CN102392046A - Transgenic method of populus simonii * p.nigra pollen plant - Google Patents
Transgenic method of populus simonii * p.nigra pollen plant Download PDFInfo
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- CN102392046A CN102392046A CN2011103851755A CN201110385175A CN102392046A CN 102392046 A CN102392046 A CN 102392046A CN 2011103851755 A CN2011103851755 A CN 2011103851755A CN 201110385175 A CN201110385175 A CN 201110385175A CN 102392046 A CN102392046 A CN 102392046A
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Abstract
The invention discloses a transgenic method of a populus simonii * p.nigra pollen plant and relates to the transgenic method of the populus simonii * p.nigra pollen plant. The transgenic method is used for solving the problem of low transgenic efficiency of the existing populus simonii * p.nigra pollen plant. The method comprises the following steps: taking a rootless seedling in a stem culture medium, transferring into a rooting culture medium and taking the plant as an explant; taking blades on the explant, respectively cutting the base part and the middle part once, abandoning blade tips and inoculating into a differentiation culture medium; picking agrobacterium tumefaciens EHA 105 carrying exogenous genes, inoculating into an LB (Luria-Bertani) culture medium for culture so as to get bacterial liquid and diluting the bacterial liquid with sterile water; placing the blades after pre-culture into the bacterial liquid for soaking, taking out the blades and sucking away the surplus bacterial liquid; inoculating the blades into the differentiation culture medium for culture; and taking out the blades, sucking dry the water, transferring the blades to a selection culture medium for culture, producing calli, placing the calli into the selection culture medium for culture and producing resistant buds for completion. By adopting the transgenic method, the more resistant buds can be obtained and the transgenic efficiency is high.
Description
Technical field
The present invention relates to a kind of little black poplar pollen plant transgenic method.
Background technology
Willow is important afforestation of China and industrial cut stock seeds.Particularly, the ecological construction of northern China and commodity woods occupy irreplaceable status in building.The willow conventional breeding cycle is long, and it is slow to take effect, and poor operability.Using gene engineering technique carries out purpose and the operability that the willow breeding has improved breeding, for a tempting new way has been opened up in the willow breeding.But there are significant difference in different genotype willow regeneration efficiency and transformation efficiency, are very important so explore its best transformation system to the different genotype willow.
Little black poplar is main greening of northern China and reproducting tree species, obtained pest-resistant, anti-salt, the little black poplar of transgenic such as antimycotic at present, but transformation efficiency is very low, has at most only obtained 13 transgenic lines (transformation efficiency is 4%).Vigorous splitted cell is the competent cell that agroinfection transforms.Explant is in the appearance that contains inducing cell division peak period on the substratum of exogenous hormone, and the aldehydes matter that is beneficial to wound discharges and accumulation, and T-DNA shifts and is incorporated in the vegetable cell genome.The reason that little black poplar transgene efficiency in the past is low is to infect the back directly to produce indefinite bud, and wound healing is too fast, is unfavorable for that T-DNA shifts to get into cell, cause agroinfection after, transformant quantity is few, transformation efficiency also reduces.
Summary of the invention
The present invention will solve the existing low problem of little black poplar pollen plant transgene efficiency, and a kind of little black poplar pollen plant transgenic method is provided.
