CN106258973B - A kind of red autumnal leaves winged euonymus polyploid cell breeding technique - Google Patents
A kind of red autumnal leaves winged euonymus polyploid cell breeding technique Download PDFInfo
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- CN106258973B CN106258973B CN201610678643.0A CN201610678643A CN106258973B CN 106258973 B CN106258973 B CN 106258973B CN 201610678643 A CN201610678643 A CN 201610678643A CN 106258973 B CN106258973 B CN 106258973B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The present invention relates to a kind of red autumnal leaves winged euonymus polyploid cell breeding methods, belong to plant ploidy breeding field, it is related to the method that triploid is cultivated in red autumnal leaves winged euonymus somatic induction, be included in the endosperm evoked callus under the conditions of sterile culture with red autumnal leaves winged euonymus seed, free protoplast and sub-elects triploid protoplast, triploid protoplast Fiber differentiation is formed to the process and method of triploid；Further include the process and method of triploid red autumnal leaves winged euonymus plant terminal bud termination of diapause.The present invention sorts enrichment three times body cell from a large amount of diploid cells of red autumnal leaves winged euonymus diploid Wild plant seed endosperm, and cultivation forms triploid plantlet and promotees growth.
Description
Technical field
The invention belongs to plant ploidy breeding fields, are red autumnal leaves winged euonymus triploid cell engineering breeding method.It is related to wild
Red autumnal leaves winged euonymus seed endosperm cell induced synthesis callus；Free protoplast, dyeing and ploidy sort triploid plasm
Body；Protoplast regeneration induction plantlet；Processes and the methods such as plantlet terminal bud termination of diapause.
Background technology
Red autumnal leaves winged euonymus（Euonymus alatus‘Compactus’）It is Celastraceae Euonymus machaka, when branch children
It is green, hairless, there is the wing of suberin in old branch.It spends pale red or light yellow.Spring and summer dark green leaf, autumn become aubergine or
Red as fire color thus claim ' flame winged euonymus ', as gardens flowers and trees isolated planting, avenue system, mass-planting by scenic spot, campus, garden, lake, in lawn,
With high in ornamental value（Liu Yanxia, 2015）, the tall sense for filling the greening of collocation group and there is the Wanshan Mountain to be popular in row upon row of trees exhaustion.Red autumnal leaves are defended
Lance drought resisting, barren-resistant, -34.4 DEG C of cold-resistant are widely cultivated in the U.S..Since the seeds are adaptable, fertility, existence energy
Power is strong, and invasive species have been classified as in 21 states.It improves in Kang Nie University of Connecticuts New England Region invasive species research centerE. alatus' Compactus ' obtains triploid, intends substitution wild diploid or the group by seed growing, declared
United States Patent is protected.
Currently, the main method of artificial culture triploid is trained in vitro by tetraploid and diploid hybrid, albuminous cell
The modes such as hybridization after screening, the induction of 2n gametes after supporting（Su Jing, etc. 2012）.Wherein endosperm as polyploid cell line and staff control,
Contain natural three times body cell in it, culture regeneration induction plant is carried out to it, can therefrom filter out triploid.This side
Method has been used for the triploid Breeding of various plants, such as citrus（Wang great Yuan, Zhang Jinren, 1978）, Kiwi berry（Huang Zhenguang, etc.,
1982）, matrimony vine（Meter Jia Li, etc. 2011）, Morinda officinalis（Yao Yan, etc. 2015）Deng.Kang Nie University of Connecticuts New England Region enters
It is, by the induction of callus and its adventitious bud, to be obtained again by the way of endosperm cultured in vitro to invade species research center
Raw plant finally carries out it ploidy screening, obtains triploid（Thammina, et al.,2011）.In this experiment,
The inductivity that ripe endosperm induction generates callus is 14.0%；Callus induction regenerated adventitious bud（Plant）Inductivity
It is 13.4%；Wherein, triploid accounts for 28.6%, and the probability that ripe albuminous cell forms triploid is 0.34%；According to
Prematurity endosperm carries out cultured in vitro, and forming the probability of triploid, the experiment flow is simple and practicable for 0.42%., convenient for behaviour
Make, but heavy workload, callus and adventitious bud induction frequency are low, and most in endosperm tissue is diploid cell, regeneration plant
Middle triploid ratio is smaller.
