CN101302507A - Establishment of genetic transformation method of heavy metals ultra-enriched plant Thlaspi caerulescens and cultivation of transgenic seedling - Google Patents
Establishment of genetic transformation method of heavy metals ultra-enriched plant Thlaspi caerulescens and cultivation of transgenic seedling Download PDFInfo
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Abstract
The invention provides a method of gene transfer mediated by agrobacterium of thlaspi caerulescens with super concentration plant and heavy metal, and a method for culturing a transgenic seedling. The method for culturing transgenic seedling comprises the following steps that: mature seeds of the thlaspi caerulescensafter being sterilized are placed on a seed germination culture medium, transferred, after germination, to a differential medium for culture until cluster buds grow and inoculated in a pre-culture medium for culture; after infected by agrobacterium, is placed in a coculture medium for coculture, transferred to a precursor cell medium until cluster buds proliferate; and then is transferred to a screening medium to differentiate resistance cluster buds; the cluster buds are placed in a basic culture medium to carry out strong seedling culture; the cluster buds are to a rooting medium for primary rootage and then are transferred to Hoagland nutrient solution to undergo water culture for further rootage culture after the cluster buds are transferred to a culture medium for rooting, an elementary rooting culture is carried out; the rooting strong seedling is transferred to a nursery substrate to be transplanted for field culture, thus the mature plant of the transgenic thlaspi caerulescens is obtained. The method has the advantages of operating in four seasons free from outside condition influence, saving seedling land occupation, reducing production cost, and obtaining a large number of hale transgenic seedlings in a short period and satisfying molecule biologic research and production application.
Description
Technical field
The invention belongs to plant biotechnology field; Be particularly related to a kind of agriculture bacillus mediated conversion of heavy metal super-enriched thlaspi caerulescens and the method for cultivation of transgenic seedling.
Technical background
Sky blue penny cress (Thlaspi caerulescens) belongs to the Cruciferae penny cress and belongs to, and is distributed widely in Northern Europe, is the hyperaccumulative plant of a kind of Zn and Cd.Therefore sky blue penny cress has become and has studied plant in the world to one of main plant of the super accumulation phenomenon of heavy metal.Sky blue penny cress claims work with it to the super enrichment of zinc, also be the super accumulation plant of cadmium and nickel simultaneously.Baker and NcGrath discover that when soil contained Zn 444mg/kg, the content of sky blue penny cress overground part Zn can reach 16 times of soil.There is sky blue penny cress that bibliographical information grows in the soil of IA and rich in mineral substances that the cumulative amount of zinc, cadmium, nickel is reached 30 respectively, 000ug Zng
-1D.wt, 14,000ug Cdg
-1D.wt, 4700ug Nig
-1D.wt.Most of plants accumulation Zn show toxicity symptom when reaching 100ppm, and sky blue penny cress can accumulate Zn and reaches 26, and 000ppm does not but show any injured symptom (Brown et al, 1995).Along with the continuous accumulation of relevant plant heavy metal accumulation phenomenon data, existing more and more researchers is transferred to related research fields such as biological chemistry, molecular biology and plant physiology in the plant accumulation heavy metal with research emphasis.
Arabidopis thaliana, tobacco etc. are the main model plants that people study new functional gene.Yet existing model plant does not have physiological function heavy metal super-enriched and that tolerance is such.So, advanced biological study technology such as utilization molecular biology, genomics, proteomics develops and new have heavy metal super-enriched and model plant tolerance, the research in this field are standardized, deepization, result of study comparability height reaches the effect of getting twice the result with half the effort.
Though sky blue penny cress possesses heavy metal accumulation and tolerance preferably, and good by domestic and international researchist, and the report relevant with its transgenic research appears at present as yet.The present invention aims to provide a kind of heavy metal super-enriched thlaspi caerulescens transgenic seedling method of cultivation, set up and improve substantially the Agrobacterium-mediated Transformation system of sky blue penny cress, have heavy metal super-enriched model plant effective technical way is provided with tolerance for it becomes.
Summary of the invention
The object of the present invention is to provide a kind of agriculture bacillus mediated method for transformation of heavy metal super-enriched thlaspi caerulescens and the cultivation of setting up transgenic seedling, to satisfy the demand of sky blue penny cress transgenic research and production practical application.
