CN103875536B - Method for obtaining populus tomentosa gene analysis material under polychlorinated biphenyl exposure condition - Google Patents
Method for obtaining populus tomentosa gene analysis material under polychlorinated biphenyl exposure condition Download PDFInfo
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- 241000249899 Populus tomentosa Species 0.000 title claims abstract description 51
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Abstract
The invention discloses a method for obtaining a gene analysis material of Chinese white poplar under polychlorinated biphenyl exposure condition, which comprises the steps of respectively preparing A, B, C, D four kinds of adventitious root differentiation solid culture media of Chinese white poplar under aroclor 1254 exposure and blank conditions, respectively filling the culture media into triangular flasks, sealing the triangular flasks and then sterilizing; selecting a tissue culture seedling of the same clone of the populus tomentosa, cutting stem sections with the same thickness between the second leaf and the third leaf under the terminal bud, respectively inoculating the stem sections into the A, B, C, D solid culture medium, and inoculating 3-5 stem sections into each triangular flask; culturing for 20-30 days in a culture room; a, B, C, D solid culture medium of Chinese white poplar material is collected and numbered separately, and the materials are packed and stored in groups. The method is simple to operate, the obtained populus tomentosa material genes have the same background and are subjected to various growth periods, the polychlorinated biphenyl influence factors are determined, the method is suitable for wide gene analysis, and a test material is provided for searching the genes with strong tolerance to the polychlorinated biphenyl.
Description
Technical field
The invention belongs to biological technical field, the acquisition methods of Chinese white poplar gene analysis material under a kind of Polychlorinated biphenyls exposure condition.
Background technology
Polychlorinated biphenyls (PCBs, aroclor 1254 is a kind of typical Polychlorinated biphenyls) be a class stable in properties, there is acute and chronic toxicity, typical lasting organic pollutant (POPs), once be used in the insulating oil of capacitor and transformer, accumulator, carbon paper, coating, lubricant, fireproof agent, bonding agent, paraffin expansion agent, the agent of agricultural chemicals prolongation of effect, all widely use in fields such as power industry, Plastics Processing Industry, chemical industry and press, by 1979, except a small amount of specific use, countries in the world stop production because of public hazards reason and use PCBs.The world estimates at 1,200,000 tons of PCBs, and wherein 65% is present in electric system, soot or is stored in land, and 31% is lost in environment.Due to its persistence and refractory organics, be still one of most important organic pollutant in environment so far.Because PCBs has strong toxicity, carcinogenicity, teratogenesis, mutagenicity, many countries have been classified as PCBs the organic pollutant list of priority acccess control.
About Environmental capacity and the soil remediation method of Polychlorinated biphenyls, mainly contain and seal landfill method, high temperature incineration method, physical method, chemical method, microbial degradation method (patent CN102550425, CN102108361, CN103252345) and phytoremediation (patent CN1663702, CN101966529) up for safekeeping.Phytoremediation, utilize plant and plant growth and the microorganism that coexists thereof to carry out method that is organic in environment purification and inorganic pollution as technological means, at present, phytoremediation mainly replaces from anaerobic-aerobic in composting (CN101966529), two inoculation (CN1663702) etc. in practice to be repaired soil, selects the crop such as alfalfa, custard squash that Polychlorinated biphenyls decomposes or accumulation ability is good during reparation; Relate generally to plant extraction (phytoextraction) in research aspect, plant fixes (phytostabilisation), plant degradation (phytodegradation), rhizosphere degrades (rhizodegradation) and root filter effect (rhizofiltration).Generally speaking, find and screen plant gene Polychlorinated biphenyls being had to tolerance, enrichment Sum decomposition ability more by force, be the key of the new variety of plant that exploitation degradation of polychlorinated biphenyl ability is strong, soil remediation efficiency is high, the theory and practice of phytoremediation is had great significance.And find and screening to Polychlorinated biphenyls tolerance, plant gene that enrichment Sum decomposition ability is strong, first and very importantly will have that genetic background is identical, exposure condition is controlled, under Polychlorinated biphenyls exposure condition, experience each growth period and have compared with the vegetable material of high-biomass and corresponding reference material for gene analysis.In current phytoremediation research, material therefor generally to be exposed the target examination seed of plant or full plants body by methods such as potted plant earth culture, vessel liquid cultivations and obtains.Potting soil culture method is because of soil constituent complexity used, the factors such as microbiological effect is unknown cause growth conditions and quality control difficulties, and then whether simple of the growth differences that cannot compare plant exposes relevant with Polychlorinated biphenyls, carrying out cultivation by vessel liquid mode makes operation partially loaded down with trivial details again, larger limitation is, if do exposed material with seed, because the genetic background of different seeds is different, the requirement carrying out gene analysis under homologous genes background cannot be met, if do exposed material with complete plant corpus, because each organ of full plants body perfects, just make gene analysis cannot disclose plant organ in Growth and Differentiation process by Polychlorinated biphenyls affect situation.Above-mentioned drawback finally causes carrying out the gene analysis under Polychlorinated biphenyls exposure condition with seed or full plants body or making gene analysis have larger limitation.Therefore, under also there is no Polychlorinated biphenyls exposure condition in prior art, for the target examination material of gene analysis and the easy acquisition methods of reference material.
