CN107996410A - A kind of method that tissue cultures are carried out using Kiwi berry blade - Google Patents
A kind of method that tissue cultures are carried out using Kiwi berry blade Download PDFInfo
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- CN107996410A CN107996410A CN201810083010.4A CN201810083010A CN107996410A CN 107996410 A CN107996410 A CN 107996410A CN 201810083010 A CN201810083010 A CN 201810083010A CN 107996410 A CN107996410 A CN 107996410A
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- kiwi berry
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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
-
- 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
Abstract
The present invention relates to one kind to utilize Kiwi berry blade explant evoked callus method, and the present invention uses vegetative manner, the merit of female parent is completely retained, and makes subculture more more excellent than maternal by Shoot-tip Grafting In Vitro.And the problem of woody tissue-cultured seedling quality of rooting difference is breached, tissue-cultured seedling well developed root system, completely, survival rate are high, without hardening, can shorten the production cycle 60 days or so.
Description
Technical field
It is more particularly to a kind of to carry out tissue cultures using Kiwi berry blade the present invention relates to field of plant tissue culture technique
Method.
Background technology
Kiwi berry (scientific name：Actinidia chinensis Planch), it is perennial bejuco, Kiwi berry
Soft texture, sweet mouthfeel.Taste is described as the mixing of strawberry, banana, pineapple three.Kiwi berry remove containing actinidine,
The organic matters such as proteolytic enzyme, tannin pectin and carbohydrate, and 17 kinds of the trace element such as calcium, potassium, selenium, zinc, germanium and needed by human body
Outside amino acid, also containing abundant vitamin C, grape acid, fructose, citric acid, malic acid, fat.
The propagation method of traditional Kiwi berry has sowing, cuttage, press strip and grafting etc..These method relative efficiencies are relatively low, mesh
Before, there are many methods using tissue cultures to carry out Kiwi berry breeding, such as：Patent CN102177847A discloses one kind
The factorial seedling-culturing method of tara vine, including the preparation of culture medium, the selection of explant and disinfection, inoculation, squamous subculture,
Sand bed transplants five steps.But this method needs to configure complicated component and the basic culture of culture medium configuration process requirement strictly
Base, inducing culture and subculture medium, operating process are cumbersome, it is necessary to more manpower and equipment cost be put into, without wide
General applicability.But in the method for existing tissue cultures.There are many shortcomings, are all to need to contain particularly in the stage of taking root
Sugar culture-medium, but sugar is pollution sources, the pollution rate in stage easy to increase of taking root, moreover, conventional method can only obtain when taking root
3-5 main root, and do not have root hair, as shown in Fig. 2, needing later stage hardening culture.
The content of the invention
For problems of the prior art, tissue is carried out using Kiwi berry blade the object of the present invention is to provide one kind
The method of culture.
In order to solve the above-mentioned technical problem, the present invention is achieved by the following scheme：
Blade explant evoked callus method
1. emerald green, thick and solid on the healthy and strong plant of selection, the blade closer from stem apex does explant material, clear water wash clean；
With 0.1% mercuric chloride solution sterilize 10min, then with 75% alcohol sterilize 5min, sterile water wash 3-5 times；
Blade after processing is cut into the square of length of side 1.5cm sizes, be put on inducing culture, illumination 800LX, daily
10h, 25 ± 2 DEG C of temperature, humidity 70%, cultivates 50 days or so and forms Cultured Actinidia deliciosa Calli.
Inducing culture is：MS culture mediums+sucrose 20g/L+ agar 6g/L+6-BA0.8mg/L+NAA (methyl α-naphthyl acetate)
The culture medium of 0.2mg/L+2,4-D (2,4 dichlorophenoxyacetic acid) 0.5mg/L；
2. induce Multiple Buds
Callus is divided into the fritter of diameter 1cm or so, disposes dead leaf tissue, be partly embedded into MS+
In the culture medium of sucrose 30g/L+ agar 6g/L+6-BA5mg/L+NAA0.5mg/L+IAA0.2mg/L, illumination 2000-2500LX,
Time 14h, 25 ± 2 DEG C of temperature, humidity 70%, callus surface forms adventitious bud after cultivating 50 days, obtains primary.
3. squamous subculture (Multiplying culture) is the adventitious bud insertion MS+ sucrose 30g/L+ agar 6g/L formed on callus
On the culture medium of+6-BA1.2mg/L+IAA0.8mg/L, illumination 2000-3000LX, 14h, 25 ± 2 DEG C of temperature, humidity 70%, is trained
Support 25 days or so, 3-5 times of appreciation rate.
4. root induction more than 3cm is reached after propagation, has the plant insertion of more than 3 cotyledons is special to take root in equipment
In the matrix in face, which is MS+ vermiculites, light application time 16h, 25 ± 2 DEG C of temperature, humidity 80%, gas concentration lwevel
900ppm-1000ppm, more than 1000 grades of culturing room's cleanliness factor.Culture forms well developed root system Kiwi berry after 20 days is completely planted
Strain.
MS+ vermiculites add 800g vermiculites for every liter of MS solution.
