CN112941100A - Genetic transformation method of thinopyrum intermedium and special primer thereof - Google Patents
Genetic transformation method of thinopyrum intermedium and special primer thereof Download PDFInfo
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Abstract
The invention provides a genetic transformation method of Elytrigia intermedium. The applicant selects 1000 varieties of elytrigia intermedium, selects a variety with good tissue culture effect, and establishes a gene gun transformation system. The method of the invention utilizes the embryonic callus of the elytrigia repens to carry out gene gun bombardment, greatly shortens the transformation period and reduces the occurrence of somatic cell variation and escape phenomenon through the optimization and adjustment of tissue culture conditions and transformation processes.
Description
Technical Field
The invention particularly relates to a genetic transformation method of Elytrigia intermedium and a special primer thereof.
Background
Currently, about 75% of the pasture in the world belongs to grass of the family gramineae. Gramineous forage grass has potential huge economic and ecological functions, and is necessary to develop basic research and application research related to molecular biology and biotechnology. An efficient gramineous forage grass transgenic technology platform is urgently needed to be established in the aspect of genetic engineering, and the speed of cultivating transgenic gramineous forage grass with important application value is accelerated by combining a gene editing technology.
Thinopyrum intermedium (Thinopyrum intermedium) is a monocotyledonous gramineous pasture with excellent quality, a plurality of stress-resistant genes exist in the Thinopyrum intermedium, and a genetic transformation system of the Thinopyrum intermedium has not been reported yet.
Disclosure of Invention
Aiming at the current situation that the genetic transformation system of Elytrigia intermedium is lacked at present, the invention aims to provide an Elytrigia intermedium genetic transformation method.
A genetic transformation method of Elytrigia intermedium comprises the following steps:
s1, inoculating the callus of the Elytrigia intermedium to a differentiation culture medium for differentiation culture until cluster buds grow, cutting off the meristem of the cluster buds, inoculating the cut meristem to an induction culture medium for induction culture, and obtaining an embryonic callus;
s2, inoculating the embryogenic callus obtained in the step S1 to a hypertonic culture medium for hypertonic treatment before bombardment; the hypertonic culture medium is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the concentration of cane sugar is 150g/L, and the pH value is 5.8 (a coagulant can be plant gel or agar and the like);
s3, bombarding the embryogenic callus processed in the step S2 with a recombinant vector containing a target gene by a gene gun;
s4, inoculating the embryogenic callus bombarded by the gene gun S3 to the hypertonic culture medium for hypertonic treatment after bombardment;
s5, transferring the embryogenic callus processed by the hypertonic processing of the step S4 to a screening culture medium in sequence for screening culture to obtain resistant callus;
s6, transferring the resistant callus induced by the step S5 to a regeneration medium for regeneration culture until a growing green head or a growing green seedling;
s7, subculturing the green heads or the green seedlings grown out in the S6 to a rooting culture medium, and culturing to obtain transformed plants; designing a primer pair aiming at a specific gene or a target gene on the plasmid, carrying out PCR amplification on DNA of a transformed plant by using the primer pair, and screening to obtain transgenic positive Elytrigia intermedium.
In the method, the gene gun is used for bombardment, and the plasmid bombarded into the embryogenic callus is a recombinant plasmid obtained by transferring a target gene by taking pCambia1300 as a starting plasmid.
In the method, the primer pair in S7 consists of single-stranded DNA named as a primer I and a primer II, wherein the primer I is the single-stranded DNA shown in SEQ ID No.1, and the primer II is the single-stranded DNA shown in SEQ ID No. 2.
In the method, the induction culture medium is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of the 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the content of the blue vitriol is 0.6mg/L, the concentration of the hydrolyzed casein is 1g/L, the concentration of the sucrose is 30g/L, and the pH value is 5.8 (the coagulator can be plant gel or agar, etc.);
the differentiation culture medium is a culture medium which takes an MS minimal medium as a basal medium, has the concentration of maltose of 30g/L, the concentration of 6-benzylaminopurine of 8.9 mu M and the pH value of 5.8 (the coagulator can select plant gel or agar, etc.);
the screening culture medium is a screening culture medium I and a screening culture medium II; the screening culture medium I is a culture medium (the coagulator can be plant gel or agar and the like) which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the content of blue vitriol is 0.6mg/L, the concentration of hydrolyzed casein is 1g/L, the concentration of sucrose is 30g/L, the concentration of silver nitrate is 10mg/L, the concentration of hygromycin is 50mg/L, and the pH value is 5.8; the screening culture medium II is a culture medium (the coagulator can be plant gel or agar and the like) which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the content of blue vitriol is 0.6mg/L, the concentration of hydrolyzed casein is 1g/L, the concentration of sucrose is 30g/L, the concentration of silver nitrate is 10mg/L, the concentration of hygromycin is 75mg/L, and the pH value is 5.8;
the regeneration culture medium is a culture medium which takes an MS minimal medium as a basal medium, has the concentration of maltose of 30g/L, the concentration of 6-benzylamino adenine of 8.9 MuM, the concentration of hygromycin of 50mg/L and the pH value of 5.8 (the coagulator can be plant gel or agar and the like);
the rooting culture medium is a culture medium which takes 1/2MS minimal medium as a basal culture medium and has the concentration of sucrose of 30g/L, pH value of 5.8 (the coagulant can be plant gel or agar and the like).
