CN109380117A - A kind of Rubus parvifolius Tissue Culture Regeneration System construction method - Google Patents
A kind of Rubus parvifolius Tissue Culture Regeneration System construction method Download PDFInfo
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- CN109380117A CN109380117A CN201811412329.3A CN201811412329A CN109380117A CN 109380117 A CN109380117 A CN 109380117A CN 201811412329 A CN201811412329 A CN 201811412329A CN 109380117 A CN109380117 A CN 109380117A
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- rubus parvifolius
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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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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of Rubus parvifolius to knit culture regenerating system construction method, and the technical method of strain stabilization Rubus parvifolius seedling is obtained by Fast Asexual Propagation Technique.The present invention is using Rubus parvifolius immature embryo as explant, Rubus parvifolius Tissue Culture Regeneration System is constructed by seed disinfection, embryo callus induction, differentiation culture, culture of rootage, hardening and transplanting and other steps, not only it had solved the problems, such as that Rubus parvifolius high quality seedling lacked, but also can lay the foundation to carry out the work of Rubus parvifolius genetic transformation from now on.
Description
Technical field
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, being related to a kind of Rubus parvifolius tissue
Cultivate regenerating system construction method.
Background technique
Rubus parvifolius alias: Japanese Raspberry Root grass, bubble in March, roadblock.Belong to shrub, is distributed in east China, south, middle part.It is raw
In hillside on the sunny side, the raised path through fields, roadside.Summer, autumn adopt cauline leaf, and the autumn and winter adopt root, and dissection is dried.It is bitter, cool in nature.With clearing heat and cooling blood,
Dispersing swelling and dissipating binds, Li Shui.Plant tissue culture technique, which has, to be accelerated breeding process, shortens repoductive time, improvement plant quality, saves
The features such as working, can producing, not limited by natural conditions throughout the year, is reduced in space, and can effectively solving Rubus parvifolius breed breeding, time-consuming
The problem of.It is made some progress in terms of Rubus parvifolius Study on tissue culture in recent years, but still remains some problems, such as explant
Browning is serious, goes out tip rate is low, it is difficult to take root, transplanting crop field survival rate is low etc., makes tissue culture technique answering in Rubus parvifolius
With being very restricted.Therefore, it is highly desirable to establish the Rubus parvifolius rapid propagation in vitro system of system, to carry out product to Rubus parvifolius from now on
Cytoplasmic inheritance improvement lays the foundation.
Summary of the invention
The purpose of the present invention is to provide a kind of Rubus parvifolius Tissue Culture Regeneration System construction method out, the present invention with Rubus parvifolius not
Mature embryo is explant, by seed disinfection, embryo callus induction, differentiation culture, culture of rootage, hardening and transplanting and other steps
Rubus parvifolius Tissue Culture Regeneration System is constructed, to realize the purpose of the present invention.
A kind of Rubus parvifolius Tissue Culture Regeneration System construction method of the invention, includes the following steps:
Step 1, prematurity drupel, the tap water punching after washing powder water immersion 25min, outwash after defoliation seed disinfection: are selected
3h is washed, still immature milky white or slightly browny seed is taken out, in superclean bench, exosper is peeled off, first with 75% ethyl alcohol
It sterilizes after 25s with sterile washing 12 times, then with 0.1% mercuric chloride solution disinfection 20min, with spare after aseptic water washing 3 times；
Step 2, embryo callus induction: prematurity embryo it will be cut into 0.6cm × 0.6cm × 0.6cm after step 1 processing and connect
It is first dark culture 40 days full under the conditions of 25~28 DEG C in kind to induced medium, it is subsequently placed in daily illumination 6 hours, illumination is strong
Degree is 25001x, until induced synthesis embryo callus；Induced medium are as follows: ER+6-BA4mg/L+NAA0.4mg/L+GA32
Mg/L+ sucrose 25g/L+ agar 5.5g/L, pH 5.3；
Step 3, differentiation culture: the greeny Cotyledon Callus that step 2 culture obtains is transferred on differential medium and is carried out
Differentiation culture, it is first dark culture 5 days full under the conditions of 25~28 DEG C after inoculation, it is subsequently placed in daily illumination 6 hours, intensity of illumination is
2500lx is placed in culture under conditions of cultivation temperature is 25~28 DEG C until formation Multiple Buds；Differential medium are as follows: ER+6-
BA8.5mg/L+IBA0.8mg/L+ sucrose 10g/L+ agar 3.3g/L, pH 5.3；
Step 4, the Multiple Buds cutting that the height that step 3 process obtains is about 1.5cm culture of rootage: is inoculated into root media
Upper carry out culture of rootage, it is first dark culture 5 days full under the conditions of 25~28 DEG C after inoculation, it is subsequently placed in daily illumination 12 hours, light
It is 1500lx according to intensity, cultivation temperature is cultivated under conditions of being 25~28 DEG C to taking root；Root media are as follows: 1/2MS+
IBA2.5mg/L+GGR2.5mg/L+KT3.5mg/L+ sucrose 13g/L+ agar 3.2g/L, pH 5.3；
Step 5, acclimatization and transplants: bottle cap is gone to be placed in natural lighting lower refining seedling 4 rooting tube plantlet for the high about 5cm that step 4 obtains
After it, test tube seedling is taken out from culture bottle, washes off root culture medium, plants in the matrix being mixed by peat soil and yellow sand mud
And it is colonized in big Tanaka.
