CN104170733B - The method of Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction - Google Patents
The method of Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction Download PDFInfo
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- CN104170733B CN104170733B CN201410436524.5A CN201410436524A CN104170733B CN 104170733 B CN104170733 B CN 104170733B CN 201410436524 A CN201410436524 A CN 201410436524A CN 104170733 B CN104170733 B CN 104170733B
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 86
- 230000006698 induction Effects 0.000 title claims abstract description 55
- 241000628997 Flos Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 45
- 230000001939 inductive effect Effects 0.000 claims abstract description 21
- 239000002609 medium Substances 0.000 claims abstract description 14
- 206010052428 Wound Diseases 0.000 claims abstract description 11
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 11
- 230000001965 increasing effect Effects 0.000 claims abstract description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 26
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 22
- 238000005286 illumination Methods 0.000 claims description 18
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 12
- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 claims description 10
- 241000245240 Lonicera Species 0.000 claims description 5
- 241000196324 Embryophyta Species 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 3
- ZPQOPVIELGIULI-UHFFFAOYSA-N 1,3-dichlorobenzene Chemical compound ClC1=CC=CC(Cl)=C1 ZPQOPVIELGIULI-UHFFFAOYSA-N 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 239000007943 implant Substances 0.000 abstract description 4
- 238000004321 preservation Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 230000032459 dedifferentiation Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000029663 wound healing Effects 0.000 description 6
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 4
- 210000004907 gland Anatomy 0.000 description 4
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 241001170080 Lonicera hypoglauca Species 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- -1 furan amino Purine Chemical compound 0.000 description 2
- 239000012567 medical material Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- AFWYNOWVYKOMGZ-UHFFFAOYSA-N 8-benzyl-7h-purine-2,6-diamine Chemical compound N=1C2=NC(N)=NC(N)=C2NC=1CC1=CC=CC=C1 AFWYNOWVYKOMGZ-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 241000345998 Calamus manan Species 0.000 description 1
- 241000100289 Lonicera confusa Species 0.000 description 1
- 241001570521 Lonicera periclymenum Species 0.000 description 1
- 241000746966 Zizania Species 0.000 description 1
- 235000002636 Zizania aquatica Nutrition 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940117389 dichlorobenzene Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 235000012950 rattan cane Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The method that the invention discloses a kind of Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction.The present invention utilizes totipotency and the plant tissue culture technique of plant cell, using the petiole of the aseptic tissue cultured seedling of Flos Lonicerae that seedling age is 20 30d as outer implant, after increasing treatment of wounds, it is placed in callus inducing medium, cultivate under appropriate conditions so that it is dedifferentiation becomes callus, and then cultivates in callus root induction culture medium, this callus induction is gone out adventitious root, and inductivity reaches 62.50%.The present invention is simple and convenient, fast and effeciently red body petiole can be induced callus in a short time, and this callus induction is gone out adventitious root, provide new Technical Reference for the preservation of Flos Lonicerae elite germplasm, fast breeding and the application in research, production.
Description
Technical field
The present invention relates to field of plant tissue culture, be specifically related to a kind of Flos Lonicerae aseptic tissue cultured seedling petiole
The method of callus root induction.
Background technology
Flos Lonicerae (Lonicera hypoglauca Miq.), plants for Caprifoliaceae Lonicera (Lonicera)
Thing, belongs to perennial half and is wound around liana or rattan shape shrub, has another name called dish gland Radix Ophiopogonis, wild rice gland Radix Ophiopogonis, gland back of the body gold
Flos Lonicerae etc., stem hollow, mainly originates in the ground such as Henan, Guangxi, Shandong, Yunnan.According to version in 2005 " in
State's pharmacopeia " regulation, Flos Lonicerae Lonicera hypoglauca Miq. and largeflower-like honeysuckle flower Lonicera
Nuwranthoides Hand.Mazz. and Lonicera confusa Lonicera confuse DC. is Flos Lonicerae (I
One of state's rare Chinese medicine) three kinds of sources.Its social required quantity is big, produces and needs supply demand gap big, its medicine
Be worth and health-care effect be enough to promote Flos Lonicerae plantation, process, the development of the industry such as sale and clinic
Applied research.
The most less about the report of Flos Lonicerae tissue culture, about the report of Flos Lonicerae Study on tissue culture
Road is few, and the research for Flos Lonicerae tissue cultured seedling petiole callus root induction not yet has been reported that.