By flow cytometer can efficient detection particulate samples institute band fluorescence intensity, thus DNA of plants content detection with
It is used widely in ploidy analysis.Although having no relevant report of the flow cytometer for the sorting of plant cell ploidy,
Someone utilizes the great-hearted protoplast of selected by flow cytometry apoptosis（Guo Yanping, etc. 2014）.Hoechst 33342 is a kind of work
Body DNA fluorescent dyes can emit blue-fluorescence under ultraviolet light with live body DNA specific bonds.In view of plant cell wall ultraviolet
Also the autofluorescence of blue is generated under light, thus the vegetable material for carrying out ploidy sorting preferably selects protoplast.And Hoechst
33342 dyeing have no effect on protoplast vigor, can cultivate to form callus（van der Valk, et al,1988）.Cause
This, carries out culture regeneration induction plant using selected by flow cytometry apoptosis three times body cell, improves ratio shared by triploid.
U.S.'s triploid red autumnal leaves winged euonymus Cultivating techniques are introduced in China's project verification in 2012.By introduction, digestion, absorption process,
Former technology is improved, optimized and is innovated be formed in breeding technique, formula and efficiency etc. facilitate improved and promoted it is new
Breeding technique system.Plantlet is directly induced therefrom to screen the breeding journey of triploid based on polyploid Callus From The Endosperms
Sequence, this research with polyploid Callus From The Endosperms dissociate protoplast, using selected by flow cytometry apoptosis triploid protoplast into
Row culture, accurate induced triploid plant regeneration, triploid obtain efficiency and are greatly promoted on the basis of 0.42%.Plantlet
Terminal bud termination of diapause is had developed plant growth regulator on the basis of with low-temperature treatment and integrates low-temperature treatment and container seedling technology, not
Releasing efficiency of sleeping is increased to 90% or more, and is container seedling, and transplanting survival rate is guaranteed.
Invention content
Main points one of the present invention provide a kind of red autumnal leaves winged euonymus Callus From The Endosperms induction mode.Its callus induction is trained
It includes minimal medium to support based component：MS inorganic salts, vitamin B1 0.1-1.0 mg/L, vitamin B6 0.1-0.5 mg/L,
Niacin 0.1-0.5 mg/L, glycine 1.0-2.0 mg/L, caseinhydrolysate 0.3-0.6 g/L, morpholino b acid 0.5-1.0
g/L；Plant growth regulator：6-BA 1.0-3.0 mg/L、NAA0.5-2.0 mg/L；Sucrose 20-30 g/L；Agar 6-8 g/
L.Embryo is removed embryo, endosperm is forwarded on callus inducing medium with indoor co-incubation under endosperm dark condition after 4 weeks
Continue dark culturing.The adjoint Callus From The Endosperms inductivity of the embryo Callus From The Endosperms inductivity adjoint far above no embryo, can
Up to 80%.
Main points two of the present invention provide the enzyme solution of the free protoplast of red autumnal leaves winged euonymus callus.Its enzymolysis liquid at
Subpackage cleaning solution containing protoplast：KH2P04 20-27.2 mg/L、KNO3 90-101 mg/L、MgSO4 ▪7H2O 180-246
mg/L、KI 0.10-0.16 mg/L、CuSO4 0.010-0.025 mg/L、CaCl2▪2H2O 1000-1480 mg/L, mannitol
0.5-0.7 M/L, morpholino b acid 5-25 mM/L；Cytohydrolist：Cellulase 1.0-2.0%, macerozyme 0.5-
1.0%, pectase 0.5-1.0%；1 M/L aqueous solutions of HCl 1 M/L and NaOH are 5.2-5.8 for adjusting pH value.It is digested
Condition is to be digested 4-8 hours for 25-29 DEG C under dark condition, and during which gentle inversion mixing is primary per hour.
Main points three of the present invention, the method for providing a kind of red autumnal leaves winged euonymus protoplast DNA dyeing and ploidy sorting.Endosperm is cured
DNA dyeing is carried out after injured tissue and Callus of Leaf respectively free protoplast, dyeing liquor is concentration 5-10 μ g/ml's
Hoechst 33342, dyeing time are 3-5 min.Plasm ploidy sorts, and protoplast is cleaned with protoplast cleaning solution
Afterwards, it compares by diploid of the protoplast of Callus of Leaf, is aseptically cured using selected by flow cytometry apoptosis endosperm
Three times body cell in injured tissue protoplast, makes it be enriched with, to improve triploid during adventitious bud inducing thereafter
The regeneration probability of plant.Note：Hoechst 33342 is live body DNA coloring agents, can be specifically bound in DNA double chain, after dyeing
Protoplast vigor is not influenced.
Main points four of the present invention provide a kind of method of red autumnal leaves winged euonymus protoplast regenerated plant, including three continuous ranks
Section.
