CN109362566A - A kind of Rabdosia amethystoides tissue culture and rapid propagation method - Google Patents
A kind of Rabdosia amethystoides tissue culture and rapid propagation method Download PDFInfo
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- CN109362566A CN109362566A CN201811429115.7A CN201811429115A CN109362566A CN 109362566 A CN109362566 A CN 109362566A CN 201811429115 A CN201811429115 A CN 201811429115A CN 109362566 A CN109362566 A CN 109362566A
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of Rabdosia amethystoides tissue culture and rapid propagation method, Rabdosia herbaceos perennial is distributed in China major part province, is born in hillside, sandy, gully more.Bitter, cold nature.With clearing heat and detoxicating, stomach invigorating, the functions such as activating blood circulation and dissipating phlegm.Rabdosia amethystoides mainly uses the traditional approach such as cuttage, grafting and sowing to carry out nursery at present, and there are seedling cost height, and nursery stock growing way is bad, irregular, breeding potentiality fail the disadvantages of being not fully exerted.The present invention is using choiceness spray as explant; Rabdosia amethystoides plant again in vitro is obtained by processes such as explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root generation, acclimatization and transplants; establish Rabdosia amethystoides tissue culture rapid propagation technique system; the merit that can keep Rabdosia amethystoides clone female parent is conducive to the large-scale production of Rabdosia amethystoides choiceness seedling.
Description
Technical field
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, being related to a kind of Rabdosia amethystoides group
Tissue culture method for fast propagation.
Background technique
Rabdosia herbaceos perennial is distributed in China major part province, is born in hillside, sandy, gully more.Taste
Bitter, cold nature.With clearing heat and detoxicating, stomach invigorating, the functions such as activating blood circulation and dissipating phlegm.Rabdosia amethystoides is the distinctive plant variety tree of south China
Kind.It grows fastly, is one of most important commodity herb of south China.Recently as collectively owned forest herd system reform propulsion and
The rise of merchantable timber price, the enthusiasm that self-employed tree cultivator manages Rabdosia amethystoides increase, the continuous improvement of management level and breeding consciousness, to perfume (or spice)
The demand of tea dish breeding strong sprout increasingly increases, and therefore, how fast-propagation superior genotypes seem very necessary.
The fast development of biotechnology, the especially foundation of tissue culture rapid propagation system in recent years are the asexual breeding of forest
Provide certain technical support.Rare and endangered species are carried out using tissue culture technique and excellent strain tree species breed, and are had
Accelerate breeding, shortens reproductive process, save space, reduce the features such as labour, whole year production.And cuttage is mainly used at present, is transferred
It the traditional approach such as connects and sows and carry out nursery, there are seedling cost height, and nursery stock growing way is bad, irregular, breeding potentiality fail
The disadvantages of being not fully exerted, so that the supply of high-quality strong sprout is also unable to satisfy the demand in market at present.In order to meet market pair
The great demand of scented tea vegetable seedling, the present invention pass through explant disinfection, Fiber differentiation, clump using choiceness spray as explant
The processes such as proliferation, adventitious root generation, acclimatization and transplants of sprouting obtain in vitro plant again, establish tissue culture rapid propagation technique body
System can not only keep female parent efficiently against some defects in traditional nursery, and to the clone nursery stock bred out
Merit plays a role, and is conducive to the large-scale production of Rabdosia amethystoides choiceness seedling.
Summary of the invention
The purpose of the present invention is to provide a kind of Rabdosia amethystoides tissue culture and rapid propagation method, the present invention is outer with choiceness spray
Implant is obtained in vitro again by processes such as explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root generation, acclimatization and transplants
Plant establishes tissue culture rapid propagation technique system, to realize the purpose of the present invention.
