CN106665357B - A method of establishing short-tube lycoris regenerating system - Google Patents
A method of establishing short-tube lycoris regenerating system Download PDFInfo
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- CN106665357B CN106665357B CN201710004973.6A CN201710004973A CN106665357B CN 106665357 B CN106665357 B CN 106665357B CN 201710004973 A CN201710004973 A CN 201710004973A CN 106665357 B CN106665357 B CN 106665357B
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- 241001633628 Lycoris Species 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000001172 regenerating effect Effects 0.000 title claims abstract description 10
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 57
- 230000006698 induction Effects 0.000 claims abstract description 23
- 230000035755 proliferation Effects 0.000 claims abstract description 22
- 230000004069 differentiation Effects 0.000 claims abstract description 19
- 230000001939 inductive effect Effects 0.000 claims abstract description 16
- 230000001954 sterilising effect Effects 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims description 47
- 229930006000 Sucrose Natural products 0.000 claims description 34
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 34
- 239000005720 sucrose Substances 0.000 claims description 34
- 229920001817 Agar Polymers 0.000 claims description 32
- 239000008272 agar Substances 0.000 claims description 32
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 claims description 20
- 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 14
- 238000005286 illumination Methods 0.000 claims description 14
- 239000000835 fiber Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 7
- 238000012549 training Methods 0.000 claims description 5
- 238000005728 strengthening Methods 0.000 claims description 3
- 239000010977 jade Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 12
- 230000001488 breeding effect Effects 0.000 abstract description 12
- 230000006872 improvement Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract 2
- 238000006243 chemical reaction Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 239000002689 soil Substances 0.000 description 14
- 238000005406 washing Methods 0.000 description 13
- 239000011159 matrix material Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 235000019362 perlite Nutrition 0.000 description 5
- 239000010451 perlite Substances 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 4
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 4
- 239000006013 carbendazim Substances 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 239000003337 fertilizer Substances 0.000 description 4
- 239000003415 peat Substances 0.000 description 4
- 244000223014 Syzygium aromaticum Species 0.000 description 3
- 235000016639 Syzygium aromaticum Nutrition 0.000 description 3
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 241000234270 Amaryllidaceae Species 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000109222 Lycoris chinensis Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002073 mitogenetic effect Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention provides a kind of methods for establishing short-tube lycoris regenerating system, comprising the following steps: 1) short-tube lycoris underground bulb is obtained newborn bulb after sterilizing, inducing Multiple Buds, proliferation adventitious bud, strong bud culture；2) newborn bulb is linked into the callus for carrying out dark culture in callus inducing medium and obtaining 1.5~2.5cm of diameter；3) callus is cut into and is inoculated into the 15~20d of differentiation for carrying out callus in bud inducement cultivation base after the agglomerate of 1~2cm of diameter and obtains Multiple Buds；4) Multiple Buds are inoculated into strong bud root media after carrying out strong bud culture of rootage and obtain tissue-cultured seedling.The Multiple Buds quantity gone out via callus induction is more, and the speed of growth is fast, and short-tube lycoris breeding coefficient can be greatly improved, while accelerating the speed of breeding；Meanwhile being expected to establish the efficient genetic conversion system of short-tube lycoris via callus, to realize the genetic improvement for carrying out molecular biology level to it.
Description
Technical field
The present invention relates to field of plant tissue culture technique more particularly to a kind of methods for establishing short-tube lycoris regenerating system.
Background technique
Lycoris (Lycoris chinensis Traub) is Amaryllidaceae (Amaryllidaceae) Lycoris
(Lycoris Herb.) herbaceos perennial.Bulb ovoid, spring go out leaf, and leaf is band-like, and umbel has little Hua 5-6 piece；
Flower yellow；Perianth sliver intensity warp and shrinkage.The month at florescence 7-8.Scape is tall and straight, and flower-shape is graceful, and pattern is gorgeous, has very high
Ornamental value has been increasingly becoming the flowering bulb of novel Fresh Cutting flower, landscape ground quilt and potting.
