CN106755058B - Wheat pollen tube transgenic breeding method - Google Patents
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Abstract
The invention belongs to the technical field of genetic breeding, and particularly relates to an improved wheat pollen tube transgenic breeding method. The method comprises the following steps: transforming expression plasmid containing target gene, obtaining recombinant strain, utilizing glass bead suspension colony method to quickly extract plasmid, utilizing pollen tube channel method to transform receptor wheat, screening and identifying. The wheat pollen tube transgenic method provided by the invention greatly improves the efficiency of obtaining a large number of plasmids and reduces the cost by optimizing the plasmid extraction method, thereby laying a foundation for the large-scale application of the pollen tube channel method in floret crops such as wheat. Compared with the application of the existing pollen tube channel method in wheat transgenic breeding, the method has the advantages of convenient and fast plasmid extraction, low cost, labor and time saving and capability of better improving the working efficiency, thereby having better promotion effect on the cultivation of new wheat varieties.
Description
Technical Field
The invention belongs to the technical field of genetic breeding, and particularly relates to an improved wheat pollen tube transgenic breeding method.
Background
Regarding the passage of pollen tube, Wang Yanjie et al (research on the mechanism of cell embryology in transgenic technology by pollen tube channel method, journal of northwest plant, 2006, 26 (3): 0628-. The pollen tube pathway method is a method in which DNA fragments of a donor organism are allowed to enter a blastocyst along the pathway of a pollen tube at a certain period after self-pollination of a recipient to transform a fertilized egg or cells (an egg cell and an early embryo cell) in front of and behind the fertilized egg into a seed.
Since the pollen tube channel method is created, the pollen tube channel method achieves remarkable performance on breeding of various crops such as corn, rice, cotton and the like, and is a simple and effective way for introducing exogenous DNA or a single gene into a plant, and the cost is low, so the pollen tube channel method is particularly suitable for being used as a new breeding method for vast breeding workers in China and is popularized and developed more rapidly in China.
Regarding the pollen tube pathway method, the following main advantages are generally considered in the prior art:
(1) transformed seeds can be directly obtained, a whole set of artificial culture process such as plant tissue culture or induced regeneration plants is not relied on, chimera is generally not formed, and the homozygosity speed is high;
(2) as the pollen tube channel is a natural common phenomenon, the genetic transformation of exogenous genes is carried out by utilizing the pollen tube channel method, and the pollen tube channel method can be basically applied to any flowering plant and can carry out the transfer of artificially synthesized genes among any species, thereby greatly expanding the source of target genes of genetic engineering and the range of receptor plants;
(3) the operation is simple and economic, and the method is easy to master by conventional breeders and is suitable for large-scale crop genetic transformation; as the transgenic plant is directly obtained, the targeted screening can be carried out aiming at the agronomic character of the transgenic plant while the identification of the transgenic target character and the transgenic molecule is carried out, so the method can be directly combined with transgenic breeding, the efficiency of forming a new variety of transgenic crops is improved, and the breeding period is greatly shortened;
(4) the transformation frequency is high, the effect is good, the variation is wide, and a large amount of valuable germplasm resources are provided for breeding;
(5) the method utilizes the egg cells, fertilized eggs or early embryo cells of the whole plant to carry out transformation, and the directly obtained product is the transgenic plant; therefore, the target character identification of transgenic expression can be completely directly carried out aiming at the phenotype of the target character, thereby avoiding the defect of large-scale selection depending on antibiotics in the identification process of plant tissue culture.
However, the pollen tube channel method also has some obvious defects, mainly including the following points:
(1) the working time for transforming the target plant with the exogenous gene is limited by the natural flowering phase, and the time and process of flowering and fertilization of each plant must be fully understood;
(2) the pollen tube channel method has strong operation experience and needs certain technical exploration and skill;
(3) the method has the advantages of high operation difficulty for the floret crops with single grains, large quantity of plasmids with high concentration, large workload and high cost, and limits the large-scale application of the method in production.
Due to the defects, the large-scale application of the pollen tube channel method in transgenic breeding, particularly in floret plant (such as wheat) breeding is severely limited to a certain extent, the invention systematizes the wheat pollen tube transgenic method, improves the plasmid extraction method, has high plasmid extraction concentration and low cost, and successfully introduces exogenous plasmids into wheat.
Disclosure of Invention
The invention aims to provide an improved wheat pollen tube transgenic breeding method, thereby providing reference and reference for application of a pollen tube channel method in crop molecular breeding.
The detailed technical scheme adopted by the invention is as follows.
