CN102925481A - Genetic transformation method for embryonic callus of photinia serrulata lamina through gene gun mediation - Google Patents
Genetic transformation method for embryonic callus of photinia serrulata lamina through gene gun mediation Download PDFInfo
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Abstract
The invention discloses a genetic transformation method for the embryonic callus of photinia serrulata lamina through gene gun mediation, comprising the following steps of: by the way of taking photinia serrulata tissue culture seedling lamina as a material, inducing the embryonic callus on an induction medium to provide an acceptor material of transgene; and introducing exogenous gene into acceptor cells by a gene gun bombardment method, and screening to obtain transgenic a plant. The method disclosed by the invention has the preferred parameters as follows: when a penetrating agent mannitol with the concentration of 0.3mol/L is added to the embryonic callus, an added DNA (deoxyribonucleic acid) precipitant prepared from 50muL 12.5mol/L of CaCl2, 50muL 0.1mol/L of spermidine is added, the bombardment distance of a gene gun is 9cm, the vacuum degree is greater than 28inHg, the gas pressure is 1100Psi, and the bombardment is performed once, the transformation efficiency is highest. The materials of the method provided by the invention are not limited by seasons, the method is higher in transformation efficiency, and suitable for the plant which is invalid for agrobacterium, and an optimized technical system capable of repeatedly transforming the photinia serrulata can be built.
Description
Technical field
The invention belongs to agricultural biological technical field or plant genetic engineering field.Specifically, relate to a kind of method by particle gun mediation Photinia glabra leaves embryo callus genetic transformation.
Background technology
Photinia glabra is the new variety of Photinia (P.glabra) and Chinese photinia (P.serralata) selection cross, evergreen dungarunga (the Zhang Hu of Rosaceae Photinia, Wang Runxian, Qiu Guojin, Deng .Tissue culture and rapid propagation of Photiniafraseri[J] .Plant Physiology Communications. Plant Physiology Communications, 2003,39 (1): 34 (in Chinese)), one of color leaf seeds of in recent years China's introduction, its blade surface light, spire is scarlet, sight is strong, be rare good Landscape Trees, extensively plant at south China, but for good Landscape Trees, its best purpose is: be suitable for national different geographical environment and plant, i.e. increase sight makes it to have stronger winter hardiness in the time of strong, and therefore, cultivation has the strong photinia glabra new variety that also have simultaneously stronger winter hardiness of sight and just is even more important.Traditional breeding method is a very very long process on forest, the application of plant genetic engineering overcome traditional breeding way deficiency (Woodson W RBiotechnology of floricultural crops[J] .HortScience, 1991,26 (8): 1029 ~ 1033).Since nineteen eighty-three obtains transfer-gen plant first, transgenic technology various plants obtained success (Wang Guanlin, Fang Hongjun. plant genetic engineering philosophy and technique [M]. Beijing: Science Press, 1998; Liang Huimin, the Xiayang, Sun Zhongxu, etc. the foundation of Genetic Transformation System of Medicago sativa Mediated by Agrobacterium tumefaciens. Journal of Agricultural Biotechnology, 2005,13 (2): 152～156; The Xiayang, Liang Huimin, Sun Zhongxu, etc. the research of tetraploid locust being changed to beet BADH gene. Scientia Agricultura Sinica, 2004,37 (8): 1208-1211; The Xiayang, Wang Taiming, Liang Huimin waits the agriculture bacillus mediated Sophora japonica transgenic of .2007. and tissue culture breeding method. the patent No.: 200710013185; Yan Li-Ping, XiaYang, Wang Fei, et al.Physiological Responses to Salt Stress of T2 Alfalfa Progenies carrying aTransgene for Betaine Aldehyde Dehydrogenase.Plant Cell, Tissue and Organ Culture.SCI; Yan Liping, Xiayang *, Mao Xiuhong, etc. the salt resistance of No. 2 kinds of transgenic rice alfalfa mountain lucerne is identified and system. seed selection Botany Gazette, 2011,46 (3): 293-301; Liu Cuilan, Yan Liping, Mao Xiuhong, Deng. No. 3 (SLM07) alfalfas of transgenic rice mountain lucerne breeding of new variety. the Chinese Grassland journal, 2011,33(3): 6-11), the research of relevant photinia glabra also only limits to the tissue culture aspect, the somatic embryo Regeneration System of plant and the report of genetic transformation few (Fang Minyan, Liang Huimin, Xiayang, Yan Liping, Liu Cuilan. the Study on influencing factors of the stripped plant regeneration frequency of photinia glabra. the northwest Botany Gazette, 2012,32(2): 0403-0408; Liang Huimin, Liu Cuilan, Xiayang, Yan Liping, Fang Minyan. induced rapid propagation culture medium for somatic embryos of leaves in vitro of photinia x frasery. the patent No.: 201110033598.0; Rafael Ramirez-Malagon1, Anatoli Borodanenko1, Jose Luis Barrera-Guerra1﹠amp; Neftali