CN106069774A - A kind of sinocalamus latiflorus stem end callus induction the method obtaining regeneration plant - Google Patents
A kind of sinocalamus latiflorus stem end callus induction the method obtaining regeneration plant Download PDFInfo
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- CN106069774A CN106069774A CN201610564784.XA CN201610564784A CN106069774A CN 106069774 A CN106069774 A CN 106069774A CN 201610564784 A CN201610564784 A CN 201610564784A CN 106069774 A CN106069774 A CN 106069774A
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- callus
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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 callus induction the method obtaining regeneration plant, belong to the fast numerous field of forest.The method include the selection of outer implant, the inducing culture of callus and enrichment culture, the induction of embryoid and sprouting, take root, strong sprout, seedling exercising and transplanting are buried.Culture medium that inducing culture that the present invention provides, proliferated culture medium, induction are sprouted and root media, make sinocalamus latiflorus differentiation efficiency height, growth coefficient height, rooting rate high, the demand of plant growth time is short, strong adaptability after cultivation, nursery stock stalwartness is tall and straight, grows fine, neat and consistent, substantially reduce nursery stock incubation, save great amount of cost.It is initial wound healing induction system that the present invention establishes by the nutritive issue of sinocalamus latiflorus, and is successfully obtained regeneration plant, and the most numerous for sinocalamus latiflorus Seedling provides a kind of technological means, and setting up for sinocalamus latiflorus transformation system in the future provides platform.
Description
Technical field
The invention belongs to the fast numerous field of forest, be specifically related to a kind of sinocalamus latiflorus stem end callus induction and obtain regeneration plant
Method.
Background technology
Sinocalamus latiflorus is grass family Bambusoideae Dendrocalamus sinocalamus latiflorus subgenus, naturally divides in portions such as Yunnan, Fujian-Taiwan Guangdong, Guangxi Guizhou
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 research that sinocalamus latiflorus tissue culture is relevant is fewer, is currently mainly main outer implant with flower pesticide and embryo.
1987, Yeh etc. was material by the embryo of sinocalamus latiflorus, successfully induces the callus of sinocalamus latiflorus and obtains regeneration plant, but by
The longest in the cycle of becoming civilized of sinocalamus latiflorus, therefore seed is difficult to obtain, and causes the restriction of the aspect of drawing materials of the method.Nineteen ninety Tsay etc.
The flower pesticide utilizing sinocalamus latiflorus obtains regeneration plant after inducing callus, but the haplobiont obtained by the method is being transplanted
I.e. death in later six months, this explanation the method feasibility on producing is less；Meanwhile, the outer implant of bamboo class is obtained by this system
Having significant limitation, the blooming cycle of such as sinocalamus latiflorus is the longest, and seldom blooms, and is therefore not readily available flower pesticide；Trained by flower pesticide
Being become monoploid by diploid from the point of view of in the regeneration plant theory raised, the later stage growth to plant causes unpredictable shadow
Ring.Hence set up the regenerating system so that sinocalamus latiflorus nutritive issue is outer implant and can crack the difficult problem that outer implant is difficult to obtain, be existing
New approach is opened up in sinocalamus latiflorus breeding for biotechnology applications.
