CN109392719A - A kind of method for building up of madder regenerating system - Google Patents
A kind of method for building up of madder regenerating system Download PDFInfo
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- CN109392719A CN109392719A CN201811412328.9A CN201811412328A CN109392719A CN 109392719 A CN109392719 A CN 109392719A CN 201811412328 A CN201811412328 A CN 201811412328A CN 109392719 A CN109392719 A CN 109392719A
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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)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of method for building up of madder regenerating system, madder is low using conventional method breeding coefficient, and growth cycle is long, while the influence vulnerable to factors such as external environmental conditions.And the method for using tissue cultures, breeding coefficient are high, can carry out selection in any growth phase and expand numerous, the condition that breeding and industrial seedling rearing to madder excellent variety are provided convenience.The present invention is using madder as explant, by differentiation culture, Multiplying culture, culture of rootage, acclimatization and transplants and etc. obtain madder Regeneration in Vitro plant, madder regenerating system is established, solid foundation is established for the cultivation of industrial seedling rearing from now on, so that it is high to solve the problems, such as that madder introduces a fine variety difficulty.
Description
Technical field
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, being related to a kind of madder regeneration
The method for building up of system.
Background technique
Madder is perennial tendril draft, is distributed the most area in the whole nation, and the autumn adopts cauline leaf, and Dong Caigen is fresh or dried.Taste
Hardship, it is cold in nature.With promoting blood circulation and hemostasis, Stasis through.As people develop the increasing of dynamics, the quantity of plant population sharply declines.
Therefore some necessary safeguard measures and method should be taken, to safeguard the quantity of Wild ornamental resources.The wild resource of madder
Extremely limited, people arbitrarily pick and the excavation of blindness in recent years, make its quantity by serious destruction.Main use is broadcast
The modes such as kind, cuttage, grafting are bred, but breeding coefficient is low, and growth cycle is long, while vulnerable to factors such as external environmental conditions
Influence.And the method for using tissue cultures, breeding coefficient are high, and it can be numerous in the progress selection expansion of any growth phase, to madder
The condition that the breeding of excellent variety and industrial seedling rearing are provided convenience.The present invention is using madder blade as explant, by differentiation
Culture, Multiplying culture, culture of rootage, acclimatization and transplants and etc. obtain Regeneration in Vitro plant, establish madder regenerating system, be
Industrial seedling rearing and domesticating and cultivating establish solid foundation from now on.
Summary of the invention
The purpose of the present invention is to provide using madder as explant, by breaking up culture, Multiplying culture, culture of rootage, refining
Transplantation of seedlings and etc. obtain madder Regeneration in Vitro plant, madder regenerating system is established, to realize a kind of regeneration of madder
The method for building up of system.
A kind of method for building up of madder regenerating system of the invention, includes the following steps:
Step 1, explant sterilizes: acquisition madder healthy plant delicacy blade dips in washing powder water with banister brush and gently scrubs, originally
Water rinses 6h and is placed in superclean bench, is first washed 8 times with after 75% ethanol disinfection 18s with sterile, then with 0.1% mercuric chloride solution
11min is sterilized, is dried again with aseptic filter paper with aseptic water washing 9 times spare after the droplet on surface；
Step 2, differentiation culture: it will cut off, be rowed dry on blade with sterile scalpel several through step 1 treated blade petiole
Knife is inoculated in differential medium in such a way that leaf back is contacted with culture medium and carries out callus and inducing clumping bud, inoculation
It is first dark culture 33 days full under the conditions of 29 DEG C afterwards, it is subsequently placed in daily illumination 19 hours, intensity of illumination 4300lx is placed in culture
Temperature counts callus induction rate after cultivating 28 days under conditions of being 29 DEG C, counts differentiation rate after 43 days；Differential medium are as follows:
MS+2.3mg/L TDZ+1.3mg/L IBA+33g/L sucrose+6.3g/L agar, pH 5.3；
Step 3, the callus with Multiple Buds that step 2 induces differentiation to generate Multiplying culture: is divided into 1.0cm's or so
Fritter is simultaneously inoculated into squamous subculture culture medium progress Multiplying culture, first dark culture 10 days full under the conditions of 29 DEG C after inoculation, then