Summary of the invention
The present invention has designed and developed a kind of Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction side
Method.The present invention utilizes totipotency and the plant tissue culture technique of plant cell, with seedling age as 20-30d
The petiole of the aseptic tissue cultured seedling of Flos Lonicerae, as outer implant, after increasing treatment of wounds, is placed in wound healing group
Knit in inducing culture, cultivate under appropriate conditions so that it is dedifferentiation becomes callus, and then more
Cultivating in injured tissue root induction culture medium, this callus induction is gone out adventitious root, inductivity reaches
62.50%.The present invention is simple and convenient, fast and effeciently red body petiole can be induced wound healing in a short time
Tissue, and this callus induction is gone out adventitious root, for the preservation of Flos Lonicerae elite germplasm, fast breeding
And the application in research, production provides new Technical Reference.
The technical scheme that the present invention provides is:
Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction method, including:
1) material prepares: by the Flos Lonicerae Lonicera aseptic tissue culture plant inoculation of hypoglauca Miq. to strong
In strong sprout in Seedling culture medium, obtain the aseptic tissue cultured seedling that seedling age is 20-30d；
2) material sampling: seedling taking be age the aseptic tissue cultured seedling of Flos Lonicerae of 20-30d with 1/4 blade
Petiole is stand-by；
3) induction of callus adventitious root: the selected stand-by petiole with 1/4 blade is increased
It is inoculated in callus inducing medium cultivation after treatment of wounds, induces petiole callus；Again will
This petiole callus is forwarded in callus root induction culture medium, and then induces adventitious root,
The cultivation temperature that wherein callus culture and root induction are cultivated is (25 ± 3) DEG C, and relative humidity is
50-75%, illumination is 12h/d, and intensity of illumination is 2000-3000Lux；
Preferably, in described Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction method,
Described 1st) step, described strong seedling culture base is addition α-naphthaleneacetic acid in every liter of 1/2MS culture medium
The culture medium that (α-NAA) 0.2mg is formed, wherein said 1/2MS culture medium is international
In MS culture medium, a great number of elements halves, other components unchanged and the culture medium that formed.
Preferably, in described Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction method,
Described 3rd) step, described process further refers to sterilized surgical scissors stand-by with 1/4
Cut off to increase wound area along depression line on petiole on the petiole of blade.
Preferably, in described Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction method,
Described 3rd) step, described callus inducing medium is containing 6-benzyl amino gland in every liter of culture medium
Purine (6-BA) 0.0-2.5mg, 2,4-Dichlorobenzene base fluoroacetic acid (2,4-D) 0.0-2.5mg, furan amino
Purine (KT) 0.1-1.0mg, α-naphthaleneacetic acid (α-NAA) 0.0-1.0mg, remaining composition is that MS cultivates
Base.
Preferably, in described Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction method,
Described 3rd) step, described callus root induction culture medium is containing 6-benzyl ammonia in every liter of culture medium
Base adenine (6-BA) 0.5-2.5mg, furan amidopurin (KT) 0.5-2.5mg, α-naphthaleneacetic acid (α-NAA)
0.1-1.0mg, remaining composition is MS culture medium.
Preferably, the method for described Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction
In, it is all illumination cultivation on the culturing rack of culturing room that described callus culture is cultivated with root induction.
The beneficial effects of the present invention is:
The petiole of the aseptic tissue cultured seedling of Flos Lonicerae that the present invention can utilize seedling age to be 20-30d lures as outer implant
Lead callus, and then promote that callus differentiates root, from petiole callus induction to callus
Root induction process only needs 50 days, can make de-point of the petiole of Flos Lonicerae in vitro cuttings in a short time
Chemical conversion callus, and induce this callus to take root, for preservation and the medical material of Flos Lonicerae germ plasm resource
Source provide new reference approach, for further utilizing these species to provide Technical Reference.
Accompanying drawing explanation
Fig. 1 Flos Lonicerae petiole callus induction design sketch
Fig. 2 Flos Lonicerae petiole callus root induction design sketch
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art's reference
Description word can be implemented according to this.
Embodiment 1
The petiole of the seedling age cultivated using this seminar aseptic tissue cultured seedling of Flos Lonicerae as 20-30d is as outer planting
Body, on superclean bench, clip is accompanied with the petiole of 1/4 blade, and cuts along this petiolar depression line
Open to increase wound area.Afterwards this treated petiole is transferred in callus inducing medium,
It is placed in illumination cultivation on the culturing rack of culturing room, cultivates and can grow rice white callus in 10 days；Treat this
(one month) (Figure 1A) after callus growth stalwartness, is transferred into Callus Root inducing culture
Base is cultivated, after cultivating 20 days, adventitious root can be grown.Callus and callus root induction
Condition of culture be: cultivation temperature: (25 ± 3) DEG C, relative humidity: 50-75%, illumination 12h/d,
Intensity of illumination 2000-3000Lux；After cultivating 30 days (Fig. 2 A), according to formula: root induction rate
(%)=(inducing the total number of petiole callus of the total number/inoculation of petiole callus of adventitious root)
× 100%, calculate root induction rate and reach 62.50%.
