CN102634541A - Agrobacterium tumefaciens gene transformation method of hybrid poplar - Google Patents
Agrobacterium tumefaciens gene transformation method of hybrid poplar Download PDFInfo
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Abstract
The invention discloses an agrobacterium tumefaciens gene transformation method of hybrid poplar. The method comprises the following steps of: (1) pre-culture; (2) strain activation and culture; (3) infection; (4) co-culture; (5) delayed selection; (5) adventitious bud induction; (7) extension culture; and (8) root induction and detection. In the method provided by the invention, NAA and 2ip act together to induce the agrobacterium tumefaciens-mediated stem section or leaf stalk to generate callus; under the effect of cytokinin TDZ, the callus redifferentiation is promoted to form adventitious buds; the bud in a vitrified state extends under the induction of BAP (Benzo A Pyrene), and the form gradually becomes normal; and the resistant regenerated poplar roots in a 1/2MS (Murashige and Skoog) culture medium containing antibiotic hygromycin. The resistant genetically-modified poplar obtained by the method has the advantages of short period and high transformation efficiency, thereby laying firm foundation and theoretical support for the practical breeding of the genetically-modified poplar.
Description
Technical field
The invention belongs to gene engineering technology field.
Background technology
Populus Populus L. has kind more than 100 approximately in the whole world, be distributed widely in Eurasia and NA.China has 62 kinds approximately, is the world maximum country of willow germ plasm resource.Poplar is fast because of its growth, flexibility is strong, of many uses, and not only playing a significant role aspect soil conservation and the maintaining ecological balance, but also having the wide industrial purposes, be the important source material of pulping and paper-making, wood veneer, wrapping material etc.
For a long time; The improvement of willow is mainly carried out through the mode of cross-breeding; But influenced by illumination, moisture, temperature several factors because of it grows, compare, many distinctive biological characteristicses are arranged with other farm crop and gardening plant; Like long growth cycle, complicated mode of reproduction, height heterozygosity etc.; Make traditional willow breeding method can not satisfy the requirement of modern breeding far away, so, utilize tissue culture technique and gene engineering method to satisfy in the practice requirement of willow breeding is had great importance.
After getting into the 1980s, along with the fast development of molecular biology theory and technology, be sign with first strain transgene tobacco appearance of nineteen eighty-three, the clone of functional gene and transgenic technology have been widely applied to the research field of plant resistance to environment stress.Although the forest genetically engineered that with the xylophyta is experiment material is bigger because of the research difficulty; Progress relatively lags behind; But because growth of poplar speed is fast, be prone to breeding, nuclear gene group (about 420Mb) and chromosome number are less relatively; Be easy to through Agrobacterium-mediated Transformation, make willow become research trees molecular biology, forestry biotechnology and the genomic model plant of trees.
At present, the genetic transforming method of on plant, using mainly contains dna direct transfer method and agrobacterium tumefaciens (Agrobactenium tumefaciens) mediated method.Wherein the latter is the most commonly used, is that application at present is the most extensive and the result is comparatively desirable, the comparatively sophisticated a kind of gene transformation method of technology.It has easy and simple to handle, and foreign gene inserts and is generally single copy or low copy, and integration site is stable; The transfer DNA fragment is clear and definite; It is little to integrate back foreign gene structure variation, and transferable large fragment DNA can directly carry out advantages such as transgenosis with different plant tissues.Be applied to the Study on Genetic Transformation of dicotyledons, existing transgenic plant such as tobacco Arabidopis thaliana tomato corn etc. obtain more than 80% by this method.
Though the genetic transformation to Populus progressively is tending towards deeply at present, these researchs are applied to seeds such as diversiform-leaved poplar, Populus nigra, comospore poplar, populus simonii more, and are less relatively to the research of white poplar group and hybridal clone thereof; And at present used agrobacterium-mediated transformation is to white poplar group and inapplicable.In view of white poplar send seeds to have to adapt to that the habitat is wide, strong stress resistance, indigenous tree is many, the species hybridization ability strong, can extensively cultivate also in the habitat range at half-dried arid zone in continent and continent humid zone etc. and can bring into play bigger unique advantages such as production effect; (P.tremula * P.alba) is an experimental subjects, has set up a kind of agriculture bacillus mediated gene transformation method with white poplar hybridal clone 717-1B4 in this research.
Summary of the invention
The object of the invention provides a kind of gene transfer method of agrobacterium of hybridizing willow.