Be placed in daily illumination 19 hours, intensity of illumination 5300lx, cultivation temperature be 29 DEG C under conditions of cultivate 53 days after with adventitious bud
Length >=1.2cm is that effective bud counts proliferation times；Subculture medium are as follows: MS+1.3mg/L TDZ+0.8mg/L IBA+33g/L
Sucrose+6.3g/L agar, pH 5.3；
Step 4, culture of rootage: the consistent tissue-cultured seedling of 3 growth conditions of selecting step is cut into the stem section of 6cm long and is seeded to and takes root
Root induction in culture, inoculation are placed on daily illumination 19 hours, intensity of illumination 5300lx, the condition that cultivation temperature is 29 DEG C
Lower culture observes the upgrowth situation of seedling and counts rooting rate after 43 days；Root media are as follows: 1/2MS+3.5mg/L IBA+
2.1mg/L ABT1+ 31g/L sucrose+6.6g/L agar, pH 5.3；
Step 5, acclimatization and transplants: choosing after culture of rootage, and the tissue-cultured seedling of well developed root system and plant strain growth stalwartness carries out hardening shifting
Plant, wash away the basifixed agar of tissue-cultured seedling first, be transplanted in the river sand matrix irrigated in advance later, backward seedling
Be sprayed, build simple plastic canopy, guarantee in canopy relative air humidity up to 99%, plastic covering film, to guarantee the hardening phase
Between humidity in canopy, gradually lift film ventilation and penetrating light later and pay attention to the humidity in canopy, after sand culture 28 days, be transplanted to by turf
Soil: fertile soil: in the mixed-matrix of perlite=3:1:1 composition, around sprinkling profoundly water after plant, when routine servicing, pays attention to guaranteeing basin soil
It is wet to avoid ponding, and spray water often to blade, transplanting counted transplanting survival rate after 23 days.
It, can be any compared with prior art the invention has the advantages that the method breeding coefficient using tissue cultures is high
Growth phase carries out selection and expands numerous, the condition that breeding and industrial seedling rearing to madder excellent variety are provided convenience.Through excessive
Change culture, Multiplying culture, culture of rootage, acclimatization and transplants and etc. obtain Regeneration in Vitro plant, establish madder regenerating system,
Solid foundation is established for industrial seedling rearing from now on and domesticating and cultivating, to solve the problems, such as that madder introduction and acclimatization difficulty is high.
Specific embodiment
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
(1) explant sterilizes: acquisition madder healthy plant delicacy blade dips in washing powder water with banister brush and gently scrubs, tap water
It rinses 4h to be placed in superclean bench, first be washed 8 times with after 75% ethanol disinfection 11s with sterile, then disappeared with 0.1% mercuric chloride solution
Malicious 8min is dried with aseptic filter paper spare after the droplet on surface again with aseptic water washing 8 times.
(2) differentiation culture: it will cut off through step (1) treated blade petiole, be rowed dry on blade with sterile scalpel
Several knives are inoculated in differential medium in such a way that leaf back is contacted with culture medium and carry out callus and inducing clumping bud.It connects
It is first dark culture 23 days full under the conditions of 26 DEG C after kind, it is subsequently placed in daily illumination 17 hours, intensity of illumination 3300lx is placed in training
Statistics differentiation rate is after statistics callus induction rate is 98%, 41 days after feeding temperature is cultivated 28 days under conditions of being 26 DEG C
98.1%.The differential medium are as follows: MS+1.8mg/L TDZ+0.6mg/L IBA+28g/L sucrose+5.8g/L agar, pH are
5.3。
(3) Multiplying culture: it is divided into 0.8cm left the callus with Multiple Buds that step (2) induce differentiation to generate
Right fritter is simultaneously inoculated into squamous subculture culture medium progress Multiplying culture.It is first dark culture 8 days full under the conditions of 26 DEG C after inoculation, so
Be placed on daily illumination 17 hours, intensity of illumination 3300lx, cultivation temperature be 26 DEG C under conditions of cultivate 31 days after with indefinite
Bud length >=1.2cm is that effective bud statistics proliferation times are 36.3.The subculture medium are as follows: MS+0.8mg/L TDZ+
0.4mg/L IBA+33g/L sucrose+3.8g/L agar, pH 5.3.
(4) culture of rootage: the consistent tissue-cultured seedling of selecting step (3) growth conditions is cut into the stem section of 6cm long and is seeded to life
Root induction in root culture.Inoculation is placed on daily illumination 17 hours, intensity of illumination 3800lx, the item that cultivation temperature is 26 DEG C
Rooting rate 94% after being cultivated 43 days under part.The root media are as follows: 1/2MS+1.8mg/L IBA+1.3mg/L ABT1+
18g/L sucrose+3.8g/L agar, pH 5.3.
