CN110463602A - A kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication - Google Patents
A kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication Download PDFInfo
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- CN110463602A CN110463602A CN201810436315.9A CN201810436315A CN110463602A CN 110463602 A CN110463602 A CN 110463602A CN 201810436315 A CN201810436315 A CN 201810436315A CN 110463602 A CN110463602 A CN 110463602A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication, it is included selection raw material, materials disinfection, inoculation, the selection of dendrobium candidum protocorm and is cultivated using static suspensions of dendrobium nobile protocorm liquid.The static suspension fast culture process of liquid of the invention can make efficient, high-quality, the inexpensive fast-propagation of dendrobium candidum protocorm, and sufficient dendrobium nobile bulb provenance is provided for dendrobium nobile seedling tissue culture, meets market to the great demand of dendrobium candidum；It is not necessarily to shaking table shake culture during the whole culture process, a large amount of capital has been saved on equipment investment and energy consumption, has effectively pushed the development of dendrobe tissue culture and dendrobium nobile related industry.
Description
Technical field
The present invention relates to plant tissue culture technical fields, static more particularly to a kind of liquid of iron sheet stone solution Protocorm Multiplication
Suspension fast culture process.
Background technique
Currently, there are many research and report to dendrobium nobile tissue culture technique, method is also varied.Tissue cultures obtain
The mode of regeneration plant is mainly include the following types: seed → protocorm → plantlet；Seed → protocorm → callus → clump
Bud → rooted seedling；Seed → protocorm → aseptic seedling stem sections → plantlet；Seed → callus → protocorm → plantlet；It is former
Bulb → artificial seed → seedling；Stem apex → callus → clump bud → rooted seedling etc..But many tissue culture enterprises are mostly using solid
Body culture medium culture dendrobium candidum protocorm, wherein main element of coagulation is agar, agar price is on every gram of 120 yuan of left sides on the market
The right side causes production cost to rise；Secondly solid medium inoculation speed is slow, and human cost is caused to rise, and solid medium increases
Dendrobium nobile protocorm is grown, proliferative amount is small, the period is long, is always the bottle for restricting dendrobe tissue culture enterprise and the development of entire dendrobium nobile industrial chain
Neck.
In the liquid suspension culture system of existing iron sheet stone solution Protocorm Multiplication, technical method are as follows: adding first
Preculture 30d in the 1/4MS culture medium of 0.5mg/LABA, is then transferred to 1/4MS+40mg/LVc fluid nutrient medium, and pH5.6 shakes
Bed revolving speed is 50r/min, and every 15d subculture is primary, dark culturing.The Quality advance of protocorm, synchronism are good.But existing iron sheet
The liquid suspension culture system of stone solution Protocorm Multiplication needs shaking table shake culture, this not only adds the investment of production equipment,
And increase energy consumption, increase production link, so that production cost be made to rise, hinder this technology of liquid suspension culture
Application and industrialization development.
Summary of the invention
The object of the present invention is to provide a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication, with
Just it during entire dendrobium nobile protocorm liquid suspension culture, is shaken without shaking table, totally stationary culture makes dendrobium candidum protocorm
Stem liquid suspension culture technology can really move towards industrialization production.
The scheme that the present invention solves technical problem is: a kind of static suspension of the liquid of iron sheet stone solution Protocorm Multiplication quickly training
Support method, it the following steps are included:
1) raw material are selected: using solid, mature, the uncracked dendrobium candidum capsule of spontaneous pollination；
2) it materials disinfection: carries out disinfection processing to mature non-dehiscent capsule；
3) it is inoculated with: aseptically by the capsule after disinfection treatment, clamping carpopodium one end with tweezers, it is with cutter that capsule is another
An osculum is cut at end, is gently shaken, and seed is spread in uniform on induction seed germination medium, bottle cap is covered immediately, is placed on training
It is cultivated feeding room；
4) selection of dendrobium candidum protocorm: the growth conditions that selection is induced by Seeds of Dendrobium Candidum are good, without differentiation, color
Bud green protocorm is material, is inoculated in workbench, and inoculation protocorm stem diameter is advisable in 0.2~0.4cm, 2~5g of quality；
5) using the static suspension culture of dendrobium nobile protocorm liquid, culture medium prescription are as follows: B5 medium+0.4~1.0g/L naphthalene second
Acid+50~150g/L potato+10~20g/L sucrose, pH value 5.5~5.8；Basic condition of culture: 20~30 DEG C of temperature, illumination 12
~15h/d, intensity of illumination are 4000lx~4500lx, and conical flask or the glass culture of 200mL are used in suspension incubation
Bottle is used as culture vessel.