Technical scheme is as follows:
The invention provides the Agrobacterium tumefaciens mediated genetic transformation of heavy metal super-enriched thlaspi caerulescens and the cultural method of transgenic seedling thereof, its step is as follows:
1) sets up first culture thing-sky blue penny cress seedling
Get sky blue penny cress mature seed, carry out surface sterilization, place mercury chloride to soak the back and take out with alcohol; Carry out aseptic water washing again, sterilize, and blot its surface-moisture with filter paper;
Place again on the seed germination substratum and cultivated 7-15 days, get first culture thing-sky blue penny cress seedling; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Described seed germination substratum is the MS substratum;
2) the sky blue penny cress cultivation of bud of growing thickly
Above-mentioned sky blue penny cress seedling is placed differentiation culture 10-14 days of the bud of growing thickly on the division culture medium, differentiate clump shape seedling bud of promptly growing thickly to eustipes part; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described division culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of division culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, and 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid two plant growth hormones, pH=5.8;
Described differentiation minimum medium is the MS substratum;
3) the sky blue penny cress pre-cultivation of bud of growing thickly
The above sky blue penny cress bud of growing thickly is tiled in and cultivated in advance on the pre-culture medium 2-3 days; Its pre-culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described pre-culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of pre-culture medium basal culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, Syringylethanone 100 mmoles and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.2;
Described pre-culture medium basal culture medium is the MS substratum;
4) activation of Agrobacterium
Picking has the single bacterium colony of Agrobacterium of dicotyledons expression vector respectively, is inoculated in 5 milliliters of LB Agrobacterium liquid substratum and cultivates 8 hours; Its culture condition is 28 ℃, 200 rev/mins;
Get the LB Agrobacterium nutrient solution of overnight incubation,, continue to cultivate 5-6 hour, to OD by in 1: 40 dilution proportion to the 20 milliliter LB liquid Agrobacterium substratum
600Be 0.6; Through 5000 rev/mins, centrifugal 5min; Collect thalline,, be diluted to OD with infecting liquid nutrient medium suspension precipitation
600Be 0.15 to infect liquid nutrient medium; Standby;
Described contained component of LB Agrobacterium liquid substratum and proportioning are: contain the Tryptoness of 10 grams in every liter of LB Agrobacterium liquid substratum, the yeast extracts of 5 grams, the sodium-chlor of 10 grams, the kantlex of 50 milligrams Rifampin and 50 milligrams; PH=7.0;
The described liquid nutrient medium that infects is a liquid MS medium, pH=5.2;
5) infect and be total to cultivation through the grow thickly Agrobacterium of bud of pre-incubated sky blue penny cress
The sky blue penny cress of cultivating the in advance 2-3 days bud of growing thickly is placed the above-mentioned OD that contains Agrobacterium
600Be 0.15 infect in the liquid nutrient medium, soak after 5-15 minute and take out; Blot with filter paper and to adhere to its surperficial bacterium liquid, be tiled on the common culture medium, under 25 ℃, secretly cultivated 2 days;
Described contained component of culture medium altogether and proportioning are: every liter contains 300 milligrams of hydrolysis milk-proteins in the substratum basal culture medium altogether, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, Syringylethanone 100 mmoles and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.2;
Described substratum basal culture medium altogether is the MS substratum;
6) preceding cultivation
Sky blue penny cress after step 5) the cultivated bud of growing thickly takes out, and places triangular flask respectively; Behind aseptic water washing 3-5 time, again with the aseptic water washing 3 times that contains Pyocianil 500 mg/litre, each 10 minutes; With the MS liquid nutrient medium flushing that contains Pyocianil 500 mg/litre 1-2 time; The bud of will growing thickly takes out, and uses the filter paper suck dry moisture, dries up in the Bechtop; Cultivated 3-15 days on the substratum before being tiled in; Its culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of culture medium and proportioning are before described: contain 300 milligrams of hydrolysis milk-proteins before every liter in the substratum basal culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, 250 milligrams of Pyocianils and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.8;
The substratum basal culture medium is the MS substratum before described;
7) screening and culturing
To take out through the preceding sky blue penny cress of the cultivating 3-15 days bud of growing thickly, be transferred on the screening culture medium; Culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃; 18 days subcultures once, the test tube bud seedling of growing thickly;
Contained component of described screening culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of screening minimum medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, 250 milligrams of Pyocianils, kantlex 5-50 milligram and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.8;
Described screening minimum medium is the MS substratum;
8) strong seedling culture
To be trimmed to the stem section of 2-3 joint of band through 3 centimetres the test tube bud seedling of growing thickly that is no less than of screening and culturing, and be inoculated in the strong seedling culture base and cultivated 18-35 days, must test-tube plantlet;
Contained component of described strong seedling culture base and proportioning are: contain sucrose 10-40 milligram every liter of strong sprout in the minimum medium, agar 5-10 milligram, inositol 50-300 milligram, 250 milligrams of Pyocianils and kantlex 5-50 milligram, pH=5.8;
Described strong sprout, minimum medium was the MS substratum;
9) root culture
To be inoculated in the root media and cultivate the seedling of to take root 18-25 days through strong seedling culture 3.5-8 centimetre test-tube plantlet; Take out from substratum, the substratum that flush away adheres to is transferred to and carries out water planting continuation root culture in the He Gelante liquid nutrient medium; Its culture condition is: as the seedling upholder, the cystose size should be consistent with water planting notch size, avoids root to see that photoconduction causes green alga and grows as far as possible with cystose; Changed Yi He Gelante liquid nutrient medium in per 5 days; 22-25 ℃, illumination 16 hours/day, humidity 80-85%;
Contained component of described root media and proportioning are: contain sucrose 10-40 milligram in the every liter of minimum medium of taking root, agar 5-10 milligram, inositol 50-300 milligram, 250 milligrams of Pyocianils and kantlex 5-50 milligram, pH=5.8;
The described minimum medium of taking root is the 1/2MS substratum;
10) survival after transplant
Treat to be no less than the taking-up of taking root of 5 centimetres water planting, be transplanted in the matrix of nursery, carry out survival after transplant through water planting root culture to root length; Described nursery matrix is to spend soil and vermiculite to form at 1: 1 by the weight part proportioning; Before the seedling replanting, nursery matrix is drenched with the He Gelante liquid nutrient medium; After the transplanting, cover, keep humidity 90-95%, note ventilating with polyvinyl chloride film; Water spray was 1 time in per 5 days, sprayed 1 time the He Gelante liquid nutrient medium in 14 days; The transplanted seedling field planting was thrown off overlay film after 10 days; Keep humidity 80% in the greenhouse again, temperature 20-30 ℃, spray water every day 3 times, sprayed the He Gelante liquid nutrient medium 1 time in 14 days, sprayed urea 1 time in 21 days, carry out speed and give birth to cultivation, finish the cultivation of sky blue penny cress transgenic seedling.
