CN105104184B - A kind of method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal - Google Patents
A kind of method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal Download PDFInfo
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Abstract
The invention discloses a kind of method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal, including：In vitro Sedum alfredii Hance plant is cut into the stem section containing complete axillary bud, is immersed in aseptic colchicine solution, seal lucifuge, and be put into vibration in the shaking table that temperature is 25 DEG C ± 1 DEG C；In vitro stem segment with axillary bud after taking-up treatment is inoculated on differential medium after aseptic water washing, and Multiple Buds are grown at culture to axillary bud；Multiple Buds are inoculated in root media, culture of rootage is whole plant；The tender leaf for taking whole plant detects the ploidy for obtaining strain on flow cytometer, selects variation plant；Variation plant is taken, stem section is cut into, is cultivated in root media and is obtained aseptic seedling；Variation plant carries out Cadmium treated with strain is compareed in the culture medium for adding cadmium, detects its internal Cd accumulation amount.The present invention doubles material by inducing Sedum alfredii Hance chromosome doubling, then process detection acquisition, improves the super accumulation ability of Sedum alfredii Hance heavy metal.
Description
Technical field
The invention belongs to plant soil restoration heavy metal pollution field, and in particular to one kind using chromosome doubling improvement
The method of zinc Cd hyperaccumulator Sedum alfredii Hance genotype.
Background technology
Cadmium Pollution in Soils is increasingly serious in recent years, and the cadmium in soil enters human body by plant (or plant arrive animal), danger
The health of victimization class.The cadmium pollution in soil how is administered and mitigates, as current urgent problem.Phytoremediation is one
Plant more environmentally friendly promising mode.Sedum alfredii Hance is a kind of zinc Cd hyperaccumulator, can be by the more absorption and accumulation of zinc cadmium
To overground part, so as to mitigate heavy metal pollution of soil.But, the biomass of Sedum alfredii Hance is relatively low, although relative accumulation amount
Height, is still difficult to meet the demand of phytoremediation.How this good resource is utilized, improve polluted soil phyto remediation efficiency particularly
It is important.
For example, the Chinese invention patent application document of Publication No. CN101444185A discloses a kind of heavy metal hyperaccumulative
The propagation method of plant Sedum alfredii Hance germchit, it is characterized in that using Callus of Leaf revulsion, by the sterilization of Sedum alfredii Hance blade,
Leaf margin, vein and petiole are cut, the small pieces of 0.2~0.3cm are cut into, it is 5.5 to be inoculated into the bottled pH of sterilized glass culture
On~5.8 inducing cultures, it is ± 25 DEG C, 1~2 week callus induction group in the incubator of no light that blake bottle is put into temperature
Knit, callus is transferred to the bottled pH of sterilized glass culture in 5.5~5.8 differential mediums, then by blake bottle
Temperature is put into for ± 25 DEG C, is cultivated 40~50 days in the incubator that the illumination 16 hours that light intensity is 3000 Luxs is kept daily,
3~5cm seedling with more than 1cm young roots high is obtained, blake bottle is then moved on into outdoor, open bottleneck, add a little distillation
After water is placed 2~3 days, culture medium is washed with water, transplant to vermiculite：In the matrix of perlite ratio 3: 1, or organic matter matrix, 2
Obtain seedling within~4 weeks.
The Chinese invention patent application document of Publication No. CN 1973617A discloses a kind of heavy metal hyperaccumulative plant east
The propagation method of southern red-spotted stonecrop seedling, using stem section cuttage, 3cm is cut into the stem section of upper band axillary bud, direct skewer by Sedum alfredii Hance
It is inserted into the soil by heavy metal pollution, is managed by Traditional Agricultural till and grown, treats plant growth to 15cm~30cm harvestings ground
Top, 3cm is cut into as seedling with the stem section of upper band axillary bud, in the soil of cuttage to pollution environment again；Or by the southeast
Red-spotted stonecrop is cut into 3cm with the stem section of upper band axillary bud, in cuttage to seedling medium or nutrient solution, is managed by Traditional Agricultural till and given birth to
It is long, obtain seedling.
The content of the invention
The present invention provides a kind of method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal, by induction
Sedum alfredii Hance chromosome doubling, then material is doubled by detection acquisition, improve the super accumulation ability of Sedum alfredii Hance heavy metal.