Under natural conditions, Lycoris and other lycoris plants are generally based on the breeding of natural bulb separation, but breeding coefficient
Low, a split every year on average bloom 3~4 years or so the time of need by mitogenetic 1~2 ball, bulbec；Even if normal solid, from kind
Son was sprouted to the time for generally requiring 5-6 of blooming；And Lycoris numerous species be not easy it is normal solid.Therefore, bulb separation is bred
Speed is slow, and the breeding cycle is long, is a problem facing in the popularization and application of entire lycoris plants.In addition to this, available
The callus generated during Regeneration System carries out the improvement of molecular biology level to it or carries out other heredity behaviour
Make.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of methods for establishing short-tube lycoris regenerating system.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of methods for establishing short-tube lycoris regenerating system, comprising the following steps: 1) passes through underground bulb
Newborn bulb is obtained after sterilizing, induction Multiple Buds, proliferation adventitious bud, strong bud culture；2) the newborn bulb is linked into callus
It organizes to carry out the callus that dark culture obtains 1.5~2.5cm of diameter in induced medium；3) callus is cut into
15~20d of differentiation that progress callus in bud inducement cultivation base is inoculated into after the agglomerate of 1.0~1.5cm of diameter is grown thickly
Bud；4) Multiple Buds are inoculated into strong bud root media carries out strong bud and culture of rootage obtains test tube seedling.
Preferably, strong bud culture described in step 1) includes 2~3 squamous subcultures, and the time of each squamous subculture is
25~35d；The temperature of the strong bud culture is independently 24~26 DEG C, 100~300 μm of olm of intensity of illumination-2·s-1, illumination
14~18h/d of time.
Preferably, bud culture is strengthened described in step 1) to be with culture medium includes 35~45g/L sucrose, 5~10g/L agar,
1.5 the MS culture medium of~2.5mg/L6- benzyl aminoadenine and 0.1~0.3mg/L methyl α-naphthyl acetate.
Preferably, callus inducing medium described in step 2) is the sucrose for including 25~35g/L, 5~10g/L
Agar, the 6- benzyl aminoadenine of 5.5~6.5mg/L and the MS culture medium of 1.0~2.0mg/L methyl α-naphthyl acetate.
Preferably, dark culture described in step 2) includes that the primary dark culture of 25~35d and the subculture of 25~35d are secretly trained
It supports, the temperature of the dark culture is 24~26 DEG C.
Preferably, induction Multiple Buds described in step 1) is carried out in bud inducement cultivation base, the bud induction training
Feeding base is the sucrose for including 25~35g/L, the agar of 5~10g/L, the 6- benzyl aminoadenine of 2.5~3.5mg/L and 1.0~
The MS culture medium of 2.0mg/L methyl α-naphthyl acetate.
Preferably, induction Multiple Buds described in step 1) includes primary Fiber differentiation and subculture Fiber differentiation, the original
Time for Fiber differentiation is 35~45d, and the time of subculture Fiber differentiation is 35~45d；Callus described in step 3)
Differentiation only includes the primary Fiber differentiation of Multiple Buds；24~26 DEG C of cultivation temperature of the induction Multiple Buds, intensity of illumination 100~
300μmol·m-2·s-1, 10~14h/d of light application time.
Preferably, proliferation adventitious bud step described in step 1) is that Multiplying culture is carried out in bud proliferated culture medium, described
Bud proliferation is the sucrose for including 25~35g/L with culture medium, and the 6- benzyl amino gland of the agar of 5~10g/L, 2.5~3.5mg/L is fast
The MS culture medium of purine and 0.5~1.5mg/L methyl α-naphthyl acetate；The proliferation Adventitious bud culture temperature is 24~26 DEG C, intensity of illumination 100
~300 μm of olm-2·s-1, 14~18h/d of light application time；The time of the proliferation Adventitious bud culture is 35~45d.
Preferably, bud root media is strengthened described in step 4) is the sucrose for including 35~45g/L, the fine jade of 5~10g/L
Rouge, the indolebutyric acid of the 6- benzyl aminoadenine of 3.5~4.5mg/L, 0.1~0.3mg/L methyl α-naphthyl acetate and 1.5~2.5mg/L
MS culture medium.
Preferably, the diameter of short-tube lycoris seedling described in step 4) is 3~5mm, and root system is 3~5.
Beneficial effects of the present invention: the method provided by the invention for establishing short-tube lycoris regenerating system, by by short-tube lycoris underground squama
After stem obtains newborn bulb after sterilizing, inducing Multiple Buds, proliferation adventitious bud, strong bud culture, newborn bulb generation is induced to be cured
Injured tissue, then go out Multiple Buds via callus induction, the Multiple Buds quantity gone out via callus induction is more, the speed of growth
Fastly, its breeding coefficient is greatly improved, while accelerating the speed of breeding, the breeding cycle is 5~6 months；And more
A variety of genetic manipulations can be carried out to it on the basis of injured tissue, improve inhereditary feature from molecular biology level.