When the incubation time described in the seed germination described in the described step 1) is cultivated was 5-9 days, effect was preferable.
Described step 3), step 5), pre-culture medium described in step 6) and the step 7), preceding culture medium, sucrose described in culture medium and the screening culture medium is the 25-40 mg/litre altogether, and kinetin is the 1.0-5 mg/litre, and the 6-benzyl purine is the 1.0-4 mg/litre, when naphthylacetic acid was the 0.5-2 mg/litre, effect was preferable.
When incubation time was 5-10 days before described in the described step 6), effect was preferable.
Adopt the inventive method to carry out the culturing process of the conversion method for agrobacterium and the transgenic seedling of stable heavy metal super-enriched thlaspi caerulescens, can obtain a large amount of healthy and strong positive transgenosis seedling in a short time, transformation efficiency is up to 50%, and it is little to be affected by the external environment, and the four seasons all can carry out.Also possesses advantage such as save the occupation of land of growing seedlings, reduce production costs simultaneously.Plant tissue culture technique is to utilize the cell totipotency, adopts a cell mass or a block organization on the plant materials, by artificial condition control, make it to form a large amount of plant, and preserved whole good characters of parent, and stabilization characteristics of genetics.The sky blue penny cress transgenosis of a large amount of stalwartnesses test-tube plantlet that obtains by this method, the speed of growth and biomass do not change, and can satisfy molecular biology research and production application fully.
Embodiment
Further describe the present invention below in conjunction with agriculture bacillus mediated sky blue penny cress genetic transformation embodiment:
Embodiment 1: the agriculture bacillus mediated sky blue efficiently penny cress genetic transformation and the cultivation of transgenic seedling
1, sets up first culture thing-sky blue penny cress seedling
Get sky blue penny cress mature seed, 70% alcohol surface sterilization placed 0.1% mercury chloride to soak 8 minutes after 30 seconds.Aseptic water washing 3-5 time, sterilization filter paper suck dry moisture; Place on the seed germination substratum and cultivated 7 days.Culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃.Described seed germination substratum is the MS substratum;
2, the sky blue penny cress cultivation of bud of growing thickly
Above-mentioned sky blue penny cress seedling is placed differentiation culture 10-14 days of the bud of growing thickly on the division culture medium, differentiate clump shape seedling bud of promptly growing thickly to eustipes part; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described division culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of division culture medium, 5 milligrams of vitamins Bs, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, and 2 milligrams of 6-benzyladenines, 0.5 milligram of naphthylacetic acid, pH=5.8;
Described differentiation minimum medium is the MS substratum;
3, the sky blue penny cress pre-cultivation of bud of growing thickly
The above sky blue penny cress bud of growing thickly is tiled in and cultivated in advance on the pre-culture medium 2-3 days; Its pre-culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described pre-culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of pre-culture medium basal culture medium, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 6 milligrams of Syringylethanone 100 mmoles and 6-benzyladenines, 2 milligrams of naphthylacetic acids, pH=5.2;
Described pre-culture medium basal culture medium is the MS substratum;
4, the activation of Agrobacterium
Picking has single bacterium colony of Agrobacterium of the dicotyledons expression vector of goal gene Bjcat5 respectively, is inoculated in 5 milliliters of LB Agrobacterium liquid substratum; 28 ℃, 200 rev/mins, cultivated 8 hours.
Get the Agrobacterium of overnight incubation,, continue to cultivate 5 hours, to OD by in 1: 40 dilution proportion to the 20 milliliter LB Agrobacterium liquid substratum
600Be 0.6; Through 5000 rev/mins, centrifugal 5min collects thalline; With infecting liquid nutrient medium suspension precipitation, be diluted to OD
600Be that 0.15 back is standby.