Described callus inducing medium is containing 2 in every liter of culture medium, 4-Dichlorobenzene base fluoroacetic acid
(2,4-D) 2.0mg, furan amidopurin (KT) 0.5mg, remaining composition is MS culture medium；Described
Callus Root inducing culture be containing containing 6-benzyl aminoadenine (6-BA) in every liter of culture medium
2.0mg, furan amidopurin (KT) 2.0mg, α-naphthaleneacetic acid (α-NAA) 0.5mg, remaining composition
For MS culture medium.
Embodiment 2
The petiole of the seedling age cultivated using this seminar aseptic tissue cultured seedling of Flos Lonicerae as 20-30d is as outer planting
Body, on superclean bench, clip is accompanied with the petiole of 1/4 blade, and cuts along this petiolar depression line
Open to increase wound area.Afterwards this treated petiole is transferred in callus inducing medium,
It is placed in illumination cultivation on the culturing rack of culturing room, cultivates and can grow rice white callus in 10 days；Treat this
(one month) (Figure 1B) after callus growth stalwartness, by switching enter Callus Root inducing culture
Cultivating in base, can grow the root of stalwartness after cultivating 20 days, wherein callus is indefinite with callus
The condition of culture of root induction is: cultivation temperature: (25 ± 3) DEG C, relative humidity: 50-75%, illumination 12h/d,
Intensity of illumination 2000-3000Lux；After cultivating 30 days (Fig. 2 B), according to indefinite shown in embodiment 1
It is 14.29% that root induction rate computing formula calculates callus root induction rate.
Described callus inducing medium is containing 2 in every liter of culture medium, 4-Dichlorobenzene base fluoroacetic acid
(2,4-D) 2.0mg, furan amidopurin (KT) 0.5mg, α-naphthaleneacetic acid (α-NAA) 0.2mg,
Remaining composition is MS culture medium；Described Callus Root inducing culture is containing 6-in every liter of culture medium
Benzyl aminoadenine (6-BA) 2.0mg, furan amidopurin (KT) 2.0mg, α-naphthaleneacetic acid (α-NAA)
0.5mg, remaining composition is MS culture medium.
Embodiment 3
The petiole of the seedling age cultivated using this seminar aseptic tissue cultured seedling of Flos Lonicerae as 20-30d is as outer planting
Body, on superclean bench, clip is accompanied with the petiole of 1/4 blade, and cuts along this petiolar depression line
Open to increase wound area.Afterwards this treated petiole is transferred in callus inducing medium,
It is placed in illumination cultivation on the culturing rack of culturing room, cultivates and can grow light green callus in 10 days；Treat this
(one month) (Fig. 1 C) after callus growth stalwartness, by switching enter in root induction culture medium cultivate,
Wherein callus with the condition of culture of callus root induction is: cultivation temperature: (25 ± 3) DEG C,
Relative humidity: 50-75%, illumination 12h/d, intensity of illumination 2000-3000Lux；After cultivating 30 days,
Callus browning is dead, calculates wound healing according to root induction rate computing formula shown in embodiment 1
Root induction rate is 0.
Described callus inducing medium is containing 6-benzyl aminoadenine (6-BA) in every liter of culture medium
2.0mg, furan amidopurin (KT) 0.5mg, remaining composition is MS culture medium；Described wound healing group
Knit root induction to cultivate as every liter of culture medium contains containing 6-benzyl aminoadenine (6-BA) 2.0mg, furan
Mutter amidopurin (KT) 2.0mg, α-naphthaleneacetic acid (α-NAA) 0.5mg, and remaining composition is that MS cultivates
Base.
Embodiment 4
The petiole of the seedling age cultivated using this seminar aseptic tissue cultured seedling of Flos Lonicerae as 20-30d is as outer planting
Body, on superclean bench, clip is accompanied with the petiole of 1/4 blade, and cuts along this petiolar depression line
Open to increase wound area.Afterwards this treated petiole is transferred in callus inducing medium,
It is placed in illumination cultivation on the culturing rack of culturing room, cultivates and can grow light green callus in 10 days；Treat this
(one month) (Fig. 1 D) after callus growth stalwartness, by switching enter in root induction culture medium cultivate,
Wherein callus with the condition of culture of callus root induction is: cultivation temperature: (25 ± 3) DEG C,
Relative humidity: 50-75%, illumination 12h/d, intensity of illumination 2000-3000Lux；After cultivating 30 days,
Callus browning is dead, calculates wound healing according to root induction rate computing formula shown in embodiment 1
Root induction rate is 0.