A wheat pollen tube transgenic breeding method comprises the following steps:
(1) transferring the expression plasmid containing the target gene into escherichia coli competent cells by adopting a heat shock method, carrying out heat shock transformation, adding a culture solution after transformation, and carrying out resistance screening to obtain a recombinant strain;
(2) the method for rapidly extracting the plasmid by using the glass bead suspension colony method comprises the following specific processes:
firstly, inoculating the recombinant strain obtained by screening in the step (1) on a plate culture medium containing resistant antibiotics for culture;
secondly, adding sterile water and sterilized glass beads on the flat plate, shaking, suspending bacterial colonies by using the glass beads, centrifuging the sterile water containing the bacterial colonies, and keeping precipitated cells;
finally, cracking the precipitated cells, and centrifugally extracting plasmids by using chloroform-isoamylol mixed solution;
(3) the method for transforming the receptor wheat by using the pollen tube channel method specifically comprises the following steps:
before wheat blossoms, ear trimming, castration and bagging are carried out according to a conventional crossbreeding method; after castration is carried out for 2-3 days, artificial pollination is carried out;
cutting off about one third of stigma after wheat pollination is carried out for 1.5-2 h, and then dropwise adding the plasmid extracted in the step (2) on the cut stigma; the dropping amount of the plasmid on each stigma can be specifically designed as follows: 100. mu.g/mL, 10. mu.L;
preferably, the plasmid is dripped on the stigma for 1 hour repeatedly after the plasmid is dripped for the first time;
and (3) dripping plasmids, bagging, marking, continuously culturing, harvesting wheat seeds after natural setting, and further culturing and screening the harvested wheat seeds to obtain corresponding transgenic plants.
The wheat pollen tube transgenic method provided by the invention greatly improves the efficiency of obtaining a large number of plasmids by optimizing the plasmid extraction method, thereby laying a foundation for the large-scale application of the pollen tube channel method in floret crops such as wheat. Compared with the application of the existing pollen tube channel method in wheat transgenic breeding, the method has the advantages of convenient and fast plasmid extraction, low cost, labor and time saving and capability of better improving the working efficiency, thereby having better promotion effect on the cultivation of new wheat varieties.
Drawings
FIG. 1 shows the 100mg/L hygromycin smear selection, with hygromycin resistance shown in the top panel and no hygromycin resistance shown in the bottom panel;
FIG. 2 shows PCR identification of transformed Tri101 gene; wherein Lane M is DNA Marker DL 2000; lanes 1-13 sample; lane 14 pCAMBIA1301S empty vector control; lane 15, positive control; lane 16, negative control; lane 17 blank (water);
FIG. 3 shows GUS staining identification of transformed plants, wherein A is positive plants and B is negative plants.
Detailed Description
The present invention is further illustrated by the following examples. Before describing specific embodiments, a brief description of some materials and test equipment involved in the present invention is provided below.
Biological material:
escherichia coli JM109 was purchased from TaKaRa;
plasmid pCAMBIA1301S, was donated by professor Tujinxing in national center laboratory for crop genetic improvement of university of agriculture in Huazhong; the plasmid vector contains a 35S promoter element, a hygromycin resistance element and a GUS gene expression element, and can be used for screening and identifying positive plants;
yu wheat 18 and Zheng wheat 366, which are commercial wheat varieties;
experimental reagent:
solution I, the components in each liter of solution are: 1M Tris-HCl (pH = 8.0), 50mL, 0.5M EDTA, 20mL, remainder ddH2O, pH =7.5, high temperature sterilization;
solution II, ddH of 0.4M NaOH2ddH of O solution with 2% by mass concentration of SDS2Equal volume of mixed solution of O solution; ddH of 0.4M NaOH2The O solution can be dissolved in 500mL ddH from 8g NaOH2ddH of SDS at 2% mass concentration prepared in O2O solution was dissolved in 500mL ddH by 10g SDS2O is prepared; it is emphasized that solution II is ready for use;
solution III, per liter of solution, was prepared as follows: ddH at 600mL2129.69g of KAc and 100mL of glacial acetic acid were added to O in this order, dissolved and mixed well, then adjusted to pH =4.8 with acetic acid, and then treated with ddH2O to volume 1L, pH = 4.8;
LB culture solution: 10g tryptone, 5g yeast extract, 10g NaCl, double distilled water to 1000mL (15 g agar was added to LB plate medium), autoclaved, and stored at 4 ℃.