The MS minimal medium is a medium obtained by removing sucrose in the MS medium.
In the above method, the differentiation culture is performed in S1 under 24 ℃ light conditions; the induction culture in S1, the hypertonic treatment before bombardment in S2, the hypertonic treatment after bombardment in S4 and the screening culture in S5 are all in the dark condition at 24 ℃; the regeneration culture is carried out in S6, the culture condition is that the temperature is 24 ℃, the photoperiod is 16h/8h, and the illumination intensity is 150 mu mol/m2/s。
The genetic transformation method of Elytrigia intermedium can also comprise the following steps:
s8, strengthening seedlings and expanding propagation and growth.
The invention also provides a primer pair for identifying the transgenic positive Elytrigia intermedium, wherein the primer pair consists of single-stranded DNAs (deoxyribonucleic acids) named as a primer I and a primer II, the primer I is the single-stranded DNA shown as SEQ ID No.1, and the primer II is the single-stranded DNA shown as SEQ ID No. 2. The Elytrigia intermedium to be identified is subjected to PCR amplification by the primer pair, and the amplified target band is the transgenic positive Elytrigia intermedium.
The invention also provides a system for genetic transformation of Elytrigia intermedium used in the method, which consists of X1, X2 and X3; the X1 is one or more or all of the induction culture medium, the differentiation culture medium, the hypertonic culture medium, the screening culture medium I, the screening culture medium II, the regeneration culture medium and the rooting culture medium, the X2 is the primer pair, and the X3 is a reagent and/or an instrument required for PCR amplification.
The invention also provides an application of any one of P1-P3:
p1 and the application of the genetic transformation method of Elytrigia intermedium in the breeding of Elytrigia intermedium;
p2, and the application of the primer pair for identifying the transgenic positive Elytrigia intermedium in the breeding of the Elytrigia intermedium;
p3 and application of the system for genetic transformation of Elytrigia intermedium in Elytrigia intermedium breeding.
In order to accelerate the research of the couch grass endogenous gene and the improvement of the quality, the applicant develops related genetic engineering research on the basis of the collection and breeding of germplasm resources for many years. The invention summarizes the experience of the traditional gramineous forage grass plant regeneration system research, screens 1000 varieties of Elytrigia intermedium according to the physiological and biochemical characteristics of Elytrigia intermedium, selects a variety with good tissue culture effect, and establishes a gene gun transformation system. The method of the invention utilizes the embryonic callus of the elytrigia repens to carry out gene gun bombardment, greatly shortens the transformation period and reduces the occurrence of somatic cell variation and escape phenomenon through the optimization and adjustment of tissue culture conditions and transformation processes.
Drawings
FIG. 1 is a physical map of plasmid pCambia1300 in example 1 of the present invention.
FIG. 2 is a photograph showing the growth of a material during transformation with Elytrigia intermedium in example 1 of the present invention. Wherein A is the induced embryogenic callus; b is the callus growth on the screening medium I; c is the growth condition of the callus after regeneration for two weeks under light; e is a resistant plant cultured for 2 weeks for roots.
FIG. 3 is the electrophoresis chart of the PCR result of hpt gene of T0 generation plant in example 1. Wherein, the leftmost strip is Marker), PC is positive control, NC is negative control, and the rest are sample strips.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Elytrigia intermedium in the following examples is described in the following documents: wang 29608j Qiongena isopentenyl transferase (1pt) gene is introduced into Elytrigia intermedium and the establishment of a tissue culture regeneration system, university of inner Mongolia, 6-month Master academic paper 2007, publicly available from Tianjin Jinopo Biotechnology Ltd.
In the following examples, the germination medium was a medium based on MS minimal medium, 2, 4-dichlorophenoxyacetic acid (2,4-D) at a concentration of 22.6. mu.M, sucrose at a concentration of 30g/L, vegetable gel at a concentration of 3g/L (as a coagulant, which may be replaced with another coagulant such as agar, the same applies hereinafter), benomyl at a concentration of 170. mu.M, and pH 5.8. The specific preparation method of the germination medium can be as follows: dissolving MS minimal medium (Duchefa M0222) with water to make the final concentration of the medium to be 4.4g/L, adding 22.6 mu M2, 4-dichlorophenoxyacetic acid and 30g/L sucrose, adding water to a constant volume of 1L, adjusting the pH value to 5.8, adding 3g/L plant gel, autoclaving at 121 ℃ for 20min, adding 170 mu M benomyl when the temperature is reduced to about 55 ℃, pouring 90mm multiplied by 15mm flat plates and 35mL per plate to prepare a solid medium, namely the germination medium. Here, "final concentration" refers to the concentration in the finally obtained medium, the same applies below.
