CN105191789B - A kind of purification and rejuvenation method of test tube seedling in Banana Tissue incubation - Google Patents
A kind of purification and rejuvenation method of test tube seedling in Banana Tissue incubation Download PDFInfo
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Abstract
The present invention relates to a kind of purification and rejuvenation method of test tube seedling in Banana Tissue incubation, including by the former shoop-tips growing point of banana by three stage cultivation detoxification rejuvenation processing acquisition rejuvenation banana plant.Rejuvenation plantlet of transplant can be recovered into the original merit of banana again into soil, it is to avoid degenerate.The transplanting banana seedlings that this method is obtained are highly consistent, robust growth, and yield is high.
Description
Technical field
The invention belongs to plant tissue culture technical field, it is related to a kind of purification and rejuvenation of test tube seedling in Banana Tissue incubation
Method.
Background technology
It is the most important Propagation Methods of current banana that Banana, which carries out factorial praluction with the method for Plant Tissue Breeding, because
The need for can not having met banana large-scale production for traditional banana propagation method, banana industrial seedling rearing has become perfume (or spice)
Important source of seedling in any of several broadleaf plants plantation, but there is a degenerate problem in the tissue-cultured seedling of banana, especially in the continuous generation of subculture 10
Left and right will generally produce degradation phenomena, and the tissue-cultured seedling plant produced after 10 generations is short and small, yield poorly, therefore tissue culture seedlings of bananas
Variety deterioration problem is always the bottleneck problem during banana is quickly bred
Therefore, the problem of presently, there are is that a kind of kind that can solve the problem that after the multiple subculture of tissue culture seedlings of bananas of research and development is moved back
The banana tissue culture method of change problem.
The content of the invention
The technical problems to be solved by the invention are that there is provided a kind of Banana Tissue incubation in view of the shortcomings of the prior art
The purification and rejuvenation method of middle test tube seedling, including the former shoop-tips growing point of banana is obtained by three stage cultivation detoxification rejuvenation processing
Obtain rejuvenation banana plant.Rejuvenation plantlet of transplant can be recovered into the original merit of banana again into soil, it is to avoid move back
Change.The banana plantlet in vitro that this method is obtained is highly ordered consistent, and differentiation rate is high, and robust growth, and yield is high；Banana can be solved
Test tube seedling quality problems of the test tube seedling in large-scale industrialized production, meet growing test tube seedling demand.
Therefore, the invention provides a kind of purification and rejuvenation method of test tube seedling in Banana Tissue incubation, including by perfume (or spice)
Any of several broadleaf plants original shoop-tips growing point obtains rejuvenation banana plant by three stage cultivation detoxification rejuvenation processing.
According to the present invention, the three stage cultivation detoxification rejuvenation processing includes：
First stage, cultivate detoxification treatment：The former shoop-tips growing point of banana is placed on culture medium in the first stage
Cultivated, obtain banana seedlings bud point；
Second stage, culture rejuvenation processing：Banana seedlings bud point is moved to and cultivated on second stage culture medium, is answered
Strong banana plantlet in vitro；
Phase III, culture rejuvenation processing：Rejuvenation banana plantlet in vitro is transferred in phase III culture medium and cultivated,
Plant obtains rejuvenation banana plant after taking root；
The former shoop-tips growing point of the banana is to be located at the cone-shaped growth point at the top of the former test tube seedling of banana, the circle
The tip of taper growing point is upward.
In the present invention, the floor space of preferably described cone-shaped growth point is 2-3mm2。
In one or more embodiments of the present invention, first stage culture medium is subject to the first rank by MS minimal mediums
The following component of section culture volume meter is constituted：
6- benzyl purines (6BA) 1-3mg/L；
Vitamin C (Vc) 1-2mg/L；And
α-naphthylacetic acid (NAA) 0.1-0.5mg/L.
In one or more embodiments of the present invention, second stage culture medium is subject to second-order by MS minimal mediums
The following component of section culture volume meter is constituted：
6- benzyl purines (6BA) 1-3mg/L；
Vitamin C (Vc) 0.01-0.1mg/L；And
α-naphthylacetic acid (NAA) 0.1-0.3mg/L.
In one or more embodiments of the present invention, phase III culture medium is subject to the by 1/2MS minimal mediums
The following component of three stage culture volume meters is constituted：
Indolebutyric acid (IBA) 0.1-0.3mg/L；And
α-naphthylacetic acid (NAA) 0.1-0.6mg/L.