5. a Kiwi berry intact plant is transplanted to inside degraded bag, warm canopy is transplanted to, 15-30 DEG C of temperature, humidity 85%, one
Crop field, survival rate more than 99% can be moved on to after week.
The present invention uses vegetative manner, the merit of female parent is completely retained, and make by Shoot-tip Grafting In Vitro
Subculture is more more excellent than maternal.And the problem of woody tissue-cultured seedling quality of rooting difference is breached, tissue-cultured seedling well developed root system, completely, into
Motility rate is high, without hardening, can shorten the production cycle 60 days or so.
Brief description of the drawings：
Fig. 1 is：State after the completion of Kiwi berry root induction in embodiment 1；
Fig. 2 is：State after the completion of traditional tissue cultures root induction；
Fig. 3：The dimensional structure diagram of embodiment 3；
Fig. 4：The cross-sectional view of embodiment 3；
Fig. 5：The overlooking the structure diagram of root bed described in embodiment 3.
Embodiment
Below in conjunction with preferred embodiment, to according to embodiment provided by the invention, details are as follows：
Embodiment 1
Using Xu Xiang kiwi fruit leafs as group training material, tissue cultures are carried out using the method for the present invention.
1. emerald green, thick and solid in the healthy and strong kiwi fruit plant of selection, the blade closer from stem apex does explant material, and clear water is washed
Totally；
With 0.1% mercuric chloride solution sterilize 10min, then with 75% alcohol sterilize 5min, sterile water wash 3-5 times；
Blade after processing is cut into the square of length of side 1.5cm sizes, is put on inducing culture,
Illumination 800LX, daily 10h, 25 ± 2 DEG C of temperature, humidity 70%, cultivates 50 days or so and forms Kiwi berry callus group
Knit.
Inducing culture is：MS culture mediums+sucrose 20g/L+ agar 6g/L+6-BA0.8mg/L+NAA (methyl α-naphthyl acetate)
The culture medium of 0.2mg/L+2,4-D (2,4 dichlorophenoxyacetic acid) 0.5mg/L；
2. induce Multiple Buds
Callus is divided into the fritter of diameter 1cm or so, disposes dead leaf tissue, be partly embedded into MS+
In the culture medium of sucrose 30g/L+ agar 6g/L+6-BA5mg/L+NAA0.5mg/L+IAA0.2mg/L, illumination 2000-2500LX,
Time 14h, 25 ± 2 DEG C of temperature, humidity 70%, callus surface forms adventitious bud after cultivating 50 days, obtains primary, such as Fig. 3
It is shown.
3. squamous subculture (Multiplying culture) is the adventitious bud insertion MS+ sucrose 30g/L+ agar 6g/L formed on callus
On the culture medium of+6-BA1.2mg/L+IAA0.8mg/L, illumination 2000-3000LX, 14h, 25 ± 2 DEG C of temperature, humidity 70%, is trained
Support 25 days, 3-5 times of appreciation rate, as shown in Figure 4.
4. more than 3cm is reached after propagation, the plant with more than 3 cotyledons is inserted into special equipment of taking root for root induction
In matrix inside (such as embodiment 3), which is MS+ vermiculites, light application time 16h, 1500-2000LX, 25 ± 2 DEG C of temperature,
Gas concentration lwevel 950ppm, humidity 80%, more than 1000 grades of culturing room's cleanliness factor.Culture forms well developed root system after 20 days
Kiwi berry intact plant.As shown in Figure 5.
5. a Kiwi berry intact plant is transplanted to inside degraded bag, warm canopy is transplanted to, 15-30 DEG C of temperature, humidity 85%, one
Crop field, survival rate more than 99% can be moved on to after week.
Embodiment 2
Using Xu Xiang kiwi fruit leafs as group training material, tissue cultures are carried out using different methods.
Experimental group：Use the method for the embodiment of the present invention 1；
Compare 1 group：Culture medium is used by root induction step：0.7mg·L-1IBA、30mg·L-1Sucrose, 7mg
L-1The 1/2MS culture mediums of agar, other are identical with experimental group.
Compare 2 groups：Culture medium is used by root induction step：In 1/2MS+NAA0.2mgL-1, other and experiment
Group is identical.
Compare 3 groups：Light application time 10h used by root induction step, 25 ± 2 DEG C of temperature, humidity 85%, other and reality
It is identical to test group.
Compare 4 groups：Gas concentration lwevel 1200ppm used by root induction step, other are identical with experimental group.
| Take root pollution rate | Transplanting survival rate | Take root situation | Whether hardening | |
| Experimental group | 0.5% | 99% | It is good | It is no |
| Compare 1 group | 5% | 86% | Generally | It is |
| Compare 2 groups | 6% | 91% | Generally | It is |
| Compare 3 groups | 1% | 95% | Preferably | It is no |
| Compare 4 groups | 1% | 93% | Generally | It is |
Embodiment 3
A kind of special equipment of taking root of tissue cultures, as shown in Figures 3 to 5, including：Box body 1, box lid 2 and root bed 3, it is described
Box lid 2 is connected by hinge 26 with box body 1, can be overturn with respect to box body 1；Described bed 3 is by some orthogonal partition plate groups
Into described bed 3 has the root bed region 30 for being used to house culture medium of several mutually independent groined types, the box lid 2
Bottom surface on be provided with LED light 20, some ventilation holes 10 are offered in the side wall of the box body 1, residing for the ventilation hole 10
Position is higher than the top surface of root bed 3.The root that macaque peach seedling is grown can all be separated by root bed 3, after the completion of avoiding training seedling, it
Warped roots in together, time-consuming and laborious when they are separated and easy damaged root system.