The little black poplar pollen plant of the present invention transgenic method; Carry out according to the following steps: one, get the no offspring that the height of cultivating in the substratum of putting forth is 1～2cm; Move into root induction in the root media; When treating seedling length to 5～8cm, the top of getting 2～3cm on the seedling moves in the root media carries out two secondary roots, chooses plant after two secondary roots of height of seedling 4～5cm as explant; Two, get that length is the new life's of 1～3cm blade on the explant of step 1, each partial application in the middle of blade base and blade discards the blade tip position, is inoculated in and cultivates 2～3d on the division culture medium in advance; Three, the picking Agrobacterium EHA105 that carries foreign gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex and cultivates 16～24h; Culture temperature is 28 ℃; Shaking speed is 220r/min; Get bacterium liquid; In bacterium liquid, add the LB substratum contain the 50mg/L kantlex and make 100 times of bacterium liquid dilutions, the OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid then reaches 0.4～0.6, and using sterilized water then is that to be diluted to the OD value be 0.1～0.2 for 0.4～0.6 bacterium liquid with the OD value; It is that 0.1～0.2 bacterium liquid soaks 5～10min that blade after cultivating in advance in the step 2 is put into OD value, takes out blade then and removes unnecessary bacterium liquid with the aseptic filter paper suction; Four, then with blade inoculation on division culture medium, under 25 ℃ of dark culture condition, cultivate 1～2d; Five, take out blade; Be placed on the aseptic thieving paper moisture is blotted; Then blade is moved to and select to cultivate 5～10d in the substratum, produce callus, callus is downcut put into new selection substratum; Produce resistant buds after cultivating 15～20d, promptly accomplish little black poplar pollen plant transgenic method; Wherein the said substratum of putting forth of step 1 is the MH substratum that contains 0.1mg/L NAA and 0.2mg/L 6-BA; The said root media of step 1 is the MH substratum that contains 0.2mg/LIBA; The described division culture medium of step 2 and step 4 is the MS substratum that contains 0.1mg/L NAA and 0.5mg/L6-BA; The described selection substratum of step 5 is the MS substratum that contains 0.1mg/L NAA, 0.5mg/L 6-BA, 40mg/L kantlex and 300mg/L CEFOTAXIME SODIUM STERILE.
Existing little black poplar transformation system is direct induction of resistance bud from the wound, and transformation efficiency is low.The present invention infects the back earlier through evoked callus, and then the induction of resistance bud, has improved transformation efficiency greatly.The inventive method is simple to operate; Be easy to grasp; Can effectively improve the genetic transformation efficiency of little black poplar pollen plant, use the Agrobacterium of carrying comospore poplar AP2 gene to infect and to obtain about 70 resistant budses, use the Agrobacterium of carrying Chinese tamarisk bZIP gene to infect and to obtain about 100 resistant budses.Present method is specially adapted to the genetic transformation of little black poplar pollen plant.
Description of drawings
The callus photo of Fig. 1 for forming in embodiment 11 step 5; Fig. 2 puts into callus for embodiment 11 photo of new selection substratum; Fig. 3 is the resistant buds photo that embodiment 11 forms.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the little black poplar pollen plant of this embodiment transgenic method; Carry out according to the following steps: one, get the no offspring that the height of cultivating in the substratum of putting forth is 1～2cm; Move into root induction in the root media; When treating seedling length to 5～8cm, the top of getting 2～3cm on the seedling moves in the root media carries out two secondary roots, chooses plant after two secondary roots of height of seedling 4～5cm as explant; Two, get that length is the new life's of 1～3cm blade on the explant of step 1, each partial application in the middle of blade base and blade discards the blade tip position, is inoculated in and cultivates 2～3d on the division culture medium in advance; Three, the picking Agrobacterium EHA105 that carries foreign gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex and cultivates 16～24h; Culture temperature is 28 ℃; Shaking speed is 220r/min; Get bacterium liquid; In bacterium liquid, add the LB substratum contain the 50mg/L kantlex and make 100 times of bacterium liquid dilutions, the OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid then reaches 0.4～0.6, and using sterilized water then is that to be diluted to the OD value be 0.1～0.2 for 0.4～0.6 bacterium liquid with the OD value; It is that 0.1～0.2 bacterium liquid soaks 5～10min that blade after cultivating in advance in the step 2 is put into OD value, takes out blade then and removes unnecessary bacterium liquid with the aseptic filter paper suction; Four, then with blade inoculation on division culture medium, under 25 ℃ of dark culture condition, cultivate 1～2d; Five, take out blade; Be placed on the aseptic thieving paper moisture is blotted; Then blade is moved to and select to cultivate 5～10d in the substratum, produce callus, callus is downcut put into new selection substratum; Produce resistant buds after cultivating 15～20d, promptly accomplish little black poplar pollen plant transgenic method; Wherein the said substratum of putting forth of step 1 is the MH substratum that contains 0.1mg/L NAA and 0.2mg/L 6-BA; The said root media of step 1 is the MH substratum that contains 0.2mg/L IBA; The described division culture medium of step 2 and step 4 is the MS substratum that contains 0.1mg/L NAA and 0.5mg/L 6-BA; The described selection substratum of step 5 is the MS substratum that contains 0.1mg/LNAA, 0.5mg/L 6-BA, 40mg/L kantlex and 300mg/L CEFOTAXIME SODIUM STERILE.