Specific embodiment
The present invention provides a kind of methods for establishing short-tube lycoris regenerating system, comprising the following steps: 1) by short-tube lycoris underground bulb
Newborn bulb is obtained after sterilizing, inducing Multiple Buds, proliferation adventitious bud, strong bud culture；2) newborn bulb is linked into callus
It organizes to carry out the callus that dark culture obtains 1.5~2.5cm of diameter in induced medium；3) callus is cut into diameter
15~20d of differentiation that progress callus in bud inducement cultivation base is inoculated into after the agglomerate of 1~2cm obtains Multiple Buds；4) by clump
It sprouts to be inoculated into strong bud root media and carries out obtaining tissue-cultured seedling after strengthening bud culture of rootage.
The present invention obtains short-tube lycoris underground bulb newborn after sterilizing, inducing Multiple Buds, proliferation adventitious bud, strong bud culture
Bulb.The short-tube lycoris kind is preferably Lycoris；The short-tube lycoris underground bulb is preferably triennial or more, more preferably 3~
Life in 5 years, the underground bulb of 1.5~4cm of diameter.The present invention preferably carries out underground bulb pre- before to the sterilizing of underground bulb
Processing obtains blocky bulb；The pretreatment includes obtaining plateau, being cleaned to plateau, stripping and slicing and washing.
The present invention preferably removably descends the outer layer scale leaf of bulb, cuts off the root of underground bulb whole and 1/2~2/3 squama
Leaf retains plateau；The cleaning preferably washes away bulb panel surface soil using hairbrush, clear water；The block that the stripping and slicing obtains
Shape plateau is preferably sized to 1.0~1.5cm3；The washing preferably concussion is washed, and cleaning solution used in the washing is preferred
For 84 thimerosals；The time of the washing is preferably 15~50min, more preferably 25~40min.After the washing, the present invention
It is preferred that rinsing 25~35min of plateau after the washing using flowing clear water.
The present invention sterilizes to the plateau after obtaining plateau.The disinfecting action is preferably in ultra-clean work
Make to carry out in platform；The sterilizing preferably includes alcohol rinsing, HgCl2Sterilizing and aseptic water washing.In the present invention, the wine
It is preferably 70~80% that essence rinsing alcohol used, which is volume fraction, and more preferably 75%；The time of the alcohol rinsing is preferred
For 25~35s, more preferably 30s.In the present invention, the HgCl2HgCl is used in sterilizing2The mass fraction of solution is preferably 0.05
~0.15% HgCl2Solution；The HgCl2The time of solution sterilization is preferably 35~45m in, more preferably 40min.At this
In invention, the number of the aseptic water washing is preferably 4~6 times, and more preferably 5 times.The present invention after the completion of aseptic water washing,
The preferred moisture that bulb panel surface is blotted using aseptic paper.
After the sterilizing, the bulb after the sterilizing is being carried out induction Multiple Buds by the present invention.It preferably will in the present invention
The blocky bulb of sterilizing is directly inoculated into bud inducement cultivation base in the downward mode of basal disc and is cultivated；The bud inducement cultivation base
Preferably comprise the sucrose of 25~35g/L, the agar of 5~10g/L, the 6- benzyl aminoadenine of 2.5~3.5mg/L and 1~
The MS culture medium of 2.0mg/L methyl α-naphthyl acetate；It more preferably include the sucrose of 30g/L, the agar of 7.5g/L, the 6- benzyl ammonia of 3.0mg/L
The MS culture medium of base adenine and 1.5mg/L methyl α-naphthyl acetate.
The process of heretofore described induction Multiple Buds preferably includes originally culture and squamous subculture, specifically: by institute
Bulb after stating sterilizing successively carries out originally culture and squamous subculture on bud inducement cultivation base.In the present invention, described primary
The time of culture is preferably 35~45d, more preferably 40d；The time of the squamous subculture is preferably 35~45d, more preferably
40d；The cultivation temperature of the induction Multiple Buds is preferably 24~26 DEG C, and more preferably 25 DEG C；The illumination of the induction Multiple Buds
Intensity is preferably 100~300 μm of olm-2·s-1, more preferably 200 μm of olm-2·s-1；The time of illumination is preferably 10~
14h/d, more preferably 12h/d.