Ochoa-Alejo.1997.Micropropagation for fraser photinia (Photinia fraseri) Plant Cell, Tissue and Organ Culture, 48:219-222).Photinia glabra is few in Agrobacterium tumefaciens mediated lower final transformant, because of after Photinia glabra leaves infected by Agrobacterium (Agrobacterium tutnefaciens), Agrobacterium is difficult to the suppressed (Liu Cuilan that causes, the Xiayang, Yan Liping, Deng. tetraploid locust, Chinese scholar tree and photinia glabra are to agrobacterium tumefaciens bacterial strain and different antibiotic sensitive Journal of Sex Research. the Shandong forestry science and technology, and 2010,190(5): 20-23).Add the pollution of endophyte (endophyticalbacteria), many studies show that, ubiquity endophyte in the plant tissue, when plant tissue carried out isolated culture, these endophytes will produce pollution, and the harm that endogenetic bacteria causes is to cause in early days cultivating unsuccessfully, cause test-tube seedling transplanting difficulty and dead (Zhou Junhui in the later stage, Zhou Hougao, Liu Huaquan. the endogenetic bacteria pollution problem [J] in the plant tissue culture. GUIHAIA .2003,23 (01): 41-47).The genetic transformation that carries out Photinia glabra leaves in view of Agrobacterium (Agrobacterium tutnefaciens) is invalid, therefore utilizes particle bombardment just can avoid the problems referred to above: the generation of different bacterium overlying pollutions.
The distinguishing feature of particle bombardment is that acceptor type is extensive, is a kind of method of mechanical transfer gene without the host range restriction, adopts to add Ca
2+, spermidine etc. makes foreign DNA be adsorbed on tungsten gold microparticle surfaces, makes the little bullet of DNA-, under the moment strength effect that particle gun excites, foreign gene is directly imported in the vegetable cell.But, yet there are no report through retrieval by the method that particle gun mediates Photinia glabra leaves embryo callus genetic transformation.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is a kind of method by particle gun mediation Photinia glabra leaves embryo callus genetic transformation.
Of the present invention by particle gun mediation Photinia glabra leaves embryo callus genetic transforming method, step comprises: 1. the blade embryonic callus induction is cultivated, 2. blade embryo callus osmotic treated, 3. carry out particle gun genetic transformation and receptor tissue's succeeding transfer culture take the blade embryo callus as acceptor, 4. transfer-gen plant is carried out Molecular Identification;
It is characterized in that:
The step 1. method of described blade embryonic callus induction cultivation is: in Bechtop, clip photinia glabra tissue cultured seedling blade cuts its blade edge, becomes 1 ± 0.5cm along the vein crosscut
2Bulk, be inoculated on the H1 substratum in advance training, then be put in the group training indoor under 25 ℃ of daytimes, night 18 ℃ of dark conditions induced embryonic callus, behind the 15d, blade edge overgrows with fine and close granular embryo callus;
The step 2. method of described blade embryo callus osmotic treated is: embryo callus is divided into 0.5 ± 0.1cm
2Piece shift and to put on the H2 substratum, interpolation concentration is that the N.F,USP MANNITOL of 0.1～0.5mol/L carries out osmotic treated 15h～16h, repeat 3 times, the embryo callus piece after the osmotic treated is closely arranged in the 9.0cm culture dish that contains the H1 substratum, every ware is arranged 25～30 callus lines;
The step 3. method of described particle gun genetic transformation is: get the bronze that particle diameter is 1.0um, sterilize stand-by, adding concentration when preparing little bullet is the RD29A plasmid DNA 3 μ l of 1ug/ul, the CaCl of 12.5mol/L
250ul+0.1mol/L the DNA precipitation agent that forms of spermidine 50ul; Bombardment parameters is 2.5cm for the distance that can split disk and carrier, carrier with stop that the distance of net is 0.8cm, vacuum tightness is greater than 28inHg, gaseous tension 1100Psi, target distance 9cm, bronze consumption 300ug/ rifle, every ware bombards 1 time, repeats 3 times;
The step 3. method of described receptor tissue succeeding transfer culture is: the material after will bombarding is at H
1Continuing dark culturing on the substratum changes over to after 5 days on the resistance culture base H3 substratum that contains 40～60mg/L kantlex, be to break up screening and culturing under the condition of 2000Lx in 25 ℃ of indoor daytimes of group training, 18 ℃ of nights, light intensity, step sizing 60～90d, the normal plant that grows is the transgenosis resistant plant; When treating resistance seedling height of seedling 1～2cm, shear to be inoculated into and carry out numerously soon on the division culture medium that does not add kantlex, when height of seedling 3-5cm, be seeded in root induction on the root media H4 substratum;
PCR method with routine is carried out Molecular Identification to the transfer-gen plant that screens with kantlex.