Described contained component of LB Agrobacterium liquid substratum and proportioning are: contain the Tryptoness of 10 grams in every liter of LB Agrobacterium liquid substratum, the yeast extracts of 5 grams, the sodium-chlor of 10 grams, the kantlex of 50 milligrams Rifampin and 50 milligrams; PH=7.0;
The described liquid nutrient medium that infects is the MS substratum, pH=5.2;
5, infect and be total to cultivation through the grow thickly Agrobacterium of bud of pre-incubated sky blue penny cress
The sky blue penny cress of cultivating the in advance 2 days bud of growing thickly is placed the above-mentioned OD that contains Agrobacterium
600Be 0.15 infect in the liquid nutrient medium, soak after 8 minutes and take out; Blot with filter paper and to adhere to its surperficial bacterium liquid, be tiled on the common culture medium, under 25 ℃, secretly cultivated 2 days;
Described contained component of culture medium altogether and proportioning are: every liter contains 300 milligrams of hydrolysis milk-proteins in the substratum basal culture medium altogether, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 6 milligrams of Syringylethanone 100 mmoles and 6-benzyladenines, 2 milligrams of naphthylacetic acids, pH=5.2;
Described substratum basal culture medium altogether is the MS substratum;
6, preceding cultivation
Sky blue penny cress after step 5 the cultivated bud of growing thickly takes out, and places triangular flask respectively; Behind aseptic water washing 3-5 time, again with the aseptic water washing 3 times that contains Pyocianil 500 mg/litre, each 10 minutes; With the MS liquid nutrient medium flushing that contains Pyocianil 500 mg/litre 2 times; The bud of will growing thickly takes out, and uses the filter paper suck dry moisture, dries up in the Bechtop; Be tiled on the preceding substratum; Its culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of culture medium and proportioning are before described: contain 300 milligrams of hydrolysis milk-proteins before every liter in the substratum basal culture medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 6 milligrams of 250 milligrams of Pyocianils and 6-benzyladenines, 2 milligrams of naphthylacetic acids, pH=5.8;
The substratum basal culture medium is the MS substratum before described;
7, screening and culturing
To take out through the sky blue penny cress of the preceding cultivation bud of growing thickly, be transferred on the screening culture medium; Culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃; 18 days subcultures once, the test tube bud seedling of growing thickly;
Contained component of described screening culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of screening minimum medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 250 milligrams of Pyocianils, 6 milligrams of 25 milligrams of kantlex and 6-benzyladenines, 2 milligrams of naphthylacetic acids, pH=5.8;
Described screening minimum medium is the MS substratum;
8, strong seedling culture
To be trimmed to the stem section of 2-3 joint of band through 3 centimetres the test tube bud seedling of growing thickly that is no less than of screening and culturing, and be inoculated in the strong seedling culture base and cultivated 18-35 days, must test-tube plantlet;
Contained component of described strong seedling culture base and proportioning are: contain 30 milligrams of sucrose every liter of strong sprout in the minimum medium, 6 milligrams in agar, 200 milligrams of inositols, 25 milligrams of 250 milligrams of Pyocianils and kantlex, pH=5.8;
Described strong sprout, minimum medium was the MS substratum;
9, root culture
To be inoculated in the root media and cultivate the seedling of to take root 18-25 days through strong seedling culture 3.5-8 centimetre test-tube plantlet; Take out from substratum, the substratum that flush away adheres to is transferred to and carries out water planting continuation root culture in the He Gelante liquid nutrient medium; Its culture condition is: as the seedling upholder, the cystose size should be consistent with water planting notch size, avoids root to see that photoconduction causes green alga and grows as far as possible with cystose; Changed Yi He Gelante liquid nutrient medium in per 5 days; 22-25 ℃, illumination 16 hours/day, humidity 80-85%;
Contained component of described root media and proportioning are: contain 30 milligrams of sucrose in the every liter of minimum medium of taking root, 5 milligrams in agar, 100 milligrams of inositols, Pyocianil 250 mg/litre and kantlex 20 mg/litre, pH=5.8;
The described minimum medium of taking root is the 1/2MS substratum;
10, survival after transplant
Treat to be no less than the taking-up of taking root of 5 centimetres water planting, be transplanted in the matrix of nursery, carry out survival after transplant through water planting root culture to root length; Described nursery matrix is to spend soil and vermiculite to form at 1: 1 by the weight part proportioning; Before the seedling replanting, nursery matrix is drenched with the He Gelante liquid nutrient medium; After the transplanting, cover, keep humidity 90-95%, note ventilating with polyvinyl chloride film; Water spray was 1 time in per 5 days, sprayed 1 time the He Gelante liquid nutrient medium in 14 days; The transplanted seedling field planting was thrown off overlay film after 10 days; Keep humidity 80% in the greenhouse again, temperature 20-30 ℃, spray water every day 3 times, sprayed the He Gelante liquid nutrient medium 1 time in 14 days, sprayed urea 1 time in 21 days, carry out speed and give birth to cultivation, finish the cultivation of sky blue penny cress transgenic seedling.
Embodiment 2: the agriculture bacillus mediated sky blue efficiently penny cress genetic transformation and the cultivation of transgenic seedling
1, sets up first culture thing-sky blue penny cress seedling
Get sky blue penny cress mature seed, 70% alcohol surface sterilization placed 0.1% mercury chloride to soak 8 minutes after 30 seconds.Aseptic water washing 3-5 time, sterilization filter paper suck dry moisture; Place on the seed germination substratum and cultivated 7 days.Culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃.Described seed germination substratum is the MS substratum;
2, the sky blue penny cress cultivation of bud of growing thickly
Above-mentioned sky blue penny cress seedling is placed differentiation culture 10-14 days of the bud of growing thickly on the division culture medium, differentiate clump shape seedling bud of promptly growing thickly to eustipes part; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described division culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of division culture medium, vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, and 2 milligrams of 6-benzyladenines, 0.5 milligram of naphthylacetic acid, pH=5.8;
Described differentiation minimum medium is the MS substratum;
3, the sky blue penny cress pre-cultivation of bud of growing thickly
The above sky blue penny cress bud of growing thickly is tiled in and cultivated in advance on the pre-culture medium 2-3 days; Its pre-culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described pre-culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of pre-culture medium basal culture medium, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 5 milligrams of Syringylethanone 100 mmoles and kinetins, 2 milligrams of 6-benzyladenines, 3 milligrams of naphthylacetic acids, pH=5.2;
Described pre-culture medium basal culture medium is the MS substratum;
4, the activation of Agrobacterium
Picking has single bacterium colony of Agrobacterium of the dicotyledons expression vector of goal gene Bjcat5 respectively, is inoculated in 5 milliliters of LB Agrobacterium liquid substratum; 28 ℃, 200 rev/mins, cultivated 8 hours.