In the present invention, preferably in the first stage in culture detoxification treatment, a periodicity of illumination is 24 hours, wherein 12 hours
Illumination/12 hour no light, intensity of illumination is 2500-3000Lux.
In the present invention, preferably in second stage culture rejuvenation processing, rejuvenation banana plantlet in vitro length is 2-3cm.
In the present invention, preferably in phase III culture rejuvenation processing, rejuvenation banana plant contains 2-3 bar roots.
In the present invention, the former test tube seedling of the banana is 8-10 banana plantlet in vitro above of subculture.
The present invention some preferred embodiments in, methods described be additionally included in the first stage culture detoxification treatment it
The step of preceding acquisition banana original shoop-tips growing point, including former test tube seedling is taken out, the blade of stem apex is peelled off, exposes and is located at
The cone-shaped growth point at top, cuts the cone-shaped growth point from base portion, that is, obtains the former shoop-tips growing point of banana.
In other preferred embodiments of the present invention, methods described is additionally included in phase III culture rejuvenation processing
Afterwards, rejuvenation banana plant is transplanted to the step in soil.
Embodiment
To make the present invention easier to understand, the present invention is described in detail below in conjunction with embodiment, these embodiments are only
Serve illustrative, it is not limited to application of the invention.
The present inventor's research finds that the growing point virus of test tube seedling is seldom, if only taking growing point to be inoculated with
Culture, then the seedling differentiated would not infect virus, and the height of seedling degree neat and consistent that such tissue culture is obtained, differentiation rate is high.This
Invention is based on what above-mentioned discovery was made, and specific method is to be carried out by the way that tissue-cultured seedling is stripped into stem apex at Shoot Tip Culture detoxification
Manage to purificate and rejuvenate to test tube seedling in Banana Tissue incubation, so as to obtain rejuvenation banana plant.
Therefore, in a kind of Banana Tissue incubation involved in the present invention test tube seedling purification and rejuvenation method, including will
Banana original shoop-tips growing point obtains rejuvenation banana plant by three stage cultivation detoxification rejuvenation processing.
Specifically, in the present invention, the three stage cultivation detoxification treatment includes：
First stage cultivates detoxification treatment, and it is enterprising that the former shoop-tips growing point of banana is placed into culture medium in the first stage
Row culture, obtains banana seedlings bud point, and the former shoop-tips growing point of the banana is to be located at the circular cone at the top of the former test tube seedling of banana
Shape growing point, the tip of the cone-shaped growth point is upward；
Second stage culture rejuvenation is handled, and banana seedlings bud point is moved to and cultivated on second stage culture medium, is answered
Strong banana plantlet in vitro；
Phase III culture rejuvenation processing, rejuvenation banana plantlet in vitro is transferred in phase III culture medium and cultivated,
Plant obtains rejuvenation banana plant (also known as transplanting banana seedlings) after taking root.
In some specific embodiments, the inventive method comprises the following steps：
(1) the former shoop-tips growing point of banana is obtained：Including former test tube seedling is taken out, the blade of stem apex is peelled off, is exposed
Cone-shaped growth point positioned at top, the floor space of the cone-shaped growth point is 2-3mm2, it is from base portion that the cone is raw
Long point is cut, that is, obtains the former shoop-tips growing point of banana；
(2) first stage culture detoxification treatment：The former shoop-tips growing point of banana is placed into culture medium in the first stage
It is upper to be cultivated, obtain banana seedlings bud point；
(3) second stage culture rejuvenation is handled：Banana seedlings bud point is moved to and cultivated on second stage culture medium, is obtained
Rejuvenation banana plantlet in vitro；
(4) phase III culture rejuvenation processing：Rejuvenation banana plantlet in vitro is transferred in phase III culture medium and trained
Support, plant obtains rejuvenation banana plant after taking root；
(5) rejuvenation banana plant is transplanted in soil.
Above-mentioned steps (2)-(4) are handled to carry out culture detoxification rejuvenation to the former shoop-tips growing point of banana in the present invention
To obtain the three phases of rejuvenation banana plant.
Each stage culture medium is the important foundation for realizing the present invention in the present invention.
In certain specific embodiments of the invention, first stage culture medium is subject to the first stage by MS minimal mediums
The following component of culture volume meter is constituted：
6BA 1-3mg/L；
Vc 1-2mg/L；And
NAA 0.1-0.5mg/L。
In other embodiments of the present invention, second stage culture medium is subject to second-order by MS minimal mediums
The following component of section culture volume meter is constituted：
6BA 1-3mg/L；
Vc 0.01-0.1mg/L；And
NAA 0.1-0.3mg/L。
There are some embodiments in the present invention, phase III culture medium is subject to the by 1/2MS minimal mediums
The following component of three stage culture volume meters is constituted：
IBA 0.1-0.3mg/L；And
NAA 0.1-0.6mg/L。
The present inventor studies by lot of experiments and found, the culture medium using the above three stage contributes to more preferably
Realization the present invention.
