CN105613288A - Construction method of rapid Euonymus japonicus L.f. aureo-marginatus Rehd propagation system - Google Patents
Construction method of rapid Euonymus japonicus L.f. aureo-marginatus Rehd propagation system Download PDFInfo
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- CN105613288A CN105613288A CN201510999046.3A CN201510999046A CN105613288A CN 105613288 A CN105613288 A CN 105613288A CN 201510999046 A CN201510999046 A CN 201510999046A CN 105613288 A CN105613288 A CN 105613288A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
Abstract
The invention belongs to the technical field of plant cell engineering and discloses a method for performing tissue culture and rapid propagation by using Euonymus japonicus L.f. aureo-marginatus Rehd stem tips and segments as explants. The method comprises the steps of conducting cluster bud induction and rooting culture on the stem tips and the stem segments with lateral buds to obtain complete regenerated plants, and sequentially conducting callus induction, callus proliferation and differentiation and rooting culture on the stem segments without lateral buds so as to obtain regenerated plants. The regenerated plants can be obtained in the two ways, operation is simple, the pollution rate and browning rate are low in the culturing process, a large amount of seedlings can be obtained within a short time without seasonal and environmental limits, and the shortcoming of cutting propagation is overcome, meanwhile conditions are provided for genetic transformation and somaclone variation breeding of the seedlings, and the method has significant practical value on theoretical research and landscape application.
Description
Technical field
The invention belongs to field of plant cell engineering technology, particularly relate to a kind of method utilizing golden-rimmed Chinese littleleaf box stem point and stem section to carry out tissue culture fast-propagation.
Background technology
Golden-rimmed Chinese littleleaf box (EuonymusJaponicuscv.Aureo-ma), it it is winged euonymus section winged euonymus platymiscium, the golden-rimmed Chinese ilex winged euonymus of another name is one of the mutation of great Ye Chinese littleleaf box, and single leaf is to life, obovate or ellipse, the blunt tooth of edge tool, surface deep green, limb edge is golden yellow, glossy, extremely there is ornamental value. Golden-rimmed Chinese littleleaf box happiness light, slightly resistance to the moon, strong adaptability, rudiment power and a branch power are strong, and resistance to pruning, is excellent afforestation seeds, all has a large amount of plantation in each big city, park, colleges and universities and community, and seedling demand is big. Golden-rimmed Chinese littleleaf box often breeds with the method for cuttage at present, but cottage propagation is by the restriction such as season, material, easy introduced disease in cuttage process, and the plant life-span obtained can shorten, especially xylophyta cuttage also there will be the phenomenon of hat partially, so long-term cottage propagation can cause, plant degenerates, the age of tree shortens and a large amount of introduced disease.
And undertaken numerous soon by tissue culture, not only not by the impact of environment in season, the shortcoming of xylophyta growth cycle length can be overcome, and can lay the foundation for golden-rimmed Chinese littleleaf box somaclonal variation breeding, selection by mutation and genetic engineering breeding, for createing the new variety offer condition of multicolour, strong stress resistance. On the other hand, axis nose part institute band virus is few, utilizes stem point to carry out tissue culture and can obtain a large amount of detoxic seedling, it is possible to reduces Pesticide use to a certain extent, not only cost-saving reducing atmospheric pollution, also golden-rimmed Chinese littleleaf box kind quality guarantee is pure, rejuvenation has significance.
Summary of the invention
Technical problem solved by the invention is to provide a kind of and carries out the method for tissue culture fast-propagation by explant of golden-rimmed Chinese littleleaf box stem point and stem section, the method can obtain golden-rimmed Chinese littleleaf box regeneration plant by callus and non-callus two kinds of approach, and industrial seedling rearing is had significance.