Technical scheme of the present invention is summarized as follows:
A kind of gene transfer method of agrobacterium of hybridizing willow comprises the steps:
(1) cultivate in advance:
(P.tremula * P.alba) axillary-bud or top-bud places successive propagation on the minimum medium, cultivates 4-6 week to obtain tissue cultured seedling with 717 willows; Cut said tissue cultured seedling 0.8-1.5cm not with the stem section of axillalry bud, draw the 0.1-0.5cm that has that causes wound for 2-3 time or cut said tissue cultured seedling along stem section y direction with blade
2The petiole of blade places M1 ' substratum, cultivates 2-3 days in advance down in 24-26 ℃ of dark condition;
(2) actication of culture and cultivation:
The Agrobacterium of getting-80 ℃ of preservations with the choicest of liquid-transfering gun rifle is inverted the dark 20-28h of cultivation for 27-29 ℃ to containing on the antibiotic YEB solid medium; Agrobacterium after picking is cultivated contains on the antibiotic YEB solid medium at another rules, and is inverted the dark 36-48h of cultivation for 27-29 ℃;
Picking list bacterium colony is secretly cultivated 12-16h for 150-200rpm 27-29 ℃ to the sterile test tube that contains antibiotic 2-4mLYEB liquid nutrient medium; Draw 0.1-1.0mL bacterium liquid to the Erlenmeyer flask that contains antibiotic 50-75mLYEB liquid nutrient medium, 150-200rpm 27-29 ℃ secretly is cultured to OD
600=0.6-0.8;
(3) infect:
The bacterium liquid that step (2) is obtained is at room temperature, the centrifugal 10-15min of 3500-4000rpm, and abandoning supernatant will precipitate with 50-75mL M liquid resuspendedly, and 24-26 ℃, 80-100rpm activation 0.5-1h obtains infecting liquid; In 30-50: the ratio of 25mL will be put into the said liquid that infects through pre-incubated stem section of step (1) or blade-carrying petiole, and 80-100rpm infects 30-60min under the 24-26 ℃ of condition;
(4) cultivate altogether:
From infect liquid, take out stem section or blade-carrying petiole, place the liquid that infects that blots remained on surface on the aseptic filter paper, be placed on the M1 solid medium, 24-26 ℃, cultivated altogether under the dark condition 2-3 days;
(5) postpone to select:
Take out stem section or blade-carrying petiole that step (4) is cultivated altogether; Earlier under 180-220rpm, wash 4-5min with deionized water; Wash 2-3 time, then use the cephamycin-deionized water solution washing 4-5min of concentration, wash 2-3 time as 350-500mg/L; Postpone to select in the substratum with blotting surface-moisture on the aseptic filter paper and being transferred to CIM; Secretly cultivated 10-11 days for 24-26 ℃, take out to change and select to continue on the substratum 24-26 ℃ of dark the cultivation 10-11 days, obtain having the stem section or the petiole of callus to new CIM delay;
(6) evoking adventive bud:
Said stem section or the petiole that has callus is transferred in the SIMa screening culture medium, cultivates under the illumination condition, changed a subculture in every 15-20 days; Wait to grow green callus 0.2-0.5cm
2, take out in green callus to the fresh SIMa screening culture medium and cultivate, treat that green callus surface begins to grow indefinite bud; The green callus that will have indefinite bud is transferred in the tissue culture bottle that contains the SIMb screening culture medium, grows to 1-2cm up to indefinite bud;
(7) elongation is cultivated:
Cutting leaves the indefinite bud that is the vitrifying state of a small amount of callus, is transferred in the tissue culture bottle that contains the SEM screening culture medium and extends cultivation, cultivates 2-4 week, obtains phenotype and is tending towards normal bud;
(8) root induction and detection:
Get the bud that step (7) obtains, cut the bottom and expand the position, change in the RM substratum, root induction, the clip blade extracts genomic dna from the growing poplar again of having taken root, and carries out full genome PCR and detect, the gene transformation of checking Agrobacterium the hybridization willow.
Consisting of of M1 ' substratum described in the step (1): MS salt 4.4g, sucrose 20-30g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.6-5.8, agar 7-7.2g adds water to 1L.
When Agrobacterium is C58/pMP90/pH7GWIWG2 (I) described in the step (2); Said microbiotic is: Rifampin (Rifampicin), qingfengmeisu qiong (Gentamicin) and spectinomycin (spectinomycin), the concentration of Rifampin is that the concentration of 25-50mg/L, qingfengmeisu qiong is that the concentration of 20-30mg/L, spectinomycin is 30-50mg/L in YEB solid medium or YEB liquid nutrient medium.
Consisting of of M liquid described in the step (3): MS salt 4.4g, sucrose 20-30g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.6-5.8, Syringylethanone 19.86mg adds water to 1L.
Consisting of of M1 solid medium described in the step (4): MS salt 4.4g, sucrose 20-30g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.6-5.8, Syringylethanone 19.86mg, agar 7-7.2g adds water to 1L.
CIM described in the step (5) postpones to select consisting of of substratum: MS salt 4.4g, and sucrose 20-30g, growth hormone NAA1.86mg, pH=5.6-5.8, phytokinin 2ip 1.02mg, cephamycin 350-500mg, agar 7-7.2g adds water to 1L.
Consisting of of SIMa screening culture medium described in the step (6): MS salt 4.4g, sucrose 20-30g, phytokinin TDZ0.05mg; PH=5.6-5.8, cephamycin 350-500mg, HYG 10mg; Agar 7-7.2g adds water to 1L, and the culture condition of cultivating under the said illumination condition is: culture temperature 22-26 ℃; Illumination 1500-2000lux, periodicity of illumination: illumination/dark is 16h/8h.
Consisting of of SIMb screening culture medium described in the step (6): MS salt 4.4g, sucrose 20-30g, phytokinin TDZ0.0022mg, cephamycin 350-500mg, HYG 10mg, pH=5.6-5.8, agar 7-7.2g adds water to 1L.
Consisting of of SEM screening culture medium described in the step (7): MS salt 4.4g, sucrose 20-30g, phytokinin BAP0.05mg, cephamycin 350-500mg, HYG 10mg, pH=5.6-5.8, agar 7-7.2g adds water to 1L.
Consisting of of RM substratum described in the step (8): MS salt 2.2g, sucrose 20-30g, cephamycin 350-500mg, HYG 10mg, pH=5.6-5.8, agar 7-7.2g adds water to 1L.
The invention provides a kind of method that willow transforms that is used to efficiently hybridize; NAA and 2ip combined action are induced through agriculture bacillus mediated stem section or petiole and are produced callus; Under phytokinin TDZ effect, impel callus to be differentiated to form indefinite bud again, the young shoot that is the vitrifying state is induced elongation down at BAP; Form is tending towards normally gradually, and the growing poplar again with resistance is taken root in containing the 1/2MS substratum of antibiotic hygromycin.The resistant transgenic willow that present method obtains, the cycle is short, and transformation efficiency is high, supports with theoretical for transgenic poplar breeding in practice provides solid basis.The present invention is the seed selection hybridization new variety such as poplar is degeneration-resistant, pest-resistant that make new advances at short notice, for the genetic improvement of forest provides a new way.
