CN105420271A - Transgenic lycium chinense hairy roots, gene transformation method and liquid culture method - Google Patents
Transgenic lycium chinense hairy roots, gene transformation method and liquid culture method Download PDFInfo
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- CN105420271A CN105420271A CN201510860926.2A CN201510860926A CN105420271A CN 105420271 A CN105420271 A CN 105420271A CN 201510860926 A CN201510860926 A CN 201510860926A CN 105420271 A CN105420271 A CN 105420271A
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Abstract
The invention discloses transgenic lycium chinense hairy roots, a gene transformation method and a liquid culture method. The gene transformation method of the transgenic lycium chinense hairy roots includes the following steps that 1, preparation of sterile lycium chinense leaves is performed; 2, activation of agrobacterium rhizogenes A4 is performed; 3, explant infection and co-culture are performed; 4, rooting culture is performed; 5, fluorescence detection is performed, wherein whether red fluorescence exists in RFP genes in hairy roots obtained in the step 4 or not is detected, and hairy roots with red fluorescence are the transgenic lycium chinense hairy roots. Through the agrobacterium rhizogenes-mediated transgenic technology, the hairy roots can be obtained within a short time, research time is shortened, and transformation efficiency is high and can reach about 60%-70%. The lycium chinense hairy roots can propagate and grow in large quantities in a liquid culture medium, the purpose of drawing materials is achieved, and growth situations are easy to observe.
Description
Technical field
The invention belongs to biological technical field, relate to plant transgenic technology, relate generally to a kind of matrimony vine Hairy root based on Agrobacterium rhizogenes mediation and transform and liquid cultivating method.
Background technology
Matrimony vine (LyciumchinenseMiller) is famous and precious medicinal material and invigorant, Chinese wolfberry skin also known as Root-bark of Chinese Wolfberry, be the root skin of matrimony vine, can be used as medicine, there is cool blood, clearing lung-heat fall fire etc. effect.At present at home, the research of matrimony vine is focused mostly in the physiological-biochemical level of over-ground part, and the gene excavating of underground organization and the report of functional analysis less; Particularly few especially to the root system research of matrimony vine.For the research of matrimony vine critical biological proterties, gene excavating and functional verification play a part indispensable, and in Chinese matrimony vine, the transformation system based on root system is not also set up, and the gene function that its root system is relevant have not been reported.Lack at present and a set ofly matrimony vine can be allowed to verify function and the analysis thereof of carotenoid genes involved by the transgenosis of root system.
Agrobacterium rhizogenes is gram negative bacterium, adopt Ri plasmid to infect plants wound and enter cell, and contain thereon one section of T-DNA is inserted in Plant Genome; The T-DNA of the Ri plasmid of Agrobacterium rhizogenes has rosA-D gene group, T-DNA has tms gene, the injury that can bring out infected plant produces a large amount of hairly root, and the gene on Ri plasmid T-DNA does not affect the regeneration of plant, transformation efficiency is high, succeeds in Radix Glycyrrhizae, tobacco etc. at present.The hairly root of Agrobacterium rhizogenes induction belongs to Hormone autotrophy, have that active constituent content is high, Physiology and biochemistry and genetic stability, be easy to carry out the features such as operation control, and the hairly root of inducing can be identified from morphological level, root is grown thickly more, multi-branched, many hairs, and apogetropism, these all can differentiate with unconverted.
In soil, obtaining boxthorn root be faced with problems, if make the increasing number of the hairly root of matrimony vine, providing safeguard for obtaining a large amount of boxthorn root effective constituent, but at present, the conversion adopting Agrobacterium rhizogenes to divide at boxthorn root pastern and cultural method are not yet reported.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of Transgenic Lycium barbarum L hairly root is provided.
Second object of the present invention is to provide a kind of gene transformation method of Transgenic Lycium barbarum L hairly root.
3rd object of the present invention is to provide a kind of liquid cultivating method of Transgenic Lycium barbarum L hairly root.
