CN106258973A - 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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- CN106258973A CN106258973A CN201610678643.0A CN201610678643A CN106258973A CN 106258973 A CN106258973 A CN 106258973A CN 201610678643 A CN201610678643 A CN 201610678643A CN 106258973 A CN106258973 A CN 106258973A
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- protoplast
- autumnal leaves
- red autumnal
- triploid
- winged euonymus
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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 technique.The invention belongs to plant ploidy breeding field, relate to the method that triploid is cultivated in red autumnal leaves winged euonymus somatic induction, be included under the conditions of aseptic culture with the endosperm callus induction of red autumnal leaves winged euonymus seed, free protoplast and sub-elect triploid protoplast, triploid protoplast inducing culture is formed process and the method for triploid；Also include process and the method for triploid red autumnal leaves winged euonymus plant terminal bud termination of diapause.The present invention sorts enrichment triploid cell from a large amount of diploid cells of red autumnal leaves winged euonymus diploid Wild plant seed endosperm, cultivates and forms triploid plantlet and promote its growth.
Description
Technical field
The invention belongs to plant ploidy breeding field, for red autumnal leaves winged euonymus triploid cell engineering breeding method.Relate to wild
Red autumnal leaves winged euonymus seed endosperm cell induction forms callus；Free protoplast, dyeing and ploidy sorting triploid protoplasm
Body；Protoplast regeneration induction plantlet；Process 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,
During branch children green, without hair, old branch has corky wing.Spend pale red or light yellow.Spring and summer, dark green leaf, became autumn
Aubergine or red as fire color thus claim ' flame winged euonymus ', as gardens HUAMU isolated planting, avenue system, mass-planting by scenic spot, campus, garden, lake,
In lawn, there is high in ornamental value (Liu Yanxia, 2015), tall fill the greening of collocation colony have the Wanshan Mountain be popular in row upon row of trees exhaustion it
Sense.Red autumnal leaves winged euonymus drought resisting, barren-resistant, cold-resistant-34.4 DEG C, widely cultivate in the U.S..Due to this seeds strong adaptability, breed energy
Power, survival ability are strong, have been classified as invasive species in 21 states.Invasive species research center, Kang Nie University of Connecticut New England Region changes
GoodE.alatus' Compactus ', it is thus achieved that triploid, intends replacing wild diploid or by seed growing
Colony, has declared United States Patent protection.
At present, the triploid main method of artificial culture is to be trained in vitro with diploid hybrid, albuminous cell by tetraploid
Screen after Yanging, the mode such as hybridization after the induction of 2n gamete (Su Jing, etc., 2012).Wherein endosperm is as polyploid cell line and staff control,
It is contained within natural triploid cell, carries out cultivating regeneration induction plant to it, can therefrom filter out triploid.This side
Method has been used for the triploid Breeding of various plants, as mandarin orange (Wang great Yuan, Zhang Jinren, 1978), Fructus actinidiae chinensis (Huang Zhenguang, etc.,
1982), Fructus Lycii (meter Jia Li, etc., 2011), Radix Morindae Officinalis (Yao Yan, etc., 2015) etc..Kang Nie University of Connecticut New England Region enters
Invading species research center is i.e. the mode using endosperm isolated culture, through callus and the induction of adventitious bud thereof, it is thus achieved that again
Raw plant, finally carries out ploidy screening to it, it is thus achieved that triploid (Thammina, et al., 2011).In this experiment,
The inductivity that ripe endosperm induction produces callus is 14.0%；The inductivity of callus induction regenerated adventitious bud (plant)
It is 13.4%；Wherein, triploid accounts for 28.6%, and it is 0.34% that mature embryo laticiferous cell forms the probability of triploid；According to
Immaturity endosperm carries out isolated culture, and the probability forming triploid is that this experiment flow of 0.42%. is simple, it is simple to behaviour
Making, but workload is big, callus and adventitious bud induction frequency are low, and in endosperm tissue, majority is diploid cell, regeneration plant
Middle triploid ratio is less.