A kind of method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal, comprises the following steps：
(1) in vitro Sedum alfredii Hance plant is cut into the stem section containing complete axillary bud, is immersed in aseptic colchicine solution,
Sealing lucifuge, and it is put into vibration in the shaking table that temperature is 25 DEG C ± 1 DEG C；
(2) the in vitro stem segment with axillary bud after step (1) treatment is taken out to be inoculated on differential medium after aseptic water washing,
Multiple Buds are grown at culture to axillary bud；
(3) Multiple Buds of gained are inoculated in root media, carry out culture of rootage；
(4) tender leaf for taking the whole plant after culture of rootage detects the ploidy for obtaining strain on flow cytometer, choosing
Select variation plant；
(5) variation plant is taken, stem section is cut into, is cultivated in root media and is obtained aseptic seedling.
It is that aseptic seedling carries out Cadmium treated with strain is compareed in the culture medium for adding cadmium by variation plant, detects its internal Cd accumulation
Amount.
The super build-up effect of Sedum alfredii Hance has benefited from the expression of its specific gene, and the doubling of chromosome can make these gene phases
To overexpression, so as to improve repairing effect；Meanwhile, its stalk can be made sturdy after doubling, biomass increase.
In the method for artificial induction's chromosome doubling, colchicine doubling techniques are fairly simple effectively, using most.Autumn waters -- limid eyes
Celestial element is that one kind extracts alkaloid from liliaceous plant colchicum, can suppress mitosis, destroys spindle, makes dyeing
Body is stuck in metaphase, and in such mitosis, although chromosome lobe, cell does not divide, it is impossible to form two
Daughter cell, thus make chromosome doubling.Polyploid after doubling generally has stronger resistance and adaptability.Sedum alfredii Hance with
Based on vegetative propagation, a large amount of axillary buds can be grown from axil, quick breeding.Axil is that Sedum alfredii Hance divides vigorous tissue area
Domain, is processed with colchicine, and effect is best.
Preferably, the mass concentration of the colchicine solution is 0.1~0.2%.Most preferably 0.2%.
Preferably, in step (1) in shaking table concussion and cultivate 24-72h.Further preferred 24h.
Preferably, the composition of the differential medium is：Basal medium MS+8.0g/L agar+30.0g/L sucrose+
3.0mg/L 6-BA+0.1mg/L NAA, pH 5.8.Preferably, the composition of the root media is：1/2MS+8.0g/L fine jades
Fat+30.0g/L sucrose+0.2mg/L 2,4-D, pH5.8.
Preferably, the method for Ploidy detection comprises the following steps in step (4)：
(1) take leaflet tablet to be placed in the culture dish of precooling, add the OttoI of precooling, chopping, dissociation；
(2) dissociation solution in aspiration step (1) culture dish, with 400 mesh membrane filtrations to centrifuge tube, in 3~5 DEG C of incubations
3~5min；
(3) abandoning supernatant is centrifuged, the OttoI of precooling is added, OttoII is added, precooling PI dye liquors are eventually adding, puts
Lucifuge dyes 8~10min under the conditions of 3~5 DEG C；The volume ratio of OttoI, OttoII and PI dye liquor is 1:4:5；
(5) loading pipe, flow cytometer detection are moved to.
Further：The composition of OttoI is：0.1mol/L citric acids, 0.5% (v/v) Tween20, pH 2.0-3.0,4
DEG C preserve；The composition of OttoII is：0.4mol/L disodium hydrogen phosphates, pH 2.0-3.0, normal temperature is preserved；PI (iodate third
Pyridine) dye liquor concentration be 0.05mg/ml, per ml add 250 μ g RNases, tinfoil parcel, lucifuge, 4 DEG C preservation.
The method of Ploidy detection further, is concretely comprised the following steps in step (4)：
1) about 0.5g blades are taken to be placed in the culture dish of precooling, the OttoI of 1ml precoolings is added, with sharp blade once
Property is quickly shredded；
2) dissociation solution in culture dish is drawn, with 400 mesh membrane filtrations to 1.5ml centrifuge tubes, is incubated in 4 DEG C of refrigerators
5min；
3) it is centrifuged, rotating speed 1000r/min, 4 DEG C of temperature, time 5min；
4) abandoning supernatant, adds the μ l of OttoI 20 of precooling, adds the μ l of OttoII 80, is eventually adding 100 μ l precoolings
Propidium iodide PI dye liquors, each sample about 200 μ l nuclei suspensions altogether.It is placed in 4 DEG C of refrigerators, lucifuge dyeing 10min；
5) loading pipe, upper machine testing are moved to.