Technical solution of the present invention is as follows:
A kind of golden-rimmed Chinese littleleaf box rapid propagation system construction process, the method carries out according to the following steps:
1) pre-treatment of explant and sterilization
Get the current-year branch of the band tender terminal bud of children, then the blade around it is cut, carry out pre-treatment and sterilization, for subsequent use;
2) explant first culture and callus induction
It is inoculated on Primary culture base to carry out first culture by the stem section of the stem disinfected point, the stem section of band axillalry bud, not band axillalry bud, it is inoculated on callus inducing medium by the stem section of not band axillalry bud evoked callus and produces; Culture condition is light intensity 800��1000lx, illumination 16��18h/d, temperature 25 DEG C;
3) grow thickly the induction of bud and callus proliferation differentiation
A, by step 2) in grow to 2��3cm length the new tip cut, be transferred on inducing clumping bud substratum, 30��45 days axillalry buds are sprouted formation in a large number and are grown thickly bud, and bud of growing thickly every 4 weeks cuts into multiple budlet clump, is seeded in succeeding transfer culture on identical substratum; In culturing process, intensity of illumination 2500��3000lx, illumination 16��18h/d, temperature 25��27 DEG C;
B, by step 2) in the callus of diameter about 3cm size cut into 1cm fritter and be seeded in callus subculture medium, emerald green can be inoculated on callus division culture medium when the callus of subculture transfers to, induced synthesis indefinite bud; In culturing process, intensity of illumination 2500��3000lx, illumination 16��18h/d, temperature 25��27 DEG C;
4) root culture
By step 3) the new tip of the stalwartness that grows into about 2��3cm cuts, is transferred on root media, carries out root induction, finally obtain complete regenerated plant, and culture condition is intensity of illumination 2000lx, 16 h light, 8 h dark, temperature 25 DEG C.
2. the construction process of a kind of golden-rimmed Chinese littleleaf box rapid propagation system according to claim 1, it is characterized in that step 1) explant pretreatment method cuts blade only to leave naked bub and naked stem, washing powder water soaks 20��40 minutes, then flowing water rinses 1��2 hour; Sterilization method soaks 20��30s for explant is placed in massfraction 75% alcohol on super clean bench, aseptic water washing one to twice, it is placed in 250mL massfraction 0.1% mercuric chloride solution again to soak 6 minutes, wherein mercuric chloride solution needs to add 0.5��1mL tween-80, the explant of sterilization to be guaranteed by mercuric chloride solution submergence, mercuric chloride sterilize after with aseptic water washing 4��5 times, finally with aseptic filter paper, unnecessary moisture is blotted.
As preferably, described step 2) in Primary culture base be: MS+6-BA2.0��3.0mg/L+NAA0.2��0.6mg/L+20��30g/L sucrose+6��8g/L agar+0.1��0.2g/L gac, Medium's PH Value is 5.8��6.0; Callus inducing medium composition is: MS+6-BA1.0��2.0mg/L+NAA0.5��1.0mg/L+30g/L sucrose+6g/L agar+0.1��0.2g/L gac, and Medium's PH Value is 5.6��5.8; During callus induction, the stem section of band axillalry bud is not wanted in horizontal insertion substratum.
As preferably, described step 3) in inducing clumping bud substratum be: MS+6-BA1.0mg/L+NAA0.05��0.2mg/L+20��30g/L sucrose+6��8g/L agar, Medium's PH Value is 5.6��5.8; Callus proliferation medium is: MS+6-BA2.0mg/L+NAA0.5��0.8mg/L+20��30g/L sucrose+6g/L agar, and Medium's PH Value is 5.6��5.8; Callus division culture medium is: 6-BA2.0��3.0mg/L+NAA0.1��0.5mg/L+20��30g/L+30g sucrose+6g/L agar, Medium's PH Value is 5.6��6.0.
As preferably, described step 4) in root media be: 1/2MS+NAA0.1��0.5mg/L+IBA0.5��1.0mg/L+20��30g/L sucrose+6g/L agar+0.2g/L gac, Medium's PH Value is 5.6��6.0; There is otch in the new slightly stem section base portion of inoculation, takes light culture 3 days after inoculation.
Compared with the prior art, its useful effect is embodied in the present invention:
1. the means of the present invention's application tissue culture carry out golden-rimmed Chinese littleleaf box and breed fast, not by environmental restraint in season, the shortcoming of easily introduced disease in golden-rimmed Chinese littleleaf box seedling cottage propagation process can be overcome, and cottage propagation often makes the plant life-span of acquisition shorten, xylophyta also there will be the phenomenon of hat partially.
2. utilize little stem tip culture not only to have detoxification efficiency, and stem point can direct seedling differentiation, growth cycle short (getting final product rooting and transplant in 2 to 3 months to survive), it is low that phenotype observes aberration rate, it is thus possible to produce disease-free in a large number and that inherited character is consistent seedling. And obtain test-tube plantlet by callus approach, it is possible to somaclonal variation can be there is, thus produce the mutant of different phenotype, it is possible to expand germ plasm resource, promote breeding of new variety.