Description of drawings
Fig. 1 time of infection is to the influence of 717 willow transformation efficiencies
The concentration of Fig. 2 Syringylethanone is to the influence of 717 willow transformation efficiencies
Fig. 3 postpones the influence of select time to 717 willow transformation efficiencies
The full genome PCR of Fig. 4 detected result
Embodiment
Embodiments of the invention are in order to enable those skilled in the art to understand better the present invention, can not to do any restriction to the present invention.
Through specific embodiment the present invention is further described below.
A kind of gene transfer method of agrobacterium of hybridizing willow comprises the steps:
(1) cultivate in advance:
(P.tremula * P.alba) axillalry bud places successive propagation on the minimum medium, cultivates 4 weeks to obtain tissue cultured seedling with 717 willows; Cut tissue cultured seedling 0.8-1.5cm not with the stem section of axillalry bud, draw 2 times with blade along stem section y direction and cause wound to place M1 ' substratum, cultivated 2 days in advance down in 24 ℃ of dark conditions;
(2) actication of culture and cultivation:
The Agrobacterium C58/pMP90/pH7GWIWG2 (I) that gets-80 ℃ of preservations with the choicest of liquid-transfering gun rifle is inverted the dark 20h of cultivation for 27 ℃ to the YEB solid medium that contains Rifampin, qingfengmeisu qiong and spectinomycin; Agrobacterium after picking is cultivated another contain with the same antibiotic YEB solid medium in front on rule, be inverted the dark 48h of cultivation for 27 ℃;
Picking list bacterium colony to the sterile test tube that contains with the same microbiotic 2mLYEB liquid nutrient medium in front, 27 ℃ of dark 16h that cultivate of 150rpm; Draw 0.1mL bacterium liquid to the Erlenmeyer flask that contains with the same antibiotic 60mLYEB liquid nutrient medium in front, 150rpm secretly is cultured to OD for 27 ℃
600=0.7;
The concentration of Rifampin is that the concentration of 50mg/L, qingfengmeisu qiong is that the concentration of 30mg/L, spectinomycin is 30mg/L in YEB solid medium or YEB liquid nutrient medium;
(3) infect:
The bacterium liquid that step (2) is obtained is at room temperature, the centrifugal 15min of 3500rpm, and abandoning supernatant will precipitate with 60mL M liquid resuspendedly, and 24 ℃, 90rpm activation 30min obtains infecting liquid; In 50: the ratio of 25mL will be put into the said liquid that infects through the pre-incubated stem section of step (1), and 90rpm infects 30min under 24 ℃ of conditions;
(4) cultivate altogether:
From infect liquid, take out the stem section, place the liquid that infects that blots remained on surface on the aseptic filter paper, be placed on the M1 solid medium, 24 ℃, cultivated altogether under the dark condition 2 days;
(5) postpone to select:
Take out the stem section that step (4) is cultivated altogether, under 180rpm, wash 5min with deionized water earlier, wash 2 times; Then use the cephamycin-deionized water solution washing 5min of concentration as 500mg/L; Wash 2 times, postpone to select in the substratum 24 ℃ of dark cultivations 11 days with blotting surface-moisture on the aseptic filter paper and being transferred to CIM; Take out to change and select to continue on the substratum 24 ℃ of dark cultivations 10 days, obtain having the stem section of callus to new CIM delay;
(6) evoking adventive bud:
The said stem section that has callus is transferred in the SIMa screening culture medium, cultivates under the illumination condition, per 20 days replacing one subcultures; Wait to grow green callus 0.2-0.5cm
2, take out in green callus to the fresh SIMa screening culture medium and cultivate, treat that green callus surface begins to grow indefinite bud; The green callus that will have indefinite bud is transferred in the tissue culture bottle that contains the SIMb screening culture medium, grows to 1-2cm up to indefinite bud;
(7) elongation is cultivated:
Cutting leaves the indefinite bud that is the vitrifying state of a small amount of callus, is transferred in the tissue culture bottle that contains the SEM screening culture medium and extends cultivation, cultivates for 3 weeks, obtains phenotype and is tending towards normal bud;
(8) root induction and detection:
Get the bud that step (7) obtains, cut the bottom and expand the position, change in the RM substratum; Root induction; The clip blade extracts genomic dna from the growing poplar again of having taken root, and carries out full genome PCR and detect, the gene transformation of checking Agrobacterium hybridization willow (see figure 4).
Minimum medium: 1/2MS, sucrose 25g, pH 5.6, agar 7.2g, surplus is a water.
Consisting of of M1 ' substratum: MS salt 4.4g, sucrose 25g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.6, agar 7.2g adds water to 1L.
Consisting of of M liquid: MS salt 4.4g, sucrose 25g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.6, Syringylethanone 19.86mg adds water to 1L.
Consisting of of M1 solid medium: MS salt 4.4g, sucrose 25g, growth hormone NAA 1.86mg, phytokinin 2ip1.02mg, pH=5.6, Syringylethanone 19.86mg, agar 7.2g adds water to 1L.
CIM postpones to select consisting of of substratum: MS salt 4.4g, and sucrose 25g, growth hormone NAA 1.86mg, pH=5.6, phytokinin 2ip 1.02mg, cephamycin 400mg, agar 7.2g adds water to 1L.