Get the Agrobacterium of overnight incubation,, continue to cultivate 5 hours, to OD by in 1: 40 dilution proportion to the 20 milliliter LB Agrobacterium liquid substratum
600Be 0.6; Through 5000 rev/mins, centrifugal 5min collects thalline; With infecting liquid nutrient medium suspension precipitation, be diluted to OD
600Be that 0.15 back is standby.
Described contained component of LB Agrobacterium liquid substratum and proportioning are: contain the Tryptoness of 10 grams in every liter of LB Agrobacterium liquid substratum, the yeast extracts of 5 grams, the sodium-chlor of 10 grams, the kantlex of 50 milligrams Rifampin and 50 milligrams; PH=7.0;
The described liquid nutrient medium that infects is the MS substratum, pH=5.2;
5, infect and be total to cultivation through the grow thickly Agrobacterium of bud of pre-incubated sky blue penny cress
The sky blue penny cress of cultivating the in advance 2 days bud of growing thickly is placed the above-mentioned OD that contains Agrobacterium
600Be 0.15 infect in the liquid nutrient medium, soak after 8 minutes and take out; Blot with filter paper and to adhere to its surperficial bacterium liquid, be tiled on the common culture medium, under 25 ℃, secretly cultivated 2 days;
Described contained component of culture medium altogether and proportioning are: every liter contains 300 milligrams of hydrolysis milk-proteins in the substratum basal culture medium altogether, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 5 milligrams of Syringylethanone 100 mmoles and kinetins, 2 milligrams of 6-benzyladenines, 3 milligrams of naphthylacetic acids, pH=5.2;
Described substratum basal culture medium altogether is the MS substratum;
6, preceding cultivation
Sky blue penny cress after step 5 the cultivated bud of growing thickly takes out, and places triangular flask respectively; Behind aseptic water washing 3-5 time, again with the aseptic water washing 3 times that contains Pyocianil 500 mg/litre, each 10 minutes; With the MS liquid nutrient medium flushing that contains Pyocianil 500 mg/litre 2 times; The bud of will growing thickly takes out, and uses the filter paper suck dry moisture, dries up in the Bechtop; Be tiled on the preceding substratum; Its culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of culture medium and proportioning are before described: contain 300 milligrams of hydrolysis milk-proteins before every liter in the substratum basal culture medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 5 milligrams of 250 milligrams of Pyocianils and kinetins, 2 milligrams of 6-benzyladenines, 3 milligrams of naphthylacetic acids, pH=5.8;
The substratum basal culture medium is the MS substratum before described;
7, screening and culturing
To take out through the sky blue penny cress of the preceding cultivation bud of growing thickly, be transferred on the screening culture medium; Culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃; 18 days subcultures once, the test tube bud seedling of growing thickly;
Contained component of described screening culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of screening minimum medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 250 milligrams of Pyocianils, 5 milligrams of 25 milligrams of kantlex and kinetins, 2 milligrams of 6-benzyladenines, 3 milligrams of naphthylacetic acids, pH=5.8;
Described screening minimum medium is the MS substratum;
8, strong seedling culture
To be trimmed to the stem section of 2-3 joint of band through 3 centimetres the test tube bud seedling of growing thickly that is no less than of screening and culturing, and be inoculated in the strong seedling culture base and cultivated 18-35 days, must test-tube plantlet;
Contained component of described strong seedling culture base and proportioning are: contain 30 milligrams of sucrose every liter of strong sprout in the minimum medium, 6 milligrams in agar, 200 milligrams of inositols, 25 milligrams of 250 milligrams of Pyocianils and kantlex, pH=5.8;
Described strong sprout, minimum medium was the MS substratum;
9, root culture
To be inoculated in the root media and cultivate the seedling of to take root 18-25 days through strong seedling culture 3.5-8 centimetre test-tube plantlet; Take out from substratum, the substratum that flush away adheres to is transferred to and carries out water planting continuation root culture in the He Gelante liquid nutrient medium; Its culture condition is: as the seedling upholder, the cystose size should be consistent with water planting notch size, avoids root to see that photoconduction causes green alga and grows as far as possible with cystose; Changed Yi He Gelante liquid nutrient medium in per 5 days; 22-25 ℃, illumination 16 hours/day, humidity 80-85%;
Contained component of described root media and proportioning are: contain 30 milligrams of sucrose in the every liter of minimum medium of taking root, 5 milligrams in agar, 100 milligrams of inositols, Pyocianil 250 mg/litre and kantlex 20 mg/litre, pH=5.8;
The described minimum medium of taking root is the 1/2MS substratum;
10, survival after transplant
Treat to be no less than the taking-up of taking root of 5 centimetres water planting, be transplanted in the matrix of nursery, carry out survival after transplant through water planting root culture to root length; Described nursery matrix is to spend soil and vermiculite to form at 1: 1 by the weight part proportioning; Before the seedling replanting, nursery matrix is drenched with the He Gelante liquid nutrient medium; After the transplanting, cover, keep humidity 90-95%, note ventilating with polyvinyl chloride film; Water spray was 1 time in per 5 days, sprayed 1 time the He Gelante liquid nutrient medium in 14 days; The transplanted seedling field planting was thrown off overlay film after 10 days; Keep humidity 80% in the greenhouse again, temperature 20-30 ℃, spray water every day 3 times, sprayed the He Gelante liquid nutrient medium 1 time in 14 days, sprayed urea 1 time in 21 days, carry out speed and give birth to cultivation, finish the cultivation of sky blue penny cress transgenic seedling.