Summary of the invention
Present invention aims to prior art not enough, it is provided that a kind of sinocalamus latiflorus stem end callus induction also obtains again
The method of raw plant.By regulating specific plant culture and different phytohormone proportionings, optimum culture condition is induced
The callus of sinocalamus latiflorus stem end, and obtain regeneration plant after induction differentiation.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of sinocalamus latiflorus stem end callus induction the method obtaining regeneration plant, specifically include following steps:
(1) selection of outer implant: taking perennial sinocalamus latiflorus edible tender branch is outer implant, rinses 2-3 hour through flowing water, 70wt%'s
Ethanol surface sterilization 45 seconds, aseptic water washing 3-5 time, the mercuric chloride concussion sterilization of rear 0.1wt%, the time of sterilization is 8 minutes,
Aseptic water washing 5-10 time；Surface moisture is blotted afterwards with aseptic paper, standby as outer implant；
(2) inducing culture of callus and enrichment culture: the aseptic explant that step (1) obtains is cut into the little of 0.5-1 cm
Section, is placed on the inducing culture of callus, and after cultivating 30-40 days under dark condition, callus initially forms；Induce
Callus present various states, separate faint yellow and fine and close embryo callus, be inoculated on proliferated culture medium, black
Cultivating under dark state, change a subculture every two weeks, the enrichment culture time is 3-4 week；
(3) induction of embryoid and sprouting: separation step (2) is bred good callus and is inoculated into the culture medium that induction is sprouted
On, to cultivate 30-50 days under illumination condition, embryoid is gradually induced and is sprouted；The condition of culture that induction is sprouted is: illumination 16 h, light
According to intensity 2000 lux；The efficiency of differentiation is at 50 %；
(4) taking root: separated from callus by the Multiple Buds induced in step (3), every 3-5 strain is that a unit is placed into
In root media；Within about 15 days, it can be seen that the sprouting of root, after 30-40 days, root is the most sturdy, and the efficiency taken root reaches 90%；
(5) strong sprout, seedling exercising and transplanting are buried: in step (4), the tissue cultured seedling in root media carries out bud while taking root
Propagation, puts in tap water in half-open ampuliform state seedling exercising 2-3 days after tissue cultured seedling takes root 3-4 root, after by plant from culture medium
Take out, after cleaning culture medium, then place 1 day；After transplanting is buried, in hot-house culture 1-2 week, hot-house culture condition is: illumination 16 h,
Intensity 2000 lux, temperature 21-26 DEG C；Carrying out astigmatism cultivation of shading subsequently, spray water every day, humidity is maintained at 40wt%-60wt%；
Survival rate 90%.
The inducing culture of the callus described in step (2): MS+BAP 0.3 mg/L+2,4-D 4 mg/L+IBA
0.2 mg/L+ 2-(N-morpholino) ethyl sulfonic acid 0.5 g/L+ sucrose 50 mg/L+ agar powder 8 g/L, PH be 5.0.
Proliferated culture medium described in step (2): MS+ sucrose 50 mg/L+PVP 0.5g/L+2,4-D 4 mg/L+ agar powder
8 g/L, PH are 5.8.
The culture medium that induction described in step (3) is sprouted: NB culture medium+sucrose 30 g/L+ 6-BA 3 mg/L+1.5
Mg/L KT+NAA 0.1 mg/L+250 mg/L PVP+ agar powder 8 g/L, PH are 5.8.
Root media described in step (4): MS+NAA 0.2 mg/L+ agar powder 8g/L, PH are 6.0.
The beneficial effects of the present invention is:
(1) establishing by the nutritive issue of sinocalamus latiflorus is initial wound healing induction system, and is successfully obtained regeneration plant, for sinocalamus latiflorus Seedling
The most numerous provide a kind of technological means；
(2) offer platform is set up for sinocalamus latiflorus transformation system in the future.
Accompanying drawing explanation
Fig. 1 is the whole process being initiateed by stem end and inducing sinocalamus latiflorus regenerating system: Figure 1A is to induce on stem end inducing culture
Go out callus；Figure 1B is callus successive transfer culture；Fig. 1 C is callus initial stage form on inducing culture；Fig. 1 D
For regeneration plant induced synthesis；Fig. 1 E is the raw growth of regeneration plant bunch；Fig. 1 F is regeneration plant root induction；Fig. 1 G is that regeneration is planted
Strain is cultivated in soil and is survived.