Consisting of of SIMa screening culture medium: MS salt 4.4g, sucrose 25g, phytokinin TDZ 0.05mg; PH=5.6, cephamycin 400mg, HYG 10mg; Agar 7.2g adds water to 1L, and the culture condition of cultivating under the said illumination condition is: 24 ℃ of culture temperature; Illumination 1500lux, periodicity of illumination: illumination/dark is 16h/8h.
Consisting of of SIMb screening culture medium: MS salt 4.4g, sucrose 25g, phytokinin TDZ 0.0022mg, cephamycin 400mg, HYG 10mg, pH=5.6, agar 7.2g adds water to 1L.
Consisting of of SEM screening culture medium: MS salt 4.4g, sucrose 25g, phytokinin BAP 0.05mg, cephamycin 400mg, HYG 10mg, pH=5.6, agar 7.2g adds water to 1L.
Consisting of of RM substratum: MS salt 2.2g, sucrose 25g, cephamycin 400mg, HYG 10mg, pH=5.6, agar 7.2g adds water to 1L.
A kind of gene transfer method of agrobacterium of hybridizing willow comprises the steps:
(1) cultivate in advance:
(P.tremula * P.alba) terminal bud places successive propagation on the minimum medium, cultivates 5 weeks to obtain tissue cultured seedling with 717 willows; Cut the 0.1-0.5cm that has of said tissue cultured seedling
2The petiole of blade places M1 ' substratum, cultivates 3 days in advance down in 26 ℃ of dark conditions;
(2) actication of culture and cultivation:
The Agrobacterium C58/pMP90/pH7GWIWG2 (I) that gets-80 ℃ of preservations with the choicest of liquid-transfering gun rifle on the YEB solid medium of qingfengmeisu qiong and spectinomycin, is inverted the dark 24h of cultivation to containing Rifampin for 28 ℃; Agrobacterium after picking is cultivated another contain with the same antibiotic YEB solid medium in front on rule, be inverted the dark 40h of cultivation for 28 ℃;
Picking list bacterium colony to the sterile test tube that contains with the same antibiotic 3mLYEB liquid nutrient medium in front, 28 ℃ of dark 14h that cultivate of 180rpm; Draw 0.5mL bacterium liquid to the Erlenmeyer flask that contains with the same antibiotic 50mLYEB liquid nutrient medium in front, 180rpm secretly is cultured to OD for 28 ℃
600=0.6;
The concentration of Rifampin is that the concentration of 25mg/L, qingfengmeisu qiong is that the concentration of 25mg/L, spectinomycin is 50mg/L in YEB solid medium or YEB liquid nutrient medium;
(3) infect:
The bacterium liquid that step (2) is obtained is at room temperature, the centrifugal 13min of 3750rpm, and abandoning supernatant will precipitate with 50mL M liquid resuspendedly, and 26 ℃, 80rpm activation 45min obtains infecting liquid; In 40: the ratio of 25mL will be put into the said liquid that infects through the pre-incubated petiole of step (1), and 80rpm infects 45min under 26 ℃ of conditions;
(4) cultivate altogether:
From infect liquid, take out petiole, place the liquid that infects that blots remained on surface on the aseptic filter paper, be placed on the M1 solid medium, 26 ℃, cultivate 60h under the dark condition altogether;
(5) postpone to select:
Take out the petiole that step (4) is cultivated altogether, under 200rpm, wash 4min with deionized water earlier, wash 3 times; Then use the cephamycin-deionized water solution washing 4min of concentration as 350mg/L; Wash 3 times, postpone to select in the substratum 26 ℃ of dark cultivations 10 days with blotting surface-moisture on the aseptic filter paper and being transferred to CIM; Take out to change and select to continue on the substratum 26 ℃ of dark cultivations 11 days, obtain having the petiole of callus to new CIM delay;
(6) evoking adventive bud:
The said petiole that has callus is transferred in the SIMa screening culture medium, cultivates under the illumination condition, per 17 days replacing one subcultures; Wait to grow green callus 0.2-0.5cm
2, take out in green callus to the fresh SIMa screening culture medium and cultivate, treat that green callus surface begins to grow indefinite bud; The green callus that will have indefinite bud is transferred in the tissue culture bottle that contains the SIMb screening culture medium, grows to 1-2cm up to indefinite bud;
(7) elongation is cultivated:
Cutting leaves the indefinite bud that is the vitrifying state of a small amount of callus, is transferred in the tissue culture bottle that contains the SEM screening culture medium and extends cultivation, cultivates for 2 weeks, obtains phenotype and is tending towards normal bud;
(8) root induction and detection:
Get the bud that step (7) obtains, cut the bottom and expand the position, change in the RM substratum, root induction, the clip blade extracts genomic dna from the growing poplar again of having taken root, and carries out full genome PCR and detect, the gene transformation of checking Agrobacterium the hybridization willow.
Minimum medium: 1/2MS, sucrose 20g, pH 5.8, agar 7.1g, surplus is a water.
Consisting of of M1 ' substratum: MS salt 4.4g, sucrose 20g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.8, agar 7.1g adds water to 1L.
Consisting of of M liquid: MS salt 4.4g, sucrose 20g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.8, Syringylethanone 19.86mg adds water to 1L.
Consisting of of M1 solid medium: MS salt 4.4g, sucrose 20g, growth hormone NAA 1.86mg, phytokinin 2ip1.02mg, pH=5.8, Syringylethanone 19.86mg, agar 7.1g adds water to 1L.
CIM postpones to select consisting of of substratum: MS salt 4.4g, and sucrose 20g, growth hormone NAA 1.86mg, pH=5.8, phytokinin 2ip 1.02mg, cephamycin 350mg, agar 7.1g adds water to 1L.