Summary of the invention
The object of this invention is to provide the acquisition methods of Chinese white poplar gene analysis material under a kind of Polychlorinated biphenyls exposure condition, the method is simple to operate, the Chinese white poplar material genetic background obtained is identical, experience each growth period, and Polychlorinated biphenyls influence factor is determined, being suitable for carrying out gene analysis widely, providing test material for finding the gene strong to Polychlorinated biphenyls tolerance.。
For achieving the above object, the present invention takes following technical scheme:
Under Polychlorinated biphenyls exposure condition, an acquisition methods for Chinese white poplar gene analysis material, comprises the steps:
A () is produced as follows A, B, C, D Chinese white poplar adventive root differentiation solid culture medium, sucrose, agar represent with the mass percent of shared solid culture medium gross mass, and 1/2MS is the MS medium that macroelement reduces by half, and IBA is indolebutyric acid:
A, 1/2MS+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
B, 1/2MS+0.5 mgL
-1iBA+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
C, 1/2MS+2.0 ~ 8.0 mgL
-1aroclor 1254+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
D, 1/2MS+0.5mg L
-1iBA+2.0 ~ 8.0 mg L
-1aroclor 1254+ 1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
Above-mentioned medium is respectively loaded in triangular flask, after sealing, carries out sterilizing;
B () selects the plantlet in vitro of Chinese white poplar same clone, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, be inoculated in described A, B, C, D solid culture medium respectively, each triangular flask inoculation 3-5 stem section;
C () adopts the LED light source of 450-680nm in culturing room, with 16 h light 8 h dark for the photoperiod, intensity of illumination is 3000-4000 Lux, and cultivation temperature is 23 ~ 27 DEG C, cultivates 20 ~ 30 days;
D () is collected the Chinese white poplar material in A, B, C, D solid culture medium respectively and is numbered, merge grouping package, storage.
Preferably, in described step (a), A, B, C, D Chinese white poplar adventive root differentiation solid culture medium is:
A, 1/2MS+1.2% sucrose+0.8% agar, pH5.6
B, 1/2MS+0.5 mgL
-1iBA+1.2% sucrose+0.8% agar, pH5.6
C, 1/2MS+4 mgL
-1aroclor 1254+1.2% sucrose+0.8% agar, pH5.6
D, 1/2MS+0.5mg L
-1iBA+4 mg L
-1aroclor 1254+ 1.2% sucrose+0.8% agar, pH5.6
Cultivate 25 days in described step (b).
The invention has the beneficial effects as follows, solid culture medium composition used is determined, Chinese white poplar stem section directly can be inserted on medium, other servicing units are not fixed, overcome soil constituent complexity, influence factor is not easily determined, water culture needs independent fixture, the drawback of complex operation, plantlet in vitro used selects same clone's sample, well eliminate genetic background difference, and stem section used experiences each vegetative stage from Organ Differentiation, eliminate full plants body and cannot to study in Organ Differentiation and process of growth vegetable material to the limitation of the reaction of exposure.The Chinese white poplar material that the inventive method obtains provides important materials for carrying out deeply gene analysis widely, contributes to finding the gene strong to Polychlorinated biphenyls tolerance.