3. present method can be regenerated by different explants, material use efficiency height, hormone in medium reasonable ratio, and test-tube plantlet germination rate height, growing way is vigorous.
4. the sterilization method that the present invention uses can take into account surviving rate, pollution rate and melting brown rate, it is possible to reaches high germination rate, low stain rate and melting brown rate.
Accompanying drawing explanation
Fig. 1 is the inventive method schema;
The golden-rimmed Chinese littleleaf box that Fig. 2 is the inventive method induction grows thickly bud;
Fig. 3 is the golden-rimmed Chinese littleleaf box callus of the inventive method induction;
Fig. 4 is the golden-rimmed Chinese littleleaf box indefinite bud of the inventive method callus differentiation.
Fig. 5 is the golden-rimmed Chinese littleleaf box test-tube plantlet of the inventive method root induction.
Embodiment
Embodiment 1
1. explant pre-treatment and sterilization:
Choosing robust growth, branch without disease and pest from nursery, the stem section choosing the tender stem point of children and band axillalry bud is as explant. Explant is removed mature leaf, soaks 40 minutes with in 4 DEG C of washing powder water, then rinse 1 hour with 4 DEG C of flowing water, explant is placed in 75% alcohol by super clean bench and soaks 30s, aseptic water washing one to twice, then it is placed in 4 DEG C of massfraction 0.1% mercuric chloride solutions immersions, soak time arranges 4min, 6min, 8min, 15min and 25min five gradients, wherein need in mercuric chloride solution to add 1mL tween-80, aseptic water washing 4��5 times, blots unnecessary moisture with aseptic filter paper; First culture base to be proceeded to is cultivated.
In order to select the suitableeest pretreatment condition, the explant of different disinfecting time process is all seeded on substratum that composition is MS+6-BA2.5mg/L+NAA0.5mg/L+30g/L sucrose+8g/L agar+0.2g/L gac, Medium's PH Value is 5.6, culture condition is light intensity 800lx, illumination 16h/d, temperature 25 DEG C, adds up melting brown rate, pollution rate, germination rate (table 1) after two weeks.
The different mercuric chloride disinfecting time of table 1 is on the impact of golden-rimmed Chinese littleleaf box terminal bud and stem segment with axillary bud first culture
Can finding from this test-results, sterilization mercuric chloride is sterilized and while ensureing high germination rate, low melting brown rate, can be reached good sterilization effect in 6 minutes.
2. first culture:
The segment that the stem section of stem point or band axillalry bud is cut on super clean bench 1��2cm, morphology upper end is upwards seeded on Primary culture base; Every bottle of inoculation 3��4 explants. Culturing room's temperature is 25 DEG C, light application time 18h/d, intensity of illumination 1000lx; Primary culture based component is MS+6-BA2.5+NAA0.5mg/L+30g/L sucrose+8g/L agar, and pH value is 5.6, is object in addition taking stem segment with axillary bud, probes into best golden-rimmed Chinese littleleaf box brown chemoprevention control means (table 2) by three kinds of process; Inoculate the terminal bud of stem point or the axillalry bud of stem section after 10 days to sprout, cultivate 30��40 days new tips and can grow to 2��3cm;
The golden-rimmed Chinese littleleaf box tissue culture He mushroom mode of table 2
Note: be 6min for examination material mercuric chloride disinfecting time.
This test-results is known: Primary culture based component is MS+6-BA1.0mg/L+NAA0.5mg/L+30g/L sucrose+8g/L agar, additional 0.2g/LAC, and pH value is 5.6, and melting brown rate is only 2.5%, and the melting brown rate adding PVP is 17.6; When light culture, melting brown rate is 22.6%
3. grow thickly the induction of bud and succeeding transfer culture:
When the explant of first culture newly slightly grows to 2��3cm, cut and received on inducing clumping bud substratum, 30��45 days a large amount of axillary bud sproutings also grow, every about 4 weeks by big bud clump segmentation and on identical substratum succeeding transfer culture, a large amount of buds of growing thickly can be obtained; Inducing clumping bud substratum is MS+6-BA1.0mg/L+NAA0.05mg/L+30g/L sucrose+7g/L agar, and Medium's PH Value is 5.8, in culturing process, and intensity of illumination 3000lx, illumination 16��18h/d, temperature 25 DEG C;
4. root culture: select the high test-tube plantlet of 3��4cm and be transferred on root media, dark treatment is cultivated under within 3 days, being placed on normal photoperiod, within 25-32 days, can obtain the seedling of taking root of more than 5 more than root length 4cm, as shown in Figure 5; Root media is 1/2MS+NAA0.2mg/L+IBA0.5mg/L+20g/L sucrose+6g/L agar+0.2g/L gac. Culture condition is: intensity of illumination 2000lx, illumination 18h/d, temperature 25 DEG C. In the root culture stage, the melting brown rate of tissue cultured seedling is lower than 2.5%.