In the following examples, the induction medium was a MS minimal medium as a basal medium, 2, 4-dichlorophenoxyacetic acid at a concentration of 22.6. mu.M, copper sulfate pentahydrate at a content of 0.6mg/L, hydrolyzed casein at a concentration of 1g/L, sucrose at a concentration of 30g/L, vegetable gel at a concentration of 3g/L, and pH at 5.8. The specific preparation method of the induction culture medium can be as follows: dissolving MS basal culture medium with water to make the final concentration of the culture medium be 4.4g/L, adding 2, 4-dichlorophenoxyacetic acid with the final concentration of 22.6 mu M, copper sulfate pentahydrate with the final concentration of 0.6mg/L, hydrolyzed casein with the final concentration of 1g/L and cane sugar with the final concentration of 30g/L, adding water to fix the volume to 1L, adjusting the pH value to 5.8, adding plant gel with the final concentration of 3g/L, autoclaving at 121 ℃ for 20min, pouring 90mm multiplied by 15mm flat plates, preparing 35mL of each plate into a solid culture medium, namely the induction culture medium.
In the following examples, the differentiation medium was a medium containing MS minimal medium as a basal medium, maltose at a concentration of 30g/L, a plant gel at a concentration of 3g/L, 6-benzylamino adenine at a concentration of 8.9. mu.M, and pH 5.8. The specific preparation method of the differentiation medium can be as follows: dissolving MS minimal medium with water to make the final concentration of the medium be 4.4g/L, adding maltose with the final concentration of 30g/L, adding water to a constant volume of 1L, adjusting the pH value to 5.8, adding plant gel with the final concentration of 3g/L, autoclaving at 121 ℃ for 20min, adding 6-benzylamino adenine with the final concentration of 8.9 mu M when the temperature is reduced to about 55 ℃, pouring 90mm multiplied by 15mm plates, and preparing a solid medium which is a differentiation medium for 35mL each plate.
In the following examples, the hypertonic medium was a medium based on MS minimal medium, 2, 4-dichlorophenoxyacetic acid 22.6. mu.M, sucrose 150g/L, plant gel 3g/L, and pH 5.8. The specific preparation method of the hypertonic culture medium can be as follows: dissolving MS basal culture medium with water to make its final concentration be 4.4g/L, adding 2, 4-dichlorophenoxyacetic acid with final concentration of 22.6 μ M and sucrose with final concentration of 150g/L, adding water to a constant volume of 1L, adjusting pH value to 5.8, adding plant gel with final concentration of 3g/L, and autoclaving at 121 deg.C for 20min to obtain solid culture medium, i.e. hyperosmotic culture medium.
In the following examples, the screening medium I was a medium comprising MS minimal medium as the basal medium, 2, 4-dichlorophenoxyacetic acid 22.6. mu.M, copper sulfate pentahydrate 0.6mg/L, hydrolyzed casein 1g/L, sucrose 30g/L, vegetable gel 3g/L, silver nitrate 10mg/L, hygromycin 50mg/L, and pH 5.8. The specific preparation method of the screening medium I can be as follows: dissolving MS basal culture medium with water to make the final concentration of the culture medium be 4.4g/L, adding 2, 4-dichlorophenoxyacetic acid with the final concentration of 22.6 mu M, copper sulfate pentahydrate with the final concentration of 0.6mg/L, hydrolyzed casein with the final concentration of 1g/L and cane sugar with the final concentration of 30g/L, adding water to fix the volume to 1L, adjusting the pH value to 5.8, adding plant gel with the final concentration of 3g/L, sterilizing at 121 ℃ for 20min under high pressure, adding silver nitrate with the final concentration of 10mg/L and hygromycin with the final concentration of 50mg/L when the temperature is reduced to about 55 ℃, pouring 90mm multiplied by 15mm plates and 35mL plates to prepare a solid culture medium, namely screening culture medium I.
In the following examples, the screening medium II was a medium comprising MS minimal medium as basal medium, 2, 4-dichlorophenoxyacetic acid 22.6. mu.M, copper sulfate pentahydrate 0.6mg/L, hydrolyzed casein 1g/L, sucrose 30g/L, vegetable gel 3g/L, silver nitrate 10mg/L, hygromycin 75mg/L, and pH 5.8. The specific preparation method of the screening culture medium II can be as follows: dissolving MS basal culture medium with water to make the final concentration of the culture medium be 4.4g/L, adding 2, 4-dichlorophenoxyacetic acid with the final concentration of 22.6 mu M, copper sulfate pentahydrate with the final concentration of 0.6mg/L, hydrolyzed casein with the final concentration of 1g/L and cane sugar with the final concentration of 30g/L, adding water to fix the volume to 1L, adjusting the pH value to 5.8, adding plant gel with the final concentration of 3g/L, sterilizing at 121 ℃ for 20min under high pressure, adding silver nitrate with the final concentration of 10mg/L and hygromycin with the final concentration of 75mg/L when the temperature is reduced to about 55 ℃, pouring 90mm multiplied by 15mm plates and 35mL plates to prepare a solid culture medium, namely screening culture medium II.