A kind of Rabdosia amethystoides tissue culture and rapid propagation method of the invention, includes the following steps:
Step 1, explant acquisition and disinfection: the full of Rabdosia amethystoides stub base portion Morphological Characterization consistent current year raw state of sprouting is taken
Full spray rinses 4h under flowing water, is soaked in 6% washing powder solution 11 minutes, with 8% washing powder solution of banister brush as explant
Gently washing material, then 91min is rinsed in the form dripped with tap water, with distilled water flushing 6 times, in superclean bench
With 75%~80% alcohol solution dipping 61s, aseptic water washing 6 times, then with 0.1~0.5% containing 0.01~0.05% Tween-20
Mercuric chloride solution sterilizes 16min, and the moisture for blotting surface with aseptic filter paper after aseptic water washing 6 times is spare;
Step 2, Fiber differentiation: by through step 1 treated spray is cut into the stem section of about 1.6cm and be inoculated into induced medium into
Row inducing clumping bud culture, inoculation be placed on daily illumination 15 hours, intensity of illumination 2100lx, be placed in cultivation temperature be 23~
25 DEG C, statistics induction situation after relative air humidity is cultivated 31 days under conditions of being 75%~80%;Induced medium are as follows: MS+
1.1mg/L NAA+8.1mg/L 6-BA+3.1mg/L KT+1.6g/L AC+31g/L sucrose+6.1g/L agar, pH 5.9；
Step 3, adventitious buds proliferation: step 2 Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud proliferation
Culture, inoculation are placed on daily illumination 15 hours, intensity of illumination 2100lx, and being placed in cultivation temperature is 23~25 DEG C, air phase
Germinative number is counted after cultivating 41 days under conditions of being 75%~80% to humidity;Proliferated culture medium are as follows: MS+1.6mg/L 2,4-D+
5.1mg/L 6-BA+1.1mg/L NAA+1.6g/L AC+31g/L sucrose+6.1g/L agar, pH 5.9；
Step 4, culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step 3 proliferation is cut from base portion and is seeded to life
Root induction is carried out in root culture medium, inoculation is placed on daily illumination 15 hours, and intensity of illumination 3100lx is placed in cultivation temperature
It is 23~25 DEG C, relative air humidity counts situation of taking root after cultivating 31 days under conditions of being 75%~80%;Root media
Are as follows: White+1.1mg/L NAA+3.1mg/L IBA+31g/L sucrose+6.1g/L agar, pH 5.9.
Step 5, acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling is moved
To after room temperature lower 6 days, opens bottle cap 4 days, the culture medium being attached on seedling root system is then washed away, in 1100 times of biological bacteria
11min is impregnated in agent, is then transplanted into the container bag for filling designed transplanting culture substrate and is cultivated, each container
Bag 1 plant of rooted seedling of transplanting, the vinyl house inside holding moisturizing temperature for being placed in the auto spraying with shading net are controlled 15~35
DEG C, humidity should be maintained at 75%~85%, avoid direct sunlight, and transplanting counted survival rate after 31 days.Transplanting culture substrate is served as reasons
Peat soil: vermiculite: pearl charcoal: tangerine bar charcoal=3:1:1:1 mixes matrix.
Compared with prior art the invention has the advantages that Rabdosia amethystoides is the distinctive plant variety in China.Recently as collection
The propulsion of body self-employed tree cultivator's system reform and the rise of merchantable timber price, the enthusiasm that self-employed tree cultivator manages increase, management level and breeding meaning
The continuous improvement of knowledge, the demand to breeding strong sprout increasingly increase.And at present mainly using traditional approach such as cuttage, grafting and sowings
Nursery is carried out, there are seedling cost height, and nursery stock growing way is bad, irregular, breeding potentiality fail the disadvantages of being not fully exerted,
So that the supply of high-quality strong sprout is also unable to satisfy the demand in market at present.In order to meet market to the great demand of scented tea vegetable seedling,
The present invention using choiceness spray as explant, by explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root occur,
The processes such as acclimatization and transplants obtain in vitro plant again, establish tissue culture rapid propagation technique system, and clone can be kept female
This merit, is conducive to the large-scale production of Rabdosia amethystoides choiceness seedling.
Specific embodiment
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
(1) explant acquisition and disinfection: the full spray of Rabdosia amethystoides base portion Morphological Characterization consistent current year raw state of sprouting is taken
As explant, 3h is rinsed under flowing water, is soaked in 3% washing powder solution 6min, is gently brushed with 3% washing powder solution of banister brush
Material is washed, then rinses 61min with tap water in the form dripped, with distilled water flushing 4 times, with 75% second in superclean bench
Alcoholic solution impregnate 21s, aseptic water washing 6 times, then with containing 0.01% Tween-20 0.1% mercuric chloride solution sterilize 6min, sterile water
The moisture for blotting surface with aseptic filter paper after rinsing 4 times is spare.
(2) Fiber differentiation: by treated that spray is cut into the stem section of about 1.6cm and is inoculated into Fiber differentiation through step (1)
Base carries out inducing clumping bud culture.Inoculation is placed on daily illumination 13 hours, intensity of illumination 1600lx, and being placed in cultivation temperature is
23 DEG C, inductivity is up to 99% after relative air humidity is cultivated 31 days under conditions of being 75%.The induced medium are as follows: MS+
0.3mg/L NAA+5.1mg/L 6-BA+1.6mg/L KT+0.9g/L AC+26g/L sucrose+5.1g/L agar, pH 5.6.
(3) adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing
Grow culture.Inoculation is placed on daily illumination 13 hours, intensity of illumination 1600lx, and being placed in cultivation temperature is 23 DEG C, and air is opposite
Humidity be 75% under conditions of cultivate 41 days after bud number 10 or more.The proliferated culture medium are as follows: MS+0.3mg/L 2,4-D+
3.6mg/L 6-BA+0.6mg/L NAA+1.1g/L AC+ 26g/L sucrose+4.6g/L agar, pH 5.6.