Described callus inducing medium is containing 6-benzyl aminoadenine (6-BA) in every liter of culture medium
2.0mg, furan amidopurin (KT) 0.5mg, α-naphthaleneacetic acid (α-NAA) 0.2mg, remaining composition
For MS culture medium；Described root induction is cultivated as containing containing 6-benzyl aminoadenine in every liter of culture medium
(6-BA) 2.0mg, furan amidopurin (KT) 2.0mg, α-naphthaleneacetic acid (α-NAA) 0.5mg,
Remaining composition is MS culture medium.
The petiole of the aseptic tissue cultured seedling of Flos Lonicerae that the present invention can utilize seedling age to be 20-30d lures as outer implant
Lead callus, and then promote that callus differentiates root, from petiole callus induction to callus
Root induction process only needs 50 days, containing 2,4-in callus inducing medium is every liter of culture medium
Dichlorobenzene base fluoroacetic acid (2,4-D) 2.0mg, furan amidopurin (KT) 0.5mg, remaining composition is
During MS culture medium, the petiole dedifferentiation of the aseptic tissue cultured seedling of Flos Lonicerae can be made in a short time to become wound healing group
Knitting, utilizing Callus Root inducing culture is containing containing 6-benzyl aminoadenine in every liter of culture medium
(6-BA) 2.0mg, furan amidopurin (KT) 2.0mg, α-naphthaleneacetic acid (α-NAA) 0.5mg,
Remaining composition is that MS culture medium induces this callus to take root, and after cultivating 30 days, root induction rate reaches
62.50%, the source of its preservation being Flos Lonicerae germ plasm resource and medical material provides new reference approach, for
These species are further utilized to provide Technical Reference.
Although embodiment of the present invention are disclosed as above, but it is not restricted to description and embodiment party
Listed utilization in formula, it can be applied to various applicable the field of the invention completely, for being familiar with ability
For the personnel in territory, be easily achieved other amendment, therefore without departing substantially from claim and etc. homotype
Enclosing under limited general concept, the present invention is not limited to specific details and shown here as the figure with description
Example.
Claims (4)
1. a method for Flos Lonicerae aseptic tissue cultured seedling petiole callus root induction, its feature exists
In, comprise the following steps:
1) material prepares: by the Flos Lonicerae Lonicera aseptic tissue culture plant inoculation of hypoglauca Miq. to strong
In strong sprout in Seedling culture medium, obtain the aseptic tissue cultured seedling that seedling age is 20-30d；
2) material sampling: seedling taking be age the aseptic tissue cultured seedling of Flos Lonicerae of 20-30d with 1/4 blade
Petiole is stand-by；
3) induction of callus adventitious root: the selected stand-by petiole with 1/4 blade is increased
It is inoculated in callus inducing medium cultivation after treatment of wounds, induces petiole callus；Again will
This petiole callus is forwarded in callus root induction culture medium, and then induces adventitious root,
The cultivation temperature that wherein callus culture and root induction are cultivated is (25 ± 3) DEG C, and relative humidity is
50-75%, illumination is 12h/d, and intensity of illumination is 2000-3000Lux；
Wherein, described callus inducing medium is containing 6-benzyl aminoadenine in every liter of culture medium
0.0-2.5mg, 2,4-Dichlorobenzene base fluoroacetic acid 0.0-2.5mg, furan amidopurin 0.1-1.0mg, α-naphthalene second
Acid 0.0-1.0mg, remaining composition is MS culture medium；Described callus root induction culture medium is
In every liter of culture medium containing 6-benzyl aminoadenine 0.5-2.5mg, furan amidopurin 0.5-2.5mg, α-
Naphthalene acetic acid 0.1-1.0mg, remaining composition is MS culture medium.
Flos Lonicerae the most according to claim 1 aseptic tissue cultured seedling petiole callus root induction
Method, it is characterised in that the described 1st) step, described strong seedling culture base is to train in every liter of 1/2MS
Supporting and add the culture medium that α-naphthaleneacetic acid 0.2mg is formed in base, wherein said 1/2MS culture medium is state
In the MS culture medium that border is general, a great number of elements halves, other components unchanged and the culture medium that formed.
Flos Lonicerae the most according to claim 1 aseptic tissue cultured seedling petiole callus root induction
Method, it is characterised in that the described 3rd) step, described carrying out increases treatment of wounds and refers to sterilized
The surgical scissors of bacterium is cut off to increase along depression line on petiole on the stand-by petiole with 1/4 blade
Wound area.
Flos Lonicerae the most according to claim 1 aseptic tissue cultured seedling petiole callus root induction
Method, it is characterised in that the cultivation of described callus and the cultivation of root induction are all in culturing room
Culturing rack on illumination cultivation.
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