In the present invention, preferably in the first stage in culture detoxification treatment, illumination is artificial lighting (fluorescent lamp or LED),
One periodicity of illumination is 21 hours, wherein 12 hours illumination/12 hour no lights, intensity of illumination is 2500-3000Lux.
Heretofore described term " transplanting banana seedlings " refers to that the rejuvenation banana plant that will be obtained according to the inventive method is moved
Plant the banana seedlings grown up in soil.
In the present invention, preferably in second stage culture rejuvenation processing, the length of rejuvenation banana plantlet in vitro is 2-3cm.
The present inventor's research is found, in phase III culture rejuvenation processing, rejuvenation banana plantlet in vitro is shifted
Cultivated into phase III culture medium, plant takes root, and when bearing 2-3 bar roots, obtain the shifting of rejuvenation banana plant
Survival rate is higher after cultivation.
Therefore, in the present invention, preferably in phase III culture rejuvenation processing, the rejuvenation banana plant contains 2-3 bars
Root.
In the present invention, the former test tube seedling of the banana is 8-10 banana plantlet in vitro above of subculture.
The inventive method can be realized by detailed description below：
(1) the former shoop-tips growing point of banana is obtained：By it is more than subculture 8-10 times banana plantlet in vitro (possess stem,
Leaf) with sterile working method test tube seedling is taken out from culture medium on superclean bench, gently peelled off with disinfecting forceps
The blade of stem apex, growth cone (the only 2-3mm until exposing top2Cone-shaped growth point), by this growing point from base
Portion is cut, and obtains the former shoop-tips growing point of banana.
(2) first stage culture detoxification treatment：The former shoop-tips growing point of banana is placed into culture medium in the first stage
It is upper to be cultivated, obtain banana seedlings bud point；The formula of the culture medium in the stage is：MS+6BA1(3mg/L)+Vc1(2mg/L)+
NAA(0.1-0.5mg/L)；It is 20-26 DEG C to cultivate room temperature, and a periodicity of illumination is 24 hours, wherein 12 hours illumination/12 are small
When no light, intensity of illumination is 2500-3000Lux；Differentiating phenomenon just occurs after culturing room cultivates 25-30 days in growth cone,
Start small bud point occur at the top of growth cone, obtain banana seedlings bud point.
(3) second stage culture rejuvenation is handled：Banana seedlings bud point is moved on second stage culture detoxification treatment culture medium
Cultivated, obtain rejuvenation banana plantlet in vitro.The small bud point (i.e. banana seedlings bud point) of differentiation is gone into second stage culture detoxification
Handle on culture medium, the culture medium prescription is：MS+6BA(1-3mg/L)+Vc(0.01-0.1mg/L)+NAA(0.1-0.3mg/
L)；It is 20-26 DEG C to cultivate room temperature, and a periodicity of illumination is 24 hours, wherein 12 hours illumination/12 hour no lights, illumination
Intensity is 2500-3000Lux；After the culture of 25-30 days, small bud point gradually grows one plant of strain banana plantlet in vitro, works as test tube
When seedling plant height reaches 2-3cm, that is, obtain rejuvenation banana plantlet in vitro.
(4) phase III culture rejuvenation processing：Rejuvenation banana plantlet in vitro is transferred to culture of rootage on superclean bench
In base (i.e. phase III culture detoxification treatment culture medium), the culture medium prescription is：1/2MS+IBA(0.1-0.3mg/L)+NAA
(0.1-0.6mg/L).It is 20-26 DEG C to cultivate room temperature, and a periodicity of illumination is 24 hours, wherein 12 hours illumination/12 hour
No light, intensity of illumination is 2500-3000Lux；By the culture of 20 days or so, banana plant just grew the root of 2-3 bars white,
Obtain rejuvenation banana plant, it is possible to which progress is transplanted.
(5) rejuvenation banana plant is transplanted in soil.
The rejuvenation banana plant of this method production is planted into soil, it is possible to recover the original Optimality of banana again
Shape, it is to avoid degenerate.The height one for the transplanting banana seedlings that the rejuvenation banana plant plantation that this method is obtained grows up into soil
Cause, robust growth, yield is high.