Technical scheme of the present invention is summarized as follows:
A gene transformation method for Transgenic Lycium barbarum L hairly root, comprises the following steps:
(1) the aseptic blade of matrimony vine is prepared: by matrimony vine blade, clear water rinses rear dehydrated alcohol wipe surfaces well, with aqueous ethanolic solution sterilization 5-10min, and aseptic distillation water rinse 3-5 time, be inoculated on MS substratum, 25 DEG C of dark Dual culture obtain the aseptic blade of matrimony vine;
(2) Agrobacterium rhizogenesA4 activation: Agrobacterium rhizogenesA4 is being rule containing on antibiotic LB solid medium, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 36-48h, picking list bacterium colony is in containing on antibiotic LB liquid nutrient medium, 28 DEG C, shaking speed 180-220rpm light culture 36-48h, obtain activating hair-root Agrobacterium A4; Containing 35S:RFP gene on the carrier of described Agrobacterium rhizogenesA4; Shown in described RFP gene SEQ.ID.NO1;
(3) infect explant, Dual culture: wound activating hair-root Agrobacterium A4 being spread upon the aseptic blade of the matrimony vine after designated port, be placed on Dual culture base, at 24 DEG C ~ 25 DEG C, illumination 16h, light culture 8h, cultivate 4-6d;
(4) hairy root culture: changed on hairy root culture base by the matrimony vine blade that step (3) obtains, sealed membrane is sealed, 24 DEG C ~ 25 DEG C, illumination 16h, light culture 8h, cultivate 25-30d, every 6-8 days and change a hairy root culture base;
(5) fluoroscopic examination; Whether the existence of the red fluorescence of the RFP gene in the hairly root that detecting step (4) obtains; The hairly root with red fluorescence is Transgenic Lycium barbarum L hairly root.
The Transgenic Lycium barbarum L hairly root that aforesaid method obtains.
The liquid cultivating method of above-mentioned Transgenic Lycium barbarum L hairly root, comprises the steps:
(1) preparation is containing the liquid nutrient medium of plant hormone: in 1/2MS liquid nutrient medium, add plant hormone IBA make concentration be 0.1-0.5mg/L, mix;
(2) what obtained in step (1) by Transgenic Lycium barbarum L hairly root contains in the liquid nutrient medium of plant hormone, and 24-28 DEG C, rotating speed 80-120rpm, cultivate; Within 15-20 days, change the liquid nutrient medium containing plant hormone that step (1) obtains, enlarged culturing is carried out to Transgenic Lycium barbarum L hairly root.
Make by following method containing antibiotic LB solid medium: in LB solid culture based formulas, add kantlex, the final concentration making kantlex is 80-120mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 80-120mg/L.
Make by following method containing antibiotic LB liquid nutrient medium: in LB liquid culture based formulas, add kantlex, the final concentration making kantlex is 80-120mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 80-120mg/L.
Advantage:
The transgenic technology that the present invention is mediated by Agrobacterium rhizogenes, can obtain hairly root at short notice, save search time, and transformation efficiency is higher, can reach about 60% ~ 70%.
The flourish that in liquid medium within, matrimony vine hairly root can be a large amount of, solves the problem of drawing materials, and is easy to observe growing state.
Accompanying drawing explanation
Fig. 1 is Transgenic Lycium barbarum L hairly root photo.
Fig. 2 is Transgenic Lycium barbarum L hairly root fluoroscopic examination result photo.
Fig. 3 is Transgenic Lycium barbarum L hairly root liquid culture photo.
Specific implementation method
Below in conjunction with specific embodiment, the present invention is further illustrated.
Shown in RFP gene SEQ.ID.NO1, RFP gene (GenBank:AF506027.1)
Agrobacterium rhizogenesA4 is bought in NTCC Type Tissue Collection (http://www.biovector.net/), in September, 2014 time buying.
The matrimony vine tree that the present invention adopts
Hebei matrimony vine tree (commercialization tree);
Lycium barbarum (commercialization tree)
Lycium barbarum (commercialization tree)
The formula of LB liquid nutrient medium: 1% peptone+0.5% yeast extract+1%Nacl;
The formula of LB solid medium: 1% peptone+0.5% yeast extract+1%Nacl+1.5% agar;
MS substratum: by MS minimum medium powder+1% agar
Dual culture substratum: MS+3% sucrose+0.7% agar
Hairy root culture base: MS+IBA0.2mg/L.