Stage 1, Protoplast cuhnre form callus.Aseptically by the protoplast of sorting and containing low melting point
Agarose（Solidification point is 26 ~ 30 DEG C）Protoplast culture medium mixing after, be poured into 5cm culture dishes, after the quartering, be transferred to
In 9cm culture bottles containing protoplast culture medium.Protoplast culture medium ingredient includes basic described in foregoing invention main points one
Culture medium, 0.5 M of mannitol, 6-BA 0.5-2.0 mg/L, NAA 0.5-1.0 mg/L.The lower 24 DEG C of concussions training of dark condition
It supports；After forming callus lines, it is forwarded on callus inducing medium.
Stage 2, callus induction regenerated adventitious bud.Callus is seeded on adventitious bud induction culture base, 24
DEG C, 36 μm of olm of intensity of illumination-2·s-1, cultivate 8 weeks under the conditions of 16 h/d of light application time, induced synthesis adventitious bud.Adventitious bud
Fiber differentiation based component includes the minimal medium described in foregoing invention main points one；Plant growth regulator 6-BA 2.0-6.0
mg/L、IBA 0.1-0.2 mg/L；Agar 0.6-0.8 g/L.
In the stage 3, adventitious bud inducing takes root for forming plantlet.Adventitious bud is with two-step method root induction.The first step, adventitious bud exist
It is cultivated 2 weeks on root media I.Root media I is WPM+ ammonium nitrate 0.4-1.0 g/L+ sucrose 20-30 g/L+IBA
1.0-3.0 mg/L, pH 5.8.Second step, the adventitious bud by first step culture are forwarded on root media II and cultivate 4
Week forms root system.II ingredient of root media is WPM+ ammonium nitrate 0.4-1.0 g/L+ sucrose 20-30 g/L+ activated carbons
2.0-4.0 g/L, pH 5.8）.
Main points five of the present invention provide a kind of releasing side for the plantlet terminal bud suspend mode that red autumnal leaves winged euonymus polyploid cell obtains
Method.The plantlet planting and inoculating of above-mentioned red autumnal leaves winged euonymus polyploid cell Fiber differentiation is containing GA32.0-4.0 mg/L and 6-BA
On the above-mentioned root media II of 1.0-2.0 mg/L, it is placed in dark treatment 30-45d at 3-8 DEG C；25 DEG C are moved to, 36 μ of illumination
mol·m-2·s-1, 15-30 d, 90% or more terminal bud termination of diapause rate are cultivated under the conditions of 16 h/d.
Description of the drawings
Fig. 1 is the callus of endosperm induction；
Fig. 2 is that callus dissociates the protoplast；
Fig. 3 is the adventitious bud that callus regeneration is formed；
Fig. 4 is flow cytometry Ploidy detection peak figure, is that triploid is glimmering at 300 wherein being diploid fluorescence intensity at 200
Luminous intensity.
Fig. 5 is the tissue-cultured seedling of taking root after bud dormancy releases.
Specific implementation mode
Embodiment one
Red autumnal leaves winged euonymus endosperm evoked callus
Red autumnal leaves winged euonymus（Euonymus alatus‘Compactus’）, mature seed, false kind of removal are acquired in late November
Skin, clear water rinse 30 min, are used in combination 70% ethyl alcohol to impregnate 60s, are placed in the thimerosal containing 1% Tween-80 and sodium dichloro cyanurate
（Effective chlorine density is 1.0%）Middle concussion handles 20 min.Aseptic water washing seed 5 times, every time 3 min.The seed that will have been sterilized
It is seeded in the test tube of the half solidification culture medium containing agar.Aseptic seed is screened after 7 days, aseptically with sterilizing（121
DEG C, 0.11 MPa, 20 min, similarly hereinafter）Scalpel and tweezers removal kind of a skin, embryo and endosperm are seeded in callus together and lured
It leads on culture medium, dark culturing at 24 DEG C, subculture is primary every two weeks.4th week removal embryo, endosperm continue dark culturing, unite within the 8th week
Callus induction rate is counted, callus induction rate is up to 80%.