In the following examples, the regeneration medium was a medium containing MS minimal medium as the basal medium, maltose at a concentration of 30g/L, plant gel at a concentration of 3g/L, 6-benzylamino adenine at a concentration of 8.9. mu.M, hygromycin at a concentration of 50mg/L, and pH 5.8. The specific preparation method of the regeneration medium can be as follows: dissolving MS basal culture medium with water to make the final concentration of the culture medium be 4.4g/L, adding maltose with the final concentration of 30g/L into the culture medium, adding water to a constant volume of 1L, adjusting the pH value to 5.8, adding plant gel with the final concentration of 3g/L, carrying out autoclaving at 121 ℃ for 20min, adding 6-benzylamino adenine with the final concentration of 8.9 mu M and hygromycin with the final concentration of 50mg/L when the temperature is reduced to about 55 ℃, pouring the mixture into a 90mm multiplied by 15mm flat plate, and preparing a solid culture medium which is a regeneration culture medium by 35mL per plate.
In the following examples, the rooting medium was a 1/2MS minimal medium, sucrose concentration was 30g/L, plant gel concentration was 3g/L, and pH was 5.8. The specific preparation method of the rooting medium can be as follows: dissolving MS minimal medium with water to make the final concentration of 4.4g/L, adding sucrose with the final concentration of 30g/L, adding water to a constant volume of 1L, adjusting pH value to 5.8, adding plant gel with the final concentration of 3g/L, autoclaving at 121 ℃ for 20min, pouring 90mm × 15mm plates, and preparing a solid medium, namely the rooting medium, for 35mL per plate.
Example 1 transformation System of Elytrigia intermedium Gene gun
Selection of strain I and strain II
On the basis of multi-year germplasm resource collection and breeding, more than 1000 thinopyrum intermedium seeds (provided by a test station of the college of agriculture of the university of inner Mongolia) are taken for tissue culture, and because the thinopyrum intermedium has a cross-crossing rate, the progeny obtained by the vegetative propagation of more than 1000 seeds is taken as more than 1000 different strains. The specific tissue culture process is as follows:
soaking the seeds in warm water at 55 deg.C for 15min to kill endophytic fungi. The seeds were then surface-sterilized with 5% by volume (based on available chlorine) sodium hypochlorite solution (approximately 10uL of Tween20 was added dropwise) for 20 min. Washed 6 times with sterile water and blotted dry with sterile filter paper.
The seeds were transferred to a germination medium and cultured in the dark at 24 ℃ for seven days. The germinated seedlings were exposed to light (temperature 24 ℃, light intensity 150. mu. mol/m)2Second) two days after the culture, about 1cm of shoot apical meristem was cut off with a scalpel in a clean bench and then dissected longitudinally into two halves. After transfer to induction medium with the incision facing downward and dark culture at 24 ℃ for 4-5 weeks (subculture every two weeks), yellow dense embryogenic callus was seen. The obtained embryogenic callus was transferred to a differentiation medium and cultured under light at 24 ℃ for 4 weeks, subcultured every two weeks. 3 calli with better differentiation are selected, and the elytrigia intermedium line of the calli is numbered as WG1, WG2 and WG 3. The differentiation frequency was calculated according to formula (1):
differentiation frequency ═ callus number differentiated green shoots/total callus number x 100% (1)
The statistical results are shown in table 1:
TABLE 1 statistical Table of differentiation frequency
| Numbering of elytrigia intermedium lines | Callus/total callus of differentiated green seedling | Frequency of differentiation |
| WG1 | 172/256 | 67.2％ |
| WG2 | 142/263 | 54.0％ |
| WG3 | 236/332 | 71.1％ |
Among them, the best differentiated line WG3 was. The genetic transformation method of Elytrigia intermedium is constructed by using the selected product with the best differentiation effect as a material.
Genetic transformation method of Elytrigia intermedium
S1, induction of embryogenic callus: inoculating callus of Elytrigia intermedium (WG3) to a differentiation culture medium, performing differentiation culture until cluster buds grow out, cutting off meristem of the cluster buds, inoculating to an induction culture medium, and performing induction culture to obtain embryonic callus; the detailed process is as follows:
the score on the callus of line WG3 was takenTransferring the green heads of the differentiated green seedlings to a differentiation medium, and irradiating at 24 deg.C (light intensity 150 μmol/m)2And/s) culturing until cluster buds grow. Meristematic tissue of the cluster buds is cut off laterally (or chopped) with a scalpel in an ultraclean bench and spread on induction medium under dark conditions at 24 ℃ with subculture every two weeks. Until embryogenic callus grows out, as shown in FIG. 2, panel A. Subcultured every two weeks, once 5-7 days before gene gun bombardment.