The composition of above-mentioned H1 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, 2,4 dichlorophenoxyacetic acid 0.5mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0; Be used for induced embryonic callus.
The composition of above-mentioned H2 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, the N.F,USP MANNITOL of 2,4 dichlorophenoxyacetic acid 0.5mg/L, 0.1～0.5mol/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0; Be used for the blade embryo callus is carried out osmotic treated.
The composition of above-mentioned H3 substratum is: MS substratum, 6-benzyl purine 1.0-1.5mg/L, kinetin (KT) 1.0-1.5mg/L, indolebutyric acid 0.4-0.6mg/L, kantlex (Kan) 40-60mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0; Be used for embryo callus and carry out differentiation and screening.
The composition of above-mentioned H4 substratum is: 1/2MS substratum, 6-benzyl purine 0.4-0.6mg/L, indolebutyric acid 0.20-0.30mg/L, naphthylacetic acid 0.10-0.11mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0; Be used for root induction.
Above-mentioned photinia glabra is that photinia glabra is the new variety of Photinia (P.glabra) and Chinese photinia (P.serralata) selection cross, and the evergreen dungarunga of Rosaceae Photinia is one of color leaf seeds of in recent years China's introduction.
Above-mentioned by in the particle gun mediation Photinia glabra leaves embryo callus genetic transforming method: along with the increasing of mannitol concentration, transformation efficiency also once increased trend, but increasing ratio is not directly proportional with increasing concentration, it is that 0.3mol/L is comparatively suitable that experiment results proved adds mannitol concentration, therefore the preferred 0.3mol/L of the concentration of described N.F,USP MANNITOL.
Particle bombardment of the present invention (particle bomrdment; Microprojectile; Biolistics; Particle gun) being the high-speed metal particle of collection introduces a kind of genetic transfoumation in the viable cell with nucleic acid molecule.Used particle gun is U.S. Bio-RAD company product among the present invention, refers to adopt high speed helium particle gun PDS-1000/He (Bio-Rad).
Test-results shows: embryo callus is 0.3mol/L adding the permeate agent mannitol concentration, adds DNA precipitation agent 12.5mol/LCaCL
250ul+0.1mol/L spermidine 50ul and particle gun target distance adopt 9cm, vacuum tightness is greater than 28inHg, gaseous tension 1100Psi, and when bombarding 1 time, transformation efficiency is the highest.
The present invention is take photinia glabra tissue cultured seedling blade as material, and induced embryonic callus on inducing culture provides genetically modified acceptor material.Adopt particle bombardment with the Studies of Transfer of Alien Genes Into Receptors cell, obtain transfer-gen plant through screening.In conversion process, the several important factors that affect the particle bombardment genetic transformation are compared in depth analyze emphatically, tentatively set up the optimisation technique system that suitable photinia glabra can repeat genetic transformation.Outstanding feature of the present invention is: 1) Photinia glabra leaves can be induced the generation embryo callus, transgenic acceptor is provided, 2) draw materials and be not subject to seasonal restrictions, 3) transformation efficiency is higher, 4) be suitable for the plant invalid to Agrobacterium, 5) the most features that keep the protogene type of transfer-gen plant, 6) tentatively set up and be fit to the optimisation technique system that photinia glabra can repeat to transform.