Examples
The fusarium graminearum Tri101 gene can encode a trichothecene 3-O-acetyltransferase, and the enzyme can degrade gibberellic disease toxin in an acetyl form, so that the incidence rate of wheat scab is reduced. In this embodiment, the operation of extracting a large amount of plasmids of a plant expression vector (recombinant plasmid pCAMBIA1301S-Tri 101) recombined with a fusarium graminearum Tri101 gene and transforming wheat by a pollen tube channel method is taken as an example, and the wheat pollen tube transgenic breeding method is specifically introduced. The detailed process is described below.
Firstly, integrating a gene (target gene Tri101 gene) segment to be transformed into a plasmid genome, then transferring an expression plasmid containing the target gene into an escherichia coli competent cell by adopting a heat shock method, carrying out heat shock transformation, adding a culture solution after transformation, and carrying out resistance screening to obtain a recombinant strain. The specific process is as follows:
(1) firstly, designing a primer pair, and amplifying by utilizing a PCR (polymerase chain reaction) technology to obtain a Tri101 gene;
the following primer pairs (primers synthesized by Shanghai biological engineering Co., Ltd.) were designed based on the Tri101 gene sequence:
Z-Tri101-F：5′-TCTAGAATGGCTTTCAAGATACAGCT-3′
(5' end TCTAGA sequence is introduced XbaI restriction site),
Z-Tri 101-R: 5'-GGATCCCTAACCAACGTACTGCGCAT-3' (5 ' GGATCC sequence is introduced BamHI site);
25 muL PCR amplification system:
10×Buffer（Mg2+free），2.5 μL；
MgCl2（25mM），2.5 μL；
dNTP（10mM），0.4 μL；
Z-Tri101-F（10µM），1 μL；
Z-Tri101-R（10µM），1 μL；
rTaq DNA polymerase (0.5U/. mu.L), 0.2. mu.L
Tri101 gene DNA (10 ng/muL), 6 muL;
ddH2O，11.4 μL；
amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, extension at 72 ℃ for 1 min, and performing 30 cycles; extension at 72 ℃ for 10min and holding at 10 ℃.
The PCR product was detected by electrophoresis on 1% agarose gel, and the desired DNA fragment was recovered by using a DNA gel recovery kit.
(2) Carrying out enzyme digestion and connection to construct a recombinant plasmid, which specifically comprises the following steps:
using XbaI and BamHI to double-enzyme-cut and recover the target DNA fragment, recovering the fragment of about 1500bp, connecting with the expression vector pCAMBIA1301S which is subjected to the same enzyme cutting and the recovered enzyme cutting product, and connecting overnight at 16 ℃ under the action of T4 ligase;
the enzyme digestion system is designed as follows:
10×Fast Digest buffer，6μL；
pCAMBIA1301S plasmid (or PCR amplification product), 2. mu.g, 20. mu.L;
XbaI，1U/L 、2μL；
BamHI，1U/L 、2μL；
ddH2O，30 μL；
the connection system is designed as follows:
10 XT 4 DNA ligation buffer, 1. mu.L;
tri101 gene fragment, 7 μ L;
pCAMBIA1301S vector, 1 μ L;
t4 DNA ligase, 1. mu.L.
(3) Thermal shock conversion: transforming the connecting product (recombinant expression plasmid containing target gene) into escherichia coli JM109 competent cells by a heat shock method, wherein the specific transformation process comprises the following steps:
adding 2 μ L of plasmid containing target gene expression into 100 μ L of competent cells, mixing, and standing on ice for 10 min;
thermally shocking the cells in a water bath at 42 deg.C for 50 s, rapidly placing on ice for 90 s, and then placing at room temperature for 10 min;
adding 100 μ L LB culture solution preheated to 37 deg.C into the tube, culturing at 37 deg.C for 1h (140 r/min shaking culture), simultaneously placing LB plate at 37 deg.C for incubation for 30 min, then adding 44 μ L IPTG/X-gal (4 μ L IPTG and 40 μ L X-gal), uniformly coating on the surface of LB plate, placing for a while, and allowing it to adsorb and penetrate;
adding the cell/SOC culture solution cultured for 1h at 37 ℃ to the surface of an LB flat plate coated with IPTG/X-gal, and placing for a moment to enable the cell/SOC culture solution to be adsorbed; finally, the plate is cultured for 12-16 h at 37 ℃.
After the culture is finished, extracting plasmids, sequencing the recombined plasmids by the company Limited in Biotechnology engineering (Shanghai), and naming the positive clone with correct sequencing as pCAMBIA1301S-Tri 101.