Accompanying drawing explanation
Fig. 1 is that the situation of taking root of Chinese white poplar stem section in A, B, C, D medium is according to figure;
Fig. 2 is that the Aroclro 1254 of various dose exposes separately and collaborative 0.5mgL
-1iBA exposes and affects Chinese white poplar plantlet in vitro adventive root Biomass Accumulation.
Embodiment
Following 1/2MS is the MS minimal medium that macroelement reduces by half, and belongs to known medium; IBA is indolebutyric acid; Aroclor 1254 is a kind of Polychlorinated biphenyls, the content of sucrose, agar represents by the mass percent of respective shared solid culture medium gross mass, the known preparation method of solid culture medium containing agar is that first adding each constituent is according to quantity made into the aqueous solution, then be heated to boiling, then be cooled to 40 DEG C below by way of agar solidification formation solid culture medium.
Embodiment one: the acquisition methods of Chinese white poplar gene analysis material under Polychlorinated biphenyls exposure condition, comprises the steps:
A () is produced as follows A, B, C, D Chinese white poplar adventive root differentiation solid culture medium:
A, 1/2MS+1.2% sucrose+0.8% agar, pH5.6
B, 1/2MS+0.5 mgL
-1iBA+1.2% sucrose+0.8% agar, pH5.6
C, 1/2MS+4.0 mgL
-1aroclor 1254+1.2% sucrose+0.8% agar, pH5.6
D, 1/2MS+0.5 mg L
-1iBA+4.0mg L
-1aroclor 1254+ 1.2% sucrose+0.8% agar, pH5.6
Above-mentioned medium is respectively loaded in triangular flask, after sealing, at 121 DEG C, carries out sterilizing 16min.
B () selects the plantlet in vitro of Chinese white poplar same clone, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, be inoculated in described A, B, C, D solid culture medium respectively, each triangular flask inoculates 3 stem sections.
C () adopts the LED light source of 600nm in culturing room, this light source can prevent ultraviolet light to the decomposition of Polychlorinated biphenyls, with 16 h light 8 h dark for the photoperiod, intensity of illumination is 3500 Lux, cultivation temperature is 25 DEG C, and cultivate 25 days, the situation of taking root in each triangular flask bottom medium as shown in Figure 1, as seen from Figure 1,4mgL is added separately in C medium
-1aroclor 1254 exposure condition under Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation significantly better than the blank of A medium.0.5mgL is added in addition in Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation and D medium in C medium
-1iBA synergistic effect is basically identical, similar with the situation of not adding aroclor 1254 in B medium, illustrate that the Chinese white poplar material obtained in C, D medium under this culture environment obtains good growth under aroclor 1254 exposure condition, adventive root biomass is higher, can as the material of gene analysis together with Chinese white poplar material in A, B medium.
D () is collected the Chinese white poplar material of A, B, C, D solid culture medium respectively and is numbered, often kind of material indicates corresponding medium, and four kinds of materials are merged grouping package, storage, as one group of gene analysis materials'use.By to the gene analysis of above-mentioned material and contrast, contributing to finding the gene strong to aroclor 1254 tolerance, preparing for developing new variety of plant further.。
Embodiment two: the acquisition methods of Chinese white poplar gene analysis material under Polychlorinated biphenyls exposure condition, comprises the steps:
A () is produced as follows A, B, C, D Chinese white poplar adventive root differentiation solid culture medium:
A, 1/2MS+1.0% sucrose+0.6% agar, pH5.0
B, 1/2MS+0.5 mgL
-1iBA+1.0% sucrose+0.6% agar, pH5.0
C, 1/2MS+2.0 mgL
-1aroclor 1254+1.0% sucrose+0.6% agar, pH5.0
D, 1/2MS+0.5 mg L
-1iBA+2.0mg L
-1aroclor 1254+ 1.0% sucrose+0.6% agar, pH5.0
Above-mentioned medium is respectively loaded in triangular flask, after sealing, at 120 DEG C, carries out sterilizing 18min.