The above described is only a preferred embodiment of the present invention, being not the limitation for making other forms to the present invention, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But it is every without departing from technical solution of the present invention content, the technical spirit according to the present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection domain of technical solution of the present invention.
Claims (3)
- A kind of 1. method that tissue cultures are carried out using Kiwi berry blade, it is characterised in that the method for root induction is：More than 3cm is reached after propagation, the plant with more than 3 cotyledons is inserted into inside special equipment of taking root (such as embodiment 3) Matrix on, which is MS+ vermiculites, light application time 16h, 1500-2000LX, 25 ± 2 DEG C of temperature, gas concentration lwevel 950ppm, humidity 80%, more than 1000 grades of culturing room's cleanliness factor；Culture forms well developed root system Kiwi berry after 20 days is completely planted Strain.
- 2. the method according to claim 1 that tissue cultures are carried out using Kiwi berry blade, it is characterised in that MS+ vermiculites Add 800g vermiculites for every liter of MS solution.
- 3. the method according to claim 1 or 2 that tissue cultures are carried out using Kiwi berry blade, it is characterised in that specific Step is：1) emerald green, thick and solid in the healthy and strong kiwi fruit plant of selection, the blade closer from stem apex does explant material, and clear water is washed dry Only；With 0.1% mercuric chloride solution sterilize 10min, then with 75% alcohol sterilize 5min, sterile water wash 3-5 times；Blade after processing is cut into the square of length of side 1.5cm sizes, is put on inducing culture,Illumination 800LX, daily 10h, 25 ± 2 DEG C of temperature, humidity 70%, cultivates 50 days or so and forms Cultured Actinidia deliciosa Calli.Inducing culture is：MS culture mediums+sucrose 20g/L+ agar 6g/L+6-BA0.8mg/L+NAA (methyl α-naphthyl acetate) 0.2mg/L+ The culture medium of 2,4-D (2,4 dichlorophenoxyacetic acid) 0.5mg/L；2) Multiple Buds are inducedCallus is divided into the fritter of diameter 1cm or so, disposes dead leaf tissue, be partly embedded into MS+ sucrose In the culture medium of 30g/L+ agar 6g/L+6-BA5mg/L+NAA0.5mg/L+IAA0.2mg/L, illumination 2000-2500LX, the time 14h, 25 ± 2 DEG C of temperature, humidity 70%, callus surface forms adventitious bud after cultivating 50 days, obtains primary；3) squamous subculture (Multiplying culture) is the adventitious bud insertion MS+ sucrose 30g/L+ agar 6g/L+6- formed on callus On the culture medium of BA1.2mg/L+IAA0.8mg/L, illumination 2000-3000LX, 14h, 25 ± 2 DEG C of temperature, humidity 70%, is cultivated 25 days, 3-5 times of appreciation rate,4) more than 3cm is reached after propagation, the plant with more than 3 cotyledons is inserted into inside special equipment of taking root for root induction In matrix, which is MS+ vermiculites, light application time 16h, 1500-2000LX, 25 ± 2 DEG C of temperature, gas concentration lwevel 950ppm, humidity 80%, more than 1000 grades of culturing room's cleanliness factor.Culture forms well developed root system Kiwi berry after 20 days is completely planted Strain；5) Kiwi berry intact plant is transplanted to inside degraded bag, is transplanted to warm canopy, 15-30 DEG C of temperature, humidity 85%, after a week Crop field can be moved on to.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107996411A (en) * | 2018-03-02 | 2018-05-08 | 宝鸡松良农业科技有限公司 | A kind of method of root induction in Chinese Gooseberry culture |
| CN110122335A (en) * | 2019-06-27 | 2019-08-16 | 西南林业大学 | The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant |
| CN114766367A (en) * | 2022-05-18 | 2022-07-22 | 西北农林科技大学 | Kiwi fruit tissue culture method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107996411A (en) * | 2018-03-02 | 2018-05-08 | 宝鸡松良农业科技有限公司 | A kind of method of root induction in Chinese Gooseberry culture |
| CN110122335A (en) * | 2019-06-27 | 2019-08-16 | 西南林业大学 | The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant |
| CN110122335B (en) * | 2019-06-27 | 2022-03-22 | 西南林业大学 | Induction method of salt-tolerant mutant of slow-fragrant kiwi fruit tissue culture seedling |
| CN114766367A (en) * | 2022-05-18 | 2022-07-22 | 西北农林科技大学 | Kiwi fruit tissue culture method |
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