(II) adopting glass bead suspension colony method to quickly extract plasmid
(1) Preparing cells, specifically:
selecting a single colony of the correctly identified recombinant expression vector pCAMBIA1301-Tri101, inoculating the single colony into a liquid culture medium containing a resistant antibiotic, and performing amplification culture (specifically, firstly inoculating the single colony into 2mL of a culture solution containing LB/Kan (the concentration of the Kan is 0.1mg/mL), culturing at 37 ℃ for 3-4 h, and performing amplification culture in a 50mL centrifuge tube for 4-6 h);
sucking the bacterial liquid after the expanded culture, coating the bacterial liquid on a plate of a solid culture medium containing the resistant antibiotics, and culturing overnight (specifically, sucking 50 mu L of the bacterial liquid after the expanded culture or diluting the bacterial liquid into 200 mu L of the bacterial liquid according to 4 times and coating the bacterial liquid on a new LB/Kan plate culture medium (the diameter of the plate is 15cm, the concentration of Kan is 0.1mg/mL), and culturing overnight (12-16 h) at 37 ℃);
adding sterile water and sterilized glass beads on the plate, shaking, and suspending colonies by using the glass beads (specifically, adding 4mL of sterile water into the culture medium full of colonies (after overnight culture, the colonies can be basically full of the plate culture medium) and then putting the sterilized glass beads into the culture medium, and shaking);
when the bacteria colony remains in the culture medium, sucking out the sterile water containing the bacteria colony by using a pipette gun, placing the sterile water into a centrifuge tube, centrifuging to remove the supernatant, and keeping the precipitate (specifically, placing the sterile water into 4 centrifuge tubes with the volume of 2mL respectively, centrifuging at 12000 g for 5min, removing the supernatant, and keeping the precipitate);
(2) lysing the cells, specifically:
adding 600 muL of solution I into the sediment after centrifugation in the step (1), violently shaking and resuspending the thalli on a shaker, and standing for 5 min;
then adding 600 muL of solution II, slightly reversing for several times, thoroughly mixing until the solution becomes clear, ensuring that the whole inner wall of the centrifuge tube is fully contacted with the solution II in the mixing process, and standing on ice for 5min after mixing;
finally, adding 600 muL of the solution III, slightly reversing for several times, flicking the centrifuge tube to make the solution III uniform when floccules are seen in the mixing process, meanwhile, taking care to make the solution III uniformly dispersed in the viscous cell lysate in the mixing process, and placing the solution III on ice for 5min after uniform mixing;
(3) extracting plasmids, specifically:
centrifuging the solution obtained in the step (2) after the solution is placed on ice (specifically, centrifuging at 4 ℃ for 10min at 12000 g), discarding the precipitate, taking the supernatant into a new 2mL centrifuge tube, adding a chloroform-isoamylol mixed solution with equal volume, uniformly mixing, and placing for 20 min; in the chloroform-isoamyl alcohol mixed solution, by volume ratio, chloroform: isoamyl alcohol = 24: 1;
centrifuging the mixed solution after standing (specifically, centrifuging at 4 ℃ and 12000 g for 10 min), taking the supernatant, placing the supernatant into a new 2mL centrifuge tube added with absolute ethyl alcohol in advance, centrifuging (specifically, centrifuging at 4 ℃ and 12000 g for 10 min), discarding the supernatant, and rinsing the precipitate after centrifuging twice with 75% volume fraction of ethyl alcohol to obtain the extracted plasmid;
the amount of the absolute ethyl alcohol added into the centrifugal tube in advance is 2 times of the volume of the supernatant, and the absolute ethyl alcohol added is precooled absolute ethyl alcohol at the temperature of-20 ℃;
the extracted plasmid was dried, dissolved in 1mL of TE buffer, diluted to a concentration of 100. mu.g/mL after concentration measurement, and stored at-80 ℃ until use.
(III) transformation of acceptor wheat by pollen tube channel method
The method specifically comprises the following steps:
taking the Henan region as an example, at the beginning of 10 months, planting Yumai 18 and Zheng mai 366 in a test field (the test field is located in Yuyang county), and managing according to normal field;
before wheat blossoms, ear trimming, castration and bagging are carried out according to a conventional crossbreeding method; after castration is carried out for 2-3 days, artificial pollination is carried out;
shearing about one third of stigmas after pollination of wheat for 2h, and then dropwise adding the plasmids extracted in the step (II) on the sheared stigmas; the dropping amount of the plasmid on each stigma is 10 mu L and 100 mu g/mL;
repeatedly dripping the plasmid for 1h after the plasmid is firstly dripped on the stigma;
and (3) dripping plasmids, bagging, marking, continuously cultivating, and harvesting wheat seeds after natural fructification.