B () selects the plantlet in vitro of Chinese white poplar same clone, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, be inoculated in described A, B, C, D solid culture medium respectively, each triangular flask inoculates 4 stem sections.
C () adopts the LED light source of 450nm in culturing room, this light source can prevent ultraviolet light to the decomposition of Polychlorinated biphenyls, and with 16 h light 8 h dark for the photoperiod, intensity of illumination is 3000 Lux, and cultivation temperature is 27 DEG C, cultivates 20 days.2mgL is added separately in C medium
-1aroclor 1254 exposure condition under Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation significantly better than the blank of A medium.0.5mgL is added in addition in Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation and D medium in C medium
-1iBA synergistic effect is basically identical, similar with the situation of not adding aroclor 1254 in B medium, illustrate that the Chinese white poplar material obtained in C, D medium under this culture environment obtains good growth under aroclor 1254 exposure condition, adventive root biomass is higher, can as the material of gene analysis together with Chinese white poplar material in A, B medium.
D () is collected the Chinese white poplar material of A, B, C, D solid culture medium respectively and is numbered, often kind of material indicates corresponding medium, and four kinds of materials are merged grouping package, storage, as one group of gene analysis materials'use.By to the gene analysis of above-mentioned material and contrast, contributing to finding the gene strong to aroclor 1254 tolerance, preparing for developing new variety of plant further.
Embodiment three: the acquisition methods of Chinese white poplar gene analysis material under Polychlorinated biphenyls exposure condition, comprises the steps:
A () is produced as follows A, B, C, D Chinese white poplar adventive root differentiation solid culture medium:
A, 1/2MS+1.5% sucrose+1.0% agar, pH6.0
B, 1/2MS+0.5 mgL
-1iBA+1.5% sucrose+1.0% agar, pH6.0
C, 1/2MS+8.0 mgL
-1aroclor 1254+1.5% sucrose+1.0% agar, pH6.0
D, 1/2MS+0.5 mg L
-1iBA+8.0mg L
-1aroclor 1254+ 1.5% sucrose+1.0% agar, pH6.0
Above-mentioned medium is respectively loaded in triangular flask, after sealing, at 125 DEG C, carries out sterilizing 15min.
B () selects the plantlet in vitro of Chinese white poplar same clone, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, be inoculated in described A, B, C, D solid culture medium respectively, each triangular flask inoculates 5 stem sections.
C () adopts the LED light source of 680nm in culturing room, this light source can prevent ultraviolet light to the decomposition of Polychlorinated biphenyls, and with 16 h light 8 h dark for the photoperiod, intensity of illumination is 4000 Lux, and cultivation temperature is 23 DEG C, cultivates 30 days.8mgL is added separately in C medium
-1aroclor 1254 exposure condition under Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation significantly better than the blank of A medium.0.5mgL is added in addition in Chinese white poplar plantlet in vitro adventive root rooting rate and adventive root upgrowth situation and D medium in C medium
-1iBA synergistic effect is basically identical, similar with the situation of not adding aroclor 1254 in B medium, illustrate that the Chinese white poplar material obtained in C, D medium under this experimental technique obtains good growth under aroclor 1254 exposure condition, adventive root biomass is higher, can as the material of gene analysis together with Chinese white poplar material in A, B medium.
D () is collected the Chinese white poplar material of A, B, C, D solid culture medium respectively and is numbered, often kind of material indicates corresponding medium, merges grouping package, storage, as one group of gene analysis materials'use through four kinds of materials.By to the gene analysis of above-mentioned material and contrast, contributing to finding the gene strong to aroclor 1254 tolerance, preparing for developing new variety of plant further.