Embodiment 1
A gene transformation method for transgenosis Hebei matrimony vine hairly root, comprises the following steps:
(1) the aseptic blade of Hebei matrimony vine is prepared: by good for fresh growing way Hebei matrimony vine blade, clear water rinses rear dehydrated alcohol wipe surfaces well, with the aqueous ethanolic solution sterilization 8min that volumetric concentration is 73%, aseptic distillation water rinse 4 times, be inoculated on MS substratum, 25 DEG C of dark Dual culture obtain the aseptic blade of Hebei matrimony vine;
(2) Agrobacterium rhizogenesA4 activation: Agrobacterium rhizogenesA4 is being rule containing on antibiotic LB solid medium, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 42h, picking list bacterium colony is in containing on antibiotic LB liquid nutrient medium, 28 DEG C, shaking speed 200rpm light culture 42h, obtain activating hair-root Agrobacterium A4; Containing 35S:RFP gene on the carrier of described Agrobacterium rhizogenesA4; Shown in described RFP gene SEQ.ID.NO1;
(3) infect explant, Dual culture: wound activating hair-root Agrobacterium A4 being spread upon the aseptic blade of Hebei matrimony vine after designated port, is placed on Dual culture base, at 25 DEG C, illumination 16h, light culture 8h, cultivate 5d;
(4) hairy root culture: Hebei matrimony vine blade step (3) obtained changes on hairy root culture base, and sealed membrane is sealed, 25 DEG C, illumination 16h, light culture 8h, cultivates 28d, within every 7 days, changes a hairy root culture base;
(5) fluoroscopic examination; Whether the existence of the red fluorescence of the RFP gene in the hairly root utilizing fluorescent microscope detecting step (4) to obtain; Result shows that the plant root visible whole piece root proceeded to 35S:RFP reporter gene all has stronger red fluorescence to be transgenosis Hebei matrimony vine hairly root.(see Fig. 1, Fig. 2)
Make by following method containing antibiotic LB solid medium: in LB solid culture based formulas, add kantlex, the final concentration making kantlex is 100mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 100mg/L.
Make by following method containing antibiotic LB liquid nutrient medium: in LB liquid culture based formulas, add kantlex, the final concentration making kantlex is 100mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 100mg/L.
Embodiment 2
A gene transformation method for transgenosis Lycium barbarum hairly root, comprises the following steps:
(1) the aseptic blade of Lycium barbarum is prepared: by matrimony vine blade, clear water rinses rear dehydrated alcohol wipe surfaces well, with the aqueous ethanolic solution sterilization 10min that volumetric concentration is 70%, aseptic distillation water rinse 3 times, be inoculated on MS substratum, 25 DEG C of dark Dual culture obtain the aseptic blade of Lycium barbarum
(2) Agrobacterium rhizogenesA4 activation: Agrobacterium rhizogenesA4 is being rule containing on antibiotic LB solid medium, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 36h, picking list bacterium colony is in containing on antibiotic LB liquid nutrient medium, 28 DEG C, shaking speed 180rpm light culture 48h, obtain activating hair-root Agrobacterium A4; Containing 35S:RFP gene on the carrier of described Agrobacterium rhizogenesA4; Shown in described RFP gene SEQ.ID.NO1;
(3) infect explant, Dual culture: wound activating hair-root Agrobacterium A4 being spread upon the aseptic blade of the Lycium barbarum after designated port, be placed on Dual culture base, at 24 DEG C, illumination 16h, light culture 8h, cultivate 4d;
(4) hairy root culture: Lycium barbarum blade exchanging step (3) obtained is on hairy root culture base, and sealed membrane is sealed, 24 DEG C, illumination 16h, light culture 8h, cultivates 25d, within every 6 days, changes a hairy root culture base;
(5) fluoroscopic examination; Whether the existence of the red fluorescence of the RFP gene in the hairly root that detecting step (4) obtains; The hairly root with red fluorescence is transgenosis Lycium barbarum hairly root.The shape of its transgenosis Lycium barbarum hairly root is similar to the shape of the transgenosis Hebei matrimony vine hairly root of embodiment 1.
Make by following method containing antibiotic LB solid medium: in LB solid culture based formulas, add kantlex, the final concentration making kantlex is 80mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 80mg/L.
Make by following method containing antibiotic LB liquid nutrient medium: in LB liquid culture based formulas, add kantlex, the final concentration making kantlex is 80mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 80mg/L.