Due to flow cytometer can efficient detection particulate samples institute band fluorescence intensity, thus DNA of plants content detection with
Ploidy analysis is used widely.Although have no flow cytometer for the relevant report that plant cell ploidy sorts, but
Someone utilizes the great-hearted protoplast of selected by flow cytometry apoptosis (Guo Yanping, etc., 2014).Hoechst 33342 is a kind of living
Body DNA fluorescent dye, can launch blue-fluorescence under ultraviolet light with live body DNA specific bond.In view of plant cell wall is in ultraviolet
Also produce the autofluorescence of blueness under light, thus the vegetable material carrying out ploidy sorting preferably selects protoplast.And Hoechst
33342 dyeing have no effect on protoplast vigor, can cultivate formation callus (van der Valk, et al, 1988).Cause
This, available selected by flow cytometry apoptosis triploid cell carries out cultivating regeneration induction plant, improves ratio shared by triploid.
U.S.'s triploid red autumnal leaves winged euonymus Cultivating techniques is introduced in China's project verification in 2012.By introduction, digestion, absorption process,
Former technology is improved, optimizes and innovates and is formed at that breeding technique, formula and efficiency etc. are convenient obtains improve and promote 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 dissociates protoplast with polyploid Callus From The Endosperms, utilizes selected by flow cytometry apoptosis triploid protoplast to enter
Row is cultivated, accurate induced triploid plant regeneration, and triploid obtains efficiency and is greatly promoted on the basis of 0.42%.Plantlet
Terminal bud termination of diapause have developed plant growth regulator on the basis of with K cryogenic treatment and integrates K cryogenic treatment and container seedling technology, stops
Releasing efficiency of sleeping brings up to more than 90%, and is container seedling, and transplanting survival rate is guaranteed.
Summary of the invention
Main points one of the present invention, it is provided that a kind of red autumnal leaves winged euonymus Callus From The Endosperms induction mode.Its callus induction is trained
Foster based component comprises minimal medium: MS inorganic salt, vitamin B1 0.1-1.0 mg/L, vitamin B6 0.1-0.5 mg/L,
Nicotinic acid 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.Indoor co-cultivation is after 4 weeks with under endosperm dark condition for embryo, and by embryo stripping, endosperm is forwarded on callus inducing medium
Continue dark culturing.The adjoint Callus From The Endosperms inductivity of embryo, can far above the Callus From The Endosperms inductivity adjoint without embryo
Reach 80%.
Main points two of the present invention, it is provided that red autumnal leaves winged euonymus callus dissociates the enzyme solution of protoplast.Its enzymolysis solution becomes
Subpackage cleaning mixture Han 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%；It is 5.2-5.8 that HCl 1 M/L and NaOH 1 M/L aqueous solution is used for regulating pH value.Its enzymolysis
Condition is 25-29 DEG C of enzymolysis 4-8 hour under dark condition, and period gentle inversion per hour mixes once.
Main points three of the present invention, it is provided that a kind of red autumnal leaves winged euonymus protoplast DNA dyeing and the method for ploidy sorting.Endosperm is more
Injured tissue and Callus of Leaf dissociate respectively and carry out DNA dyeing after protoplast, and dyeing liquor is concentration 5-10 μ g/ml
Hoechst 33342, dyeing time is 3-5 min.Protoplasm ploidy sorts, and protoplast protoplast cleaning mixture cleans
After, compare for diploid with the protoplast of Callus of Leaf, aseptically utilize selected by flow cytometry apoptosis endosperm more
Triploid cell in injured tissue protoplast so that it is be enriched with, thus improve triploid during Induce aerosor thereafter
The regeneration probability of plant.Note: Hoechst 33342 is live body DNA stain, can be specific binding on DNA double chain, after dyeing
Do not affect protoplast vigor.
Main points four of the present invention, it is provided that a kind of method of red autumnal leaves winged euonymus protoplast regenerated plant, including three continuous rank
Section.