Preferably, when the plant in step (3) grow to 5cm it is high after, on flow cytometer detect.
The inventive method treatment conditions are simple and easy to control, and repeatability is high, time saving and energy saving.
Brief description of the drawings
Figure 1A is flow cytomery Sedum alfredii Hance blade relative dna content point after Otto ' s extractions, the dyeing of PI dye liquors
Cloth histogram, Figure 1B is that Otto ' s are extracted, flow cytomery doubles Sedum alfredii Hance blade and contains with respect to DNA after the dyeing of PI dye liquors
Amount distribution histogram.
Fig. 2 compares figure for Sedum alfredii Hance plant, and Fig. 2A is adjoining tree aspect graph, and Fig. 2 B are that ploidy increases plant forms
Figure.
Fig. 3 is the nucleus that basis of microscopic observation is arrived after using Otto ' s to extract PI dyeing.
Fig. 4 is Sedum alfredii Hance adjoining tree and cadmium content in ploidy increase plant body under Cd stress.
Fig. 5 A are Tris.MgCl2Flow cytomery Sedum alfredii Hance blade is relative after extractant is extracted, PI dye liquors are dyeed
DNA content distribution histogram, Fig. 5 B are Tris.MgCl2Flow cytomery doubles east after extractant is extracted, PI dye liquors are dyeed
Southern red-spotted stonecrop blade relative dna content distribution histogram.
Specific embodiment
Embodiment 1
(1) acquisition of aseptic seedling：After Sedum alfredii Hance upper semisection plant is taken through flowing water flushing 20 minutes, soaked with 75% ethanol
Bubble sterilization 60s, then the HgCl for being placed in 0.1%2Sterilization 5min, with aseptic water washing 4-5 times after, lain in autoclaving mistake
Filter paper on, piece is cut into small pieces with aseptic operation knife, then access MS+8.0g/L agar+30.0g/L sucrose+3.0mg/L
Squamous subculture on the culture medium of 6-BA+0.1mg/L NAA, growth obtains a large amount of Multiple Buds after a period of time, then is transferred to 1/2MS+
8.0g/L agar+30.0g/L sucrose+0.2mg/L 2, carries out culture of rootage in the culture medium of 4-D, obtain substantial amounts of aseptic seedling and supply
Experiment is selected.
(2) colchicine is doubled：Choose healthy and strong aseptic seedling to be placed on aseptic filter paper, cut with the big blade of aseptic operation hilt
Half, then cane is cut into stem section, complete axillary bud is included in stem section, it is then placed in autoclaving is crossed 0.2% colchicine
Soaked in solution.The conical flask for containing in vitro stem segment with axillary bud and colchicine solution is sealed with sealed membrane and rubber band
Mouthful, it is ensured that it is isolated from the outside, and lucifuge is wrapped up with tinfoil, is put into temperature to vibrate 24h in 25 DEG C ± 1 DEG C of shaking table, and rotating speed is
100rpm。
(3) Multiplying culture：On superclean bench, treated stem segment with axillary bud is taken out, aseptic water washing 4-5 times,
Residual moisture is blotted with aseptic filter paper again, MS+8.0g/L agar+30.0g/L sucrose+3.0mg/L 6-BA+0.1mg/L are inoculated in
In the culture medium of NAA, notice that culture medium is inserted in morphology upper end upward, in 25 DEG C of temperature, light intensity is the special LED of 1500lx tissue cultures
Illumination 16h under lamp, until growing a large amount of axillary buds.
(4) culture of rootage：In order to quickly breed and obtain a large amount of plant, axillary bud is cut out, be inoculated in 1/2MS+8.0g/L
Taken root in agar+30.0g/L sucrose+0.2mg/L 2,4-D culture mediums growth.
(5) Ploidy detection：Culture about one month, take tender leaf sample preparation detect on flow cytometer obtain strain again
Property, induced mutation rate now can be up to 27.3%.