Carry the preparation method of the Agrobacterium EHA105 of foreign gene in the step 3: be to change among the Agrobacterium EHA105 through the plant expression vector that the triparental mating method will carry foreign gene comospore poplar AP2 gene or Chinese tamarisk bZIP gene.Agrobacterium EHA105 obtains for buying.
NAA is a naphthylacetic acid; 6-BA is a 6-benzyladenine; IBA is an indolebutyric acid.
Existing little black poplar transformation system is direct induction of resistance bud from the wound, and transformation efficiency is low.This embodiment is to infect the back earlier through evoked callus, and then the induction of resistance bud, has improved transformation efficiency greatly.This embodiment method is simple to operate; Be easy to grasp; Can effectively improve the genetic transformation efficiency of little black poplar pollen plant, use the Agrobacterium of carrying comospore poplar AP2 gene to infect and to obtain about 70 resistant budses, use the Agrobacterium of carrying Chinese tamarisk bZIP gene to infect and to obtain about 100 resistant budses.Present method is specially adapted to the genetic transformation of little black poplar pollen plant.
Embodiment two: what this embodiment and embodiment one were different is: get in the step 2 that length is the new life's of 2cm blade on the explant of step 1.Other is identical with embodiment one.
Embodiment three: what this embodiment was different with embodiment one or two is: the foreign gene that step 3 is said carries among the Agrobacterium EHA105 of foreign gene is comospore poplar AP2 gene or Chinese tamarisk bZIP gene.Other is identical with embodiment one or two.
Embodiment four: what this embodiment was different with one of embodiment one to three is: the Agrobacterium EHA105 that picking carries foreign gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex and cultivates 18～22h.Other is identical with one of embodiment one to three.
Embodiment five: what this embodiment was different with one of embodiment one to four is: the OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid in the step 3 reaches 0.5.Other is identical with one of embodiment one to four.
Embodiment six: what this embodiment was different with one of embodiment one to five is: soak 8min in the step 3.Other is identical with one of embodiment one to five.
Embodiment seven: what this embodiment was different with one of embodiment one to six is: in the step 5 blade moved to and select to cultivate 6～9d in the substratum.Other is identical with one of embodiment one to six.
Embodiment eight: what this embodiment was different with one of embodiment one to six is: in the step 5 blade moved to and select to cultivate 7～8d in the substratum.Other is identical with one of embodiment one to six.
Embodiment nine: what this embodiment was different with one of embodiment one to eight is: in the step 5 new selection substratum is put in the callus cutting-out, produced resistant buds behind cultivation 16～19d.Other is identical with one of embodiment one to eight.
Embodiment ten: what this embodiment was different with one of embodiment one to eight is: in the step 5 new selection substratum is put in the callus cutting-out, produced resistant buds behind cultivation 17～18d.Other is identical with one of embodiment one to eight.