It is compared with prior art the invention has the advantages that domestic at present not yet mature about Rubus parvifolius group culturation rapid propagating technology, and
The present invention has the characteristics that simple, easy, economical.The Rubus parvifolius test tube seedling transplanting survival rate being bred as through the invention reach 89% with
On, it can effectively solve the problems, such as that Rubus parvifolius seedling lacks.
Specific embodiment
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
(1) prematurity small nut fruit, the tap water flushing after washing powder water immersion 5min, outwash after defoliation seed disinfection: are selected
2h takes out still immature milky white or slightly browny seed, in superclean bench, peels off exosper, is first disappeared with 75% ethyl alcohol
With sterile washing 2 times after malicious 4s, then with 0.1% mercuric chloride solution 5min is sterilized, with spare after aseptic water washing 3 times, is inoculated with after a week
Counting pollution rate can be down to 1%.
(2) embryo callus induction: 0.4cm × 0.4cm × 0.4cm will be cut by prematurity embryo after step (1) is handled
It is inoculated into induced medium, it is first dark culture 25 days full under the conditions of 28 DEG C, it is subsequently placed in daily illumination 5 hours, intensity of illumination
For 1900x, until induced synthesis embryo callus, callus induction rate 69%.The induced medium is ER+6-
BA1.0mg/L+NAA0.5mg/L+GA35 mg/L+ sucrose 12g/L+ agar 3.4g/L, pH 5.5.
(3) it is enterprising that the greeny Cotyledon Callus that step (2) culture obtains differentiation culture: is transferred to differential medium
Row differentiation culture, it is first dark culture 6 days full under the conditions of 28 DEG C after inoculation, it is subsequently placed in daily illumination 10 hours, intensity of illumination is
2500lx is placed in culture under conditions of cultivation temperature is 28 DEG C until formation Multiple Buds.The differential medium is ER+6-
BA1.0mg/L+IBA0.1mg/L+ sucrose 13g/L+ agar 3.2g/L, pH 5.3.
(4) the Multiple Buds cutting that the height that step (3) process obtains is about 1.5cm culture of rootage: is inoculated into training of taking root
It supports and carries out culture of rootage on base, it is first dark culture 6 days full under the conditions of 28 DEG C after inoculation, it is subsequently placed in daily illumination 13 hours, light
It is 1800lx according to intensity, cultivation temperature is cultivated under conditions of being 28 DEG C to taking root, rooting rate 86%.The root media
For 1/2MS+IBA0.2mg/L+GGR1.0mg/L+KT1.0mg/L+ sucrose 12g/L+ agar 2.5g/L, pH 5.3.
(5) it acclimatization and transplants: goes bottle cap to be placed under natural lighting the rooting tube plantlet for the high about 5cm that step (4) obtain and refines
After seedling 4 days, test tube seedling is taken out from culture bottle, washes off root culture medium, plants into the base being mixed by peat soil and yellow sand mud
In matter and it is colonized in big Tanaka, transplanting survival rate is up to 91.8%.
Embodiment 2:
(1) prematurity small nut fruit, the tap water flushing after washing powder water immersion 10min, outwash after defoliation seed disinfection: are selected
1h takes out still immature milky white or slightly browny seed, in superclean bench, peels off exosper, is first disappeared with 75% ethyl alcohol
With sterile washing 2 times after malicious 5s, then with 0.1% mercuric chloride solution 10min is sterilized, with spare after aseptic water washing 3 times, inoculation one week
Statistics pollution rate can be down to 7% afterwards.
(2) embryo callus induction: 0.3cm × 0.3cm × 0.3cm will be cut by prematurity embryo after step (1) is handled
It is inoculated into induced medium, it is first dark culture 28 days full under the conditions of 28 DEG C, it is subsequently placed in daily illumination 10 hours, intensity of illumination
For 1200x, until induced synthesis embryo callus, callus induction rate 64%.The induced medium is ER+6-
BA1.0mg/L+NAA0.2mg/L+GA33 mg/L+ sucrose 12g/L+ agar 1.8g/L, pH 5.3.
(3) it is enterprising that the greeny Cotyledon Callus that step (2) culture obtains differentiation culture: is transferred to differential medium
Row differentiation culture, it is first dark culture 7 days full under the conditions of 28 DEG C after inoculation, it is subsequently placed in daily illumination 10 hours, intensity of illumination is
3500lx is placed in culture under conditions of cultivation temperature is 28 DEG C until formation Multiple Buds.The differential medium is ER+6-
BA1.2mg/L+IBA0.5mg/L+ sucrose 13g/L+ agar 1.8g/L, pH 5.9.