Consisting of of SIMa screening culture medium: MS salt 4.4g, sucrose 20g, phytokinin TDZ 0.05mg; PH=5.8, cephamycin 350mg, HYG 10mg; Agar 7.1g adds water to 1L, and the culture condition of cultivating under the said illumination condition is: 26 ℃ of culture temperature; Illumination 1800lux, periodicity of illumination: illumination/dark is 16h/8h.
Consisting of of SIMb screening culture medium: MS salt 4.4g, sucrose 20g, phytokinin TDZ 0.0022mg, cephamycin 350mg, HYG 10mg, pH=5.8, agar 7.1g adds water to 1L.
Consisting of of SEM screening culture medium: MS salt 4.4g, sucrose 20g, phytokinin BAP 0.05mg, cephamycin 350mg, HYG 10mg, pH=5.8, agar 7.1g adds water to 1L.
Consisting of of RM substratum: MS salt 2.2g, sucrose 20g, cephamycin 350mg, HYG 10mg, pH=5.8, agar 7.1g adds water to 1L.
Embodiment 3
A kind of gene transfer method of agrobacterium of hybridizing willow comprises the steps:
(1) cultivate in advance:
(P.tremula * P.alba) axillalry bud places successive propagation on the minimum medium, cultivates 6 weeks to obtain tissue cultured seedling with 717 willows; Cut said tissue cultured seedling 0.8-1.5cm not with the stem section of axillalry bud, draw 3 times with blade along stem section y direction and cause wound to place M1 ' substratum, cultivated 3 days in advance down in 25 ℃ of dark conditions;
(2) actication of culture and cultivation:
The Agrobacterium C58/pMP90/pH7GWIWG2 (I) that gets-80 ℃ of preservations with the choicest of liquid-transfering gun rifle on the YEB solid medium of qingfengmeisu qiong and spectinomycin, is inverted the dark 28h of cultivation to containing Rifampin for 29 ℃; Agrobacterium after picking is cultivated another contain with the same antibiotic YEB solid medium in front on rule, be inverted the dark 36h of cultivation for 29 ℃;
Picking list bacterium colony to the sterile test tube that contains with the same antibiotic 4mLYEB liquid nutrient medium in front, the 200rpm29 ℃ of dark 12h that cultivates; Draw 1mL bacterium liquid to the Erlenmeyer flask that contains with the same antibiotic 75mLYEB liquid nutrient medium in front, 200rpm secretly is cultured to OD for 29 ℃
600=0.8;
The concentration of Rifampin is that the concentration of 30mg/L, qingfengmeisu qiong is that 20mg/L, spectinomycin concentration are 40mg/L in YEB solid medium or YEB liquid nutrient medium;
(3) infect:
The bacterium liquid that step (2) is obtained is at room temperature, the centrifugal 10min of 4000rpm, and abandoning supernatant will precipitate with 75mL M liquid resuspendedly, and 25 ℃, 100rpm activation 60min obtains infecting liquid; In 30: the ratio of 25mL will be put into the said liquid that infects through the pre-incubated stem section of step (1), and 100rpm infects 60min under 25 ℃ of conditions;
(4) cultivate altogether:
From infect liquid, take out the stem section, place the liquid that infects that blots remained on surface on the aseptic filter paper, be placed on the M1 solid medium, 25 ℃, cultivated altogether under the dark condition 3 days;
(5) postpone to select:
Take out the stem section that step (4) is cultivated altogether, under 220rpm, wash 5min with deionized water earlier, wash 2 times; Then use the cephamycin-deionized water solution washing 5min of concentration as 500mg/L; Wash 2 times, postpone to select in the substratum 25 ℃ of dark cultivations 10 days with blotting surface-moisture on the aseptic filter paper and being transferred to CIM; Take out to change and select to continue on the substratum 25 ℃ of dark cultivations 11 days, obtain having the stem section of callus to new CIM delay;
(6) evoking adventive bud:
The said stem section that has callus is transferred in the SIMa screening culture medium, cultivates under the illumination condition, per 15 days replacing one subcultures; Wait to grow green callus 0.2-0.5cm
2, take out in green callus to the fresh SIMa screening culture medium and cultivate, treat that green callus surface begins to grow indefinite bud; The green callus that will have indefinite bud is transferred in the tissue culture bottle that contains the SIMb screening culture medium, grows to 1-2cm up to indefinite bud;
(7) elongation is cultivated:
Cutting leaves the indefinite bud that is the vitrifying state of a small amount of callus, is transferred in the tissue culture bottle that contains the SEM screening culture medium and extends cultivation, cultivates for 4 weeks, obtains phenotype and is tending towards normal bud;
(8) root induction and detection:
Get the bud that step (7) obtains, cut the bottom and expand the position, change in the RM substratum, root induction, the clip blade extracts genomic dna from the growing poplar again of having taken root, and carries out full genome PCR and detect, the gene transformation of checking Agrobacterium the hybridization willow.
Minimum medium: 1/2MS, sucrose 30g, pH 5.7, agar 7.0g, surplus is a water.
Consisting of of M1 ' substratum: MS salt 4.4g, sucrose 30g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.7, agar 7.0g adds water to 1L.
Consisting of of M liquid: MS salt 4.4g, sucrose 30g, growth hormone NAA 1.86mg, phytokinin 2ip 1.02mg, pH=5.7, Syringylethanone 19.86mg adds water to 1L.
Consisting of of M1 solid medium: MS salt 4.4g, sucrose 30g, growth hormone NAA 1.86mg, phytokinin 2ip1.02mg, pH=5.7, Syringylethanone 19.86mg, agar 7.0g adds water to 1L.