The present invention carries out proliferation adventitious bud after inducing Multiple Buds, by obtained bulb Multiple Buds, specially lures described
That leads that Multiple Buds obtain is transplanted in bud proliferated culture medium, carries out adventitious bud proliferation.In the present invention, the bud proliferated culture medium
Preferably comprise the sucrose of 25~35g/L, the agar of 5~10g/L, the 6- benzyl aminoadenine of 2.5~3.5mg/L and 0.5~
The MS culture medium of 1.5mg/L methyl α-naphthyl acetate more preferably includes the sucrose of 30g/L, the agar of 7.5g/L, the 6- benzyl ammonia of 3.0mg/L
The MS culture medium of base adenine and 1.0mg/L methyl α-naphthyl acetate.
In the present invention, the cultivation temperature of the proliferation adventitious bud is preferably 24~26 DEG C, and more preferably 25 DEG C；The increasing
The intensity of illumination for growing adventitious bud is preferably 100~300 μm of olm-2·s-1, more preferably 200 μm of olm-2·s-1；The light
According to time be preferably 14~18h/d, more preferably 16h/d；The time of the proliferation Adventitious bud culture is preferably 35~45d,
More preferably 40d.
The present invention carries out strong bud culture after completing adventitious bud proliferation, by obtained bulb adventitious bud, obtains newborn bulb.
In the present invention, the strong bud culture preferably includes 2~3 squamous subcultures, and the time of each squamous subculture is preferably 25
~35d；More preferably 30d.In the present invention, the temperature of the strong bud culture is preferably 24~26 DEG C, and more preferably 25 DEG C；It is described
The intensity of illumination of strong bud culture is preferably 100~300 μm of olm-2·s-1, more preferably 200 μm of olm-2·s-1；It is described strong
The light application time of bud culture is preferably 14~18h/d, more preferably 16h/d.
In the present invention, the strong bud culture specifically: the bulb adventitious bud that adventitious bud obtains will be proliferated and be transplanted to strong bud
Strong bud culture is carried out on culture medium.In the present invention, the strong bud culture preferably comprises the sugarcane of 35~45g/L with culture medium
Sugar, the agar of 5~10g/L, the 6- benzyl aminoadenine of 1.5~2.5mg/L
With the MS culture medium of 0.1~0.3mg/L methyl α-naphthyl acetate；More preferably include 30g/L sucrose, the agar of 7.5g/L,
The 6- benzyl aminoadenine of 2.0mg/L and the MS culture medium of 0.2mg/L methyl α-naphthyl acetate.
After the completion of strong bud culture of the present invention, the newborn bulb of 2~4mm of diameter is obtained.
Newborn bulb is linked into callus inducing medium after obtaining newborn bulb and carries out dark culture by the present invention
Obtain callus.Heretofore described callus inducing medium preferably comprises the sucrose of 25~35g/L, 5~
The agar of 10g/L, the 6- benzyl aminoadenine of 5.5~6.5mg/L and the MS culture medium of 1.0~2.0mg/L methyl α-naphthyl acetate；More preferably
It is the sucrose for including 30g/L, the agar of 7.5g/L, the MS culture of the 6- benzyl aminoadenine and 1.5mg/L methyl α-naphthyl acetate of 6.0mg/L
Base.
In the present invention, the dark culture preferably includes originally culture and squamous subculture, the originally culture and after being commissioned to train
Feeding time independent preferably 25~35d, more preferably 30d.The temperature of heretofore described dark culture is preferably 24~26
DEG C, more preferably 25 DEG C.There is white protrusion after dark culture 15d in newborn bulb in the present invention around bulb, is callus
It sprouts, occurs flaxen tissue after 20d around white protrusion, the callus at visible flaxen initial stage, subculture after 30d
It can get the callus of 1.5~2.5cm of diameter after culture.
Callus is preferably cut into agglomerate and is inoculated into bud inducement cultivation base by the present invention after obtaining callus
The differentiation for carrying out callus, obtains Multiple Buds.After the callus is cut into the agglomerate of 1~2cm of diameter by the present invention
Inoculation differentiation is carried out again.In the present invention, the callus differentiation is lured with bud inducement cultivation base with described in above-mentioned technical proposal
It leads within the scope of Multiple Buds bud inducement cultivation base and voluntarily selects；The condition and induction Multiple Buds step of callus differentiation culture
Condition in rapid is identical, and details are not described herein again.
