CN109452171A - A kind of tissue culture method of the sterile induction plant regeneration of pale reddish brown trident bletilla striata seeds - Google Patents
A kind of tissue culture method of the sterile induction plant regeneration of pale reddish brown trident bletilla striata seeds Download PDFInfo
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- CN109452171A CN109452171A CN201811419709.XA CN201811419709A CN109452171A CN 109452171 A CN109452171 A CN 109452171A CN 201811419709 A CN201811419709 A CN 201811419709A CN 109452171 A CN109452171 A CN 109452171A
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- bletilla striata
- reddish brown
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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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- 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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Abstract
The present invention relates to pale reddish brown trident bletilla striata technical field of tissue culture, more particularly to a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds, the following steps are included: step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, 3min first is rinsed with deionized water, is subsequently placed in 70% alcohol and impregnates 5min, then handle 6-10min in dislocation bactericidal liquid, it is then rinsed 4-6 times, is placed in spare in gnotobasis using sterile distilled water.The tissue culture method of the sterile induction plant regeneration of the pale reddish brown trident bletilla striata seeds of invention, relative in routine techniques, the present invention is in sterilization process, first 5min is impregnated in 70% alcohol, it is handled in dislocation bactericidal liquid again, the bactericidal liquid that bactericidal liquid is formed using graphene solution, chitosan, montmorillonite dispersions and potassium permanganate.
Description
Technical field
The present invention relates to pale reddish brown trident bletilla striata technical field of tissue culture, and in particular to a kind of pale reddish brown trident bletilla striata seeds without
The tissue culture method of bacterium induction plant regeneration.
Background technique
The bletilla striata also known as Lian Jicao, Gan Gen, give free of charge, Zhu Lan, purple orchid etc. are orchid family bletilla striata category perennial herb plant bulbs, white
The medicinal effects of splendid achnatherum are divided into dry tuber, mild-natured, slightly cold, bitter, sweet, puckery, there is astringing to arrest bleeding effect, be mainly used for spitting blood,
Traumatic hemorrhage, chapped skin etc., bletilla striata kind are roughly divided into two major class, and one kind is the pale reddish brown bletilla striata, flower aubergine, leaf drape over one's shoulders needle or
Wide lanceolar, apex is tapering, and base portion is downward to embrace stem at sheath shape, includes big kind, two group of microspecies, and another kind of is the chrysanthemum bletilla striata,
Flower yellow, leaf strip lanceolar, the pale reddish brown trident bletilla striata it is too big for requirement difference, cause largely acquire and consumption plant resources,
Along with the Hills afforestation rate of southern each province further increases, wild bletilla striata yield can also be aggravated and declined every year, when
When acquisition and consumption are more than the power of regeneration of natural resources, the in imminent danger of species will lead to.
The also non-full maturity of existing sterile its plant regeneration technique of induction of pale reddish brown trident bletilla striata seeds, is planted during tissue culture
Son sense bacterium rate is higher, while seed germination rate is poor, therefore the present invention is further studied for this status, improves sterile lure
Lead plant regeneration technique.
Summary of the invention
The purpose of the present invention is to provide a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds, packets
Include following steps:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handle 6-10min in dislocation bactericidal liquid, then adopts
It is rinsed 4-6 times, is placed in spare in gnotobasis with sterile distilled water；
The spare seed of step 1 is first carried out radiation treatment, handles 30-40s, be then inoculated in Initial culture by step 2
On base MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
Be as further scheme of the invention: in the step 1 sterilize liquid and preparation method thereof be by Graphene powder, go from
Sub- water is mixed according to 3:7, is configured to graphene solution, and the chitosan that graphene solution total amount 10% is then added is molten
Liquid continues to stir 5-15min, until chitosan sufficiently dissolves, it is molten then to add the potassium permanganate that mass fraction is 10-20%
Liquid is eventually adding the montmorillonite dispersions of chitosan total amount 70-80%, stirs evenly.
Be as further scheme of the invention: the montmorillonite dispersions preparation method is to grind montmorillonite,
20-100 mesh is crossed, heating water bath is then carried out, heating temperature is 65-75 DEG C, heats 25-35min, subsequent re-ultrasonic dispersion
15-25min.
Be as further scheme of the invention: radiation treatment condition is using ultraviolet irradiation, spoke in the step 2
Firing association dosage 2.0-3.0kGy, irradiatometer dose rate are 20-30Gy/min.