Concretely comprise the following steps：
1) about 0.5g blades are taken to be placed in the culture dish of precooling, the OttoI of 1ml precoolings is added, with sharp blade once
Property is quickly shredded；
2) dissociation solution in culture dish is drawn, with 400 mesh membrane filtrations to 1.5ml centrifuge tubes, is incubated in 4 DEG C of refrigerators
5min；
3) it is centrifuged, rotating speed 1000r/min, 4 DEG C of temperature, time 5min；
4) abandoning supernatant, adds the μ l of OttoI 20 of precooling, adds the μ l of OttoII 80, is eventually adding 100 μ l precoolings
Propidium iodide PI dye liquors, each sample about 200 μ l nuclei suspensions altogether are placed in 4 DEG C of refrigerators, lucifuge dyeing 10min；
5) loading pipe, upper machine testing are moved to.
The composition of OttoI is：0.1mol/L citric acids, 0.5% (v/v) Tween20, pH 2.0-3.0,4 DEG C of preservations；
The composition of OttoII is：0.4mol/L disodium hydrogen phosphates, pH 2.0-3.0, normal temperature is preserved；PI (propidium iodide) dye liquor
Concentration is 0.05mg/ml, and 250 μ g RNases, tinfoil parcel, lucifuge, 4 DEG C of preservations are added per ml.
After extracting PI dyeing with Otto ' s, the nucleus that basis of microscopic observation is arrived is as shown in Figure 3.
(6) expand numerous：The ploidy that to have determined that increases plant and largely expands numerous, is cut into stem section, 1/2MS+8.0g/L agar+
30.0g/L sucrose+0.2mg/L 2, cultivates in 4-D culture mediums, preserves aseptic seedling.
Figure 1A is flow cytomery Sedum alfredii Hance blade relative dna content distribution histogram；Figure 1B is fluidic cell
Instrument detection doubles Sedum alfredii Hance blade relative dna content distribution histogram.
Fig. 2 compares figure for Sedum alfredii Hance plant, and Fig. 2A is adjoining tree aspect graph, and Fig. 2 B are that ploidy increases plant forms
Figure.
Plant cell ploidy is detected using flow cytometer, check plant there are four peaks, illustrates without colchicum
The Sedum alfredii Hance that element is doubled in vivo containing four kinds of cells of DNA content, i.e., itself is exactly in itself mixoplod.Variant is carried out
DNA content is determined, and the ploidy that discovery has five peaks, chromosome increases, but it is still mixoplod.In formalness, variant
Stalk than compareing is sturdy, and blade is abundant, well developed root system.The increased seedling of ploidy is substantially strengthened in control seedling under condition of tissue culture, biological
Amount is big.Under condition of tissue culture, level seedling, the weight that ploidy increases seedling is approximately 2-3 times of control.
Fig. 5 A are Tris.MgCl2Flow cytomery Sedum alfredii Hance blade is relative after extractant is extracted, PI dye liquors are dyeed
DNA content distribution histogram, Fig. 5 B are Tris.MgCl2Flow cytomery doubles east after extractant is extracted, PI dye liquors are dyeed
Southern red-spotted stonecrop blade relative dna content distribution histogram.Use Tris.MgCl2Extractant is extracted, and is dyeed with PI, through flow cytometer
Analysis, some peaks are not it is obvious that and having many background fragments.Compare, Otto ' s extracting method effects are more preferable.
Tris.MgCl2：0.2mol/L Tris, 4mmol/L magnesium chloride hexahydrates, 0.5% (v/v) Triton X-100, pH
7.5,4 DEG C of preservations.
Embodiment 2
(1) Cadmium treated：Selection growing way identical ploidy increases seedling and compares, and moves into MS+8.0g/L agar+30.0g/L sugarcanes
In sugar+0.2mg/L 2,4-D culture medium, the concentration of cadmium is respectively 0,50,100,200 μM in culture medium, each three weight for the treatment of
It is multiple.
(2) sample is received：Sampling is harvested after test process 30d.When harvesting plant, 20mM Na are placed a plant into2EDTA solution is handed over
Change 15 minutes, remove the Cd of adsorption2+；Then washed respectively three times with running water and deionized water.
(3) cadmium content is determined：The plant that will be collected finishes 30min at being placed in 105 DEG C, is then dried to perseverance at 65 DEG C
Weight；It is with mortar that plant sample is levigate, for determination of elemental analysis.0.1g plant drying powder is weighed, HNO is used3/HClO4It is double
Acid system disappear boiling.Disappear after the deionized water constant volume of the solution after boiling, cadmium content is carried out using ICP-MS (Agilent 7500a)
Determine.