S2, hypertonic treatment before bombardment: inoculating the embryogenic callus obtained in step S1 to a hyperosmotic medium for pre-bombardment hyperosmotic treatment. The detailed process is as follows:
inoculating small embryogenic callus on hypertonic culture medium, sequentially arranging in a circular area of 2.0-3.0cm in the center of a 60mm × 15mm culture dish, sealing with Parafilm, and performing hypertonic culture at 24 deg.C in dark for 4 hr.
S3, gene gun bombardment: the embryogenic callus treated in step S2 was bombarded by gene gun, and the plasmid bombarded into the embryogenic callus was pCambia1300 (SEQ ID NO: 1) with specific hpt gene on pCambia1300 and hygromycin resistance. The detailed process is as follows:
1) preparation of gold powder
Weighing 40-60mg gold powder with particle size of 0.6 μm in a sterilized 1.5ml centrifuge tube, adding 1ml 75% ethanol, vortexing for 15min, centrifuging for 5min at 3000g, removing supernatant, and repeatedly cleaning for 3 times. Sterile water was added to a final concentration of 40mg/ml and stored at-20 ℃.
2) Preparation of plasmid and coating of gold powder
pCambia1300 was used as the plasmid for the experiment.
In a 1.5ml sterile centrifuge tube were added in the following order: 50 ul gold powder solution (40mg/ml), 10ul DNA (1 ug/ul) and 50 ul CaCl mixed in advance2(2.5M) and 20. mu.l spermidine (0.1M).
Gently swirling for 3min, mixing, standing on ice for 15min-1h, centrifuging at 3000g for 10s to remove supernatant, adding sterilized anhydrous ethanol for cleaning for 2 times, centrifuging at 3000g for 1min to remove supernatant, adding 200 μ l anhydrous ethanol, gently swirling for 2min, mixing, and uniformly coating in the central 2cm area of 10 carrier films at a ratio of 15 μ l/piece.
3) Bombardment with gene gun
Using pCambia1300 as a plasmid, a PDS1000/He gene gun from Bio-Red was used, with a bombardment pressure of 1100psi, a bombardment distance of 6cm, a vacuum of 26-28inHg, and a bombardment of one material per dish.
S4, hypertonic treatment after bombardment: inoculating the embryogenic callus bombarded by the gene gun in the step S3 to a hypertonic culture medium for hypertonic treatment after bombardment, wherein the detailed process is as follows:
after bombardment by a gene gun, continuously inoculating the seeds into a hypertonic culture medium under the dark condition of 24 ℃ and carrying out hypertonic culture for 16 h.
S5, screening of callus: and transferring the embryogenic callus after the hypertonic treatment in the step S4 to a screening culture medium I and a screening culture medium II containing hygromycin in sequence for screening culture to obtain the resistant callus. The detailed process is as follows:
the bombarded calli were transferred to selection medium I, 25 cells per dish in 90mm 15mm petri dishes and incubated in the dark at 24 ℃ for 2 weeks, as shown in FIG. 2B. The plumule and fibrous root are removed in time during the culture process, and the polluted material is discarded. Transferring the bombarded callus to a screening culture medium II, cutting 7-9 clones with the diameter of 90 multiplied by 15mm per dish, and culturing in the dark at the temperature of 24 ℃ for 2 weeks to obtain the resistant callus.
S6, regeneration culture: transferring the resistant callus induced by the step S5 to a regeneration medium for regeneration culture until a green head or a green seedling grows out. The detailed steps are as follows:
transferring the induced resistant callus to regeneration medium, and irradiating at 24 deg.C and 150 μmol/m light intensity2And culturing for 4 weeks (subcultured every two weeks) under the conditions of the photoperiod of 16h/8h (light/dark) at the second time to grow green heads or seedlings. After the first two weeks, the calli from each clone were divided into four pieces, pooled and cultured, 10 clones per dish in 90mm by 15mm petri dishes, as shown in FIG. 2C. The contaminated and stressed dead material was discarded in time during the cultivation.
S7, rooting culture: subculturing the green heads or the green seedlings grown out from the S6 to a rooting culture medium, and culturing to obtain a transformed plant; designing a primer pair aiming at a specific gene or a target gene on the plasmid, carrying out PCR amplification on DNA of a transformed plant by using the primer pair, and screening to obtain transgenic positive Elytrigia intermedium. The detailed process is as follows:
and subculturing the grown green heads or green seedlings to a rooting culture medium. Tissue material from each clone was pooled, 3-9 in 90mm by 15mm petri dishes at 24 ℃ with 150. mu. mol/m light intensity2The cells were cultured for 2 to 4 weeks under the conditions of 16h/8h photoperiod (light/dark), and the photograph after 2 weeks of culture is shown in FIG. 2D. The contaminated and stressed dead material was discarded in time during the cultivation. Culturing to obtain transformed plant.