Embodiment
Embodiment 1：
(1) the former shoop-tips growing point of banana is obtained：By banana plantlet in vitro (the boat any of several broadleaf plants of subculture 10 times in laboratory
No. 1) use the method for sterile working to take out on super quiet workbench, the blade of stem apex, Zhi Daolu are gently peelled off with disinfecting forceps
Go out a growth cone (only 2-3mm at top2Cone-shaped growth point), will be about at least dialling 3-5 piece blades, this grown
Point is cut from base portion, obtains the former shoop-tips growing point of banana.Banana original shoop-tips growing point is very tiny, tissue children
It is tender, it is careful operation.
(2) first stage culture detoxification treatment：The former shoop-tips growing point of banana is placed into culture medium in the first stage
It is upper to be cultivated, obtain banana seedlings bud point；The formula of the culture medium in the stage is：MS+6BA(2.5mg/L)+Vc(2mg/L)+
NAA(0.3mg/L)；Cultivation temperature is 25 DEG C, and periodicity of illumination is 12 hours bright/12 hours no lights, and intensity of illumination is
2500Lux；Growth cone just occurs after culturing room cultivates 25-30 days starts small bud point occur at the top of differentiating phenomenon, growth cone,
Obtain banana seedlings bud point.
(3) second stage culture rejuvenation is handled：Banana seedlings bud point is moved on second stage culture detoxification treatment culture medium
Cultivated, obtain rejuvenation banana plantlet in vitro.The small bud point (i.e. banana seedlings bud point) of differentiation is gone into second stage culture again to take off
On poison processing culture medium, the culture medium prescription is：MS+6BA(3mg/L)+Vc(0.01mg/L)+NAA(0.1mg/L)；Deng differentiation
When the seedling length gone out is to 3 centimetres, that is, obtain rejuvenation banana plantlet in vitro.
(4) phase III culture rejuvenation processing：Rejuvenation banana plantlet in vitro is cut at root on superclean bench and is transferred to
In root media (i.e. phase III culture detoxification treatment culture medium)；The culture medium prescription is：1/2MS+IBA(0.1mg/L)+
NAA(0.6mg/L)；By the culture of 20 days or so, banana plant just grew the root of 2-3 bars white, obtained rejuvenation banana plant,
It can be carried out transplanting.
(5) the rejuvenation banana plant produced through this method is transplanted to big Tanaka, it is possible to recover banana again original
Merit, it is to avoid degenerate.
The rejuvenation banana plant that this method is obtained is transplanted to big Tanaka, the height of the transplanting banana seedlings grown up in big Tanaka
Unanimously, robust growth, yield is high.
The rejuvenation banana plant that purification and rejuvenation acquisition is carried out using the inventive method was planted in crop field in 2 months 2011,
December is with regard to result then, and yield is high, as a result early.140 rejuvenation banana plants have been planted altogether, and 20,000 jin of perfume (or spice) are obtained altogether
Any of several broadleaf plants, average 140 jin of single plant yield, 1 times is improved relative to the plant yield not produced using the inventive method, and in good taste.
The banana plantlet in vitro not produced using the inventive method was planted in big as the comparison example of the present invention in 2 months 2011
Field, in March, the 2nd, result, and yielded poorly, and fruit ear is short, and single fruit weight is small, 70 jin of single plant yield.
Embodiment 2：
(1) the former shoop-tips growing point of banana is obtained：By the banana plantlet in vitro of subculture 10 times navigates any of several broadleaf plants 2 in laboratory
Taken out using the method for sterile working number on super quiet workbench, the blade of stem apex is gently peelled off with disinfecting forceps, until exposing
A growth cone (only 2-3mm at top2Cone-shaped growth point), will be about at least dialling 3-5 piece blades, by this growing point
Cut from base portion, obtain the former shoop-tips growing point of banana.Banana original shoop-tips growing point is very tiny, tissue children
It is tender, it is careful operation.
(2) first stage culture detoxification treatment：The former shoop-tips growing point of banana is placed into culture medium in the first stage
It is upper to be cultivated, obtain banana seedlings bud point；The formula that the culture medium in the stage is：MS+6BA(1mg/L)+Vc(1mg/L)+NAA
(0.03mg/L)；Cultivation temperature is 25 DEG C, and periodicity of illumination is 12 hours illumination/12 hour no lights, and intensity of illumination is
3000Lux；Growth cone just occurs after culturing room cultivates 25-30 days starts small bud point occur at the top of differentiating phenomenon, growth cone,
Obtain banana seedlings bud point.