CIM postpones to select consisting of of substratum: MS salt 4.4g, and sucrose 30g, growth hormone NAA 1.86mg, pH=5.7, phytokinin 2ip 1.02mg, cephamycin 500mg, agar 7.0g adds water to 1L.
Consisting of of SIMa screening culture medium: MS salt 4.4g, sucrose 30g, phytokinin TDZ 0.05mg; PH=5.7, cephamycin 500mg, HYG 10mg; Agar 7.0g adds water to 1L, and the culture condition of cultivating under the said illumination condition is: 22 ℃ of culture temperature; Illumination 2000lux, periodicity of illumination: illumination/dark is 16h/8h.
Consisting of of SIMb screening culture medium: MS salt 4.4g, sucrose 30g, phytokinin TDZ 0.0022mg, cephamycin 500mg, HYG 10mg, pH=5.7, agar 7.0g adds water to 1L.
Consisting of of SEM screening culture medium: MS salt 4.4g, sucrose 30g, phytokinin BAP 0.05mg, cephamycin 500mg, HYG 10mg, pH=5.7, agar 7.0g adds water to 1L.
Consisting of of RM substratum: MS salt 2.2g, sucrose 30g, cephamycin 500mg, HYG 10mg, pH=5.7, agar 7.0g adds water to 1L.
Above the related MS salt (Duchefa Biochemie B.V. company) of each embodiment
YEB solid medium among each embodiment: beef extract 5.0g/L, Tryptones 5.0g/L, yeast extract 1.0g/L, sucrose 5.0g/L, MgSO
4.7H
2O 0.5g/L, agar 15g/L; PH=7.0, surplus is a water.
YEB liquid nutrient medium among each embodiment: beef extract 5.0g/L, Tryptones 5.0g/L, yeast extract 1.0g/L, sucrose 5.0g/L, MgSO
4.7H
2O 0.5g/L, pH=7.0, surplus is a water.
Embodiment 4: (P.tremula * P.alba) willow produces the influence of positive resistant buds to the different explants type to 717
This experiment adopts 717 willows not with the stem section of axillalry bud, has 0.1-0.5cm
2The petiole of blade, 1-2cm
2The explant material that infects as Agrobacterium of three kinds of different tissues of blade.Experimental procedure and condition all adopt step and the condition of embodiment 1, and the result shows (table 1), under the identical situation of other conversion condition; The frequency of the positive regeneration bud of anti-Totomycin that different explant materials produces is different; Wherein the transformation efficiency of stem section is the highest, and petiole takes second place, but difference is not remarkable; And the frequency of the regeneration bud that is produced by blade is minimum, does not almost have regeneration bud to produce.Therefore the present invention adopts not with the stem section of axillalry bud, has 0.1-0.5cm
2The petiole of blade is the transformation receptor material as suitable transformation receptor material and do not select prior art blade commonly used for use.
This transformation system that shows among the present invention to be set up is fit to the conversion of stem section and petiole, to blade as the conversion of explant and inapplicable.
The different acceptor materials of table 1 produce the influence of resistance indefinite bud to 717 willows
Embodiment 5: dissimilar Agrobacteriums produces the influence of positive resistant buds to 717 willows
In the conversion of willow, the Agrobacterium of often using has two types on nopaline and octopine type.Many experiment showed, with respect to octopine type Agrobacterium, Cathay poplar group, it is more responsive to nopaline type Agrobacterium to reach the intraspecific cross poplar between the kind of black poplar group.717 used willows of this experiment are the species hybridization poplar of white poplar group, have confirmed that also nopaline type Agrobacterium C58 (pMP90) is higher than octopine type Agrobacterium LBA4404 transformation efficiency.
The dissimilar Agrobacteriums of table 2 produce the influence of positive resistant buds to 717 willows
| The Agrobacterium type | Explant number (individual) | Produce resistant buds explant number (individual) | Transformation efficiency (%) |
| LBA4404/pH7GWIWG2(I) | 60 | 10 | 16.7 |
| C58/pMP90/pH7GWIWG2(I) | 60 | 17 | 28.3 |
The Agrobacterium that the present invention adopts is C58/pMP90/pH7GWIWG2 (I), and C58 has rifampicin resistance, and auxiliary Ti-plasmids pMP90 has the qingfengmeisu qiong resistance; Expression vector pH7GWIWG2 (I) through the Gateway technique construction has the spectinomycin resistance, and the hygromycin phosphotransferase gene on its T-DNA is as the selectable marker gene of transgenic poplar, and proteins encoded has hygromycin resistance.Regrowth with hygromycin resistance carries out full genome PCR with Auele Specific Primer and detects, and the product size is 456bp, and checking purpose fragment changes in 717 willows.
Embodiment 6: incubation time is to the influence of 717 willow transformation efficiencies in advance
In the genetic transformation of plant, pre-incubated effect is the division that promotes cell, and the cell under the splitting status is integrated foreign DNA more easily; Improve transformation efficiency, this experimental result is illustrated in the certain hour section, along with the prolongation of preparatory incubation time; Transformation frequency improves, and when preparatory incubation time was 48h, transformation efficiency was the highest; Be 28.3%, but to handle transformation efficiency difference down not remarkable with 72h, 96h, is 48h so this tests preparatory incubation time the best.