(4) culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step (3) proliferation is cut and is seeded to from base portion
Root induction is carried out in root media.Inoculation is placed on daily illumination 15 hours, intensity of illumination 2100lx, is placed in culture temperature
Degree is 23 DEG C, and relative air humidity is taken root after cultivating 31 days under conditions of being 75% reaches 96% or more.The root media
Are as follows: White+0.4mg/L NAA+1.6mg/L IBA+19g/L sucrose+3.9g/L agar, pH 5.6.
(5) acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often
Temperature is opened bottle cap 3 days after lower 5 days, the culture medium being attached on seedling root system is then washed away, in 1000 times of bacteria agent
6min is impregnated, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag transplanting
1 plant of rooted seedling.It is placed in the vinyl house inside holding moisturizing of the auto spraying with shading net, temperature is controlled at 25 DEG C, and humidity is answered
It is maintained at 75%~85% or so, avoids direct sunlight, survival rate reaches 93% or more after transplanting 31 days.The transplanting culture
Matrix is by peat soil: vermiculite: pearl charcoal: tangerine bar charcoal=3:1:1:1 mixes matrix.
Embodiment 2:
(1) explant acquisition and disinfection: Rabdosia amethystoides stub base portion Morphological Characterization consistent current year raw the full of state of sprouting is taken
Spray rinses 5h under flowing water as explant, is soaked in 3% washing powder solution 10min, light with 4% washing powder solution of banister brush
Light washing material, then rinse 72min with tap water in the form dripped, with distilled water flushing 6 times, in superclean bench with
78% alcohol solution dipping 32s, aseptic water washing 9 times, then 7min is sterilized with 0.1% mercuric chloride solution containing 0.01% Tween-20,
The moisture for blotting surface with aseptic filter paper after aseptic water washing 7 times is spare.
(2) Fiber differentiation: by treated that spray is cut into the stem section of about 1.7cm and is inoculated into Fiber differentiation through step (1)
Base carries out inducing clumping bud culture.Inoculation is placed on daily illumination 15 hours, intensity of illumination 2200lx, and being placed in cultivation temperature is
25 DEG C, inductivity is up to 98.5% after relative air humidity is cultivated 32 days under conditions of being 75%.The induced medium are as follows: MS+
0.5mg/LNAA+6.2mg/L 6-BA+2.2mg/L KT+1.1g/L AC+32g/L sucrose+5.2g/L agar, pH 5.7.
(3) adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing
Grow culture.Inoculation is placed on daily illumination 15 hours, intensity of illumination 2700lx, and being placed in cultivation temperature is 25 DEG C, and air is opposite
Humidity be 75% under conditions of cultivate 42 days after bud number 11 or more.The proliferated culture medium are as follows: MS+0.4mg/L 2,4-D+
3.7mg/L 6-BA+0.7mg/L NAA+1.2g/L AC+ 27g/L sucrose+4.7g/L agar, pH 5.7.
(4) culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step (3) proliferation is cut and is seeded to from base portion
Root induction is carried out in root media.Inoculation is placed on daily illumination 15 hours, intensity of illumination 2200lx, is placed in culture temperature
Degree is 23 DEG C, and relative air humidity is taken root after cultivating 32 days under conditions of being 75% reaches 97% or more.The root media
Are as follows: White+1.0mg/L NAA+2.2mg/L IBA+25g/L sucrose+4.7g/L agar, pH 5.7.
(5) acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often
Temperature is opened bottle cap 5 days after lower 6 days, then washes away the culture medium being attached on seedling root system, soak in 1000 times of bacteria agent
9min is steeped, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag transplants 1 plant
Rooted seedling.It is placed in the vinyl house inside holding moisturizing of the auto spraying with shading net, temperature is controlled at 25 DEG C, and humidity should protect
It holds 75%~85% or so, avoids direct sunlight, survival rate reaches 98% or more after transplanting 32 days.The transplanting culture medium
Matter is by peat soil: vermiculite: pearl charcoal: tangerine bar charcoal=3:2:1:1 mixes matrix.