(3) second stage culture rejuvenation is handled：Banana seedlings bud point is moved on second stage culture detoxification treatment culture medium
Cultivated, obtain rejuvenation banana plantlet in vitro.The small bud point (i.e. banana seedlings bud point) of differentiation is gone into second stage culture again to take off
On poison processing culture medium, the culture medium prescription is：MS+6BA(1.5mg/L)+Vc(0.01mg/L)+NAA(0.1mg/L)；Decile
When the seedling length dissolved is to 3 centimetres, that is, obtain rejuvenation banana plantlet in vitro.
(4) phase III culture rejuvenation processing：Rejuvenation banana plantlet in vitro is cut at root on superclean bench and is transferred to
In root media (i.e. phase III culture detoxification treatment culture medium), the culture medium prescription is：1/2MS+IBA(0.2mg/L)+
NAA(0.2mg/L)；By the culture of 20 days or so, banana plant just grew the root of 2-3 bars white, obtained rejuvenation banana plant,
It can be carried out transplanting.
The rejuvenation banana plant obtained through this Methods For Purification rejuvenation for 2 months (5) 2010 years is planted in a piece of hillside fields in Shenzhen
On, November then, this batch transplants banana seedlings successful result, and fruit ear neat and consistent, yield is high, and average single plant yield is up to 110 jin, phase
60% is improved for the plant yield not produced using the inventive method.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (6)
1. a kind of purification and rejuvenation method of test tube seedling in Banana Tissue incubation, including by the former shoop-tips growing point of banana
Rejuvenation banana plant is obtained by three stage cultivation detoxification rejuvenation processing, the three stage cultivation detoxification rejuvenation processing includes：
First stage, cultivate detoxification treatment：The former shoop-tips growing point of banana is placed and carried out on culture medium in the first stage
Culture, obtains banana seedlings bud point；
Second stage, culture rejuvenation processing：Banana seedlings bud point is moved to and cultivated on second stage culture medium, rejuvenation is obtained fragrant
Any of several broadleaf plants test tube seedling；
Phase III, culture rejuvenation processing：Rejuvenation banana plantlet in vitro is transferred in phase III culture medium and cultivated, plant
Rejuvenation banana plant is obtained after taking root；
The former shoop-tips growing point of the banana is to be located at the cone-shaped growth point at the top of the former test tube seedling of banana, the cone
The tip of growing point is upward；
The following component that first stage culture medium is subject to first stage culture volume meter by MS minimal mediums is constituted：
6- benzyl purines 1-3mg/L；
Vitamin C 1-2mg/L；And
α-naphthylacetic acid 0.1-0.5mg/L；
The following component that second stage culture medium is subject to second stage culture volume meter by MS minimal mediums is constituted：
6- benzyl purines 1-3mg/L；
Vitamin C 0.01-0.1mg/L；And
α-naphthylacetic acid 0.1-0.3mg/L；
The following component that phase III culture medium is subject to phase III culture volume meter by 1/2MS minimal mediums is constituted：
Indolebutyric acid 0.1-0.3mg/L；And
α-naphthylacetic acid 0.1-0.6mg/L.
2. according to the method described in claim 1, it is characterised in that the floor space of the cone-shaped growth point is 2-3mm2。
3. method according to claim 1 or 2, it is characterised in that cultivate in the first stage in detoxification treatment, an illumination
Cycle is 24 hours, wherein 12 hours illumination/12 hour no lights；Intensity of illumination is 2500-3000Lux.
4. method according to claim 1 or 2, it is characterised in that in second stage culture rejuvenation processing, rejuvenation banana
Test tube seedling length is 2-3cm.
5. method according to claim 1 or 2, it is characterised in that in phase III culture rejuvenation processing, rejuvenation banana
Plant contains 2-3 bar roots.
6. method according to claim 1 or 2, it is characterised in that methods described is additionally included in cultivates de- in the first stage
The step of banana original shoop-tips growing point is obtained before poison processing, including former test tube seedling is taken out, the blade of stem apex is peelled off,
Expose the cone-shaped growth point positioned at top, cut the cone-shaped growth point from base portion, that is, obtain the former test tube seedling stem of banana
Sharp growing point.
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| CN114258857A (en) * | 2021-12-08 | 2022-04-01 | 天津农学院 | Comprehensive method for preventing and treating aging of tissue culture seedlings and application |
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| CN104686323B (en) * | 2014-12-31 | 2017-05-17 | 四川省农业科学院园艺研究所 | Method for cultivating strawberry seedlings by secondary detoxification method |
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