(5) acclimatization and transplants: choosing after culture of rootage, and the tissue-cultured seedling of well developed root system and plant strain growth stalwartness carries out hardening shifting
Plant, wash away the basifixed agar of tissue-cultured seedling first, be transplanted in the river sand matrix irrigated in advance later, backward seedling
Be sprayed, build simple plastic canopy, guarantee in canopy relative air humidity up to 99%, plastic covering film, to guarantee the hardening phase
Between humidity in canopy, gradually lift film ventilation and penetrating light later and pay attention to the humidity in canopy, after sand culture 18 days, be transplanted to by turf
Soil: fertile soil: in the mixed-matrix of perlite=3:1:1 composition, around sprinkling profoundly water after plant, when routine servicing, pays attention to guaranteeing basin soil
It is wet to avoid ponding, and spray water often to blade, statistics transplanting survival rate is 94.1% after transplanting 23 days.
Embodiment 2:
(1) explant sterilizes: acquisition madder healthy plant delicacy blade dips in washing powder water with banister brush and gently scrubs, tap water
It rinses 5h to be placed in superclean bench, first be used sterile water 6 times with after 75% ethanol disinfection 12s, then sterilized with 0.1% mercuric chloride solution
5min is dried with aseptic filter paper spare after the droplet on surface again with aseptic water washing 7 times.
(2) differentiation culture: it will cut off through step (1) treated blade petiole, be rowed dry on blade with sterile scalpel
Several knives are inoculated in differential medium in such a way that leaf back is contacted with culture medium and carry out callus and inducing clumping bud.It connects
It is first dark culture 27 days full under the conditions of 28 DEG C after kind, it is subsequently placed in daily illumination 17 hours, intensity of illumination 3700lx is placed in training
Statistics differentiation rate is after statistics callus induction rate is 94%, 43 days after feeding temperature is cultivated 26 days under conditions of being 28 DEG C
92.5%.The differential medium are as follows: MS+2.4mg/L TDZ+0.4mg/L IBA+22g/L sucrose+4.5g/L agar, pH are
5.7。
(3) Multiplying culture: it is divided into 0.7cm left the callus with Multiple Buds that step (2) induce differentiation to generate
Right fritter is simultaneously inoculated into squamous subculture culture medium progress Multiplying culture.It is first dark culture 9 days full under the conditions of 28 DEG C after inoculation, so
Be placed on daily illumination 17 hours, intensity of illumination 4200lx, cultivation temperature be 28 DEG C under conditions of cultivate 52 days after with indefinite
Bud length >=1.2cm is that effective bud statistics proliferation times are 34.The subculture medium are as follows: MS+1.2mg/L TDZ+
0.4mg/LIBA+32g/L sucrose+4.5g/L agar, pH 5.7.
(4) culture of rootage: the consistent tissue-cultured seedling of selecting step (3) growth conditions is cut into the stem section of 5cm long and is seeded to life
Root induction in root culture.Inoculation is placed on daily illumination 15 hours, intensity of illumination 3700lx, the item that cultivation temperature is 28 DEG C
Rooting rate 96% after being cultivated 42 days under part.The root media are as follows: 1/2MS+2.0mg/L IBA+1.2mg/L ABT1+
32g/L sucrose+5.6g/L agar, pH 5.7.
(5) acclimatization and transplants: choosing after culture of rootage, and the tissue-cultured seedling of well developed root system and plant strain growth stalwartness carries out hardening shifting
Plant, wash away the basifixed agar of tissue-cultured seedling first, be transplanted in the river sand matrix irrigated in advance later, backward seedling
Be sprayed, build simple plastic canopy, guarantee in canopy relative air humidity up to 99%, plastic covering film, to guarantee the hardening phase
Between humidity in canopy, gradually lift film ventilation and penetrating light later and pay attention to the humidity in canopy, after sand culture 27 days, be transplanted to by turf
Soil: fertile soil: in the mixed-matrix of perlite=2:2:1 composition, around sprinkling profoundly water after plant, when routine servicing, pays attention to guaranteeing basin soil
It is wet to avoid ponding, and spray water often to blade, transplanting survival rate 97% is counted after transplanting 22 days.