Be as further scheme of the invention: radiation treatment condition is using ultraviolet irradiation, spoke in the step 2
Firing association dosage 2.5kGy, irradiatometer dose rate are 25Gy/min.
Be as further scheme of the invention: culturing room's condition is that intensity of illumination is 2000Lx, humidity 55-
65%, time 12h/d.
Be as further scheme of the invention: the proliferated culture medium is MS+6-BA 1.2mg/L+IBA 0.1mg/L+
GA31.5mg/L。
Be as further scheme of the invention: the root media is 1/2MS+AC 0.05g/l+IBA 1.0mg/L
+ sucrose 15g/L.
Compared with prior art, the present invention have it is following the utility model has the advantages that
The tissue culture method of the sterile induction plant regeneration of pale reddish brown trident bletilla striata seeds of the invention, relative in routine techniques,
The present invention is first impregnated in 70% alcohol and is handled in 5min, then dislocation bactericidal liquid in sterilization process, and bactericidal liquid uses graphene
The bactericidal liquid that solution, chitosan, montmorillonite dispersions and potassium permanganate form, the bactericidal liquid are formed using inorganic raw material, are killed
Bacterium is high-efficient, and environmentally protective, easily cleaned, then can induce seed by radiation treatment again, improve seed activity, into
And seed is promoted to sprout, proliferated culture medium, the root media of use can be improved pale reddish brown trident bletilla striata seeds and take root in the later period
Physiological status, seed germination rate of the invention reach 98.9%, and sense bacterium rate reaches 0.1%, it is known that, the present invention is relative to routine
Tissue culture method improvement is very significant.
Specific embodiment
Combined with specific embodiments below, technical scheme in the embodiment of the invention is clearly and completely described, shows
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1:
A kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds of the present embodiment, including following step
It is rapid:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handles 6min in dislocation bactericidal liquid, then uses nothing
It bacterium distilled water flushing 4 times, is placed in spare in gnotobasis；
The spare seed of step 1 is first carried out radiation treatment, handles 30s, be then inoculated in initial culture base by step 2
On MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
It is to mix Graphene powder, deionized water according to 3:7 that liquid and preparation method thereof is sterilized in the step of the present embodiment one
Stirring, is configured to graphene solution, and the chitosan solution of graphene solution total amount 10% is then added, and continues to stir 5min, until
Chitosan sufficiently dissolves, and then adds the liquor potassic permanganate that mass fraction is 10%, is eventually adding chitosan total amount 70%
Montmorillonite dispersions, stir evenly.
The montmorillonite dispersions preparation method of the present embodiment is to grind montmorillonite, crosses 20 meshes, then carries out water
Bath heating, heating temperature are 65 DEG C, heat 25-35min, subsequent re-ultrasonic dispersion 15min.
Radiation treatment condition is using ultraviolet irradiation, total radiation dose 2.0kGy, irradiation in the step of the present embodiment two
Dosage rate is 20Gy/min.
Culturing room's condition of the present embodiment is that intensity of illumination is 2000Lx, humidity 55%, time 12h/d.
The proliferated culture medium of the present embodiment is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
The root media of the present embodiment is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
Embodiment 2:
A kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds of the present embodiment, including following step
It is rapid:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handles 10min in dislocation bactericidal liquid, then uses
Sterile distilled water rinses 6 times, is placed in spare in gnotobasis；
The spare seed of step 1 is first carried out radiation treatment, handles 40s, be then inoculated in initial culture base by step 2
On MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
It is to mix Graphene powder, deionized water according to 3:7 that liquid and preparation method thereof is sterilized in the step of the present embodiment one
Stirring, is configured to graphene solution, and the chitosan solution of graphene solution total amount 10% is then added, and continues to stir 15min, until
Chitosan sufficiently dissolves, and then adds the liquor potassic permanganate that mass fraction is 20%, is eventually adding chitosan total amount 80%
Montmorillonite dispersions, stir evenly.
The montmorillonite dispersions preparation method of the present embodiment is to grind montmorillonite, sieves with 100 mesh sieve, then carries out water
Bath heating, heating temperature are 75 DEG C, heat 35min, subsequent re-ultrasonic dispersion 25min.