(4) the Multiple Buds cutting that the height that step (3) process obtains is about 2.5cm culture of rootage: is inoculated into training of taking root
It supports and carries out culture of rootage on base, it is first dark culture 4 days full under the conditions of 28 DEG C after inoculation, it is subsequently placed in daily illumination 11 hours, light
It is 2400lx according to intensity, cultivation temperature is cultivated under conditions of being 28 DEG C to taking root, rooting rate 85%.The root media
For 1/2MS+IBA0.4mg/L+GGR1.5mg/L+KT1.0mg/L+ sucrose 12g/L+ agar 2.0g/L, pH 5.9.
(5) it acclimatization and transplants: goes bottle cap to be placed under natural lighting the rooting tube plantlet for the high about 6cm that step (4) obtain and refines
After seedling 8 days, test tube seedling is taken out from culture bottle, washes off root culture medium, plants into the base being mixed by peat soil and yellow sand mud
In matter and it is colonized in big Tanaka, transplanting survival rate is up to 90%.
Claims (1)
1. a kind of Rubus parvifolius Tissue Culture Regeneration System construction method, it is characterised in that the following steps are included:
Step 1, prematurity drupel, the tap water punching after washing powder water immersion 25min, outwash after defoliation seed disinfection: are selected
3h is washed, still immature milky white or slightly browny seed is taken out, in superclean bench, exosper is peeled off, first with 75% ethyl alcohol
It sterilizes after 25s with sterile washing 12 times, then with 0.1% mercuric chloride solution disinfection 20min, with spare after aseptic water washing 3 times；
Step 2, embryo callus induction: prematurity embryo it will be cut into 0.6cm × 0.6cm × 0.6cm after step 1 processing and connect
It is first dark culture 40 days full under the conditions of 25~28 DEG C in kind to induced medium, it is subsequently placed in daily illumination 6 hours, illumination is strong
Degree is 25001x, until induced synthesis embryo callus；Induced medium are as follows: ER+6-BA4mg/L+NAA0.4mg/L+GA32
Mg/L+ sucrose 25g/L+ agar 5.5g/L, pH 5.3；
Step 3, differentiation culture: the greeny Cotyledon Callus that step 2 culture obtains is transferred on differential medium and is carried out
Differentiation culture, it is first dark culture 5 days full under the conditions of 25~28 DEG C after inoculation, it is subsequently placed in daily illumination 6 hours, intensity of illumination is
2500lx is placed in culture under conditions of cultivation temperature is 25~28 DEG C until formation Multiple Buds；Differential medium are as follows: ER+6-
BA8.5mg/L+IBA0.8mg/L+ sucrose 10g/L+ agar 3.3g/L, pH 5.3；
Step 4, the Multiple Buds cutting that the height that step 3 process obtains is about 1.5cm culture of rootage: is inoculated into root media
Upper carry out culture of rootage, it is first dark culture 5 days full under the conditions of 25~28 DEG C after inoculation, it is subsequently placed in daily illumination 12 hours, light
It is 1500lx according to intensity, cultivation temperature is cultivated under conditions of being 25~28 DEG C to taking root；Root media are as follows: 1/2MS+
IBA2.5mg/L+GGR2.5mg/L+KT3.5mg/L+ sucrose 13g/L+ agar 3.2g/L, pH 5.3；
Step 5, acclimatization and transplants: bottle cap is gone to be placed in natural lighting lower refining seedling 4 rooting tube plantlet for the high about 5cm that step 4 obtains
After it, test tube seedling is taken out from culture bottle, washes off root culture medium, plants in the matrix being mixed by peat soil and yellow sand mud
And it is colonized in big Tanaka.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116138168A (en) * | 2023-02-17 | 2023-05-23 | 北京林业大学 | Culture medium for promoting germination of raspberry seeds and tissue culture seedling method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104255500A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Rubus sumatranus tissue culture and intermediate propagation method |
| CN104686332A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Method for establishing tissue culture regeneration system of camellia sinensis L. |
| CN106135005A (en) * | 2016-08-26 | 2016-11-23 | 李军 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
| CN106718872A (en) * | 2016-11-14 | 2017-05-31 | 黑龙江省林业科学研究所 | A kind of tissue culture method for expanding breeding culture medium and ripple Rana for raspberry ripple Rana |
-
2018
- 2018-11-25 CN CN201811412329.3A patent/CN109380117A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104255500A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Rubus sumatranus tissue culture and intermediate propagation method |
| CN104686332A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Method for establishing tissue culture regeneration system of camellia sinensis L. |
| CN106135005A (en) * | 2016-08-26 | 2016-11-23 | 李军 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
| CN106718872A (en) * | 2016-11-14 | 2017-05-31 | 黑龙江省林业科学研究所 | A kind of tissue culture method for expanding breeding culture medium and ripple Rana for raspberry ripple Rana |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116138168A (en) * | 2023-02-17 | 2023-05-23 | 北京林业大学 | Culture medium for promoting germination of raspberry seeds and tissue culture seedling method |
| CN116138168B (en) * | 2023-02-17 | 2024-04-05 | 北京林业大学 | Culture medium for promoting germination of raspberry seeds and tissue culture seedling method |
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Application publication date: 20190226 |
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