In the stage 1, Protoplast cuhnre forms callus.Aseptically by the protoplast of sorting and containing low melting point
After the protoplast culture medium mixing of agarose (freezing point is 26 ~ 30 DEG C), it is poured in 5cm culture dish, after the quartering, proceeds to
In 9cm culture bottle containing protoplast culture medium.It is basic that protoplast culture medium composition includes described in foregoing invention main points one
Culture medium, mannitol 0.5 M, 6-BA 0.5-2.0 mg/L, NAA 0.5-1.0 mg/L.The lower 24 DEG C of concussion trainings of dark condition
Support；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, intensity of illumination 36 μm ol m-2·s-1, cultivate 8 weeks under the conditions of light application time 16 h/d, induced synthesis adventitious bud.Adventitious bud
Inducing culture 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, Induce aerosor is taken root formation plantlet.Adventitious bud is with two-step method root induction.The first step, adventitious bud exists
Cultivate 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 are 5.8.Second step, the adventitious bud cultivated through the first step is forwarded on root media II cultivate 4
Week forms root system.Root media II composition is WPM+ ammonium nitrate 0.4-1.0 g/L+ sucrose 20-30 g/L+ activated carbon
2.0-4.0 g/L, pH are 5.8).
Main points five of the present invention, it is provided that the releasing side of the plantlet terminal bud dormancy that a kind of red autumnal leaves winged euonymus polyploid cell obtains
Method.The plantlet planting and inoculating of above-mentioned red autumnal leaves winged euonymus polyploid cell inducing culture 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, dark treatment 30-45d at being placed in 3-8 DEG C；Move to 25 DEG C, illumination 36 μ
mol·m-2·s-1, under the conditions of 16 h/d, cultivate 15-30 d, terminal bud termination of diapause rate more than 90%.
Accompanying drawing explanation
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 wherein diploid fluorescence intensity at 200, is that triploid fluorescence is strong at 300
Degree.
Fig. 5 is the tissue cultured seedling of taking root after bud dormancy releases.
Detailed description of the invention
Embodiment one
Red autumnal leaves winged euonymus endosperm callus induction
Red autumnal leaves winged euonymus (Euonymus alatus' Compactus '), gather mature seed in late November, go
Except aril, clear water rinses 30 min, and by 70% soak with ethanol 60s, is placed in containing 1% tween 80 and sodium dichloro cyanurate
In disinfectant solution (effective chlorine density is 1.0%), concussion processes 20 min.Aseptic water washing seed 5 times, each 3 min.To sterilize
Seed be seeded in the test tube of the half solidification culture medium containing agar.Aseptic seed is screened, aseptically with sterilizing after 7 days
(121 DEG C, 0.11 MPa, 20 min, lower with) dissecting knife and tweezers remove seed coat, embryo and endosperm are together seeded in wound healing group
Knitting on inducing culture, dark culturing at 24 DEG C, subculture is once every two weeks.4th week removal embryo, endosperm continuation dark culturing, the 8th
Zhou Tongji callus induction rate, callus induction rate is up to 80%.
Embodiment two
Callus dissociates protoplast
Red autumnal leaves winged euonymus (Euonymus alatus' Compactus ') Callus From The Endosperms, choose flaxen embryo
Breast callus, dark treatment 24h at 4 DEG C, aseptically cuts into slices callus, at electricity with sterilizing tweezers and dissecting knife
Callus 0.5g is weighed on sub-balance.Callus is placed in the 50 ml centrifuge tubes containing 10 ml enzymolysis solutions, with sealed membrane
Sealing, dark processing 4 h at 27 DEG C, the most reverse mixing is once.After ferment treatment, it is sieved through filter with 300 mesh cells,
Sealing in filtrate inserts new centrifuge tube and with sealed membrane, be centrifuged with refrigerated centrifuge, centrifugal force is 100 g, and centrifugation time is
3min.Supernatant is abandoned in suction, and it is resuspended to add protoplast cleaning mixture, inhale abandon-resuspended in triplicate.Suspension is settled to 500 μ l, takes
A little suspension, uses Trypan Blue.Stin of thickness is 0.04%, and dyeing time is 5 min.Suspension dropping after dyeing
To cell counting count board, examine under a microscope and add up protoplasm free number and survival rate.1 g callus can dissociate
Go out 2 × 106Individual protoplast, survival rate is 91%.