Embodiment 3
A gene transformation method for transgenosis lycium barbarum hairly root, comprises the following steps:
(1) the aseptic blade of lycium barbarum is prepared: by lycium barbarum blade, clear water rinses rear dehydrated alcohol wipe surfaces well, with the aqueous ethanolic solution sterilization 5min that volumetric concentration is 75%, aseptic distillation water rinse 5 times, be inoculated on MS substratum, 25 DEG C of dark Dual culture obtain the aseptic blade of lycium barbarum;
(2) Agrobacterium rhizogenesA4 activation: Agrobacterium rhizogenesA4 is being rule containing on antibiotic LB solid medium, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 48h, picking list bacterium colony is in containing on antibiotic LB liquid nutrient medium, 28 DEG C, shaking speed 220rpm light culture 36h, obtain activating hair-root Agrobacterium A4; Containing 35S:RFP gene on the carrier of described Agrobacterium rhizogenesA4; Shown in described RFP gene SEQ.ID.NO1;
(3) infect explant, Dual culture: wound activating hair-root Agrobacterium A4 being spread upon the aseptic blade of the lycium barbarum after designated port, be placed on Dual culture base, at 25 DEG C, illumination 16h, light culture 8h, cultivate 6d;
(4) hairy root culture: changed on hairy root culture base by the matrimony vine blade that step (3) obtains, sealed membrane is sealed, 25 DEG C, illumination 16h, light culture 8h, cultivates 30d, within every 8 days, changes a hairy root culture base;
(5) fluoroscopic examination; Whether the existence of the red fluorescence of the RFP gene in the hairly root that detecting step (4) obtains; The hairly root with red fluorescence is transgenosis lycium barbarum hairly root.The shape of its transgenosis lycium barbarum hairly root is similar to the shape of the transgenosis Hebei matrimony vine hairly root of embodiment 1.
Make by following method containing antibiotic LB solid medium: in LB solid culture based formulas, add kantlex, the final concentration making kantlex is 120mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 120mg/L.
Make by following method containing antibiotic LB liquid nutrient medium: in LB liquid culture based formulas, add kantlex, the final concentration making kantlex is 120mg/L, adds spectinomycin, makes the final concentration of spectinomycin be 120mg/L.
Embodiment 4
The liquid cultivating method of Transgenic Lycium barbarum L hairly root, comprises the steps:
(1) preparation is containing the liquid nutrient medium of plant hormone: in 1/2MS liquid nutrient medium, add plant hormone IBA make concentration be 0.3mg/L, mix;
(2) what Transgenic Lycium barbarum L hairly root embodiment 1 obtained obtained in step (1) contains in the liquid nutrient medium of plant hormone, 26 DEG C, and rotating speed 100rpm, cultivates; The liquid nutrient medium containing plant hormone that replacing in 18 days step (1) obtains, carries out enlarged culturing to Transgenic Lycium barbarum L hairly root.(see Fig. 3).
Embodiment 5
The liquid cultivating method of Transgenic Lycium barbarum L hairly root, comprises the steps:
(1) preparation is containing the liquid nutrient medium of plant hormone: in 1/2MS liquid nutrient medium, add plant hormone IBA make concentration be 0.1mg/L, mix;
(2) what Transgenic Lycium barbarum L hairly root embodiment 2 obtained obtained in step (1) contains in the liquid nutrient medium of plant hormone, 24 DEG C, and rotating speed 80rpm, cultivates; The liquid nutrient medium containing plant hormone that replacing in 15 days step (1) obtains, carries out enlarged culturing to Transgenic Lycium barbarum L hairly root.Result is similar to embodiment 4.
Embodiment 6
The liquid cultivating method of Transgenic Lycium barbarum L hairly root, comprises the steps:
(1) preparation is containing the liquid nutrient medium of plant hormone: in 1/2MS liquid nutrient medium, add plant hormone IBA make concentration be 0.5mg/L, mix;
(2) what Transgenic Lycium barbarum L hairly root embodiment 3 obtained obtained in step (1) contains in the liquid nutrient medium of plant hormone, 28 DEG C, and rotating speed 120rpm, cultivates; The liquid nutrient medium containing plant hormone that replacing in 20 days step (1) obtains, carries out enlarged culturing to Transgenic Lycium barbarum L hairly root.Result is similar to embodiment 4.
Method of the present invention, utilizes the Agrobacterium rhizogenesA4 containing 35S:RFP reporter gene to infect the blade of matrimony vine aseptic seedling, obtains cotransformation plant, and then obtain transformed hairy root.Adopt method of the present invention not only greatly can shorten the transformation period, also can see experimental result intuitively; The research carrying out root system development aspect with the in vitro hairly root system of matrimony vine can manual control culture condition, eliminates the impact that environmental factors is brought; In addition, the method expands numerous cultivation in conjunction with liquid can overcome matrimony vine seedling root system because edatope is to observing, sampling the difficulty brought, thus is expected to change the present situation that matrimony vine root system research falls behind relatively.The present invention is that the checking of matrimony vine gene function provides a kind of fast and reliable method, also for the genetic improvement of matrimony vine gene provides a kind of new method for transformation.