Embodiment 11: the little black poplar pollen plant of this embodiment transgenic method; Carry out according to the following steps: one, get the no offspring that the height of cultivating in the substratum of putting forth is 2cm; Move into root induction in the root media; Treat that seedling is long during to 6cm, the top of getting 2cm on the seedling moves in the root media carries out two secondary roots, chooses plant after two secondary roots of height of seedling 4cm as explant; Two, get that length is the new life's of 2cm blade on the explant of step 1, each partial application in the middle of blade base and blade discards the blade tip position, is inoculated in and cultivates 3d on the division culture medium in advance; Three, the picking Agrobacterium EHA105 that carries Chinese tamarisk bZIP gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex and cultivates 20h; Culture temperature is 28 ℃; Shaking speed is 220r/min, gets bacterium liquid, in bacterium liquid, adds the LB substratum that contains the 50mg/L kantlex and makes 100 times of bacterium liquid dilutions; The OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid then reaches 0.4～0.6, and using sterilized water then is that to be diluted to the OD value be 0.2 for 0.5 bacterium liquid with the OD value; It is that 0.2 bacterium liquid soaks 5min that blade after cultivating in advance in the step 2 is put into OD value, takes out blade then and removes unnecessary bacterium liquid with the aseptic filter paper suction; Four, then with blade inoculation on division culture medium, under 25 ℃ of dark culture condition, cultivate 1d; Five, take out blade, be placed on the aseptic thieving paper moisture is blotted, blade is moved to select to cultivate 7d in the substratum then; Produce callus; New selection substratum is put in the callus cutting-out, produced resistant buds behind the cultivation 15d, promptly accomplish little black poplar pollen plant transgenic method; Wherein the said substratum of putting forth of step 1 is the MH substratum that contains 0.1mg/L NAA and 0.2mg/L 6-BA; The said root media of step 1 is the MH substratum that contains 0.2mg/L IBA; The described division culture medium of step 2 and step 4 is the MS substratum that contains 0.1mg/L NAA and 0.5mg/L 6-BA; The described selection substratum of step 5 is the MS substratum that contains 0.1mg/L NAA, 0.5mg/L 6-BA, 40mg/L kantlex and 300mg/L CEFOTAXIME SODIUM STERILE.
Carry the preparation method of the Agrobacterium EHA105 of foreign gene in the step 3: be to change among the Agrobacterium EHA105 through the plant expression vector that the triparental mating method will carry foreign gene Chinese tamarisk bZIP gene.Agrobacterium EHA105 obtains for buying.
60 resistant budses of the final acquisition of this embodiment.
The callus that forms in this embodiment step 5 is as shown in Figure 1, and the photo of callus being put into new selection substratum is as shown in Figure 2, and the resistant buds of formation is as shown in Figure 3.
Claims (10)
1. one kind little black poplar pollen plant transgenic method; It is characterized in that little black poplar pollen plant transgenic method; Carry out according to the following steps: one, get the no offspring that the height of cultivating in the substratum of putting forth is 1～2cm, move into root induction in the root media, when treating seedling length to 5～8cm; The top of getting 2～3cm on the seedling moves in the root media carries out two secondary roots, chooses plant after two secondary roots of height of seedling 4～5cm as explant; Two, get that length is the new life's of 1～3cm blade on the explant of step 1, each partial application in the middle of blade base and blade discards the blade tip position, is inoculated in and cultivates 2～3d on the division culture medium in advance; Three, the picking Agrobacterium EHA105 that carries foreign gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex and cultivates 16～24h; Culture temperature is 28 ℃; Shaking speed is 220r/min; Get bacterium liquid; In bacterium liquid, add the LB substratum contain the 50mg/L kantlex and make 100 times of bacterium liquid dilutions, the OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid then reaches 0.4～0.6, and using sterilized water then is that to be diluted to the OD value be 0.1～0.2 for 0.4～0.6 bacterium liquid with the OD value; It is that 0.1～0.2 bacterium liquid soaks 5～10min that blade after cultivating in advance in the step 2 is put into OD value, takes out blade then and removes unnecessary bacterium liquid with the aseptic filter paper suction; Four, then with blade inoculation on division culture medium, under 25 ℃ of dark culture condition, cultivate 1～2d; Five, take out blade; Be placed on the aseptic thieving paper moisture is blotted; Then blade is moved to and select to cultivate 5～10d in the substratum, produce callus, callus is downcut put into new selection substratum; Produce resistant buds after cultivating 15～20d, promptly accomplish little black poplar pollen plant transgenic method; Wherein the said substratum of putting forth of step 1 is the MH substratum that contains 0.1mg/L NAA and 0.2mg/L 6-BA; The said root media of step 1 is the MH substratum that contains 0.2mg/L IBA; The described division culture medium of step 2 and step 4 is the MS substratum that contains 0.1mg/LNAA and 0.5mg/L 6-BA; The described selection substratum of step 5 is the MS substratum that contains 0.1mg/L NAA, 0.5mg/L 6-BA, 40mg/L kantlex and 300mg/L CEFOTAXIME SODIUM STERILE.