It is described to carry out disinfection processing method to mature non-dehiscent capsule in step 2 are as follows: mature non-dehiscent capsule → from
Water rinses → 75% alcohol and impregnates the mercuric chloride solution of 1min → 0.1% immersion 4 times → aseptic filter paper of 15min → aseptic water washing
It blots rear spare.
In step 3), the induction seed germination medium and condition of culture are as follows: with MS minimal medium, add agar
0.4~0.6mg/L of 0.68%, 6- benzylaminopurine, 0.1~0.4mg/L of methyl α-naphthyl acetate, 80~100g/L of potato, indolebutyric acid
0.2~0.5mg/L, 20~30g/L of sucrose；Medium's PH Value 5.4~5.8；Condition of culture: 25~28 DEG C of temperature, illumination 12h/
D, intensity of illumination 2000lx.
In step 5), specific incubation the following steps are included:
A. B5 basal medium mother liquor is prepared, wherein a great number of elements A is 20 times of concentrations；Microelement B is 1000 times of concentrations；Molysite
C is 100 times of concentrations；Organic D is 1000 times of concentrations；Inositol is 500 times of concentrations；
B. to prepare 5L as column, the graduated cylinder of 5L is selected, the water of 1L is first added, is then successively proportionally added into 0.4~1.0g/L naphthalene
Acetic acid, 50~150g/L potato and 10~20g/L sucrose；
C. murphy juice is added, is removed the peel after potato washing, weighs requirement by formula, is filtered after juicing, remove soil dynamic test and upper layer
Foam；
D. after adding medium component, deionized water is added, then constant volume to 5L adjusts pH value；
E. it bottles by formula ratio, autoclave, 121 DEG C of 20~25min of high temperature and pressure moist heat sterilization is put into after sealing；
F. culture medium is taken out, clean, relatively sterile transfer room is put into, places 5~7 days, is followed by culture medium without microbiological contamination phenomenon
Kind；
G. it is inoculated in workbench, before culture medium is put into workbench, is sprayed and sterilized with 75% alcohol mist；
H. it the dendrobium candidum protocorm of select, is caught broken into sterilized tweezers appropriately sized, transfers and trained into liquid suspension
It supports in base, inoculum concentration is 1~3g；
I. it seals, after dark treatment 1~3 day, is put into the culture of illumination cultivation room.
The invention has the advantages that: the static suspension fast culture process of liquid of the invention can make dendrobium candidum
Efficient, high-quality, the inexpensive fast-propagation of protocorm provides sufficient dendrobium nobile bulb provenance for dendrobium nobile seedling tissue culture, meets market pair
The great demand of dendrobium candidum；It is not necessarily to shaking table shake culture during the whole culture process, is saved on equipment investment and energy consumption
A large amount of capital has effectively pushed the development of dendrobe tissue culture and dendrobium nobile related industry, can be used for the protocorm and class of orchid family Dendrobium
Protocorm Multiplication, to achieve the purpose that fast-propagation and conservation.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
Embodiment
The static suspension fast culture process of liquid of the invention, it the following steps are included:
1) raw material are selected: using solid, mature, the uncracked dendrobium candidum capsule of spontaneous pollination；
2) it materials disinfection: carries out disinfection processing to mature non-dehiscent capsule；
3) it is inoculated with: aseptically by the capsule after disinfection treatment, clamping carpopodium one end with tweezers, it is with cutter that capsule is another
An osculum is cut at end, is gently shaken, and seed is spread in uniform on induction seed germination medium, bottle cap is covered immediately, is placed on training
It is cultivated feeding room；
4) selection of dendrobium candidum protocorm: the growth conditions that selection is induced by Seeds of Dendrobium Candidum are good, without differentiation, color
Bud green protocorm is material, is inoculated in workbench, and inoculation protocorm stem diameter is advisable in 0.2cm, quality 3g；
5) using the static suspension culture of dendrobium nobile protocorm liquid, culture medium prescription are as follows: B5 medium+1.0g/L methyl α-naphthyl acetate+
100g/L potato+20g/L sucrose, pH value 5.6；Basic condition of culture: 25 DEG C of temperature, illumination 13h/d, intensity of illumination is
4000lx uses the conical flask of 200mL as culture vessel in suspension incubation.