Radiation treatment condition is using ultraviolet irradiation, total radiation dose 3.0kGy, irradiation in the step of the present embodiment two
Dosage rate is 30Gy/min.
Culturing room's condition of the present embodiment is that intensity of illumination is 2000Lx, humidity 65%, time 12h/d.
The proliferated culture medium of the present embodiment is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
The root media of the present embodiment is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
Embodiment 3:
A kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds of the present embodiment, including following step
It is rapid:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handle 6-10min in dislocation bactericidal liquid, then adopts
It is rinsed 5 times, is placed in spare in gnotobasis with sterile distilled water；
The spare seed of step 1 is first carried out radiation treatment, handles 30-40s, be then inoculated in Initial culture by step 2
On base MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
It is to mix Graphene powder, deionized water according to 3:7 that liquid and preparation method thereof is sterilized in the step of the present embodiment one
Stirring, is configured to graphene solution, and the chitosan solution of graphene solution total amount 10% is then added, and continues to stir 10min, until
Chitosan sufficiently dissolves, and then adds the liquor potassic permanganate that mass fraction is 15%, is eventually adding chitosan total amount 70-
80% montmorillonite dispersions, stir evenly.
The montmorillonite dispersions preparation method of the present embodiment is to grind montmorillonite, crosses 60 meshes, then carries out water
Bath heating, heating temperature are 70 DEG C, heat 30min, subsequent re-ultrasonic dispersion 20min.
Radiation treatment condition is using ultraviolet irradiation, total radiation dose 2.5kGy, irradiation in the step of the present embodiment two
Dosage rate is 25Gy/min.
Culturing room's condition of the present embodiment is that intensity of illumination is 2000Lx, humidity 60%, time 12h/d.
The proliferated culture medium of the present embodiment is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
The root media of the present embodiment is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
Embodiment 4:
A kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds of the present embodiment, including following step
It is rapid:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handles 4min in dislocation bactericidal liquid, then uses nothing
It bacterium distilled water flushing 3 times, is placed in spare in gnotobasis；
The spare seed of step 1 is first carried out radiation treatment, handles 25s, be then inoculated in initial culture base by step 2
On MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
It is to mix Graphene powder, deionized water according to 3:7 that liquid and preparation method thereof is sterilized in the step of the present embodiment one
Stirring, is configured to graphene solution, and the chitosan solution of graphene solution total amount 10% is then added, and continues to stir 5-15min,
It is sufficiently dissolved to chitosan, then adds the liquor potassic permanganate that mass fraction is 8%, be eventually adding chitosan total amount 70-
80% montmorillonite dispersions, stir evenly.
The montmorillonite dispersions preparation method of the present embodiment is to grind montmorillonite, crosses 10 meshes, then carries out water
Bath heating, heating temperature are 60 DEG C, heat 20min, subsequent re-ultrasonic dispersion 10min.
Radiation treatment condition is using ultraviolet irradiation, total radiation dose 1.8kGy, irradiation in the step of the present embodiment two
Dosage rate is 15Gy/min.
Culturing room's condition of the present embodiment is that intensity of illumination is 2000Lx, humidity 40%, time 12h/d.
The proliferated culture medium of the present embodiment is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
The root media of the present embodiment is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
Embodiment 5:
A kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds of the present embodiment, including following step
It is rapid:
Step 1, selection full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first uses
Deionized water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handles 12min in dislocation bactericidal liquid, then uses
Sterile distilled water rinses 7 times, is placed in spare in gnotobasis；
The spare seed of step 1 is first carried out radiation treatment, handles 45s, be then inoculated in initial culture base by step 2
On MS, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, culture of rootage are then transferred to after 60d by step 3
In base, after cultivating 40d, then acclimatization and transplants.
It is to mix Graphene powder, deionized water according to 3:7 that liquid and preparation method thereof is sterilized in the step of the present embodiment one
Stirring, is configured to graphene solution, and the chitosan solution of graphene solution total amount 10% is then added, and continues to stir 20min, until
Chitosan sufficiently dissolves, and then adds the liquor potassic permanganate that mass fraction is 25%, is eventually adding chitosan total amount 85%
Montmorillonite dispersions, stir evenly.
The montmorillonite dispersions preparation method of the present embodiment is to grind montmorillonite, crosses 120 meshes, then carries out water
Bath heating, heating temperature are 80 DEG C, heat 40min, subsequent re-ultrasonic dispersion 30min.