Claims (1)
1. a kind of Rabdosia amethystoides tissue culture and rapid propagation method, it is characterised in that the following steps are included:
Step 1, explant acquisition and disinfection: the full of Rabdosia amethystoides stub base portion Morphological Characterization consistent current year raw state of sprouting is taken
Full spray rinses 4h under flowing water, is soaked in 6% washing powder solution 11 minutes, with 8% washing powder solution of banister brush as explant
Gently washing material, then 91min is rinsed in the form dripped with tap water, with distilled water flushing 6 times, in superclean bench
With 75%~80% alcohol solution dipping 61s, aseptic water washing 6 times, then with 0.1~0.5% containing 0.01~0.05% Tween-20
Mercuric chloride solution sterilizes 16min, and the moisture for blotting surface with aseptic filter paper after aseptic water washing 6 times is spare;
Step 2, Fiber differentiation: by through step 1 treated spray is cut into the stem section of about 1.6cm and be inoculated into induced medium into
Row inducing clumping bud culture, inoculation be placed on daily illumination 15 hours, intensity of illumination 2100lx, be placed in cultivation temperature be 23~
25 DEG C, statistics induction situation after relative air humidity is cultivated 31 days under conditions of being 75%~80%;Induced medium are as follows: MS+
1.1mg/L NAA+8.1mg/L 6-BA+3.1mg/L KT+1.6g/L AC+31g/L sucrose+6.1g/L agar, pH 5.9；
Step 3, adventitious buds proliferation: step 2 Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud proliferation
Culture, inoculation are placed on daily illumination 15 hours, intensity of illumination 2100lx, and being placed in cultivation temperature is 23~25 DEG C, air phase
Germinative number is counted after cultivating 41 days under conditions of being 75%~80% to humidity;Proliferated culture medium are as follows: MS+1.6mg/L 2,4-D+
5.1mg/L 6-BA+1.1mg/L NAA+1.6g/L AC+31g/L sucrose+6.1g/L agar, pH 5.9；
Step 4, culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step 3 proliferation is cut from base portion and is seeded to life
Root induction is carried out in root culture medium, inoculation is placed on daily illumination 15 hours, and intensity of illumination 3100lx is placed in cultivation temperature
It is 23~25 DEG C, relative air humidity counts situation of taking root after cultivating 31 days under conditions of being 75%~80%;Root media
Are as follows: White+1.1mg/L NAA+3.1mg/L IBA+31g/L sucrose+6.1g/L agar, pH 5.9；
Step 5, acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often
Temperature is opened bottle cap 4 days after lower 6 days, the culture medium being attached on seedling root system is then washed away, in 1100 times of bacteria agent
11min is impregnated, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag is moved
1 plant of rooted seedling is planted, the vinyl house inside holding moisturizing temperature for being placed in the auto spraying with shading net is controlled at 15~35 DEG C,
Humidity should be maintained at 75%~85%, avoid direct sunlight, and transplanting counts survival rate after 31 days, transplanting culture substrate is by mud
Charcoal soil: vermiculite: pearl charcoal: tangerine bar charcoal=3:1:1:1 mixes matrix.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110972949A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | In-bottle rooting method for tissue culture seedlings of rabdosia nervosa |
| CN110972946A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | Subculture method of rabdosia amethystoides |
| CN110972829A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | Method for transplanting and hardening tissue culture seedlings of rabdosia nervosa |
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| CN104663453A (en) * | 2015-03-10 | 2015-06-03 | 朱海燕 | Tissue culture and rapid propagation method for Cunninghamia lanceolate |
| CN105309314A (en) * | 2015-11-23 | 2016-02-10 | 内蒙古科技大学 | Method for setting up tissue culture regeneration system of Mongolian medicine dracocephalum moldavica |
| CN108308037A (en) * | 2018-05-08 | 2018-07-24 | 新疆维吾尔自治区药物研究所 | A kind of new tower flower quick breeding method for tissue culture |
| CN108849526A (en) * | 2018-08-20 | 2018-11-23 | 王远能 | The tissue cultivating method of glaucocalyx rabdosia herb |
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2018
- 2018-11-27 CN CN201811429115.7A patent/CN109362566A/en active Pending
Patent Citations (4)
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| CN104663453A (en) * | 2015-03-10 | 2015-06-03 | 朱海燕 | Tissue culture and rapid propagation method for Cunninghamia lanceolate |
| CN105309314A (en) * | 2015-11-23 | 2016-02-10 | 内蒙古科技大学 | Method for setting up tissue culture regeneration system of Mongolian medicine dracocephalum moldavica |
| CN108308037A (en) * | 2018-05-08 | 2018-07-24 | 新疆维吾尔自治区药物研究所 | A kind of new tower flower quick breeding method for tissue culture |
| CN108849526A (en) * | 2018-08-20 | 2018-11-23 | 王远能 | The tissue cultivating method of glaucocalyx rabdosia herb |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110972949A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | In-bottle rooting method for tissue culture seedlings of rabdosia nervosa |
| CN110972946A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | Subculture method of rabdosia amethystoides |
| CN110972829A (en) * | 2019-12-19 | 2020-04-10 | 美尚生态景观股份有限公司 | Method for transplanting and hardening tissue culture seedlings of rabdosia nervosa |
| CN110972946B (en) * | 2019-12-19 | 2022-04-01 | 美尚生态景观股份有限公司 | Subculture method of rabdosia amethystoides |
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