It is described to carry out disinfection processing method to mature non-dehiscent capsule in step 2 are as follows: mature non-dehiscent capsule → from
Water rinses → 75% alcohol and impregnates immersion 4 times → aseptic filter paper of the 15min → aseptic water washing suction of the mercuric chloride solution of 1min → 0.1%
It is spare after dry.
In step 3), the induction seed germination medium and condition of culture are as follows: with MS minimal medium, add agar
0.68%, 6- benzylaminopurine 0.6mg/L, methyl α-naphthyl acetate 0.4mg/L, potato 100g/L, indolebutyric acid 0.5mg/L, sucrose
25g/L；Medium's PH Value 5.6；Condition of culture: 28 DEG C of temperature, illumination 12h/d, intensity of illumination 2000lx.
4. a kind of static suspension fast culture side of liquid of iron sheet stone solution Protocorm Multiplication according to claim 1
Method, it is characterised in that: in step 5), specific incubation the following steps are included:
A. B5 basal medium mother liquor is prepared, wherein a great number of elements A is 20 times of concentrations；Microelement B is 1000 times of concentrations；Molysite
C is 100 times of concentrations；Organic D is 1000 times of concentrations；Inositol is 500 times of concentrations；
B. with prepare 5L for column, select the graduated cylinder of 5L, first be added 1L water, be then successively proportionally added into 0.6g/L methyl α-naphthyl acetate,
100g/L potato and 15g/L sucrose；
C. murphy juice is added, is removed the peel after potato washing, weighs requirement by formula, is filtered after juicing, remove soil dynamic test and upper layer
Foam；
D. after adding medium component, deionized water is added, then constant volume to 5L adjusts pH value；
E. it bottles by formula ratio, autoclave, 121 DEG C of 20~25min of high temperature and pressure moist heat sterilization is put into after sealing；
F. culture medium is taken out, clean, relatively sterile transfer room is put into, places 6 days, is inoculated with after culture medium is without microbiological contamination phenomenon；
G. it is inoculated in workbench, before culture medium is put into workbench, is sprayed and sterilized with 75% alcohol mist；
H. it the dendrobium candidum protocorm of select, is caught broken into sterilized tweezers appropriately sized, transfers and trained into liquid suspension
It supports in base, inoculum concentration 2g；
I. it seals, after dark treatment 2 days, is put into the culture of illumination cultivation room.
The static suspension fast culture process of liquid of the invention can make dendrobium candidum protocorm efficient, high-quality, inexpensive fast
Speed expansion is numerous, and sufficient dendrobium nobile bulb provenance is provided for dendrobium nobile seedling tissue culture, meets market to the great demand of dendrobium candidum；Entire
It is not necessarily to shaking table shake culture in incubation, a large amount of capital has been saved on equipment investment and energy consumption, has effectively pushed dendrobium nobile group
Training and the development of dendrobium nobile related industry can be used for the protocorm and protocorms proliferation of orchid family Dendrobium, to reach fast-propagation
With the purpose of conservation.