Radiation treatment condition is using ultraviolet irradiation, total radiation dose 3.5kGy, irradiation in the step of the present embodiment two
Dosage rate is 35Gy/min.
Culturing room's condition of the present embodiment is that intensity of illumination is 2000Lx, humidity 70%, time 12h/d.
The proliferated culture medium of the present embodiment is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
The root media of the present embodiment is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
Comparative example 1.
It is essentially identical with the material and preparation process of embodiment 3, it has only the difference is that being handled without bactericidal liquid.
Comparative example 2.
It is essentially identical with the material and preparation process of embodiment 3, it has only the difference is that bactericidal liquid is not added with montmorillonite.
Comparative example 3.
Using conventional tissue culture method.
Embodiment 1-5 and comparative example 1-3 performance measurements are as follows
| Feel bacterium rate (%) | Germination rate (%) | |
| Embodiment 1 | 0.5 | 98.1 |
| Embodiment 2 | 0.7 | 98.4 |
| Embodiment 3 | 0.1 | 98.9 |
| Embodiment 4 | 1.3 | 97.6 |
| Embodiment 5 | 1.6 | 97.3 |
| Comparative example 1 | 3.2 | 92.3 |
| Comparative example 2 | 2.4 | 93.5 |
| Comparative example 3 | 5.4 | 90.2 |
Obtained from embodiment 1-5 and comparative example 1-3, tissue culture method of the invention relative to comparative example 1-3, feel bacterium rate,
Germination rate has and significantly improves, it is known that, the present invention obtains very big complete relative to conventional sterile induction plant regeneration technique
It is kind.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (8)
1. a kind of tissue culture method of the sterile induction plant regeneration of pale reddish brown trident bletilla striata seeds, which comprises the following steps:
Step 1, choose full grains, color is normal, size is uniform, the pale reddish brown trident bletilla striata seeds of robust growth, first spend from
Sub- water rinses 3min, is subsequently placed in 70% alcohol and impregnates 5min, then handles 6-10min in dislocation bactericidal liquid, then uses nothing
Bacterium distilled water flushing 4-6 times is placed in spare in gnotobasis；
The spare seed of step 1 is first carried out radiation treatment, handles 30-40s, be then inoculated in initial culture base MS by step 2
On, it is placed in culturing room, observes rudiment and upgrowth situation after 30d；
The aseptic seedlings that step 2 is sprouted are transferred in proliferated culture medium, root media are then transferred to after 60d by step 3
In, after cultivating 40d, then acclimatization and transplants.
2. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 1, special
Sign is that it is that Graphene powder, deionized water are mixed according to 3:7 that liquid and preparation method thereof is sterilized in the step 1, matches
Graphene solution is made, the chitosan solution of graphene solution total amount 10% is then added, continues to stir 5-15min, until shell is poly-
Sugared sufficiently dissolution, then adds the liquor potassic permanganate that mass fraction is 10-20%, is eventually adding chitosan total amount 70-
80% montmorillonite dispersions, stir evenly.
3. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 2, special
Sign is that the montmorillonite dispersions preparation method is to grind montmorillonite, crosses 20-100 mesh, then carries out water-bath and adds
Heat, heating temperature are 65-75 DEG C, heat 25-35min, subsequent re-ultrasonic dispersion 15-25min.
4. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 1, special
Sign is that radiation treatment condition is using ultraviolet irradiation, total radiation dose 2.0-3.0kGy, radiation resistance in the step 2
Rate is 20-30Gy/min.
5. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 4, special
Sign is that radiation treatment condition is using ultraviolet irradiation, total radiation dose 2.5kGy in the step 2, and irradiatometer dose rate is
25Gy/min。
6. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 1, special
Sign is that culturing room's condition is that intensity of illumination is 2000Lx, humidity 55-65%, time 12h/d.
7. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 1, special
Sign is that the proliferated culture medium is MS+6-BA 1.2mg/L+IBA 0.1mg/L+GA31.5mg/L。
8. a kind of tissue culture method of pale reddish brown sterile induction plant regeneration of trident bletilla striata seeds according to claim 1, special
Sign is that the root media is 1/2MS+AC 0.05g/l+IBA 1.0mg/L+ sucrose 15g/L.
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| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
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