Description of drawings
Fig. 1: A is the Photinia glabra leaves induced embryonic callus; B is the screening of kalamycin resistance seedling and differentiation; C is that the transgenosis resistant plant is numerous soon; D is that transfer-gen plant is taken root.
Fig. 2: transfer-gen plant pcr analysis, M:DNA molecular weight among the figure; P is plasmid (positive control); The negative contrast of CK; 1-6 is transfer-gen plant; 7-8 is unconverted plant.
Embodiment
Embodiment 1: method and the utilization of the embryo callus genetic transformation of crossing by particle gun mediation different concns N.F,USP MANNITOL osmotic treated
In Bechtop, clip photinia glabra tissue cultured seedling blade cuts its blade edge, becomes 1 ± 0.5cm along the vein crosscut
2Bulk, be inoculated on the H1 substratum in advance training, then be put in the group training indoor under 25 ℃ of daytimes, night 18 ℃ of dark conditions induced embryonic callus, behind the 15d, blade edge overgrows with fine and close granular embryo callus;
Embryo callus is divided into 0.5 ± 0.1cm
2Piece shift and to put on the H2 substratum, add concentration and be 0.1,0.3, the N.F,USP MANNITOL of 0.5mol/L carries out osmotic treated 15h～16h, every processing repeats 3 times, embryo callus piece after the osmotic treated is closely arranged in the 9.0cm culture dish that contains the H1 substratum, every ware is arranged 30 callus lines, waits to bombard;
Plasmid and preparation thereof: plasmid pCAMBIA2300, about length 8.7Kb, contain approximately 2.1Kb of salt tolerant, cold-resistant gene RD29A() and anti-Kan gene NPT II (approximately 0.7Kb), by Shandong Province's forest tree genetic improvement key lab provide (research [D] is induced in the structure and the tetraploid locust regeneration that see the beautiful duckweed .RD29A of swallow gene plant expression vector for details. Gansu Agriculture University,, 2005).Adopt alkaline lysis method of extracting plasmid DNA, and carry out purifying (Sambrook with the PEG precipitator method, Fritsch E F, Maniatis.Molecular Cloning:A Laboratory Manual, 2nd ed ［ M ］ .New York:Cold Spring Horbor LaborartoryPress., 1989).
The making of DNA bronze bullet:
1), to take by weighing granular size be that the bronze 300ug of 1.0um is put in the Eppendorf centrifuge tube of 1.5ml, adds the alcohol of 1ml 70%, violent vortex 3-5min, static 15min; The centrifugal 10s of 14000rpm removes supernatant liquor; Add the 1ml sterilized water, violent vortex 1min, static 1min; The centrifugal 10s of 14000rpm removes supernatant liquor, and repeated washing 3 times carries out disinfection to bronze;
2), add the aseptic glycerine of 50ul, violent vortex 5min breaks aggegation, is divided into the 50ul/ pipe, bronze amount 300ug/ pipe directly uses or storage;
3) add successively RD29A plasmid DNA 3 μ l(concentration 1ug/ul after, bronze disperses), different DNA precipitation agent: i.e. CaCl
2(12.5mol/L) 50ul, spermidine (0.1mol/L) 50ul or CaCl
2(12.5mol/L) 50ul+ spermidine (0.1mol/L) 50ul, vortex limit, limit application of sample; Continue vortex 2-3min, static 1min; Remove supernatant liquor behind the centrifugal 2s of 14000rpm, add 140ul 70% washing with alcohol, remove supernatant liquor behind the centrifugal 2s of 14000rpm; Suspend with the 48ul dehydrated alcohol at last;
4), the bronze test tube to suspending, flick tube wall 5 times, vortex 2-3s under the low speed is coated with bronze suspension and DNA, then sampling is added on little missile-borne body, is used for each bombardment.
Particle gun bombardment: adopt high speed helium particle gun PDS-1000/He (Bio-Rad) to bombard.Bombardment parameters is 2.5cm for the distance that can split disk and carrier, carrier and stop that the distance of net is 0.8cm, and vacuum tightness is greater than 28inHg, gaseous tension 1100Psi, target distance 6cm or 9cm or 12cm, preferred 9cm, bronze consumption 300ug/ rifle, every ware bombardment 1 time repeats 3 times.