In the present invention, the Multiple Buds growing way broken up using callus is healthy and strong, and large number of, the speed of growth is fast, because
This present invention improves short-tube lycoris breeding coefficient using the Multiple Buds that callus breaks up, and accelerates the speed of its breeding.
The Multiple Buds are inoculated into after obtaining Multiple Buds and carry out strong bud in strong bud training root media and take root by the present invention
Tissue-cultured seedling is obtained after culture.The strong bud culture of rootage used medium is the sucrose for including 35~45g/L in the present invention,
The agar of 5~10g/L, 6- benzyl aminoadenine, 0.1~0.3mg/L methyl α-naphthyl acetate and the 1.5~2.5mg/L of 3.5~4.5mg/L
Indolebutyric acid MS culture medium.It more preferably include the sucrose of 30g/L, the agar of 7.5g/L, the 6- benzyl amino gland of 3.0mg/L
The MS culture medium of purine, 0.2mg/L methyl α-naphthyl acetate and 2.0mg/L indolebutyric acid.
In the present invention, the condition of culture and above-mentioned strong bud culture for strengthening bud culture of rootage obtain new bulb bearing condition of culture
Identical, this is repeated no more.
The diameter for the short-tube lycoris tissue-cultured seedling seedling that the present invention obtains is preferably 3~5mm, more preferably 4mm；The root of the short-tube lycoris seedling
Preferably 3~5, more preferably 4, system.
The present invention preferably carries out hardening and transplanting to it after obtaining tissue-cultured seedling.In the present invention, the hardening is indoors
It carries out, short-tube lycoris seedling is placed under natural scattered light, place 2~3d and complete hardening.
Short-tube lycoris transplantation of seedlings of present invention after the completion of hardening, after carrying out the hardening.In the present invention, the transplanting is preferred
The culture medium for washing away short-tube lycoris seedling root, is transplanted in hole tray, and the specification of the hole tray is preferably 10 × 10.
In the present invention, the cultivation matrix after short-tube lycoris transplantation of seedlings preferably includes peat soil, perlite and rural area soil；The mud
The mass ratio of charcoal soil, perlite and rural area soil is preferably 1:1:1；Heretofore described cultivation matrix preferably passes through 800~
It is used after 1000 times of carbendazim disinfection treatments.
In the present invention, it after the completion of tissue culture transplantation of seedlings, carries out normal water and fertilizer management and obtains mature short-tube lycoris seedling.
It is described in detail below with reference to the method that embodiment quickly breeds culture to short-tube lycoris provided by the invention, still
They cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The Lycoris underground bulb for taking diameter 2.0cm, health peels off outer layer scale leaf, cuts off whole roots and bulb upper half
Part 1/2, retains plateau, and hairbrush cleans the soil on root surface, is cut into 4 pieces according to plateau size, every block size
1.0cm2, 84 detergent concussion washing 30min, flowing water rinses 30min, spare.
Above-mentioned material rinses 30s, 0.1%HgCl with 75% alcohol on superclean bench2Sterilize 40min, sterile water
It rinses 4 times, sterile blotting paper blots the moisture on surface.
By sterilized bulb block, directly it is inoculated on bud inducement cultivation base in the downward mode of basal disc, the group of culture medium
It is divided into: sucrose 30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.5mg/L NAA.After the culture of 28d, from explant
Budlet is grown between the scale of body.Squamous subculture is carried out after 40d.25 DEG C of cultivation temperature, 200 μm of olm of intensity of illumination-2·s-1, light
According to time 12h/d.
After adventitious bud squamous subculture 40d, it is transferred on bud proliferated culture medium and carries out Multiplying culture.Culture medium prescription is sucrose
30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.0mg/L NAA.25 DEG C of cultivation temperature, 250 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.25 DEG C of cultivation temperature, intensity of illumination 200 μm of olm-2s-1, light application time 16h/d.
Strong bud culture is carried out after adventitious buds proliferation culture 40d, will be inoculated into sucrose 40g/L+ agar 7.5g/L+MS from sprouting
On+2.0mg/L 6-BA+0.2mg/L NAA culture medium, every 30d subculture is primary, co-cultures 90d, and Multiple Buds grow up to diameter 3mm's
Newborn bulb.25 DEG C of cultivation temperature, 270 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.