Fig. 4 is Sedum alfredii Hance adjoining tree and cadmium content in ploidy increase plant body under Cd stress.
Claims (5)
1. it is a kind of improve the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal method, it is characterised in that including as follows
Step：
(1) in vitro Sedum alfredii Hance plant is cut into the stem section containing complete axillary bud, is immersed in aseptic colchicine solution, sealing
Lucifuge, and 20~24h of vibration in the shaking table that temperature is 25 DEG C ± 1 DEG C is put into, the mass concentration of the colchicine solution is
0.1-0.2%；
(2) the in vitro stem segment with axillary bud after step (1) treatment is taken out, is inoculated on differential medium after aseptic water washing, trained
Support and Multiple Buds are grown to axillary bud, the composition of the differential medium is：Basal medium MS+8.0g/L agar+30.0g/L sugarcanes
Sugar+3.0mg/L 6-BA+0.1mg/L NAA, pH 5.8；
(3) Multiple Buds of gained are inoculated in root media, carry out culture of rootage, the composition of the root media is：
1/2MS+8.0g/L agar+30.0g/L sucrose+0.2mg/L 2,4-D, pH5.8；
(4) tender leaf for taking the whole plant after culture of rootage detects the ploidy for obtaining strain, selection dye on flow cytometer
The increased variation plant of colour solid ploidy；
(5) variation plant is taken, stem section is cut into, is cultivated in root media and is obtained aseptic seedling.
2. the method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal according to claim 1, its feature
It is that the method for Ploidy detection comprises the following steps in step (4)：
(1) take leaflet tablet to be placed in the culture dish of precooling, add the OttoI of precooling, chopping, dissociation；
(2) dissociation solution in aspiration step (1) culture dish, with 400 mesh membrane filtrations to centrifuge tube, it is incubated 3 in 3~5 DEG C~
5min；
(3) abandoning supernatant is centrifuged, the OttoI of precooling is added, OttoII is added, precooling PI dye liquors are eventually adding, it is placed in 3~
Lucifuge dyes 8~10min under the conditions of 5 DEG C；The volume ratio of OttoI, OttoII and PI dye liquor is 1:4:5；
(5) loading pipe, flow cytometer detection are moved to.
3. the method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal according to claim 2, its feature
It is that the composition of the OttoI is：0.1mol/L citric acids, volume by volume concentration is 0.5% Tween20, pH 2.0-3.0；
The composition of the OttoII is：0.4mol/L disodium hydrogen phosphates, pH 2.0-3.0.
4. the method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal according to claim 2, its feature
It is that the concentration of the PI dye liquors is 0.05mg/ml, and 250 μ g RNases are added per ml dye liquors.
5. the method for improving the super accumulation ability of hyperaccumulative plant Sedum alfredii Hance heavy metal according to claim 1, its feature
Be, when the plant in step (3) grow to 5cm it is high after, on flow cytometer detect detection.
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| CN1608408A (en) * | 2003-10-24 | 2005-04-27 | 程金龙 | Breeding method of drought-resistant polyploid linear stonecrop herb |
| CN1973617A (en) * | 2006-12-15 | 2007-06-06 | 浙江大学 | Germchit propagation process of Alfred stonecrop as heavy metal super accumulating plant |
| CN101444185A (en) * | 2006-12-15 | 2009-06-03 | 浙江大学 | Method for breeding heavy metal hyperaccumulative plant Sedum alfredii Hance germchit |
| CN101670362A (en) * | 2009-10-30 | 2010-03-17 | 四川农业大学 | Application of grain amaranth in repairing mine soil and sludge polluted by heavy metal cadmium |
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2015
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| CN1608408A (en) * | 2003-10-24 | 2005-04-27 | 程金龙 | Breeding method of drought-resistant polyploid linear stonecrop herb |
| CN1973617A (en) * | 2006-12-15 | 2007-06-06 | 浙江大学 | Germchit propagation process of Alfred stonecrop as heavy metal super accumulating plant |
| CN101444185A (en) * | 2006-12-15 | 2009-06-03 | 浙江大学 | Method for breeding heavy metal hyperaccumulative plant Sedum alfredii Hance germchit |
| CN101670362A (en) * | 2009-10-30 | 2010-03-17 | 四川农业大学 | Application of grain amaranth in repairing mine soil and sludge polluted by heavy metal cadmium |
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