The harvested wheat seeds are further cultivated and screened to obtain corresponding transgenic plants.
(IV) identification and screening of wheat Positive individuals
And (3) identifying and screening positive single plants of the harvested wheat seeds, wherein the identification and screening mainly comprises primary hygromycin resistance screening, subsequent PCR identification and GUS staining identification, and wheat seeds Yumai 18 (CK) and Zheng mai 366 (CK) which are not subjected to pollen tube transformation are set as controls in the identification process.
In the wheat planting process, the cultivation conditions are as follows:
the photoperiod is 16 hours of light and 8 hours of dark, and the light intensity is 500 mu mol/m2/s，
The temperature in the seedling stage is 20 ℃ under illumination and 15 ℃ under dark;
the temperature in the light of the booting and grouting period is 30 ℃, and the temperature in the dark is 20 ℃.
The specific screening process and results are as follows:
(1) preliminary screening for hygromycin resistance
And (3) smearing the fourth leaf of the wheat seedling transplanted in the greenhouse for 3 weeks with 100mg/L hygromycin, and continuing normal cultivation after smearing. After one week, the leaves of hygromycin-coated seedlings were observed for changes.
The non-transgenic individual plant begins to gradually yellow after being coated with hygromycin leaves, and the yellow area reaches more than 80% after two weeks (14 days); the transgenic positive individual plants are coated with hygromycin leaves and keep green growth all the time.
Wheat seedlings (initially considered to be hygromycin resistant) that remained green in color for all leaves were marked on the wall.
The identification result is shown in figure 1, which initially shows that the method can be used for transforming exogenous DNA into receptor wheat.
(2) PCR identification of target genes
And (2) carrying out leaf DNA extraction on the transgenic wheat obtained by primary screening in the step (1), and then carrying out PCR amplification identification on a target gene Tri 101.
The PCR amplification set up an empty vector (plasmid pCAMBIA 1301S) control and a blank control (water).
The PCR amplification primer sequence, the amplification system and the PCR reaction program refer to the obtaining process of the Tri101 gene in the step (I).
The PCR amplification products were detected by electrophoresis on a 1% agarose gel.
The correlation result is shown in fig. 2, the transgenic plant containing the Tri101 gene can amplify a band of about 1500bp, and the empty vector control, the negative control and the blank control (water) have no target band.
(3) GUS staining identification
Soaking the transgenic wheat seedling leaves positive in PCR identification in GUS dye solution, and dyeing overnight at 37 ℃; and then decolorizing for 2-3 times by using 70% ethanol until the negative control material is white.
The result of microscopic observation is shown in FIG. 3, and the position with significant blue coloration in the image is the GUS expression site. The result shows that the GUS gene is expressed in the wheat plant. The improved pollen tube mediated transgenic method can ensure that the exogenous DNA segment Tri101 is successfully transferred into a receptor plant.
Frequency and efficiency of transformation statistics
The transformation efficiency of each wheat variety was counted, and the results are shown in the following table. Wherein: conversion efficiency = (obtaining T)0Generation PCR positive plant/survival plant) x 100%;
the above results show that positive plants can be obtained in both Yumai 18 and Zheng mai 366 as the receptor wheat varieties by pollen tube mediated wheat transgenic method, and the transformation rates are 0.97% and 1.15% respectively.
Regarding the gene transformation frequency, the transformation frequency of the existing conventional gene gun transgenic method is greatly different due to the difference of varieties and gene shapes, but is generally lower than 1.0%. For example, Wanghai wind successfully introduces ZmalI gene into wheat plant by gene gun-mediated transgenic method, bombards about 3000 young embryos with gene gun to obtain 59 regenerated plants, and identifies 16 positive seedlings by PCR detection, wherein the transformation rate is 0.53% (Proc. of ZmalI gene transfer by gene gun method, proceedings of northwest university of agriculture and forestry, 2011 (8): 90-94); bombarding about 1680 small elytrigia 22 young embryo callus by using a particle gun method to successfully obtain 6 bar gene-transferred positive plants and 4 KN2 gene-transferred positive plants, wherein the transformation rates of the positive plants are 0.36 percent and 0.24 percent respectively (the particle gun method mediates the acquisition and identification of KN2 gene wheat plants, the university of agricultural and forestry science and technology in northwest, 2014, 42 (1): 83-88); the Naojunli and the like successfully introduce HAL1 gene into 3 excellent varieties (lines) of Xiaoyan I, Qianjiao and 9848 in Huang-Huai-Mai region by using a high-efficiency sucrose hyperosmotic gene gun transformation receptor system, and the average transformation efficiency reaches about 0.90% (the establishment of a common wheat gene gun transformation high-efficiency receptor system, the academic newspaper of northwest agriculture and forestry science and technology university, 2007, 35 (7): 117-122).