Embodiment four: the cumulative effect of adventive root biomass under different aroclor 1254 reconditioning
Make the solid culture medium that some aroclor 1254 content is different: 1/2MS+1.2% sucrose+0.8% agar+XmgL
-1, pH5.6, X get 2,4,6,8,10,20mgL
-1, obtain aroclor 1254 series; The solid culture medium that aroclor 1254 content of making containing IBA is different again: 1/2MS+1.2% sucrose+0.8% agar+0.5 mg L
-1iBA+XmgL
-1, pH5.6, X get 2,4,6,8,10,20mgL
-1, obtain aroclor 1254+IBA series.Select the plantlet in vitro of the same clone of Chinese white poplar, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, insert in the medium of above-mentioned two series respectively, adopt the LED light source of 600nm in culturing room, with 16 h light 8 h dark for the photoperiod, intensity of illumination is 3500 Lux, cultivation temperature is 25 DEG C, cultivates 25 days, record produce the biomass data of adventive root, then map, as shown in Figure 2.As seen from Figure 2, Chinese white poplar plantlet in vitro adventive root differentiation and process of growth in 2.0 ~ 8.0 mgL
-1the independent exposure of dosage aroclor 1254 and collaborative 0.5mgL
-1iBA exposes, and all promote the accumulation of adventive root biomass, its highest effect dose is 4mgL
-1.
Claims (2)
1. the acquisition methods of Chinese white poplar gene analysis material under Polychlorinated biphenyls exposure condition, comprises the steps:
A () is produced as follows A, B, C, D Chinese white poplar adventive root differentiation solid culture medium, sucrose, agar represent with the mass percent of shared solid culture medium gross mass, and 1/2MS is the MS medium that macroelement reduces by half, and IBA is indolebutyric acid:
A, 1/2MS+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
B, 1/2MS+0.5 mgL
-1iBA+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
C, 1/2MS+2.0 ~ 8.0 mgL
-1aroclor 1254+1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
D, 1/2MS+0.5mg L
-1iBA+2.0 ~ 8.0 mg L
-1aroclor 1254+ 1.0 ~ 1.5% sucrose+0.6 ~ 1.0% agar, pH5.0 ~ 6.0
Above-mentioned medium is respectively loaded in triangular flask, after sealing, carries out sterilizing;
B () selects the plantlet in vitro of Chinese white poplar same clone, to cut under terminal bud the consistent stem section of thickness between second to the 3rd leaf, be inoculated in described A, B, C, D solid culture medium respectively, each triangular flask inoculation 3 ~ 5 stem sections;
C () adopts the LED light source of 450-680nm in culturing room, with 16 h light 8 h dark for the photoperiod, intensity of illumination is 3000-4000 Lux, and cultivation temperature is 23 ~ 27 DEG C, cultivates 20 ~ 30 days;
D () is collected the Chinese white poplar material in A, B, C, D solid culture medium respectively and is numbered, merge grouping package, storage.
2. the acquisition methods of Chinese white poplar gene analysis material under Polychlorinated biphenyls exposure condition as claimed in claim 1, it is characterized in that, in described step (a), A, B, C, D Chinese white poplar adventive root differentiation solid culture medium is:
A, 1/2MS+1.2% sucrose+0.8% agar, pH5.6
B, 1/2MS+0.5 mgL
-1iBA+1.2% sucrose+0.8% agar, pH5.6
C, 1/2MS+4 mgL
-1aroclor 1254+1.2% sucrose+0.8% agar, pH5.6
D, 1/2MS+0.5mg L
-1iBA+4 mg L
-1aroclor 1254+ 1.2% sucrose+0.8% agar, pH5.6
Cultivate 25 days in described step (c).
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| JP2920185B2 (en) * | 1994-05-18 | 1999-07-19 | 日本製紙株式会社 | Plant production method of plants belonging to the sedge section |
| CN101897296B (en) * | 2010-07-15 | 2012-11-21 | 东北林业大学 | Tissue culture rapid propagation method of populus tremula multiplied by P. tremuloides |
| CN102392046B (en) * | 2011-11-28 | 2013-01-09 | 东北林业大学 | Transgenic method of pollen plant of populus simonii x p.nigra |
| CN103583371B (en) * | 2013-11-29 | 2015-03-18 | 黑龙江省林业科学研究所 | Tissue culture method for improved variety of Populus davidiana Dode--P. alba*P. davidian CL. 1333 |
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