Detailed description of the invention
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 callus induction the method obtaining regeneration plant, specifically include following steps:
(1) selection of outer implant: taking perennial sinocalamus latiflorus edible tender branch is outer implant, rinses 3 hours through flowing water, the second of 70wt%
Alcohol surface sterilization 45 seconds, aseptic water washing 4 times, the mercuric chloride concussion sterilization of rear 0.1wt%, the time of sterilization is 8 minutes, aseptic
Water rinses 8 times；Surface moisture is blotted afterwards with aseptic paper, standby as outer implant；
(2) inducing culture of callus and enrichment culture: the aseptic explant that step (1) obtains is cut into the little of 0.5 cm
Section, is placed on the inducing culture of callus, and after cultivating 30 days under dark condition, callus initially forms；Induce heals
Injured tissue presents various states, separates faint yellow and fine and close embryo callus, is inoculated on proliferated culture medium, at dark shape
Cultivating under state, change a subculture every two weeks, the enrichment culture time is 3 weeks；
(3) induction of embryoid and sprouting: separation step (2) is bred good callus and is inoculated into the culture medium that induction is sprouted
On, to cultivate 30 days under illumination condition, embryoid is gradually induced and is sprouted；The condition of culture that induction is sprouted is: illumination 16 h, illumination is strong
Spend 2000 lux；The efficiency of differentiation is at 50 %；
(4) taking root: separated from callus by the Multiple Buds induced in step (3), every 3 strains are that a unit is placed into life
In root culture medium；The efficiency taken root reaches 90%；
(5) strong sprout, seedling exercising and transplanting are buried: in step (4), the tissue cultured seedling in root media carries out bud while taking root
Propagation, takes root at tissue cultured seedling and puts into after 3 in tap water in half-open ampuliform state seedling exercising 2 days, after plant is taken out from culture medium,
After cleaning culture medium, then place 1 day；After transplanting is buried, hot-house culture 1 week, hot-house culture condition is: illumination 16 h, intensity
2000 lux, temperature 25 DEG C；Carrying out astigmatism cultivation of shading subsequently, spray water every day, humidity is maintained at 40wt%；Survival rate 90%.
The inducing culture of the callus described in step (2): MS+BAP 0.3 mg/L+2,4-D 4 mg/L+IBA
0.2 mg/L+ 2-(N-morpholino) ethyl sulfonic acid 0.5 g/L+ sucrose 50 mg/L+ agar powder 8 g/L, PH be 5.0.
Proliferated culture medium described in step (2): MS+ sucrose 50 mg/L+PVP 0.5g/L+2,4-D 4 mg/L+ agar powder
8 g/L, PH are 5.8.
The culture medium that induction described in step (3) is sprouted: NB culture medium+sucrose 30 g/L+ 6-BA 3 mg/L+1.5
Mg/L KT+NAA 0.1 mg/L+250 mg/L PVP+ agar powder 8 g/L, PH are 5.8.
Root media described in step (4): MS+NAA 0.2 mg/L+ agar powder 8g/L, PH are 6.0.
Embodiment 2
A kind of sinocalamus latiflorus stem end callus induction the method obtaining regeneration plant, specifically include following steps:
(1) selection of outer implant: taking perennial sinocalamus latiflorus edible tender branch is outer implant, rinses 2 hours through flowing water, the second of 70wt%
Alcohol surface sterilization 45 seconds, aseptic water washing 5 times, the mercuric chloride concussion sterilization of rear 0.1wt%, the time of sterilization is 8 minutes, aseptic
Water rinses 10 times；Surface moisture is blotted afterwards with aseptic paper, standby as outer implant；
(2) inducing culture of callus and enrichment culture: the aseptic explant that step (1) obtains is cut into the segment of 1 cm,
Being placed on the inducing culture of callus, after cultivating 40 days under dark condition, callus initially forms；The wound healing induced
Tissue presents various states, separates faint yellow and fine and close embryo callus, is inoculated on proliferated culture medium, at dark state
Under cultivate, change a subculture every two weeks, the enrichment culture time is 4 weeks；
(3) induction of embryoid and sprouting: separation step (2) is bred good callus and is inoculated into the culture medium that induction is sprouted
On, to cultivate 50 days under illumination condition, embryoid is gradually induced and is sprouted；The condition of culture that induction is sprouted is: illumination 16 h, illumination is strong
Spend 2000 lux；The efficiency of differentiation is at 50 %；
(4) taking root: separated from callus by the Multiple Buds induced in step (3), every 5 strains are that a unit is placed into life
In root culture medium；The efficiency taken root reaches 90%；
(5) strong sprout, seedling exercising and transplanting are buried: in step (4), the tissue cultured seedling in root media carries out bud while taking root
Propagation, takes root at tissue cultured seedling and puts into after 4 in tap water in half-open ampuliform state seedling exercising 3 days, after plant is taken out from culture medium,
After cleaning culture medium, then place 1 day；After transplanting is buried, hot-house culture 2 weeks, hot-house culture condition is: illumination 16 h, intensity
2000 lux, temperature 26 DEG C；Carrying out astigmatism cultivation of shading subsequently, spray water every day, humidity is maintained at 60wt%；Survival rate 90%.