Claims (5)
1. a gene transformation method for Transgenic Lycium barbarum L hairly root, is characterized in that comprising the following steps:
(1) the aseptic blade of matrimony vine is prepared: by matrimony vine blade, clear water rinses rear dehydrated alcohol wipe surfaces well, with aqueous ethanolic solution sterilization 5-10min, and aseptic distillation water rinse 3-5 time, be inoculated on MS substratum, 25 DEG C of dark Dual culture obtain the aseptic blade of matrimony vine;
(2) Agrobacterium rhizogenesA4 activation: Agrobacterium rhizogenesA4 is being rule containing on antibiotic LB solid medium, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 36-48h, picking list bacterium colony is in containing on antibiotic LB liquid nutrient medium, 28 DEG C, shaking speed 180-220rpm light culture 36-48h, obtain activating hair-root Agrobacterium A4; Containing 35S:RFP gene on the carrier of described Agrobacterium rhizogenesA4; Shown in described RFP gene SEQ.ID.NO1;
(3) infect explant, Dual culture: wound activating hair-root Agrobacterium A4 being spread upon the aseptic blade of the matrimony vine after designated port, be placed on Dual culture base, at 24 DEG C ~ 25 DEG C, illumination 16h, light culture 8h, cultivate 4-6d;
(4) hairy root culture: changed on hairy root culture base by the matrimony vine blade that step (3) obtains, sealed membrane is sealed, 24 DEG C ~ 25 DEG C, illumination 16h, light culture 8h, cultivate 25-30d, every 6-8 days and change a hairy root culture base;
(5) fluoroscopic examination; Whether the existence of the red fluorescence of the RFP gene in the hairly root that detecting step (4) obtains; The hairly root with red fluorescence is Transgenic Lycium barbarum L hairly root.
2. the Transgenic Lycium barbarum L hairly root of the method acquisition of claim 1.
3. the liquid cultivating method of the Transgenic Lycium barbarum L hairly root of claim 2, is characterized in that comprising the steps:
(1) preparation is containing the liquid nutrient medium of plant hormone: in 1/2MS liquid nutrient medium, add plant hormone IBA make concentration be 0.1-0.5mg/L, mix;
(2) what obtained in step (1) by Transgenic Lycium barbarum L hairly root contains in the liquid nutrient medium of plant hormone, and 24-28 DEG C, rotating speed 80-120rpm, cultivate; Within 15-20 days, change the liquid nutrient medium containing plant hormone that step (1) obtains, enlarged culturing is carried out to Transgenic Lycium barbarum L hairly root.
4. the gene transformation method of a kind of Transgenic Lycium barbarum L hairly root according to claim 1, it is characterized in that described is make by following method containing antibiotic LB solid medium: in LB solid culture based formulas, add kantlex, the final concentration of kantlex is made to be 80-120mg/L, add spectinomycin, make the final concentration of spectinomycin be 80-120mg/L.
5. the gene transformation method of a kind of Transgenic Lycium barbarum L hairly root according to claim 1, it is characterized in that described is make by following method containing antibiotic LB liquid nutrient medium: in LB liquid culture based formulas, add kantlex, the final concentration of kantlex is made to be 80-120mg/L, add spectinomycin, make the final concentration of spectinomycin be 80-120mg/L.
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| CN105734024A (en) * | 2016-04-08 | 2016-07-06 | 天津大学 | Wolfberry glutamyl cysteine synthetase and encoding gene and application |
| CN114958904A (en) * | 2022-05-20 | 2022-08-30 | 南京农业大学 | Method for rapidly obtaining radish transgenic material |
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| CN102321664A (en) * | 2011-10-21 | 2012-01-18 | 福建农林大学 | Method for inducing production of tripterygium wilfordii hairy root by agrobacterium rhizogenes |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734024A (en) * | 2016-04-08 | 2016-07-06 | 天津大学 | Wolfberry glutamyl cysteine synthetase and encoding gene and application |
| CN114958904A (en) * | 2022-05-20 | 2022-08-30 | 南京农业大学 | Method for rapidly obtaining radish transgenic material |
| CN114958904B (en) * | 2022-05-20 | 2023-10-24 | 南京农业大学 | Method for rapidly obtaining radish transgenic material |
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