Claims (4)
1. a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication, it is characterised in that: it includes following
Step:
1) raw material are selected: using solid, mature, the uncracked dendrobium candidum capsule of spontaneous pollination；
2) it materials disinfection: carries out disinfection processing to mature non-dehiscent capsule；
3) it is inoculated with: aseptically by the capsule after disinfection treatment, clamping carpopodium one end with tweezers, it is with cutter that capsule is another
An osculum is cut at end, is gently shaken, and seed is spread in uniform on induction seed germination medium, bottle cap is covered immediately, is placed on training
It is cultivated feeding room；
4) selection of dendrobium candidum protocorm: the growth conditions that selection is induced by Seeds of Dendrobium Candidum are good, without differentiation, color
Bud green protocorm is material, is inoculated in workbench, and inoculation protocorm stem diameter is advisable in 0.2~0.4cm, 2~5g of quality；
5) using the static suspension culture of dendrobium nobile protocorm liquid, culture medium prescription are as follows: B5 medium+0.4~1.0g/L naphthalene second
Acid+50~150g/L potato+10~20g/L sucrose, pH value 5.5~5.8；Basic condition of culture: 20~30 DEG C of temperature, illumination 12
~15h/d, intensity of illumination are 4000lx~4500lx, and conical flask or the glass culture of 200mL are used in suspension incubation
Bottle is used as culture vessel.
2. a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication according to claim 1,
It is characterized in that: described to carry out disinfection processing method to mature non-dehiscent capsule in step 2 are as follows: mature non-dehiscent capsule → from
Water rinses → 75% alcohol and impregnates the mercuric chloride solution of 1min → 0.1% immersion 4 times → aseptic filter paper of 15min → aseptic water washing
It blots rear spare.
3. a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication according to claim 1,
It is characterized in that: in step 3), the induction seed germination medium and condition of culture are as follows: with MS minimal medium, add fine jade
Rouge 0.68%, 0.4~0.6mg/L of 6- benzylaminopurine, 0.1~0.4mg/L of methyl α-naphthyl acetate, 80~100g/L of potato, indoles fourth
0.2~0.5mg/L of acid, 20~30g/L of sucrose；Medium's PH Value 5.4~5.8；Condition of culture: 25~28 DEG C of temperature, illumination
12h/d, intensity of illumination 2000lx.
4. a kind of static suspension fast culture process of liquid of iron sheet stone solution Protocorm Multiplication according to claim 1,
Be characterized in that: in step 5), specific incubation the following steps are included:
A. B5 basal medium mother liquor is prepared, wherein a great number of elements A is 20 times of concentrations；Microelement B is 1000 times of concentrations；Molysite
C is 100 times of concentrations；Organic D is 1000 times of concentrations；Inositol is 500 times of concentrations；
B. to prepare 5L as column, the graduated cylinder of 5L is selected, the water of 1L is first added, is then successively proportionally added into 0.4~1.0g/L naphthalene
Acetic acid, 50~150g/L potato and 10~20g/L sucrose；
C. murphy juice is added, is removed the peel after potato washing, weighs requirement by formula, is filtered after juicing, remove soil dynamic test and upper layer
Foam；
D. after adding medium component, deionized water is added, then constant volume to 5L adjusts pH value；
E. it bottles by formula ratio, autoclave, 121 DEG C of 20~25min of high temperature and pressure moist heat sterilization is put into after sealing；
F. culture medium is taken out, clean, relatively sterile transfer room is put into, places 5~7 days, is followed by culture medium without microbiological contamination phenomenon
Kind；
G. it is inoculated in workbench, before culture medium is put into workbench, is sprayed and sterilized with 75% alcohol mist；
H. it the dendrobium candidum protocorm of select, is caught broken into sterilized tweezers appropriately sized, transfers and trained into liquid suspension
It supports in base, inoculum concentration is 1~3g；
I. it seals, after dark treatment 1~3 day, is put into the culture of illumination cultivation room.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113331053A (en) * | 2021-05-26 | 2021-09-03 | 海南大学 | Separation breeding method of dendrobium pure-color flowers |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113331053A (en) * | 2021-05-26 | 2021-09-03 | 海南大学 | Separation breeding method of dendrobium pure-color flowers |
| CN113331053B (en) * | 2021-05-26 | 2022-05-24 | 海南大学 | Separation breeding method of dendrobium pure-color flowers |
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Application publication date: 20191119 |
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