Embodiment two
Callus dissociates protoplast
Red autumnal leaves winged euonymus（Euonymus alatus‘Compactus’）Callus From The Endosperms are chosen flaxen endosperm and are cured
Injured tissue, callus for 24 hours, is aseptically sliced with sterilizing tweezers and scalpel, in electronics day by dark treatment at 4 DEG C
Callus 0.5g is weighed on flat.Callus is placed in the 50 ml centrifuge tubes containing 10 ml enzymolysis liquids, is sealed with sealed membrane,
4 h of dark processing at 27 DEG C, gently reverse mixing is primary per hour.It after enzymatic treatment, is filtered with 300 mesh cell sieves, filtrate is set
Enter in new centrifuge tube and sealed with sealed membrane, centrifuged with refrigerated centrifuge, centrifugal force is 100 g, centrifugation time 3min.It inhales
Supernatant is abandoned, protoplast cleaning solution is added and is resuspended, suction is abandoned-is resuspended in triplicate.Suspension is settled to 500 μ l, is taken a little outstanding
Supernatant liquid uses Trypan Blue.Stin of thickness is 0.04%, and dyeing time is 5 min.Hanging drop after dyeing adds to cell
On tally, protoplasm free number and survival rate are observed and counted under the microscope.1 g callus can dissociate 2 ×
106A protoplast, survival rate 91%.
Embodiment three
Protoplast ploidy sorts and plant regeneration
Red autumnal leaves winged euonymus（Euonymus alatus‘Compactus’）Protoplast with 33342 dyeing liquors of Hoechst into
Row dyeing.Stin of thickness is 5 μ g/ml, and dyeing time is 5 min.100 g centrifuge 3 min, and suction is abandoned dyeing liquor, is used in combination primary
Plastid culture medium is resuspended, in triplicate.Protoplast is collected, fluidic cell sorting is aseptically carried out, is made according to instrument
Triploid protoplast is sorted with specification.
Gained triploid protoplast will be sorted and contain low melting-point agarose（Solidification point is 26 ~ 30 DEG C）, temperature 30-40
DEG C protoplast culture medium mixing, pour into the culture dish of 5 cm of diameter.After to be solidified, four are cut into sterilized scalpel
Decile is transferred in the culture bottle of 9 cm of diameter of the liquid containing Protoplast cuhnre, soft shake culture.Callus lines to be formed
Afterwards, it is seeded on callus inducing medium, in 36 μm of olm of intensity of illumination-2·s-1, 16 h/ of photoperiod, 8 h
（Light dark）Green fine and close callus is seeded on adventitious bud induction culture base and cultivates by lower culture 4 weeks, evoking adventive bud
Occur and grows.
Diploid tissue culture seedling leaf and each 50mg of regenerated adventitious bud blade are taken, is placed in the 5ml plastic culture dish of precooling, and
It is separately added into 500 μ l of dissociation dyeing liquor.It includes 50 μ g/ml of PI, sodium citrate 0.1% and Triton-X to dissociate dyeing liquor
100 0.3 μl/ml.In in 1min with double-edged razor blade fly-cutting blade at fragment, be used in combination 200 mesh filter screens to filter, filtrate is placed in
In flow cytometer loading pipe.After hatching 5min on ice, upper machine testing is control with diploid cell core, and detection regeneration is indefinite
Bud ploidy.Ploidy detection is carried out to 7 regenerated adventitious buds, wherein 6 sample ploidies are triploid, ratio is shared by triploid
85.7%。
Example IV
Red autumnal leaves winged euonymus adventitious bud rooting and bud dormancy release
The adventitious bud for choosing the red autumnal leaves winged euonymus triploid protoplast regeneration of sturdy plant height 2-3cm, is inoculated in training of taking root
It supports on base I, induces root restriction.It is forwarded to after 2 weeks on root media II, carries out root growth.The tissue culture of taking root of red autumnal leaves winged euonymus
Generally there is bud dormancy phenomenon in seedling, and transplanting hardening survival rate is low.Rooted seedling is inoculated in containing 3.0 mg/L GA3 and 1.0 mg/L
On the root media II of 6-BA, it is placed at 5 DEG C after dark treatment 30d, moves to illumination cultivation at 25 DEG C.It is reachable that bud dormancy abolishes rate
90% or more.