Embodiment 3: the agriculture bacillus mediated sky blue penny cress genetic transformation and the cultivation of transgenic seedling
1, sets up first culture thing-sky blue penny cress seedling
Get sky blue penny cress mature seed, 70% alcohol surface sterilization placed 0.1% mercury chloride to soak 8 minutes after 30 seconds.Aseptic water washing 3-5 time, sterilization filter paper suck dry moisture; Place on the seed germination substratum and cultivated 7 days.Culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃.Described seed germination substratum is the MS substratum;
2, the sky blue penny cress cultivation of bud of growing thickly
Above-mentioned sky blue penny cress seedling is placed differentiation culture 10-14 days of the bud of growing thickly on the division culture medium, differentiate clump shape seedling bud of promptly growing thickly to eustipes part; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described division culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of division culture medium, vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, and 2 milligrams of 6-benzyladenines, 0.5 milligram of naphthylacetic acid, pH=5.8;
Described differentiation minimum medium is the MS substratum;
3, the sky blue penny cress pre-cultivation of bud of growing thickly
The above sky blue penny cress bud of growing thickly is tiled in and cultivated in advance on the pre-culture medium 2-3 days; Its pre-culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described pre-culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of pre-culture medium basal culture medium, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 1 milligram of Syringylethanone 100 mmoles and 6-benzyladenine, 0.2 milligram of naphthylacetic acid, pH=5.2;
Described pre-culture medium basal culture medium is the MS substratum;
4, the activation of Agrobacterium
Picking has single bacterium colony of Agrobacterium of the dicotyledons expression vector of goal gene Bjcat5 respectively, is inoculated in 5 milliliters of LB Agrobacterium liquid substratum; 28 ℃, 200 rev/mins, cultivated 8 hours.
Get the Agrobacterium of overnight incubation,, continue to cultivate 5 hours, to OD by in 1: 40 dilution proportion to the 20 milliliter LB Agrobacterium liquid substratum
600Be 0.6; Through 5000 rev/mins, centrifugal 5min collects thalline; With infecting liquid nutrient medium suspension precipitation, be diluted to OD
600Be that 0.15 back is standby.
Described contained component of LB Agrobacterium liquid substratum and proportioning are: contain the Tryptoness of 10 grams in every liter of LB Agrobacterium liquid substratum, the yeast extracts of 5 grams, the sodium-chlor of 10 grams, the kantlex of 50 milligrams Rifampin and 50 milligrams; PH=7.0;
The described liquid nutrient medium that infects is the MS substratum, pH=5.2;
5, infect and be total to cultivation through the grow thickly Agrobacterium of bud of pre-incubated sky blue penny cress
The sky blue penny cress of cultivating the in advance 2 days bud of growing thickly is placed the above-mentioned OD that contains Agrobacterium
600Be 0.15 infect in the liquid nutrient medium, soak after 8 minutes and take out; Blot with filter paper and to adhere to its surperficial bacterium liquid, be tiled on the common culture medium, under 25 ℃, secretly cultivated 2 days;
Described contained component of culture medium altogether and proportioning are: every liter contains 300 milligrams of hydrolysis milk-proteins in the substratum basal culture medium altogether, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 1 milligram of Syringylethanone 100 mmoles and 6-benzyladenine, 0.2 milligram of naphthylacetic acid, pH=5.2;
Described substratum basal culture medium altogether is the MS substratum;
6, preceding cultivation
Sky blue penny cress after step 5 the cultivated bud of growing thickly takes out, and places triangular flask respectively; Behind aseptic water washing 3-5 time, again with the aseptic water washing 3 times that contains Pyocianil 500 mg/litre, each 10 minutes; With the MS liquid nutrient medium flushing that contains Pyocianil 500 mg/litre 2 times; Blade is taken out, use the filter paper suck dry moisture, dry up in the Bechtop; Be tiled on the preceding substratum; Its culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of culture medium and proportioning are before described: contain 300 milligrams of hydrolysis milk-proteins before every liter in the substratum basal culture medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 1 milligram of 250 milligrams of Pyocianils and 6-benzyladenine, 0.2 milligram of naphthylacetic acid, pH=5.8;
The substratum basal culture medium is the MS substratum before described;
7, screening and culturing
To take out through the sky blue penny cress of the preceding cultivation bud of growing thickly, be transferred on the screening culture medium; Culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃; 18 days subcultures once, the test tube bud seedling of growing thickly;
Contained component of described screening culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of screening minimum medium, the vitamin B5 milligram, 200 milligrams of inositols, 30 milligrams of sucrose, 6 milligrams in agar, 250 milligrams of Pyocianils, 1 milligram of 25 milligrams of kantlex and 6-benzyladenine, 0.2 milligram of naphthylacetic acid, pH=5.8;
Described screening minimum medium is the MS substratum;
8, strong seedling culture
To be trimmed to the stem section of 2-3 joint of band through 3 centimetres the test tube bud seedling of growing thickly that is no less than of screening and culturing, and be inoculated in the strong seedling culture base and cultivated 18-35 days, must test-tube plantlet;
Contained component of described strong seedling culture base and proportioning are: contain 30 milligrams of sucrose every liter of strong sprout in the minimum medium, 6 milligrams in agar, 200 milligrams of inositols, 25 milligrams of 250 milligrams of Pyocianils and kantlex, pH=5.8;
Described strong sprout, minimum medium was the MS substratum;
