+2, 4-D 4 mg/L+IBA
0.2 mg/L+ 2-（N- morpholinoes）0.5 g/L+ sucrose of ethanesulfonic acid, 50 mg/L+ agar powders 8 g/L, PH 5.0.
Step（2）The proliferated culture medium：50 mg/L+PVP 0.5g/L+2,4-D of MS+ sucrose, 4 mg/L+ agar powders
8 g/L, PH 5.8.
Step（3）The culture medium that the induction is sprouted：NB culture mediums+30 g/L+ 6-BA of sucrose, 3 mg/L+1.5
0.1 mg/L+250 mg/L PVP+ agar powders of mg/L KT+NAA 8 g/L, PH 5.8.
Step（4）Described in root media：0.2 mg/L+ agar powders 8g/L, PH 6.0 of MS+NAA.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (1)
1. a kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant, it is characterised in that：Specifically include following step
Suddenly：
（1）The selection of explant：Perennial sinocalamus latiflorus edible tender branch is taken to be rinsed 2-3 hours through flowing water for explant, 70wt%'s
Ethyl alcohol surface sterilization 45 seconds, aseptic water washing 3-5 times, the rear mercuric chloride concussion disinfection with 0.1wt%, the time of disinfection is 8 minutes,
Aseptic water washing 5-10 times；Surface moisture is blotted with aseptic paper afterwards, it is spare as explant；
（2）The Fiber differentiation and Multiplying culture of callus：By step（1）Obtained aseptic explant is cut into the small of 0.5-1 cm
Section, is placed on the inducing culture of callus, callus initially forms after being cultivated 30-40 days under dark condition；It induces
Callus various states are presented, the faint yellow and fine and close embryo callus of separation is inoculated on proliferated culture medium, black
It is cultivated under dark state, replaces a subculture every two weeks, the Multiplying culture time is 3-4 weeks；
（3）The induction and sprouting of embryoid：Separation step（2）It is proliferated good callus and is inoculated into the culture medium that induction is sprouted
On, it is cultivated 30-50 days under illumination condition, embryoid gradually sprout by induction；Inducing the condition of culture sprouted is：16 h of illumination, light
According to 2000 lux of intensity；
（4）It takes root：By step（3）In the Multiple Buds that induce detached from callus, be that a unit is placed into per 3-5 plant
In root media；
（5）Strong sprout, hardening and transplanting are buried：Step（4）Tissue-cultured seedling in middle root media carries out bud while taking root
Proliferation is put into tap water after tissue culture seedling rooting 3-4 roots in half-open ampuliform state hardening 2-3 days, after by plant from culture medium
It takes out, after cleaning culture medium, then places 1 day；It transplants after burying, hot-house culture 1-2 weeks, hot-house culture condition is：16 h of illumination,
2000 lux of intensity, 21-26 DEG C of temperature；Shade astigmatism culture is then carried out, is sprayed water daily, humidity is maintained at 40wt%-60wt%；
Step（2）The inducing culture of the callus：MS+BAP 0.3 mg/L+2, 4-D 4 mg/L+IBA 0.2
mg/L+ 2-（N- morpholinoes）0.5 g/L+ sucrose of ethanesulfonic acid, 50 mg/L+ agar powders 8 g/L, pH 5.0；
Step（2）The proliferated culture medium：50 mg/L+PVP 0.5g/L+2,4-D of MS+ sucrose, 4 mg/L+ agar powders, 8 g/
L, pH 5.8；
Step（3）The culture medium that the induction is sprouted：NB culture mediums+30 g/L+ 6-BA of sucrose, 3 mg/L+1.5 mg/L
0.1 mg/L+250 mg/L PVP+ agar powders of KT+NAA 8 g/L, pH 5.8；
Step（4）Described in root media：0.2 mg/L+ agar powders 8g/L, pH 6.0 of MS+NAA.
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| CN113692969A (en) * | 2021-03-11 | 2021-11-26 | 吉林农业大学 | Tissue culture method of dendrocalamus latiflorus |
| CN113115709B (en) * | 2021-05-17 | 2022-07-29 | 中国林业科学研究院亚热带林业研究所 | In-vitro regeneration method for immature embryos of dendrocalamus latiflorus |
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|---|---|---|---|---|
| CN103155862A (en) * | 2011-12-12 | 2013-06-19 | 蒋晶 | Method of dendrocalamuslatiflorus Munro anther somatic embryo induction and for acquiring regenerated plant |
| CN104285788A (en) * | 2014-09-19 | 2015-01-21 | 南京林业大学 | Method for building green bamboo regeneration system by somatic embryogenesis approach |
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| CN103155862A (en) * | 2011-12-12 | 2013-06-19 | 蒋晶 | Method of dendrocalamuslatiflorus Munro anther somatic embryo induction and for acquiring regenerated plant |
| CN104285788A (en) * | 2014-09-19 | 2015-01-21 | 南京林业大学 | Method for building green bamboo regeneration system by somatic embryogenesis approach |
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