The preparatory preparatory incubation time of incubation time of table 3 is to the influence of 717 willow transformation efficiencies
| Preparatory incubation time (h) | Explant number (individual) | Produce resistant buds explant number (individual) | Transformation efficiency (%) |
| ?0 | 35 | 6 | 17.1 |
| ?12 | 40 | 7 | 17.5 |
| ?24 | 40 | 10 | 25.0 |
| ?48 | 60 | 17 | 28.3 |
| ?72 | 60 | 15 | 25.0 |
| ?96 | 40 | 11 | 27.5 |
Embodiment 7: infect and the altogether incubation time influence that 717 willows produced positive resistant buds
Suitable time of infection is to transform successful key, and time of infection is too short to be unfavorable for conversion, and overlong time makes brownization of explant easily, causes death.Four kinds of different times of infection (Fig. 1) have been adopted in this experiment, and 1/2h is infected in result's demonstration, 1h all can produce higher transformation efficiency, is best time of infection; But along with the prolongation of time of infection, when for example time of infection was 16h, the generation frequency of resistant buds obviously reduced, and is merely 47% of highest frequency.
The common culturing process of Agrobacterium and explant is a most important link in the whole transformation experiment, and the length of incubation time directly influences T-DNA transfer, the quantity of integration and transformant altogether.If incubation time is too short altogether, then Agrobacterium can not fully be infected the otch cell, and transformation efficiency is low; If incubation time is long altogether, then the Agrobacterium growth too much can cause transforming explant death.The best of this experiment (table 4) incubation time altogether is 2d, transforms stem section or petiole and substratum contact position in the petridish this moment and the visible bacterium colony of naked eyes occurs, and the frequency of the hygromycin resistance bud that obtains at last is up to 29.2%.
Table 4 is total to incubation time to producing the influence of positive resistant buds
| Be total to incubation time (d) | Explant number (individual) | Produce resistant buds explant number (individual) | Transformation efficiency (%) |
| ?1 | 40 | 8 | 20 |
| ?2 | 65 | 19 | 29.2 |
| ?3 | 60 | 16 | 26.7 |
| ?4 | 35 | 6 | 17.1 |
| ?5 | 35 | 3 | 8.6 |
Embodiment 8: the inductor Syringylethanone produces the influence of positive resistant buds to 717 willows
Discover that the transfer of agrobacterium tumefaciens T-DNA and integration need vir expression of gene regulation and control in the Ti-plasmids.Some compound of plant injured cell excretory, like Syringylethanone, glycoloyl syringone, glucose, N.F,USP MANNITOL can be induced Agrobacterium Vir district gene to be activated and expressed, and promote T-DNA to shift, and wherein Syringylethanone induces effect best.This experimental study different concns Syringylethanone to regeneration resistant buds influence; Can find out by Fig. 2; The Syringylethanone that in being total to culture medium, adds 50 μ M (9.93mg/L); The frequency of the positive resistant buds that produces is compared with contrast does not have obvious variation, but when the concentration of Syringylethanone was brought up to 100 μ M (19.86mg/L), its transformation efficiency had improved nearly 60% with respect to the transformation efficiency that does not add Syringylethanone.
Embodiment 9: postpone the influence that selection produces positive resistant buds to 717 willows
How screening transformant is the important step in the plant genetic conversion process, in conversion process, takes the effective choice method most important.Morning and evening according to the selective agent adding is divided into system of selection the selection in early stage, postpones to select and the later stage selection.Select early stage is after explant is cultivated altogether, and in Induce aerosor pressures that bring Selection In at the very start, it is the selection on the cell levels, and its advantage is to help the competition of transformant to non-transformed cell, the raising transformation efficiency; Later stage is selected promptly to regenerate earlier afterwards to select, and is meant the agent that when callus induction and differentiation adventitious buds, do not bring Selection In, and when regeneration bud is taken root, screens usually, and it is the selection on the cell levels, but the false positive that this method produces is more, and later stage screening task is heavy; Postpone to select to fall between, be meant to change over to earlier after common cultivation finishes and cultivate for some time in the recovery media and change in the screening culture medium again and cultivate, so not only helped the growth of transformant but also can not produce too much mosaic.As shown in Figure 3, this test mid-early stage selection is unfavorable for obtaining resistant buds, postpones to select the resistant buds acquisition more and adopt, and wherein delay selects the 21d transformation efficiency the highest; Postpone to select 31d, transformation efficiency significantly reduces, and its reason possibly be not have under the condition of selecting to press owing to being in for a long time; The quantity of non-transformed cell and transformant form competition; And, impel the resistance seedling of formation to reduce, thereby reduce transformation efficiency its generation restraining effect.