9, root culture
To be inoculated in the root media and cultivate the seedling of to take root 18-25 days through strong seedling culture 3.5-8 centimetre test-tube plantlet; Take out from substratum, the substratum that flush away adheres to is transferred to and carries out water planting continuation root culture in the He Gelante liquid nutrient medium; Its culture condition is: as the seedling upholder, the cystose size should be consistent with water planting notch size, avoids root to see that photoconduction causes green alga and grows as far as possible with cystose; Changed Yi He Gelante liquid nutrient medium in per 5 days; 22-25 ℃, illumination 16 hours/day, humidity 80-85%;
Contained component of described root media and proportioning are: contain 30 milligrams of sucrose in the every liter of minimum medium of taking root, 5 milligrams in agar, 100 milligrams of inositols, Pyocianil 250 mg/litre and kantlex 20 mg/litre, pH=5.8;
The described minimum medium of taking root is the 1/2MS substratum;
10, survival after transplant
Treat to be no less than the taking-up of taking root of 5 centimetres water planting, be transplanted in the matrix of nursery, carry out survival after transplant through water planting root culture to root length; Described nursery matrix is to spend soil and vermiculite to form at 1: 1 by the weight part proportioning; Before the seedling replanting, nursery matrix is drenched with the He Gelante liquid nutrient medium; After the transplanting, cover, keep humidity 90-95%, note ventilating with polyvinyl chloride film; Water spray was 1 time in per 5 days, sprayed 1 time the He Gelante liquid nutrient medium in 14 days; The transplanted seedling field planting was thrown off overlay film after 10 days; Keep humidity 80% in the greenhouse again, temperature 20-30 ℃, spray water every day 3 times, sprayed the He Gelante liquid nutrient medium 1 time in 14 days, sprayed urea 1 time in 21 days, carry out speed and give birth to cultivation, finish the cultivation of sky blue penny cress transgenic seedling.
Claims (4)
1, the cultivation of a kind of foundation of genetic transformation method of heavy metals ultra-enriched plant Thlaspi caerulescens and transgenic seedling, its step is as follows:
1) sets up first culture thing-sky blue penny cress seedling
Get sky blue penny cress mature seed, carry out surface sterilization, afterwards, place mercury chloride to soak the back and take out with alcohol; Carry out aseptic water washing again, sterilize, and blot its surface-moisture with filter paper;
Place again on the seed germination substratum and cultivated 7-15 days, get first culture thing-sky blue penny cress seedling; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Described seed germination substratum is the MS substratum;
2) the sky blue penny cress cultivation of bud of growing thickly
Above-mentioned sky blue penny cress seedling is placed differentiation culture 10-14 days of the bud of growing thickly on the division culture medium, differentiate clump shape seedling bud of promptly growing thickly to eustipes part; Its culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described division culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of division culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, and 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid two plant growth hormones, pH=5.8;
Described differentiation minimum medium is the MS substratum;
3) the sky blue penny cress pre-cultivation of bud of growing thickly
The above sky blue penny cress bud of growing thickly is tiled in and cultivated in advance on the pre-culture medium 2-3 days; Its pre-culture condition is: intensity of illumination is the 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of described pre-culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of pre-culture medium basal culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, Syringylethanone 100 mmoles and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.2;
Described pre-culture medium basal culture medium is the MS substratum;
4) activation of Agrobacterium
Picking has single bacterium colony of Agrobacterium of the dicotyledons expression vector of goal gene Bjcat5 respectively, is inoculated in 5 milliliters of LB Agrobacterium liquid substratum to cultivate 8 hours; Its culture condition is 28 ℃, 200 rev/mins;
Get the LB Agrobacterium nutrient solution of overnight incubation,, continue to cultivate 5-6 hour, to OD by in 1: 40 dilution proportion to the 20 milliliter LB liquid Agrobacterium substratum
600Be 0.6; Through 5000 rev/mins, centrifugal 5min; Collect thalline,, be diluted to OD with infecting liquid nutrient medium suspension precipitation
600Be 0.15 to infect liquid nutrient medium; Standby;
Described contained component of LB Agrobacterium liquid substratum and proportioning are: contain the Tryptoness of 10 grams in every liter of LB Agrobacterium liquid substratum, the yeast extracts of 5 grams, the sodium-chlor of 10 grams, the kantlex of 50 milligrams Rifampin and 50 milligrams; PH=7.0;
The described liquid nutrient medium that infects is the MS substratum, pH=5.2;
5) infect and be total to cultivation through the grow thickly Agrobacterium of bud of pre-incubated sky blue penny cress
The sky blue penny cress of cultivating the in advance 2-3 days bud of growing thickly is placed the above-mentioned OD that contains Agrobacterium
600Be 0.15 infect in the liquid nutrient medium, soak after 5-15 minute and take out; Blot with filter paper and to adhere to its surperficial bacterium liquid, be tiled on the common culture medium, under 25 ℃, secretly cultivated 2 days;
Described contained component of culture medium altogether and proportioning are: every liter contains 300 milligrams of hydrolysis milk-proteins in the substratum basal culture medium altogether, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, Syringylethanone 100 mmoles and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.2;
Described substratum basal culture medium altogether is the MS substratum;