The newborn bulb grown after strong bud culture is transferred in culture medium: sucrose 30g/L+ agar 7.5g/L+MS+
After 6.0mg/L 6-BA+1.5mg/L NAA2 week, occurs the protrusion of white around clove, for sprouting for callus, 20d
Occurs flaxen tissue around the protrusion of visible white, visible flaxen callus agglomerate after 30d carries out again at this time
Squamous subculture, until inducing the callus agglomerate that diameter is more than 1cm.Cultivation temperature is 25 DEG C, dark culture.
Callus is cut, the fritter of diameter 1.5cm is divided into, is transferred on above-mentioned bud inducement cultivation base, grows thickly
The induction of bud.Cultural method is identical as bud induction step.Every piece of callus differentiates 9 budlets.
Above-mentioned Multiple Buds are transferred on strong bud root media, strong bud culture of rootage is carried out.26 DEG C of cultivation temperature, illumination
100~300 μm of olm of intensity-2·s-1, light application time 16h.Strong bud root media is sucrose 30g/L+ agar 7.5g/L+MS
+ 2.0mg/L 6-BA+0.2mg/L NAA+2.0mg/L IBA can grow up to the tissue culture of diameter 4mm, root system 4 after cultivating 30d
Seedling.
Tissue-cultured seedling seedling is subjected to hardening and transplanting, hardening carry out indoors.Tissue culture bottle is uncapped, natural scattered light is placed in
Under, place 3d.After the completion of hardening, short-tube lycoris seedling is taken out with tweezers, the culture medium of root is washed away, is transplanted to 10 × 10 hole tray
In.Cultivation matrix is peat soil: perlite: rural area soil=1:1:1, and cultivation matrix must be disinfected through 800 times of carbendazim.Later
It carries out normal water and fertilizer management and obtains mature Lycoris.
The whole cycle that the present embodiment obtains short-tube lycoris seedling is about 5~6 months.
Embodiment 2
The Lycoris underground bulb for taking diameter 3.0cm, health pair peels off outer layer scale leaf, cuts off on whole roots and bulb
Half part 2/3, retains plateau, and hairbrush cleans the soil on root surface, is cut into 4 pieces according to plateau size, every block size
1.0cm2, 84 detergent concussion washing 28min, flowing water rinses 33min, spare.
Above-mentioned material rinses 33s, 0.1%HgCl with 75% alcohol on superclean bench2Sterilize 42min, sterile water
It rinses 5 times, sterile blotting paper blots the moisture on surface.
By sterilized bulb block, directly it is inoculated on bud inducement cultivation base in the downward mode of basal disc, the group of culture medium
It is divided into: sucrose 30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.5mg/L NAA.After the culture of 28d, from explant
Budlet is grown between the scale of body.Squamous subculture is carried out after 40d.26 DEG C of cultivation temperature, 220 μm of olm of intensity of illumination-2·s-1, light
According to time 12h/d.
After adventitious bud squamous subculture 40d, it is transferred on bud proliferated culture medium and carries out Multiplying culture.Culture medium prescription is sucrose
30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.0mg/L NAA.24 DEG C of cultivation temperature, 250 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.25 DEG C of cultivation temperature, intensity of illumination 200 μm of olm-2s-1, light application time 16h/d.
Strong bud culture is carried out after adventitious buds proliferation culture 40d, will be inoculated into sucrose 40g/L+ agar 7.5g/L+MS from sprouting
On+2.0mg/L 6-BA+0.2mg/L NAA culture medium, every 30d subculture is primary, co-cultures 60d, and Multiple Buds grow up to diameter 3mm's
Newborn bulb.25 DEG C of cultivation temperature, 270 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.
The newborn bulb grown after strong bud culture is transferred in culture medium: sucrose 30g/L+ agar 7.5g/L+MS+
After 6.0mg/L 6-BA+1.5mg/L NAA2 week, occurs the protrusion of white around clove, for sprouting for callus, 20d
Occurs flaxen tissue around the protrusion of visible white, visible flaxen callus agglomerate after 30d carries out again at this time
Squamous subculture, until inducing the callus agglomerate of diameter 5cm.Cultivation temperature is 25 DEG C, dark culture.
Callus is cut, the fritter of diameter 1.5cm is divided into, is transferred on above-mentioned bud inducement cultivation base, grows thickly
The induction of bud.Cultural method is identical as bud induction step.Every piece callus differentiation budding 11.