Embodiment three
The sorting of protoplast ploidy and plant regeneration
Red autumnal leaves winged euonymus (Euonymus alatus' Compactus ') protoplast is with Hoechst 33342 dyeing liquor
Dye.Stin of thickness is 5 μ g/ml, and dyeing time is 5 min.100 g are centrifuged 3 min, inhale and abandon dyeing liquor, and with former
Raw plastid culture medium is resuspended, in triplicate.Collect protoplast, aseptically carry out fluidic cell sorting, according to instrument
Operation instructions sorting triploid protoplast.
Gained triploid protoplast will be sorted and containing low melting-point agarose (freezing point is 26 ~ 30 DEG C), temperature will be 30-40
DEG C protoplast culture medium mixing, pour in the culture dish of diameter 5 cm.After to be solidified, it is cut into four with sterilized dissecting knife
Decile, proceeds to, in the culture bottle of diameter 9 cm containing Protoplast cuhnre liquid, softly shake cultivation.Callus lines to be formed
After, it is seeded on callus inducing medium, at intensity of illumination 36 μm ol m-2·s-1, photoperiod 16 h/ 8 h
Cultivate 4 weeks under (light dark), green fine and close callus is seeded on adventitious bud induction culture base cultivation, evoking adventive bud
Occur and growth.
Take diploid tissue cultured seedling blade and each 50mg of regenerated adventitious bud blade, be placed in the 5ml plastic culture dish of pre-cooling, and
It is separately added into the dyeing liquor 500 μ l that dissociates.The dyeing liquor that dissociates comprises PI 50 μ g/ml, sodium citrate 0.1% and Triton-X
100 0.3 μl/ml.Becoming fragment with double-edged razor blade fly-cutting blade in 1min, and filter with 200 mesh filter screens, filtrate is placed in
In flow cytometer loading pipe.On ice after hatching 5min, upper machine testing, with diploid cell core for comparison, detection regeneration is indefinite
Bud ploidy.7 regenerated adventitious buds carry out Ploidy detection, and wherein 6 sample ploidies are triploid, and ratio shared by triploid is
85.7%。
Embodiment four
Red autumnal leaves winged euonymus adventitious bud rooting and bud dormancy release
Choose the adventitious bud of the red autumnal leaves winged euonymus triploid protoplast regeneration of sturdy plant height 2-3cm, be inoculated in root media I
On, induce root restriction.It is forwarded to after 2 weeks on root media II, carries out root growth.The tissue cultured seedling of taking root of red autumnal leaves winged euonymus is universal
Bud dormancy phenomenon occurs, transplants seedling exercising survival rate low.Seedling of taking root is inoculated in containing 3.0 mg/L GA3's and 1.0 mg/L 6-BA
On root media II, it is placed at 5 DEG C after dark treatment 30d, moves to illumination cultivation at 25 DEG C.Bud dormancy abolish rate up to 90% with
On.
Claims (7)
1. a red autumnal leaves winged euonymus polyploid cell breeding technique, it is characterised in that: a) wild red autumnal leaves winged euonymus seed endosperm lures in vitro
Lead callus；B) Protoplast cuhnre forms plantlet；C) plantlet terminal bud termination of diapause.
The most in accordance with the method for claim 1, it is characterised in that a kind of callus inducing medium, basic cultivation is comprised
Base: MS inorganic salt, vitamin B1 0.1-1.0 mg/L, vitamin B6 0.1-0.5 mg/L, nicotinic acid 0.1-0.5 mg/L, sweet
Propylhomoserin 1.0-2.0 mg/L, caseinhydrolysate 0.3-0.6 g/L, morpholino b acid 0.5-1.0 g/L, sucrose 20-30 g/
L；Plant growth regulator: 6-BA 1.0-3.0 mg/L and NAA 0.5-2.0 mg/L；Agar 6-8 g/L.