Molecular identification and screening:
and extracting T0 generation transformed plant genome DNA by using a TIANGEN rapid plant genome DNA extraction system kit.
Primers of specific hpt genes and target genes are respectively designed, and positive plants are detected by PCR.
The primer pair of the hpt gene used in the test is a primer pair consisting of an upstream primer I and a downstream primer II:
primer I: 5'-ATGCCTCCGCTCGAAGTAGC-3' (SEQ ID NO: 2 of the sequence Listing).
And (3) primer II: 5'-GGGTGTCACGTTGCAAGACC-3' (SEQ ID NO: 3 of the sequence Listing);
the size of the amplified fragment of the hpt gene is 472bp, and the specific amplification result is shown in FIG. 3, which shows that 41 positive bands are contained in 51 samples, and the positive rate is 80.39%.
Please show the primer sequence and detection result of the target gene
S8, strengthening seedlings and expanding propagation growth
When the overground part of the transformed plant grows to about 10cm and more than 2 strong roots are induced, transplanting the transformed plant into a flowerpot with the size of 12cm multiplied by 12cm, and illuminating at the illumination intensity of 500 mu mol/m2And/s, a photoperiod of 16h/8h (light/dark), day and night temperature of 20 ℃/15 ℃ in a seedling stage, and day and night temperature of 30 ℃/20 ℃ in a booting stage and a grouting stage.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced with equivalent parameters and conditions within a wide range without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In summary, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<120> Elytrigia intermedium genetic transformation method and special primer thereof
<110> Tianjin Jinocuowa Biotech Co., Ltd
<130> GNCSY210897
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8958
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca 60
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 120
cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 180
ccttcccaac agttgcgcag cctgaatggc gaatgctaga gcagcttgag cttggatcag 240
attgtcgttt cccgccttca gtttaaacta tcagtgtttg acaggatata ttggcgggta 300
aacctaagag aaaagagcgt ttattagaat aacggatatt taaaagggcg tgaaaaggtt 360
tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta 420
ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca gtgcagccgt 480
cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc gccctgccct 540
tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact tgcgactaga 600
accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg 660
tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg gccggctgca 720
ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag ctggccagga 780
tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac cgcctggccc 840
gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc gcgggcctgc 900
gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg gtgttgaccg 960
tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc 1020
gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc accccggcac 1080
agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg 1140
cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc gaggaagtga 1200
cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc gaggccgacg 1260
ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc accaggacgg 1320
ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg cggccgggta 1380
cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc tggccggttt 1440
gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa ccgagcgccg 1500
ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta 1560
tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt tatcgctgta 1620
cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc ccgcgccctg 1680
caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag tgcccgcgat 1740
tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt 1800
gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg agcgccccag 1860
gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat tccggtgcag 1920
ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa gcagcgcatt 1980
gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg 2040
cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat tcttgagtcc 2100
cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac cgttcttgaa 2160
tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga aattaaatca 2220
aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg 2280
ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc 2340
cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag ctgaagatgt 2400
acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc gcgcagctac 2460
cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa aggaggcggc 2520
atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg tggaggaacg 2580
ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg cactggaacc 2640
cccaagcccg aggaatcggc gtgacggtcg caaaccatcc ggcccggtac aaatcggcgc 2700
ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc agcggcaacg 2760
catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa 2820
agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc ccaagggcga 2880
cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag 2940
catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat 3000
ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg gcatggccag 3060
tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat ccatgaaccg 3120
ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt 3180
actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg 3240
cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca agaacggccg 3300
cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg taaagagcga 3360
aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc gcgagatcac 3420
agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga tcgatcccgg 3480
catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag aagccagatg 3540
gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga agttctgttt 3600
caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga aggaggaggc 3660
ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc 3720
cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag gggaaaaagg 3780
tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc cgtacattgg 3840
gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt cacacatgta 3900
agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt taaaacttat 3960
taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca cagccgaaga 4020
gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg 4080
gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca atctaccagg 4140
gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag gcaccctgcc 4200
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 4260
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 4320
ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 4380
gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 4440
accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac 4500
tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4560
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 4620
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4680
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4740
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4800
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4860
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4920
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4980
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 5040
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 5100
aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 5160
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 5220
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 5280
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt ctaggtacta 5340
aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct tgatccccag 5400
taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga tcgaccggac 5460
gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat caataaagcc 5520
acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt gggaaaagac 5580
aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa 5640
tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat tcagtaagta 5700
atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg atatgtcgat 5760
ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag ggctttgttc 5820
atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca gattgctcca 5880
gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt ccagccatag 5940
catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc tgtccgtcat 6000
ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag gagacattcc 6060
ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg gtgatattct 6120
cattttagcc atttattatt tccttcctct tttctacagt atttaaagat accccaagaa 6180
gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa ccttaaatac 6240
cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt atcgacggag 6300
ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta caatcaacat 6360
gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc gttcttccga 6420
atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg ctgacgccgt 6480
cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga 6540
gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac 6600
ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta attcggggga 6660
tctggatttt agtactggat tttggtttta ggaattagaa attttattga tagaagtatt 6720
ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag cgaaacccta 6780
taggaaccct aattccctta tctgggaact actcacacat tattatggag aaactcgagc 6840
ttgtcgatcg acagatccgg tcggcatcta ctctatttct ttgccctcgg acgagtgctg 6900
gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg 6960
cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg 7020
catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt 7080
tggtcaagac caatgcggag catatacgcc cggagtcgtg gcgatcctgc aagctccgga 7140
tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag 7200
aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc 7260
gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg 7320
tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg 7380
gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc 7440
gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg 7500
aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatagcctc cgcgaccggt 7560
tgtagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg 7620
cgggagatgc aataggtcag gctctcgcta aactccccaa tgtcaagcac ttccggaatc 7680
gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg 7740
cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat 7800
tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac 7860
ttctcgacag acgtcgcggt gagttcaggc tttttcatat ctcattgccc cccgggatct 7920
gcgaaagctc gagagagata gatttgtaga gagagactgg tgatttcagc gtgtcctctc 7980
caaatgaaat gaacttcctt atatagagga aggtcttgcg aaggatagtg ggattgtgcg 8040
tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga cgtggttgga 8100
acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg 8160
gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt gtaggtgcca 8220
ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg gaatccgagg 8280
aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc ttctgagact 8340
gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt atcacatcaa 8400
tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt 8460
gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta 8520
tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga 8580
cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc 8640
tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg 8700
tcgtgctcca ccatgttggc aagctgctct agccaatacg caaaccgcct ctccccgcgc 8760
gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 8820
agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 8880
tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 8940
gctatgacca tgattacg 8958
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgcctccgc tcgaagtagc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gggtgtcacg ttgcaagacc 20
Claims (9)
1. A genetic transformation method of Elytrigia intermedium is characterized in that: the method comprises the following steps:
s1, inoculating the callus of the Elytrigia intermedium to a differentiation culture medium for differentiation culture until cluster buds grow, cutting off the meristem of the cluster buds, inoculating the cut meristem to an induction culture medium for induction culture, and obtaining an embryonic callus;
s2, inoculating the embryogenic callus obtained in the step S1 to a hypertonic culture medium for hypertonic treatment before bombardment; the hypertonic culture medium is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the concentration of sucrose is 150g/L, and the pH value is 5.8;
s3, bombarding the embryogenic callus processed in the step S2 with a recombinant vector containing a target gene by a gene gun;
s4, inoculating the embryogenic callus bombarded by the gene gun S3 to the hypertonic culture medium for hypertonic treatment after bombardment;
s5, transferring the embryogenic callus processed by the hypertonic processing of the step S4 to a screening culture medium in sequence for screening culture to obtain resistant callus;
s6, transferring the resistant callus induced by the step S5 to a regeneration medium for regeneration culture until a growing green head or a growing green seedling;
s7, subculturing the green heads or the green seedlings grown out in the S6 to a rooting culture medium, and culturing to obtain transformed plants; designing a primer pair aiming at a specific gene or a target gene on the plasmid, carrying out PCR amplification on DNA of a transformed plant by using the primer pair, and screening to obtain transgenic positive Elytrigia intermedium.
2. The method of claim 1, wherein: the plasmid bombarded into the embryogenic callus is a recombinant plasmid obtained by transferring a target gene by taking pCambia1300 as a starting plasmid.
3. The method of claim 2, wherein: the primer pair in S7 is composed of single-stranded DNA named as a primer I and a primer II, wherein the primer I is the single-stranded DNA shown in SEQ ID No.1, and the primer II is the single-stranded DNA shown in SEQ ID No. 2.
4. The method of claim 2, wherein: the induction culture medium is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorphenoxyacetic acid is 22.6 mu M, the content of blue vitriol is 0.6mg/L, the concentration of hydrolyzed casein is 1g/L, and the concentration of sucrose is 30g/L, pH and the value is 5.8;
the differentiation culture medium is a culture medium which takes an MS minimal medium as a basal medium, the concentration of maltose is 30g/L, the concentration of 6-benzylamino adenine is 8.9 mu M, and the pH value is 5.8;
the screening culture medium is a screening culture medium I and a screening culture medium II; the screening culture medium I is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorphenoxyacetic acid is 22.6 mu M, the content of blue vitriol is 0.6mg/L, the concentration of hydrolyzed casein is 1g/L, the concentration of sucrose is 30g/L, the concentration of silver nitrate is 10mg/L, the concentration of hygromycin is 50mg/L, and the pH value is 5.8; the screening culture medium II is a culture medium which takes an MS basic culture medium as a basic culture medium, the concentration of 2, 4-dichlorophenoxyacetic acid is 22.6 mu M, the content of blue vitriol is 0.6mg/L, the concentration of hydrolyzed casein is 1g/L, the concentration of sucrose is 30g/L, the concentration of silver nitrate is 10mg/L, the concentration of hygromycin is 75mg/L, and the pH value is 5.8;
the regeneration culture medium is a culture medium which takes an MS minimal medium as a basal medium, the concentration of maltose is 30g/L, the concentration of 6-benzylamino adenine is 8.9 mu M, the concentration of hygromycin is 50mg/L, and the pH value is 5.8;
the rooting culture medium is a culture medium which takes 1/2MS minimal medium as a basal culture medium and has the concentration of sucrose of 30g/L, pH value of 5.8.