Claims (1)
1. a kind of method for building up of madder regenerating system, it is characterised in that the following steps are included:
Step 1, explant sterilizes: acquisition madder healthy plant delicacy blade dips in washing powder water with banister brush and gently scrubs, originally
Water rinses 6h and is placed in superclean bench, is first washed 8 times with after 75% ethanol disinfection 18s with sterile, then with 0.1% mercuric chloride solution
11min is sterilized, is dried again with aseptic filter paper with aseptic water washing 9 times spare after the droplet on surface；
Step 2, differentiation culture: it will cut off, be rowed dry on blade with sterile scalpel several through step 1 treated blade petiole
Knife is inoculated in differential medium in such a way that leaf back is contacted with culture medium and carries out callus and inducing clumping bud, inoculation
It is first dark culture 33 days full under the conditions of 29 DEG C afterwards, it is subsequently placed in daily illumination 19 hours, intensity of illumination 4300lx is placed in culture
Temperature counts callus induction rate after cultivating 28 days under conditions of being 29 DEG C, counts differentiation rate after 43 days；Differential medium are as follows:
MS+2.3mg/L TDZ+1.3mg/L IBA+33g/L sucrose+6.3g/L agar, pH 5.3；
Step 3, the callus with Multiple Buds that step 2 induces differentiation to generate Multiplying culture: is divided into 1.0cm's or so
Fritter is simultaneously inoculated into squamous subculture culture medium progress Multiplying culture, first dark culture 10 days full under the conditions of 29 DEG C after inoculation, then
Be placed in daily illumination 19 hours, intensity of illumination 5300lx, cultivation temperature be 29 DEG C under conditions of cultivate 53 days after with adventitious bud
Length >=1.2cm is that effective bud counts proliferation times；Subculture medium are as follows: MS+1.3mg/L TDZ+0.8mg/L IBA+33g/L
Sucrose+6.3g/L agar, pH 5.3；
Step 4, culture of rootage: the consistent tissue-cultured seedling of 3 growth conditions of selecting step is cut into the stem section of 6cm long and is seeded to and takes root
Root induction in culture, inoculation are placed on daily illumination 19 hours, intensity of illumination 5300lx, the condition that cultivation temperature is 29 DEG C
Lower culture observes the upgrowth situation of seedling and counts rooting rate after 43 days；Root media are as follows: 1/2MS+3.5mg/L IBA+
2.1mg/L ABT1+ 31g/L sucrose+6.6g/L agar, pH 5.3；
Step 5, acclimatization and transplants: choosing after culture of rootage, and the tissue-cultured seedling of well developed root system and plant strain growth stalwartness carries out hardening shifting
Plant, wash away the basifixed agar of tissue-cultured seedling first, be transplanted in the river sand matrix irrigated in advance later, backward seedling
Be sprayed, build simple plastic canopy, guarantee in canopy relative air humidity up to 99%, plastic covering film, to guarantee the hardening phase
Between humidity in canopy, gradually lift film ventilation and penetrating light later and pay attention to the humidity in canopy, after sand culture 28 days, be transplanted to by turf
Soil: fertile soil: in the mixed-matrix of perlite=3:1:1 composition, around sprinkling profoundly water after plant, when routine servicing, pays attention to guaranteeing basin soil
It is wet to avoid ponding, and spray water often to blade, transplanting counted transplanting survival rate after 23 days.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110800561A (en) * | 2019-11-14 | 2020-02-18 | 韦东 | Improvement method of arrowhead variety |
| CN114190275A (en) * | 2021-11-30 | 2022-03-18 | 云南省农业科学院药用植物研究所 | Tissue culture rapid propagation method for one-step seedling formation of rubia yunnanensis |
| CN116326479A (en) * | 2023-03-17 | 2023-06-27 | 德兴市中医研究院试验培训基地 | Culture method for obtaining madder tissue culture seedlings through callus induction |
| CN116616181A (en) * | 2023-06-13 | 2023-08-22 | 攀枝花市农林科学研究院 | Tissue culture method for bergamot trees |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110800561A (en) * | 2019-11-14 | 2020-02-18 | 韦东 | Improvement method of arrowhead variety |
| CN114190275A (en) * | 2021-11-30 | 2022-03-18 | 云南省农业科学院药用植物研究所 | Tissue culture rapid propagation method for one-step seedling formation of rubia yunnanensis |
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Application publication date: 20190301 |