Embodiment 2
1. explant pre-treatment and sterilization:
The stem section choosing the golden-rimmed Chinese littleleaf box tender not band axillalry bud of children is as explant, soak 30 minutes with in 4 DEG C of washing powder water, 1 hour is rinsed again with 4 DEG C of flowing water, explant is placed in massfraction 75% alcohol by super clean bench and soaks 30s, aseptic water washing one to twice, it is placed in 4 DEG C of 0.1% mercuric chloride solution again to soak, soak time arranges 5min, 8min, 12min, 15min, 25min five gradients, wherein needing in mercuric chloride solution to add 1mL tween-80, aseptic water washing 4��5 times, blots unnecessary moisture with aseptic filter paper. Explant is all seeded on the substratum that composition is MS+6-BA2.0+NAA0.5mg/L+30g/L sucrose+6g/L agar+0.2g/L gac. Culture condition, with embodiment 1, adds up pollution rate, melting brown rate and healing rate in two weeks.
The different mercuric chloride disinfecting time of table 3 is on the impact of golden-rimmed Chinese littleleaf box without axillary bud stem section first culture
By this test it will be seen that mercuric chloride sterilization 8min is that golden-rimmed Chinese littleleaf box is without the best disinfecting time of axillary bud stem section.
2. the induction of callus:
The segment that stem section is cut into 1��2cm on super clean bench, horizontal is connected on callus inducing medium, every bottle of inoculation 3��4 explants. Culturing room's temperature is 25 DEG C, light application time 18h/d, intensity of illumination 800lx; Callus inducing medium composition is MS+6-BA2.0+NAA1.0mg/L+30g/L sucrose+6g/L agar+0.2g/L gac, and Medium's PH Value is 5.6. 3. the subculture of callus and differentiation:
The callus lines growing to diameter about 3cm is cut into the fritter of diameter about 1cm, it is placed on callus proliferation medium, within every about 30 days, in same medium, subculture is once, subculture medium is MS+6-BA2.0mg/L+NAA0.5mg/L+30g/L sucrose+6g/L agar, and Medium's PH Value is 5.8; In subculture process, callus can be transferred to emerald green by yellow-green colour, transferred in division culture medium, bud point can occur for 20��35 days, being divided into indefinite bud subsequently, callus division culture medium is 6-BA2.5mg/L+NAA0.1mg/L+20��30g/L sucrose+6g/L agar, and Medium's PH Value is 5.8, culture condition is: intensity of illumination 2500lx, illumination 16 h light, 8 h dark, temperature 25 DEG C.
4. root culture:
The new tip cutting about 3cm robust growth is transferred on root media, and dark treatment is transferred after 3 days again and cultivated under light, cultivates and within about 3 weeks, obtains more than 4 the complete regrowths of more than root length 4cm; Root media is 1/2MS+NAA0.2mg/L+IBA0.5mg/L+20g/L sucrose+6g/L agar+0.2g/L gac, and Medium's PH Value is 5.8. Culture condition is: intensity of illumination 2000lx, illumination 18h/d, temperature 25 DEG C, and in the root culture stage, the melting brown rate of tissue cultured seedling is lower than 2.5%.
After conventional hardening, the regrowth of above-described embodiment 1 and embodiment 2 gained can be obtained transplanted seedling transplant respectively. The transplanted seedling of embodiment 1 is observed through phenotype after strain and is found, the hereditary shape of its phenotype and seedling is consistent substantially, therefore can produce disease-free in a large number and that inherited character is consistent seedling. And through phenotype observation, the transplanted seedling obtained through embodiment 2 method finds that aberration rate is higher after strain, can be used for expanding germ plasm resource, promote breeding of new variety.