Claims (10)
1. gene transfer method of agrobacterium of hybridizing willow, its characteristic comprises the steps:
(1) cultivate in advance:
717 willow axillary-bud or top-buds are placed successive propagation on the minimum medium, cultivate and 4-6 week obtain tissue cultured seedling; Cut said tissue cultured seedling 0.8-1.5cm not with the stem section of axillalry bud, draw the 0.1-0.5cm that has that causes wound for 2-3 time or cut said tissue cultured seedling along stem section y direction with blade
2The petiole of blade places M1 ' substratum, cultivates 2-3 days in advance down in 24-26 ℃ of dark condition;
(2) actication of culture and cultivation:
The Agrobacterium of getting-80 ℃ of preservations with the choicest of liquid-transfering gun rifle is inverted the dark 20-28h of cultivation for 27-29 ℃ to containing on the antibiotic YEB solid medium; Agrobacterium after picking is cultivated contains on the antibiotic YEB solid medium at another rules, and is inverted the dark 36-48h of cultivation for 27-29 ℃;
Picking list bacterium colony is secretly cultivated 12-16h for 150-200rpm 27-29 ℃ to the sterile test tube that contains antibiotic 2-4mLYEB liquid nutrient medium; Draw 0.1-1.0mL bacterium liquid to the Erlenmeyer flask that contains antibiotic 50-75mLYEB liquid nutrient medium, 150-200rpm 27-29 ℃ secretly is cultured to OD
600=0.6-0.8;
(3) infect:
The bacterium liquid that step (2) is obtained is at room temperature, the centrifugal 10-15min of 3500-4000rpm, and abandoning supernatant will precipitate with 50-75mL M liquid resuspendedly, and 24-26 ℃, 80-100rpm activation 0.5-1h obtains infecting liquid; In 30-50: the ratio of 25mL will be put into the said liquid that infects through pre-incubated stem section of step (1) or blade-carrying petiole, and 80-100rpm infects 30-60min under the 24-26 ℃ of condition;
(4) cultivate altogether:
From infect liquid, take out stem section or blade-carrying petiole, place the liquid that infects that blots remained on surface on the aseptic filter paper, be placed on the M1 solid medium, 24-26 ℃, cultivated altogether under the dark condition 2-3 days;
(5) postpone to select:
Take out stem section or blade-carrying petiole that step (4) is cultivated altogether; Earlier under 180-220rpm, wash 4-5min with deionized water; Wash 2-3 time, then use the cephamycin-deionized water solution washing 4-5min of concentration, wash 2-3 time as 350-500mg/L; Postpone to select in the substratum with blotting surface-moisture on the aseptic filter paper and being transferred to CIM; Secretly cultivated 10-11 days for 24-26 ℃, take out to change and select to continue on the substratum 24-26 ℃ of dark the cultivation 10-11 days, obtain having the stem section or the petiole of callus to new CIM delay;
(6) evoking adventive bud:
Said stem section or the petiole that has callus is transferred in the SIMa screening culture medium, cultivates under the illumination condition, changed a subculture in every 15-20 days; Wait to grow green callus 0.2-0.5cm
2, take out in green callus to the fresh SIMa screening culture medium and cultivate, treat that green callus surface begins to grow indefinite bud; The green callus that will have indefinite bud is transferred in the tissue culture bottle that contains the SIMb screening culture medium, grows to 1-2cm up to indefinite bud;
(7) elongation is cultivated:
Cutting leaves the indefinite bud that is the vitrifying state of a small amount of callus, is transferred in the tissue culture bottle that contains the SEM screening culture medium and extends cultivation, cultivates 2-4 week, obtains phenotype and is tending towards normal bud;
(8) root induction and detection:
Get the bud that step (7) obtains, cut the bottom and expand the position, change in the RM substratum, root induction, the clip blade extracts genomic dna from the growing poplar again of having taken root, and carries out full genome PCR and detect, the gene transformation of checking Agrobacterium the hybridization willow.
2. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1; It is characterized in that consisting of of M1 ' substratum described in the step (1): MS salt 4.4g, sucrose 20-30g, growth hormone NAA 1.86mg; Phytokinin 2ip1.02mg; PH=5.6-5.8, agar 7-7.2g adds water to 1L.
3. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1; It is characterized in that Agrobacterium described in the step (2) is C58/pMP90/pH7GWIWG2 (I); Said microbiotic is: Rifampin, qingfengmeisu qiong and spectinomycin, the concentration of Rifampin is that the concentration of 25-50mg/L, qingfengmeisu qiong is that the concentration of 20-30mg/L, spectinomycin is 30-50mg/L in YEB solid medium or YEB liquid nutrient medium.
4. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1; It is characterized in that consisting of of M liquid described in the step (3): MS salt 4.4g, sucrose 20-30g, growth hormone NAA 1.86mg; Phytokinin 2ip 1.02mg; PH=5.6-5.8, Syringylethanone 19.86mg adds water to 1L.
5. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1 is characterized in that consisting of of M1 solid medium described in the step (4): MS salt 4.4g, sucrose 20-30g; Growth hormone NAA 1.86mg; Phytokinin 2ip1.02mg, pH=5.6-5.8, Syringylethanone 19.86mg; Agar 7-7.2g adds water to 1L.
6. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1 is characterized in that CIM described in the step (5) postpones to select consisting of of substratum: MS salt 4.4g, sucrose 20-30g; Growth hormone NAA 1.86mg; PH=5.6-5.8, phytokinin 2ip 1.02mg, cephamycin 350-500mg; Agar 7-7.2g adds water to 1L.
7. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1 is characterized in that consisting of of SIMa screening culture medium described in the step (6): MS salt 4.4g, sucrose 20-30g; Phytokinin TDZ 0.05mg, pH=5.6-5.8, cephamycin 350-500mg; HYG 10mg; Agar 7-7.2g adds water to 1L, and the culture condition of cultivating under the said illumination condition is: culture temperature 22-26 ℃; Illumination 1500-2000lux, periodicity of illumination: illumination/dark is 16h/8h.
8. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1 is characterized in that consisting of in SIMb screening culture medium described in the step (6): MS salt 4.4g, sucrose 20-30g; Phytokinin TDZ 0.0022mg; Cephamycin 350-500mg, HYG 10mg, pH=5.6-5.8; Agar 7-7.2g adds water to 1L.
9. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1 is characterized in that consisting of in SEM screening culture medium described in the step (7): MS salt 4.4g, sucrose 20-30g; Phytokinin BAP 0.05mg; Cephamycin 350-500mg, HYG 10mg, pH=5.6-5.8; Agar 7-7.2g adds water to 1L.
10. a kind of gene transfer method of agrobacterium of hybridizing willow according to claim 1; It is characterized in that consisting of: MS salt 2.2g, sucrose 20-30g, cephamycin 350-500mg at RM substratum described in the step (8); HYG 10mg; PH=5.6-5.8, agar 7-7.2g adds water to 1L.
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