From the comparison, the transgenic operation method provided by the application has relatively high gene transformation frequency without considering the difference of the gene and the crop variety. Meanwhile, compared with the higher operation cost of the existing gene gun operation method, the operation process is optimized by improving the plasmid extraction operation method, the operation cost is well reduced, the labor and the time are saved, and the working efficiency is well improved. And the natural advantages of the pollen tube mediated transgenic operation method in large-scale crop genetic breeding are added, so that the application has better application value in new wheat variety cultivation.
Claims (4)
1. A method for cultivating a new wheat variety with resistance to gibberellic disease is characterized in that the resistance to gibberellic disease of wheat is improved by transforming Tri101 gene with gene engineering gene, and the method comprises the following steps:
(1) transforming an expression plasmid containing a target gene to obtain a recombinant strain;
the target gene is Tri101 gene; the strain adopts escherichia coli;
(2) the method for extracting the plasmid by using the glass bead suspension colony method comprises the following specific processes:
firstly, inoculating the recombinant strain obtained by screening in the step (1) on a plate culture medium containing resistant antibiotics for culture;
secondly, adding sterile water and sterilized glass beads on the flat plate, shaking, suspending bacterial colonies by using the glass beads, centrifuging the sterile water containing the bacterial colonies, and keeping precipitated cells;
finally, cracking the precipitated cells, and centrifugally extracting plasmids;
(3) the method for transforming the receptor wheat by using the pollen tube channel method specifically comprises the following steps:
before wheat blossoms, ear trimming, castration and bagging are carried out according to a conventional crossbreeding method; after castration is carried out for 2-3 days, artificial pollination is carried out;
shearing part of stigmas after wheat pollination is carried out for 1.5-2 h, and then dropwise adding the plasmids extracted in the step (2) on the sheared stigmas;
and (3) dripping plasmids, bagging, marking, harvesting wheat seeds after natural fructification, further culturing and screening the harvested wheat seeds to obtain corresponding transgenic plants.
2. A method of breeding a new variety of wheat with resistance to gibberellic disease as claimed in claim 1, wherein in step (3), one third of the stigma is cut off.
3. The method for breeding a new wheat variety with resistance to gibberellic disease as claimed in claim 1, wherein the dropping of the plasmid is repeated once 1 hour after the initial dropping of the plasmid on the stigma.
4. The method for breeding a new wheat variety with resistance to gibberellic disease according to claim 1, wherein in step (3), the amount of plasmid added per stigma is: 100. mu.g/mL, 10. mu.L.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532028A (en) * | 2009-04-21 | 2009-09-16 | 江南大学 | Method for breeding high-yield glucoamylase industrial production strains |
| CN105441477A (en) * | 2016-01-07 | 2016-03-30 | 西北农林科技大学 | Optimization method of wheat pollen cell electric-shock-method transformation system and application of optimization method |
-
2016
- 2016-11-11 CN CN201610992109.7A patent/CN106755058B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532028A (en) * | 2009-04-21 | 2009-09-16 | 江南大学 | Method for breeding high-yield glucoamylase industrial production strains |
| CN105441477A (en) * | 2016-01-07 | 2016-03-30 | 西北农林科技大学 | Optimization method of wheat pollen cell electric-shock-method transformation system and application of optimization method |
Non-Patent Citations (4)
| Title |
|---|
| 利用花粉管通道法将cry Ia基因导入小麦;侯文胜等;《作物学报》;20030630;第806-809页 * |
| 小麦抗蚜虫基因PPA的花粉管通道法转化;范冬冬等;《中国优秀硕士学位论文全文数据库农业科技辑》;20131215;第D047-30页 * |
| 用花粉管通道法导人Prd 29A：DRER 1A融合基因;梁高峰等;《核农学报》;20080630;第22卷(第3期);第265-259页 * |
| 花粉管通道法介导小麦抗病相关基因的转化和抗锈性鉴定;张立等;《麦类作物学报》;20121225;第33卷(第1期);第29-34页 * |
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