With the material after the bombardment not with the H of N.F,USP MANNITOL
1(dark culturing helps to repair the damage that the bronze particle causes to continue dark culturing on the substratum, and impel callus continue to grow) change on the resistance culture base H3 substratum that contains the 50mg/L kantlex behind the 5d, be to break up screening and culturing under the condition of 2000Lx in 25 ℃ of indoor daytimes of group training, 18 ℃ of nights, light intensity, step sizing 60～90d, the normal plant that grows is the transgenosis resistant plant; The material that is equipped with not bombardment in above-mentioned steps compares.
When treating resistance seedling height of seedling 1～2cm, shear to be inoculated into and carry out numerously soon on the division culture medium that does not add kantlex, when height of seedling 3-5cm, be seeded in root induction on the root media H4 substratum.The results are shown in Figure 1.
The composition of above-mentioned H1 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, 2,4 dichlorophenoxyacetic acid 0.5mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H2 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, the N.F,USP MANNITOL of 2,4 dichlorophenoxyacetic acid 0.5mg/L, 0.3mol/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H3 substratum is: MS substratum, 6-benzyl purine 1.0mg/L, kinetin (KT) 1.0mg/L, indolebutyric acid 0.5mg/L, kantlex (Kan) 50mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H4 substratum is: 1/2MS substratum, 6-benzyl purine 0.5mg/L, indolebutyric acid 0.25mg/L, naphthylacetic acid 0.10mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0.
The Molecular Identification of transfer-gen plant: get the resistant plant blade, extract total DNA(king General Guan Yu's Tomb, Fang Hongjun by the CTAB method. plant genetic engineering philosophy and technique [M]. Beijing: Science Press, 1998), if the negative contrast of unconverted plant DNA is increased with the primer of RD29A gene.
The upstream primer sequence is (PI): 5 '-CGAGGATCCTCTCTTAAAGCTCCTT 3 ',
The downstream primer sequence is (P II): 5 '-TCTAGGGTACC GTGGAAAATGGATC – 3 ' (it is synthetic that worker bio-engineering corporation is given birth in Shanghai), amplified fragments is the long 2100bp of molecule.
PCR reaction system cumulative volume is 25 μ L, 2 * Taq PCR MasterMix 12.5uL (day root biochemical technology company limited), F(10mmol/L) 1uL, R(10mmol/L) 1uL, ddH2O10.5uL.Amplification program is: 95 ℃ of denaturation 4min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 2min, 35 circulations; 72 ℃ are extended 10min.The results are shown in Figure 2.
Above-mentioned MS medium component is:
Above-mentioned 1/2MS medium component is:
Claims (2)
1. one kind by particle gun mediation Photinia glabra leaves embryo callus genetic transforming method, step comprises: 1. the blade embryonic callus induction is cultivated, 2. blade embryo callus osmotic treated, 3. carry out particle gun genetic transformation and receptor tissue's succeeding transfer culture take the blade embryo callus as acceptor, 4. transfer-gen plant is carried out Molecular Identification;
It is characterized in that:
The step 1. method of described blade embryonic callus induction cultivation is: in Bechtop, clip photinia glabra tissue cultured seedling blade cuts its blade edge, becomes 1 ± 0.5cm along the vein crosscut
2Bulk, be inoculated on the H1 substratum in advance training, then be put in the group training indoor under 25 ℃ of daytimes, night 18 ℃ of dark conditions induced embryonic callus, behind the 15d, blade edge overgrows with fine and close granular embryo callus;
The step 2. method of described blade embryo callus osmotic treated is: embryo callus is divided into 0.5 ± 0.1cm
2Piece shift and to put on the H2 substratum, interpolation concentration is that the N.F,USP MANNITOL of 0.1～0.5mol/L carries out osmotic treated 15h～16h, repeat 3 times, the embryo callus piece after the osmotic treated is closely arranged in the 9.0cm culture dish that contains the H1 substratum, every ware is arranged 25～30 callus lines;
The step 3. method of described particle gun genetic transformation is: get the bronze that particle diameter is 1.0um, sterilize stand-by, adding concentration when preparing little bullet is the RD29A plasmid DNA 3 μ l of 1ug/ul, the CaCl of 12.5mol/L