Claims (4)
1. a kind of red autumnal leaves winged euonymus polyploid cell breeding method, it is characterised in that：
A) the adjoint endosperm in-vitro inducing callus of wild red autumnal leaves winged euonymus seed embryo；
B) protoplasm free and sorting, and cultivate and form plantlet；
C) low temperature is handled with hormons, releases plantlet terminal bud suspend mode,
Wherein a) it is characterized in that in the method：Seed removal kind skin, red autumnal leaves winged euonymus endosperm callus is seeded in by embryo and endosperm
It is cultivated 4 weeks on tissue inducing culture, embryo stripping, endosperm continues to cultivate evoked callus；
The wherein described callus inducing medium includes minimal medium：MS inorganic salts, vitamin B1 0.1-1.0mg/L, dimension
Raw element B6 0.1-0.5mg/L, niacin 0.1-0.5mg/L, glycine 1.0-2.0mg/L, caseinhydrolysate 0.3-0.6g/L,
Quinoline ethanesulfonic acid 0.5-1.0g/L, sucrose 20-30g/L；Plant growth regulator：6-BA 1.0-3.0mg/L and NAA 0.5-
2.0mg/L；Agar 6-8g/L；
It b) is characterized in that in the method：Dark processing is after 24 hours at 4 DEG C for Callus From The Endosperms, for the plasm that dissociates
Body, endosperm slice are placed in red autumnal leaves winged euonymus callus enzymolysis liquid 4-8 hours；After free red autumnal leaves winged euonymus protoplast, carry out
The dyeing of Hoechst 33342, flow cytometry screening and low melting point agarose embedding culture, are trained in red autumnal leaves winged euonymus adventitious bud inducing
It supports after forming adventitious bud on base, is forwarded on red autumnal leaves winged euonymus root induction culture medium and forms plantlet；
The wherein described enzymolysis liquid includes：
Protoplast cleaning solution：KH2P04 20-27.2mg/L、KNO3 90-101mg/L、MgSO4▪ 7H2O 180-246mg/L、
KI 0.10-0.16mg/L、CuSO4 0.010-0.025mg/L、CaCl2▪ 2H2O 1000-1480mg/L, mannitol 0.5-
0.7M/L, morpholino b acid 5-25mM/L；
Cytohydrolist：Cellulase 1.0-2.0%, macerozyme 0.5-1.0%, pectase 0.5-1.0%；And
HCl 1M/L and NaOH 1M/L aqueous solutions are 5.2-5.8 for adjusting pH value；
The wherein described adventitious bud induction culture base includes the minimal medium；Plant growth regulator 6-BA 2.0-6.0mg/
L、IBA 0.1-0.2mg/L；Agar 0.6-0.8g/L；
The wherein described root induction culture medium includes：Root media I, it includes 1/2WPM+ ammonium nitrate 0.4-1.0g/L+ sugarcanes
Sugared 20-30g/L+IBA 1.0-3.0mg/L；With root media II, it includes 1/2WPM+ ammonium nitrate 0.4-1.0g/L+ sucrose
20-30g/L+ activated carbons 2.0-4.0g/L；
It c) is characterized in that in the method：Tissue cultures plantlet is containing plant growth regulator GA3With the training of taking root of 6-BA
It supports on base II, the light culture 30-45d at 3-8 DEG C moves to 25 DEG C, 36 μm of olm of illumination-2·s-1, solution is cultivated under the conditions of 16h/d
Except terminal bud suspend mode.
2. a kind of red autumnal leaves winged euonymus polyploid cell breeding method described in accordance with the claim 1, it is characterised in that：Protoplast
DNA is dyed, and dyeing liquor is the Hoechst 33342, dyeing time 3-5min of concentration 5-10 μ g/ml；Plasm ploidy sorts,
The protoplast of Protoplast cuhnre liquid cleaning dyeing aseptically sub-elects the primary of triploid with flow cytometer
Plastid.
3. according to a kind of red autumnal leaves winged euonymus polyploid cell breeding method as claimed in claim 1 or 2, it is characterised in that：Including three
A successive stages：(1) Protoplast cuhnre, aseptically by the protoplast of sorting with containing the primary of low melting-point agarose
It after plastid culture medium mixing, is poured into 5cm culture dishes, the quartering after solidification, is transferred to the 9cm cultures containing protoplast culture medium
In bottle, the lower 24 DEG C of shake cultures of dark condition 2 weeks；After callus to be formed, it is forwarded to callus inducing medium
On；(2) callus is seeded on adventitious bud induction culture base by callus induction regenerated adventitious bud, and at 24 DEG C, illumination is strong
Spend 36 μm of olm-2·s-1, cultivate 8 weeks under the conditions of light application time 16h/d, induced synthesis adventitious bud；(3) adventitious bud inducing is given birth to
Root, adventitious bud are cultivated 2 weeks on root media I, induce root restriction；It is forwarded on root media II, cultivates 4 weeks shapes
At root system.
4. according to a kind of red autumnal leaves winged euonymus polyploid cell breeding method as claimed in claim 1 or 2, it is characterised in that：Terminal bud is stopped
The release method of dormancy is that the tissue cultures plantlet of acquisition is planted in containing GA31.0-3.0mg/L and 6-BA 0.5-
On the root media II of 2.0mg/L, it is placed in dark treatment 30-45d at 3-8 DEG C, moves to 25 DEG C, 36 μm of olm of illumination-2·s-1,
It is cultivated under the conditions of 16h/d, 90% or more terminal bud termination of diapause rate.
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