Above-mentioned Multiple Buds are transferred on strong bud root media, strong bud culture of rootage is carried out.25 DEG C of cultivation temperature, illumination
100~300 μm of olm of intensity-2·s-1, light application time 16h.Strong bud root media is sucrose 30g/L+ agar 7.5g/L+MS
+ 2.0mg/L IBA+0.2mg/L NAA+2.0mg/L 6-BA can grow up to the tissue culture of diameter 3mm, root system 4 after cultivating 30d
Seedling.
Tissue-cultured seedling is subjected to hardening and transplanting, hardening carry out indoors.Tissue culture bottle is uncapped, is placed under natural scattered light,
Place 2d.After the completion of hardening, short-tube lycoris seedling is taken out with tweezers, the culture medium of root is washed away, is transplanted in 10 × 10 hole tray.It plants
Training matrix is peat soil: perlite: rural area soil=1:1:1, and cultivation matrix must be disinfected through 900 times of carbendazim.It carries out later
Normal water and fertilizer management obtains mature Lycoris.
The whole cycle that the present embodiment obtains short-tube lycoris seedling is about 5~6 months.
Embodiment 3
The Lycoris underground bulb for taking diameter 4.0cm, health pair peels off outer layer scale leaf, cuts off on whole roots and bulb
Half part 3/5 retains plateau, and hairbrush cleans the soil on root surface, according to plateau size stripping and slicing, every block size
1.2cm2, 84 detergent concussion washing 33min, flowing water rinses 30min, spare.
Above-mentioned material rinses 28s, 0.1%HgCl with 75% alcohol on superclean bench2Sterilize 35min, sterile water
It rinses 3 times, sterile blotting paper blots the moisture on surface.
By sterilized bulb block, directly it is inoculated on bud inducement cultivation base in the downward mode of basal disc, the group of culture medium
It is divided into: sucrose 30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.5mg/L NAA.After the culture of 28d, from explant
Budlet is grown between the scale of body.Squamous subculture is carried out after 40d.26 DEG C of cultivation temperature, 220 μm of olm of intensity of illumination-2·s-1, light
According to time 12h/d.
After adventitious bud squamous subculture 40d, it is transferred on bud proliferated culture medium and carries out Multiplying culture.Culture medium prescription is sucrose
30g/L+ agar 7.5g/L+MS+3.0mg/L 6-BA+1.0mg/L NAA.24 DEG C of cultivation temperature, 150 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.25 DEG C of cultivation temperature, intensity of illumination 260 μm of olm-2s-1, light application time 16h/d.
Strong bud culture is carried out after adventitious buds proliferation culture 40d, will be inoculated into sucrose 40g/L+ agar 7.5g/L+MS from sprouting
On+2.0mg/L 6-BA+0.2mg/L NAA culture medium, every 30d subculture is primary, co-cultures 90d, and Multiple Buds grow up to diameter 2.5mm
Newborn bulb.25 DEG C of cultivation temperature, 300 μm of olm of intensity of illumination-2·s-1, light application time 16h/d.
The newborn bulb grown after strong bud culture is transferred in culture medium: sucrose 30g/L+ agar 7.5g/L+MS+
After 6.0mg/L 6-BA+1.5mg/L NAA2 week, occurs the protrusion of white around clove, for sprouting for callus, 20d
Occurs flaxen tissue around the protrusion of visible white, visible flaxen callus agglomerate after 30d carries out again at this time
Squamous subculture, until inducing the callus agglomerate of diameter 5cm.Cultivation temperature is 25 DEG C, dark culture.
Callus is cut, the fritter of diameter 1.2cm is divided into, is transferred on above-mentioned bud inducement cultivation base, grows thickly
The induction of bud, every piece of callus differentiate 10 buds.Cultural method is identical as bud induction step.
It is transferred to above-mentioned on strong bud root media from sprouting, carries out strong bud culture of rootage.25 DEG C of cultivation temperature, illumination
100~300 μm of olm of intensity-2·s-1, light application time 16h.Strong bud root media is sucrose 30g/L+ agar 7.5g/L+MS
+ 2.0mg/L IBA+0.2mg/L NAA+2.0mg/L 6-BA can grow up to the tissue culture of diameter 5mm, root system 3 after cultivating 30d
Seedling.