5. The method of claim 4, wherein: s1, wherein the culture condition is 24 ℃ illumination condition; the induction culture in S1, the hypertonic treatment before bombardment in S2, the hypertonic treatment after bombardment in S4 and the screening culture in S5 are all in the dark condition at 24 ℃; the regeneration culture is carried out in S6, the culture condition is that the temperature is 24 ℃, the photoperiod is 16h/8h, and the illumination intensity is 150 mu mol/m2/s。
6. The method according to any one of claims 1 to 5, wherein: the method further comprises the following steps:
s8, strengthening seedlings and expanding propagation and growth.
7. A primer pair for identifying a transgenic positive Elytrigia intermedium as claimed in any of claims 1 to 6, characterized in that: the primer pair consists of single-stranded DNA (deoxyribonucleic acid) named as a primer I and a primer II, wherein the primer I is the single-stranded DNA shown in SEQ ID No.1, and the primer II is the single-stranded DNA shown in SEQ ID No. 2.
8. A system for genetic transformation of Elytrigia intermedium for use in the method of any one of claims 1-6, characterized in that: consists of X1, X2 and X3; the X1 is one or more or all of the induction culture medium, the differentiation culture medium, the hypertonic culture medium, the screening culture medium I, the screening culture medium II, the regeneration culture medium and the rooting culture medium, the X2 is the primer pair, and the X3 is a reagent and/or an instrument required for PCR amplification.
The use of any one of P1-P3:
use of P1, the method of genetic transformation of elytrigia intermedium according to any one of claims 1 to 6 in the breeding of elytrigia intermedium;
the application of P2 and the primer pair for identifying the transgenic positive Elytrigia intermedium of claim 7 in the breeding of Elytrigia intermedium;
the use of P3, the system for genetic transformation of Elytrigia intermedium of claim 8in the breeding of Elytrigia intermedium.
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| CN202110240390.XA CN112941100B (en) | 2021-03-04 | 2021-03-04 | Genetic transformation method of elytrigia intermedium and special primer thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001062890A2 (en) * | 2000-02-25 | 2001-08-30 | Villoo Morawala Patell | A process for generating genetically modified pearl millet through agrobacterium and biolistic transformation |
| AU2001255050B2 (en) * | 2000-02-25 | 2005-07-14 | Avestha Gengraine Technologies Pvt. Ltd. | A process for generating genetically modified pearl millet through agrobacteriumand biolistic transformation |
| CN103710378A (en) * | 2013-12-04 | 2014-04-09 | 西北农林科技大学 | Wheat mature embryo callus gene gun genetic transformation method |
| CN103766208A (en) * | 2012-10-22 | 2014-05-07 | 李静 | Elytigia intermedium and application thereof to wheat genetic improvement |
| CN103766207A (en) * | 2012-10-22 | 2014-05-07 | 李静 | Elytigia intermedium and application thereof to wheat genetic improvement |
-
2021
- 2021-03-04 CN CN202110240390.XA patent/CN112941100B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001062890A2 (en) * | 2000-02-25 | 2001-08-30 | Villoo Morawala Patell | A process for generating genetically modified pearl millet through agrobacterium and biolistic transformation |
| AU2001255050B2 (en) * | 2000-02-25 | 2005-07-14 | Avestha Gengraine Technologies Pvt. Ltd. | A process for generating genetically modified pearl millet through agrobacteriumand biolistic transformation |
| CN103766208A (en) * | 2012-10-22 | 2014-05-07 | 李静 | Elytigia intermedium and application thereof to wheat genetic improvement |
| CN103766207A (en) * | 2012-10-22 | 2014-05-07 | 李静 | Elytigia intermedium and application thereof to wheat genetic improvement |
| CN103710378A (en) * | 2013-12-04 | 2014-04-09 | 西北农林科技大学 | Wheat mature embryo callus gene gun genetic transformation method |
Non-Patent Citations (2)
| Title |
|---|
| HAJDUKIEWICZ,P.等: "Binary vector pCAMBIA-1300, complete sequence", 《NCBI》 * |
| 王珏琼: "异戊烯基转移酶（ipt）基因导入中间偃麦草及组织培养再生体系的建立", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
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