The inducing culture of the callus described in step (2): MS+BAP 0.3 mg/L+2,4-D 4 mg/L+IBA
0.2 mg/L+ 2-(N-morpholino) ethyl sulfonic acid 0.5 g/L+ sucrose 50 mg/L+ agar powder 8 g/L, PH be 5.0.
Proliferated culture medium described in step (2): MS+ sucrose 50 mg/L+PVP 0.5g/L+2,4-D 4 mg/L+ agar powder
8 g/L, PH are 5.8.
The culture medium that induction described in step (3) is sprouted: NB culture medium+sucrose 30 g/L+ 6-BA 3 mg/L+1.5
Mg/L KT+NAA 0.1 mg/L+250 mg/L PVP+ agar powder 8 g/L, PH are 5.8.
Root media described in step (4): MS+NAA 0.2 mg/L+ agar powder 8g/L, PH are 6.0.
Embodiment 3
A kind of sinocalamus latiflorus stem end callus induction the method obtaining regeneration plant, specifically include following steps:
(1) selection of outer implant: taking perennial sinocalamus latiflorus edible tender branch is outer implant, rinses 3 hours through flowing water, the second of 70wt%
Alcohol surface sterilization 45 seconds, aseptic water washing 3 times, the mercuric chloride concussion sterilization of rear 0.1wt%, the time of sterilization is 8 minutes, aseptic
Water rinses 8 times；Surface moisture is blotted afterwards with aseptic paper, standby as outer implant；
(2) inducing culture of callus and enrichment culture: the aseptic explant that step (1) obtains is cut into the little of 0.8 cm
Section, is placed on the inducing culture of callus, and after cultivating 35 days under dark condition, callus initially forms；Induce heals
Injured tissue presents various states, separates faint yellow and fine and close embryo callus, is inoculated on proliferated culture medium, at dark shape
Cultivating under state, change a subculture every two weeks, the enrichment culture time is 3 weeks；
(3) induction of embryoid and sprouting: separation step (2) is bred good callus and is inoculated into the culture medium that induction is sprouted
On, to cultivate 40 days under illumination condition, embryoid is gradually induced and is sprouted；The condition of culture that induction is sprouted is: illumination 16 h, illumination is strong
Spend 2000 lux；The efficiency of differentiation is at 50 %；
(4) taking root: separated from callus by the Multiple Buds induced in step (3), every 4 strains are that a unit is placed into life
In root culture medium；The efficiency taken root reaches 90%；
(5) strong sprout, seedling exercising and transplanting are buried: in step (4), the tissue cultured seedling in root media carries out bud while taking root
Propagation, takes root at tissue cultured seedling and puts into after 3 in tap water in half-open ampuliform state seedling exercising 2 days, after plant is taken out from culture medium,
After cleaning culture medium, then place 1 day；After transplanting is buried, hot-house culture 2 weeks, hot-house culture condition is: illumination 16 h, intensity
2000 lux, temperature 21 DEG C；Carrying out astigmatism cultivation of shading subsequently, spray water every day, humidity is maintained at 50wt%；Survival rate 90%.
The inducing culture of the callus described in step (2): MS+BAP 0.3 mg/L+2,4-D 4 mg/L+IBA
0.2 mg/L+ 2-(N-morpholino) ethyl sulfonic acid 0.5 g/L+ sucrose 50 mg/L+ agar powder 8 g/L, PH be 5.0.
Proliferated culture medium described in step (2): MS+ sucrose 50 mg/L+PVP 0.5g/L+2,4-D 4 mg/L+ agar powder
8 g/L, PH are 5.8.
The culture medium that induction described in step (3) is sprouted: NB culture medium+sucrose 30 g/L+ 6-BA 3 mg/L+1.5
Mg/L KT+NAA 0.1 mg/L+250 mg/L PVP+ agar powder 8 g/L, PH are 5.8.