Claims (5)
1. a golden-rimmed Chinese littleleaf box rapid propagation system construction process, it is characterised in that the method carries out according to the following steps:
1) pre-treatment of explant and sterilization
Get the current-year branch of the band tender terminal bud of children, the blade around it is cut, carries out pre-treatment and sterilization, for subsequent use;
2) explant first culture and callus induction
It is inoculated on Primary culture base to carry out first culture by the stem section of the stem disinfected point, band axillalry bud, it is inoculated on callus inducing medium by the stem section part of not band axillalry bud evoked callus and produces; Culture condition is light intensity 800��1000lx, illumination 16��18h/d, temperature 25 DEG C;
3) grow thickly the induction of bud and callus proliferation differentiation
A, by step 2) in grow into 2��3cm length the new tip cut, it is transferred on inducing clumping bud substratum, 30��45 days axillalry buds are sprouted formation in a large number and are grown thickly bud, and bud of growing thickly every 4 weeks cuts into multiple budlet clump, is seeded in succeeding transfer culture on identical substratum; In culturing process, intensity of illumination 2500��3000lx, illumination 16��18h/d, temperature 25��27 DEG C;
B, by step 2) in the callus of diameter about 3cm size cut into 1cm fritter and be seeded in callus subculture medium, emerald green can be inoculated on callus division culture medium when the callus of subculture transfers to, induced synthesis indefinite bud; In culturing process, intensity of illumination 2500��3000lx, illumination 16��18h/d, temperature 25��27 DEG C;
4) root culture
By step 3) the new tip of the stalwartness that grows into about 2��3cm cuts, is transferred on root media, carries out root induction, finally obtain complete regenerated plant, and culture condition is intensity of illumination 2000lx, 16 h light, 8 h dark, temperature 25 DEG C.
2. the construction process of a kind of golden-rimmed Chinese littleleaf box rapid propagation system according to claim 1, it is characterized in that step 1) explant pretreatment method be by blade remove only leave naked bub and naked stem, washing powder water soaks 20��40 minutes, then flowing water rinses 1��2 hour; Sterilization method soaks 20��30s for explant is placed in massfraction 75% alcohol on super clean bench, aseptic water washing one to twice, it is placed in 250mL massfraction 0.1% mercuric chloride solution again to soak 6 minutes, wherein mercuric chloride solution needs to add 0.5��1mL tween-80, unnecessary moisture again with aseptic water washing 4��5 times, is finally blotted after sterilizing by mercuric chloride with aseptic filter paper.
3. the construction process of a kind of golden-rimmed Chinese littleleaf box rapid propagation system according to claim 1, it is characterized in that step 2) in Primary culture base be: MS+6-BA2.0��3.0mg/L+NAA0.2��0.6mg/L+20��30g/L sucrose+6��8g/L agar+0.1��0.2g/L gac, Medium's PH Value is 5.8��6.0; Callus inducing medium composition is: MS+6-BA1.0��2.0mg/L+NAA0.5��1.0mg/L+30g/L sucrose+6g/L agar+0.1��0.2g/L gac, and Medium's PH Value is 5.6��5.8; During callus induction, the stem section of band axillalry bud is not wanted in horizontal insertion substratum.
4. the construction process of a kind of golden-rimmed Chinese littleleaf box rapid propagation system according to claim 1, it is characterized in that step 3) in inducing clumping bud substratum be: MS+6-BA1.0mg/L+NAA0.05��0.2mg/L+20��30g/L sucrose+6��8g/L agar, Medium's PH Value is 5.6��5.8; Callus proliferation medium is: MS+6-BA2.0mg/L+NAA0.5��0.8mg/L+20��30g/L sucrose+6g/L agar, and Medium's PH Value is 5.6��5.8; Callus division culture medium is: 6-BA2.0��3.0mg/L+NAA0.1��0.5mg/L+20��30g/L+30g sucrose+6g/L agar, Medium's PH Value is 5.6��6.0.
5. the construction process of a kind of golden-rimmed Chinese littleleaf box rapid propagation system according to claim 1, it is characterized in that step 4) in root media be: 1/2MS+NAA0.1��0.5mg/L+IBA0.5��1.0mg/L+20��30g/L sucrose+6g/L agar+0.2g/L gac, Medium's PH Value is 5.6��6.0; There is otch in the new slightly stem section base portion of inoculation, takes light culture 3 days after inoculation.
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