6) preceding cultivation
Sky blue penny cress after step 5) the cultivated bud of growing thickly takes out, and places triangular flask respectively; Behind aseptic water washing 3-5 time, again with the aseptic water washing 3 times that contains Pyocianil 500 mg/litre, each 10 minutes; With the MS liquid nutrient medium flushing that contains Pyocianil 500 mg/litre 1-2 time; The bud of will growing thickly takes out, and uses the filter paper suck dry moisture, dries up in the Bechtop; Cultivated 3-15 days on the substratum before being tiled in; Its culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃;
Contained component of culture medium and proportioning are before described: contain 300 milligrams of hydrolysis milk-proteins before every liter in the substratum basal culture medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, 250 milligrams of Pyocianils and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.8;
The substratum basal culture medium is the MS substratum before described;
7) screening and culturing
To take out through the preceding sky blue penny cress of the cultivating 3-15 days bud of growing thickly, be transferred on the screening culture medium; Culture condition: intensity of illumination 3000-5000 lux, light application time 16 hours/day, temperature 20-25 ℃; 18 days subcultures once, the test tube bud seedling of growing thickly;
Contained component of described screening culture medium and proportioning are: contain 300 milligrams of hydrolysis milk-proteins in every liter of screening minimum medium, vitamins B 0-8 milligram, inositol 50-300 milligram, sucrose 10-40 milligram, agar 5-10 milligram, 250 milligrams of Pyocianils, kantlex 5-50 milligram and kinetin, 6-benzyladenine, each 0.1-8 milligram of naphthylacetic acid three plant growth hormones, pH=5.8;
Described screening minimum medium is the MS substratum;
8) strong seedling culture
To be trimmed to the stem section of 2-3 joint of band through 3 centimetres the test tube bud seedling of growing thickly that is no less than of screening and culturing, and be inoculated in the strong seedling culture base and cultivated 18-35 days, must test-tube plantlet;
Contained component of described strong seedling culture base and proportioning are: contain sucrose 10-40 milligram every liter of strong sprout in the minimum medium, agar 5-10 milligram, inositol 50-300 milligram, 250 milligrams of Pyocianils and kantlex 5-50 milligram, pH=5.8;
Described strong sprout, minimum medium was the MS substratum;
9) root culture
To be inoculated in the root media and cultivate the seedling of to take root 18-25 days through strong seedling culture 3.5-8 centimetre test-tube plantlet; Take out from substratum, the substratum that flush away adheres to is transferred to and carries out water planting continuation root culture in the He Gelante liquid nutrient medium; Its culture condition is: as the seedling upholder, the cystose size should be consistent with water planting notch size, avoids root to see that photoconduction causes green alga and grows as far as possible with cystose; Changed Yi He Gelante liquid nutrient medium in per 5 days; 22-25 ℃, illumination 16 hours/day, humidity 80-85%;
Contained component of described root media and proportioning are: contain sucrose 10-40 milligram in the every liter of minimum medium of taking root, agar 5-10 milligram, inositol 50-300 milligram, 250 milligrams of Pyocianils and kantlex 5-50 milligram, pH=5.8;
The described minimum medium of taking root is the 1/2MS substratum;
10) survival after transplant
Treat to be no less than the taking-up of taking root of 5 centimetres water planting, be transplanted in the matrix of nursery, carry out survival after transplant through water planting root culture to root length; Described nursery matrix is to spend soil and vermiculite to form at 1: 1 by the weight part proportioning; Before the seedling replanting, nursery matrix is drenched with the He Gelante liquid nutrient medium; After the transplanting, cover, keep humidity 90-95%, note ventilating with polyvinyl chloride film; Water spray was 1 time in per 5 days, sprayed 1 time the He Gelante liquid nutrient medium in 14 days; The transplanted seedling field planting was thrown off overlay film after 10 days; Keep humidity 80% in the greenhouse again, temperature 20-30 ℃, spray water every day 3 times, sprayed the He Gelante liquid nutrient medium 1 time in 14 days, sprayed urea 1 time in 21 days, carry out speed and give birth to cultivation, finish the cultivation of sky blue penny cress transgenic seedling.
By the described method of claim 1, it is characterized in that 2, the incubation time described in the seed germination described in the described step 1) is cultivated is 5-9 days.
3, by the described method of claim 1, it is characterized in that, described step 3), step 5), the pre-culture medium described in step 6) and the step 7), preceding culture medium, vitamins B described in culture medium and the screening culture medium is the 3-7 mg/litre altogether, and sucrose is the 25-40 mg/litre, and kinetin is the 0.1-8 mg/litre, 6-benzyladenine is the 1-4 mg/litre, and naphthylacetic acid is the 0.1-8 mg/litre.
By the described method of claim 1, it is characterized in that 4, preceding incubation time is 5-10 days described in the described step 6).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105960969A (en) * | 2016-05-04 | 2016-09-28 | 枞阳县熊天然生态农业有限公司 | Cultivation method for Chinese herbaceous peony |
| CN115836641A (en) * | 2022-12-27 | 2023-03-24 | 广东省农业科学院农业质量标准与监测技术研究所 | Water culture seedling raising method for cruciferae hyper-enriched plants |
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2007
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105960969A (en) * | 2016-05-04 | 2016-09-28 | 枞阳县熊天然生态农业有限公司 | Cultivation method for Chinese herbaceous peony |
| CN115836641A (en) * | 2022-12-27 | 2023-03-24 | 广东省农业科学院农业质量标准与监测技术研究所 | Water culture seedling raising method for cruciferae hyper-enriched plants |
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