Tissue-cultured seedling is subjected to hardening and transplanting, hardening carry out indoors.Tissue culture bottle is uncapped, is placed under natural scattered light,
Place 2d.After the completion of hardening, seedling is taken out with tweezers, washes away the culture medium of root, is transplanted in 10 × 10 hole tray.Cultivation base
Matter is peat soil: perlite: rural area soil=1:1:1, and cultivation matrix must be disinfected through 1000 times of carbendazim.It carries out later normal
Water and fertilizer management obtain mature Lycoris.
The whole cycle that the present embodiment obtains short-tube lycoris seedling is about 5~6 months.
As seen from the above embodiment, the method that short-tube lycoris provided by the invention quickly breeds culture, by by short-tube lycoris underground squama
After stem obtains newborn bulb after sterilizing, inducing Multiple Buds, proliferation adventitious bud, strong bud culture, by the induction of newborn bulb for more
Injured tissue, then go out Multiple Buds via callus induction, the Multiple Buds quantity gone out via callus induction is more, every piece of callus group
It knits and differentiates 9~11 buds, the speed of growth is fast, short-tube lycoris breeding coefficient can be greatly improved, while accelerating the speed of breeding.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (2)
1. a kind of method for establishing short-tube lycoris regenerating system, comprising the following steps:
1) short-tube lycoris bulb is obtained into newborn bulb after sterilizing, inducing Multiple Buds, proliferation adventitious bud and strong bud culture；
2) the newborn bulb is linked into callus inducing medium and carries out dark culture to obtain diameter being 1.5~2.5cm
Callus；
3) callus is cut into after the agglomerate of 1~2cm of diameter to be inoculated into bud inducement cultivation base and carries out callus
15~20d of differentiation obtain Multiple Buds；
4) Multiple Buds are inoculated into strong bud root media and carry out strong bud culture of rootage, obtain short-tube lycoris seedling；
Strong bud culture described in step 1) includes 2~3 squamous subcultures, and the time of each squamous subculture independently is 25~
35d；The temperature of the strong bud culture is 24~26 DEG C, 100~300 μm of olm of intensity of illumination-2·s-1, light application time 14~
18h/d；
Strengthening bud culture described in step 1) to be with culture medium includes 35~45g/L sucrose, 5~10g/L agar, 1.5~2.5mg/
The MS culture medium of L6- benzyl aminoadenine and 0.1~0.3mg/L methyl α-naphthyl acetate；
Callus inducing medium described in step 2) is the sucrose for including 25~35g/L, the agar of 5~10g/L, 5.5
The 6- benzyl aminoadenine of~6.5mg/L and the MS culture medium of 1.0~2.0mg/L methyl α-naphthyl acetate；
Dark culture described in step 2) includes the primary dark culture of 25~35d and the subculture dark culture of 25~35d, the dark training
Feeding temperature is 24~26 DEG C；
Bud inducement cultivation base used in induction Multiple Buds described in step 1) includes the sucrose of 25~35g/L, the fine jade of 5~10g/L
Rouge, the 6- benzyl aminoadenine of 2.5~3.5mg/L and the MS culture medium of 1.0~2.0mg/L methyl α-naphthyl acetate；
The differentiation of callus described in step 3) only includes the primary Fiber differentiation of Multiple Buds；The time of the primary Fiber differentiation
For 35~45d, the time of subculture Fiber differentiation is 35~45d；24~26 DEG C of cultivation temperature, 100~300 μm of ol of intensity of illumination
m-2·s-1, 10~14h/d of light application time；
Proliferation adventitious bud described in step 1) is the sucrose for including 25~35g/L with bud proliferated culture medium, the agar of 5~10g/L,
The 6- benzyl aminoadenine of 2.5~3.5mg/L and the MS culture medium of 0.5~1.5mg/L methyl α-naphthyl acetate；The training of the proliferation adventitious bud
Supporting temperature is 24~26 DEG C, 100~300 μm of olm of intensity of illumination-2·s-1, 14~18h/d of light application time；The proliferation is indefinite
The incubation time of bud is 35~45d；
It is the sucrose for including 35~45g/L that bud root media is strengthened described in step 4), the agar of 5~10g/L, 1.5~
The MS culture medium of the indolebutyric acid of the 6- benzyl aminoadenine of 2.5mg/L, 0.1~0.3mg/L methyl α-naphthyl acetate and 1.5~2.5mg/L.
2. the method according to claim 1 for establishing short-tube lycoris regenerating system, which is characterized in that short-tube lycoris seedling described in step 4)
Diameter be 3~5mm, root system be 3~5.
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