2. a kind of little black poplar pollen plant transgenic method according to claim 1 is characterized in that getting in the step 2 that length is the new life's of 2cm blade on the explant of step 1.
3. a kind of little black poplar pollen plant transgenic method according to claim 1 and 2 is characterized in that the foreign gene among the said Agrobacterium EHA105 that carries foreign gene of step 3 is comospore poplar AP2 gene or Chinese tamarisk bZIP gene.
4. a kind of little black poplar pollen plant transgenic method according to claim 3 is characterized in that Agrobacterium EHA105 that picking carries foreign gene is inoculated in the LB substratum that contains 50mg/L Rifampin and 50mg/L kantlex to cultivate 18～22h.
5. a kind of little black poplar pollen plant transgenic method according to claim 4 is characterized in that in the step 3 that the OD value that under 28 ℃, 220r/min rotating speed, is cultured to bacterium liquid reaches 0.5.
6. a kind of little black poplar pollen plant transgenic method according to claim 5 is characterized in that soaking in the step 3 8min.
7. a kind of little black poplar pollen plant transgenic method according to claim 6 is characterized in that in the step 5 blade moved to and selects to cultivate 6～9d in the substratum.
8. a kind of little black poplar pollen plant transgenic method according to claim 6 is characterized in that in the step 5 blade moved to and selects to cultivate 7～8d in the substratum.
9. a kind of little black poplar pollen plant transgenic method according to claim 7 is characterized in that in the step 5 new selection substratum being put in the callus cutting-out, produces resistant buds behind cultivation 16～19d.
10. a kind of little black poplar pollen plant transgenic method according to claim 7 is characterized in that in the step 5 new selection substratum being put in the callus cutting-out, produces resistant buds behind cultivation 17～18d.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103461123A (en) * | 2013-09-13 | 2013-12-25 | 东北林业大学 | Method for inducing regeneration of adventitious bud of western balsam poplar by histone deacetylase inhibitor |
| CN103583362A (en) * | 2013-11-11 | 2014-02-19 | 东北林业大学 | Method for producing various secondary products by tamarix chinensis tissue culture system |
| CN103875536A (en) * | 2014-04-16 | 2014-06-25 | 河北农业大学 | Method for obtaining populus tomentosa gene analysis material under polychlorinated biphenyl exposure condition |
| CN104561090A (en) * | 2015-01-14 | 2015-04-29 | 北京市农林科学院 | Method for improving agrobacterium tumefacien-mediated genetic transformation efficiency of poplar |
| CN108342411A (en) * | 2018-02-26 | 2018-07-31 | 北京林业大学 | A kind of genetic transforming method of willow |
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| CN103461123A (en) * | 2013-09-13 | 2013-12-25 | 东北林业大学 | Method for inducing regeneration of adventitious bud of western balsam poplar by histone deacetylase inhibitor |
| CN103461123B (en) * | 2013-09-13 | 2015-06-10 | 东北林业大学 | Method for inducing regeneration of adventitious bud of western balsam poplar by histone deacetylase inhibitor |
| CN103583362A (en) * | 2013-11-11 | 2014-02-19 | 东北林业大学 | Method for producing various secondary products by tamarix chinensis tissue culture system |
| CN103583362B (en) * | 2013-11-11 | 2018-07-24 | 东北林业大学 | A method of producing a variety of secondary metabolites using Chinese tamarisk tissue culture system |
| CN103875536A (en) * | 2014-04-16 | 2014-06-25 | 河北农业大学 | Method for obtaining populus tomentosa gene analysis material under polychlorinated biphenyl exposure condition |
| CN104561090A (en) * | 2015-01-14 | 2015-04-29 | 北京市农林科学院 | Method for improving agrobacterium tumefacien-mediated genetic transformation efficiency of poplar |
| CN104561090B (en) * | 2015-01-14 | 2017-09-22 | 北京市农林科学院 | A set of method for improving Agrobacterium tumefaciens mediated willow genetic transformation efficiency |
| CN108342411A (en) * | 2018-02-26 | 2018-07-31 | 北京林业大学 | A kind of genetic transforming method of willow |
| CN108342411B (en) * | 2018-02-26 | 2021-11-23 | 北京林业大学 | Genetic transformation method of poplar |
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