The most in accordance with the method for claim 1, it is characterised in that a kind of red autumnal leaves winged euonymus callus dissociates the enzyme of protoplast
Solution method, enzymolysis solution composition comprises protoplast cleaning mixture: 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%；HCl 1 M/L and NaOH 1 M/L aqueous solution is used for regulating pH value
For 5.2-5.8.
4. according to the method described in claim 1 and 3, it is characterised in that the sorting side of a kind of red autumnal leaves winged euonymus protoplast ploidy
Method, protoplast DNA dyes, and dyeing liquor is the Hoechst 33342 of concentration 5-10 μ g/ml, and dyeing time is 3-5 min；
Protoplasm ploidy sorts, and Protoplast cuhnre liquid cleans the protoplast of dyeing, with flow cytometer, aseptically sorts
Go out triploid protoplast.
5. according to the method described in claim 1-4, it is characterised in that a kind of culture medium inducing protoplast to form plantlet,
Including (1) adventitious bud induction culture base, its composition is the minimal medium described in the claims 2；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；(2) root induction culture medium, i.e. takes root
Culture medium I comprises WPM+ ammonium nitrate 0.4-1.0 g/L+ sucrose 20-30 g/L+IBA 1.0-3.0 mg/L；Root media
II composition is WPM+ ammonium nitrate 0.4-1.0 g/L+ sucrose 20-30 g/L+ activated carbon 2.0-4.0 g/L.
6. according to the method described in claim 1 ~ 5, it is characterised in that a kind of method of red autumnal leaves winged euonymus protoplast regenerated plant,
Including three successive stages: (1) Protoplast cuhnre, aseptically by the protoplast of sorting and containing low melting-point agarose
After the protoplast culture medium mixing of (freezing point is 26 ~ 30 DEG C), it is poured in 5cm culture dish, the quartering after solidification, proceeds to contain
In the 9cm culture bottle of protoplast culture medium, the lower 24 DEG C of concussions of dark condition are cultivated 2 weeks；After callus to be formed, by its turn
It is connected on callus inducing medium；(2) callus induction regenerated adventitious bud, is seeded in Induce aerosor by callus
In culture medium, at 24 DEG C, intensity of illumination 36 μm ol m-2·s-1, cultivate 8 weeks under the conditions of light application time 16 h/d, induced synthesis
Adventitious bud；(3) Induce aerosor is taken root, and adventitious bud is cultivated 2 weeks on root media I, induces root restriction；It is forwarded to raw
In root culture medium II, cultivate 4 weeks and form root system.
7. according to the method described in claim 1 and 6, it is characterised in that the plantlet terminal bud dormancy of a kind of red autumnal leaves winged euonymus tissue culture
Release method, said process obtain tissue culture's plantlet be planted in containing GA31.0-3.0 mg/L and 6-BA 0.5-
On the root media II of 2.0 mg/L, dark treatment at being placed in 3-8 DEG C.
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| CN111670807A (en) * | 2020-04-27 | 2020-09-18 | 江苏农林职业技术学院 | Tissue culture method of euonymus alatus |
| CN111670807B (en) * | 2020-04-27 | 2022-03-11 | 江苏农林职业技术学院 | Tissue culture method of euonymus alatus |
| CN114099547A (en) * | 2021-12-07 | 2022-03-01 | 洛阳市三启生物技术有限公司 | Stem cell exosome preparation for skin repair |
| CN114099547B (en) * | 2021-12-07 | 2023-11-21 | 洛阳市三启生物技术有限公司 | Stem cell exosome preparation for skin repair |
| CN114532228A (en) * | 2022-03-21 | 2022-05-27 | 玉林师范学院 | Method for cultivating plukenetia volubilis hexaploid plants |
| CN114831025A (en) * | 2022-05-23 | 2022-08-02 | 安康市农业科学研究院 | Rapid induction method of konjac polyploids |
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