250ul+0.1mol/L the DNA precipitation agent that forms of spermidine 50ul; Bombardment parameters is 2.5cm for the distance that can split disk and carrier, carrier with stop that the distance of net is 0.8cm, vacuum tightness is greater than 28inHg, gaseous tension 1100Psi, target distance 9cm, bronze consumption 300ug/ rifle, every ware bombards 1 time, repeats 3 times;
The step 3. method of described receptor tissue succeeding transfer culture is: the material after will bombarding is at H
1Continuing dark culturing on the substratum changes over to after 5 days on the resistance culture base H3 substratum that contains 40～60mg/L kantlex, be to break up screening and culturing under the condition of 2000Lx in 25 ℃ of indoor daytimes of group training, 18 ℃ of nights, light intensity, step sizing 60～90d, the normal plant that grows is the transgenosis resistant plant; When treating resistance seedling height of seedling 1～2cm, shear to be inoculated into and carry out numerously soon on the division culture medium that does not add kantlex, when height of seedling 3-5cm, be seeded in root induction on the root media H4 substratum;
The composition of above-mentioned H1 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, 2,4 dichlorophenoxyacetic acid 0.5mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H2 substratum is: MS substratum, 6-benzyl purine 2.0mg/L, naphthylacetic acid 5.0mg/L, the N.F,USP MANNITOL of 2,4 dichlorophenoxyacetic acid 0.5mg/L, 0.1～0.5mol/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H3 substratum is: MS substratum, 6-benzyl purine 1.0-1.5mg/L, kinetin 1.0-1.5mg/L, indolebutyric acid 0.4-0.6mg/L, kantlex 40-60mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0;
The composition of above-mentioned H4 substratum is: 1/2MS substratum, 6-benzyl purine 0.4-0.6mg/L, indolebutyric acid 0.20-0.30mg/L, naphthylacetic acid 0.10-0.11mg/L, agar 5.5g/L, sucrose 30g/L, pH value 5.8-6.0.
2. as claimed in claim 1 by particle gun mediation Photinia glabra leaves embryo callus genetic transforming method, it is characterized in that: the concentration of described N.F,USP MANNITOL is 0.3mol/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805631A (en) * | 2014-01-07 | 2014-05-21 | 泰安市泰山林业科学研究院 | Method for efficient gene transformation of photinia serrulata |
| CN108148871A (en) * | 2017-12-22 | 2018-06-12 | 中国中医科学院中药研究所 | A kind of tripterygium wilfordii genetic transforming method of particle gun mediation |
| CN112481307A (en) * | 2020-11-30 | 2021-03-12 | 内蒙古农业大学 | Gene gun mediated flax genetic transformation method |
| CN112772416A (en) * | 2021-01-28 | 2021-05-11 | 宜宾金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124954A (en) * | 2011-01-31 | 2011-07-20 | 江苏农林职业技术学院 | Induced rapid propagation culture medium for somatic embryos of leaves in vitro of photinia x frasery |
| CN102124955A (en) * | 2011-01-31 | 2011-07-20 | 江苏农林职业技术学院 | Induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos |
| CN102586269A (en) * | 2011-01-06 | 2012-07-18 | 泰安市泰山林业科学研究院 | Ammopiptanthus mongolicus cold resistant gene AmGS |
-
2012
- 2012-11-30 CN CN2012105014162A patent/CN102925481A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586269A (en) * | 2011-01-06 | 2012-07-18 | 泰安市泰山林业科学研究院 | Ammopiptanthus mongolicus cold resistant gene AmGS |
| CN102124954A (en) * | 2011-01-31 | 2011-07-20 | 江苏农林职业技术学院 | Induced rapid propagation culture medium for somatic embryos of leaves in vitro of photinia x frasery |
| CN102124955A (en) * | 2011-01-31 | 2011-07-20 | 江苏农林职业技术学院 | Induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805631A (en) * | 2014-01-07 | 2014-05-21 | 泰安市泰山林业科学研究院 | Method for efficient gene transformation of photinia serrulata |
| CN108148871A (en) * | 2017-12-22 | 2018-06-12 | 中国中医科学院中药研究所 | A kind of tripterygium wilfordii genetic transforming method of particle gun mediation |
| CN112481307A (en) * | 2020-11-30 | 2021-03-12 | 内蒙古农业大学 | Gene gun mediated flax genetic transformation method |
| CN112772416A (en) * | 2021-01-28 | 2021-05-11 | 宜宾金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
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Application publication date: 20130213 |