Root media described in step (4): MS+NAA 0.2 mg/L+ agar powder 8g/L, PH are 6.0.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
Claims (5)
1. a sinocalamus latiflorus stem end callus induction the method that obtains regeneration plant, it is characterised in that: specifically include following step
Rapid:
(1) selection of outer implant: taking perennial sinocalamus latiflorus edible tender branch is outer implant, rinses 2-3 hour through flowing water, 70wt%'s
Ethanol surface sterilization 45 seconds, aseptic water washing 3-5 time, the mercuric chloride concussion sterilization of rear 0.1wt%, the time of sterilization is 8 minutes,
Aseptic water washing 5-10 time；Surface moisture is blotted afterwards with aseptic paper, standby as outer implant；
(2) inducing culture of callus and enrichment culture: the aseptic explant that step (1) obtains is cut into the little of 0.5-1 cm
Section, is placed on the inducing culture of callus, and after cultivating 30-40 days under dark condition, callus initially forms；Induce
Callus present various states, separate faint yellow and fine and close embryo callus, be inoculated on proliferated culture medium, black
Cultivating under dark state, change a subculture every two weeks, the enrichment culture time is 3-4 week；
(3) induction of embryoid and sprouting: separation step (2) is bred good callus and is inoculated into the culture medium that induction is sprouted
On, to cultivate 30-50 days under illumination condition, embryoid is gradually induced and is sprouted；The condition of culture that induction is sprouted is: illumination 16 h, light
According to intensity 2000 lux；
(4) taking root: separated from callus by the Multiple Buds induced in step (3), every 3-5 strain is that a unit is placed into
In root media；
(5) strong sprout, seedling exercising and transplanting are buried: in step (4), the tissue cultured seedling in root media carries out bud while taking root
Propagation, puts in tap water in half-open ampuliform state seedling exercising 2-3 days after tissue cultured seedling takes root 3-4 root, after by plant from culture medium
Take out, after cleaning culture medium, then place 1 day；After transplanting is buried, in hot-house culture 1-2 week, hot-house culture condition is: illumination 16 h,
Intensity 2000 lux, temperature 21-26 DEG C；Carrying out astigmatism cultivation of shading subsequently, spray water every day, humidity is maintained at 40wt%-60wt%.
Sinocalamus latiflorus stem end callus induction the most according to claim 1 the method obtaining regeneration plant, its feature exists
In: the inducing culture of the callus described in step (2): MS+BAP 0.3 mg/L+2,4-D 4 mg/L+IBA 0.2
Mg/L+ 2-(N-morpholino) ethyl sulfonic acid 0.5 g/L+ sucrose 50 mg/L+ agar powder 8 g/L, PH be 5.0.
Sinocalamus latiflorus stem end callus induction the most according to claim 1 the method obtaining regeneration plant, its feature exists
In: the proliferated culture medium described in step (2): MS+ sucrose 50 mg/L+PVP 0.5g/L+2,4-D 4 mg/L+ agar powder 8 g/
L, PH are 5.8.
Sinocalamus latiflorus stem end callus induction the most according to claim 1 the method obtaining regeneration plant, its feature exists
In: the culture medium that the induction described in step (3) is sprouted: NB culture medium+sucrose 30 g/L+ 6-BA 3 mg/L+1.5 mg/L
KT+NAA 0.1 mg/L+250 mg/L PVP+ agar powder 8 g/L, PH are 5.8.
Sinocalamus latiflorus stem end callus induction the most according to claim 1 the method obtaining regeneration plant, its feature exists
In: the root media described in step (4): MS+NAA 0.2 mg/L+ agar powder 8g/L, PH are 6.0.
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| CN201610564784.XA CN106069774B (en) | 2016-07-19 | 2016-07-19 | A kind of sinocalamus latiflorus stem end evoked callus and the method for obtaining regeneration plant |
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| CN113115709A (en) * | 2021-05-17 | 2021-07-16 | 中国林业科学研究院亚热带林业研究所 | In-vitro regeneration method for immature embryo of dendrocalamus latiflorus |
| CN113692969A (en) * | 2021-03-11 | 2021-11-26 | 吉林农业大学 | Tissue culture method 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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113692969A (en) * | 2021-03-11 | 2021-11-26 | 吉林农业大学 | Tissue culture method of dendrocalamus latiflorus |
| CN113115709A (en) * | 2021-05-17 | 2021-07-16 | 中国林业科学研究院亚热带林业研究所 | In-vitro regeneration method for immature embryo of dendrocalamus latiflorus |
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| CN106069774B (en) | 2018-06-29 |
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