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WO2002066599A2 - Procede efficace permettant de mettre au point des plantes transgeniques par manipulation genetique - Google Patents

Procede efficace permettant de mettre au point des plantes transgeniques par manipulation genetique Download PDF

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Publication number
WO2002066599A2
WO2002066599A2 PCT/KR2002/000232 KR0200232W WO02066599A2 WO 2002066599 A2 WO2002066599 A2 WO 2002066599A2 KR 0200232 W KR0200232 W KR 0200232W WO 02066599 A2 WO02066599 A2 WO 02066599A2
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Prior art keywords
gene
target
transformation
plant tissue
group
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PCT/KR2002/000232
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English (en)
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WO2002066599A3 (fr
Inventor
Yang-Do Choi
Hak-Soo Seo
Renago XUE
Jae-Yun Lim
Mee-Yeon Park
Ju-Kon KIM
Kwang-Soon Choi
Jong-Joo Cheong
Jong-Seob Lee
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Scigen Harvest Co., Ltd.
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Priority to AU2002234979A priority Critical patent/AU2002234979A1/en
Publication of WO2002066599A2 publication Critical patent/WO2002066599A2/fr
Publication of WO2002066599A3 publication Critical patent/WO2002066599A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Definitions

  • the present invention relates to a method for transforming plants using a gene manipulation technique, and more particularly, to an improved transformation method of plants, comprising insertion of a useful target gene for plants, in particular soybean plant, into an appropriate target plant tissue using an effective wounding method, and a transformant produced using the method.
  • Soybean is one of the most important crops in the world. It is widely cultivated on more than 50 million hectares of farmland, and soybean of more than 100 million-ton is produced every year. New breeds improved in terms of disease resistance, herbicide resistance and nutrition will provide substantial economic advantages. Up until now, conventional breeding techniques such as mutation, hybridization and natural screening have been employed to improve a breed of soybean, but have failed to provide satisfactory results. Therefore, development of new strategies for breeding of soybeans is needed. Methods that functional genes are transformed into plants and expressed using a molecular breeding method, thereby new plant breeds expressing desired characteristics being obtained, have been developed.
  • the molecular breeding method is different from the conventional hybridization breeding method, in that genetic characteristics of good breeds are kept intact during transplantation of new functional genes, and efficient breeding is accomplished within a short time (Altenbach et al., Plant Mol. Biol., 13: 513-522, 1989).
  • Methods for efficient transformation of plant cells include the method for delivering foreign DNA into plant cell using a biological vector such as Agrobacterium spp., and methods for delivering directly foreign DNA into plant cell such as microprojectile bombardment, electroporation, ultrasonic treatment and microinjection (Hansen and Wright, Trends Plant Sci., 4:226-231, 1999).
  • Agrobacterium tumefaciens-mediated transformation.
  • Agrobacterium has the ability to transfer its T-DNA into a plant's genome.
  • the T-DNA is present in the tumor- inducing (Ti) or root-inducing (Ri) plasmid of the bacteria. Therefore, if a target gene is inserted into the T-DNA of Agrobacterium and the target gene-inserted Agrobacterium and the plant cell or tissue are co-cultivated, the plant cell or tissue can be transformed with the target gene (Binns et al, Ann Rev Microbiol, 42:575-606, 1988).
  • the inventors developed the following procedure and completed the present invention: the inventors wounded the cotyledonary node of cultivar soybean Jungery, which had been germinated for 1 day, as a target plant tissue, and inserted a target gene thereinto, thereby gene delivery efficiency being increased.
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for transforming plants with Agrobacterium tumefaciens vector containing a target gene.
  • chimera means a hybrid plant consisting of genetically different cells, i.e., a cell with a target gene and a cell with no target gene.
  • a target gene means a gene encoding a useful protein, and which is introduced into a plant by using Agrobacterium.
  • transformant or “transformed plant” means a plant into which Agrobacterium containing the target gene is introduced.
  • FIG. 1 is a schematic view showing a recombinant plasmid pGB, constructed by introducing the bar gene and the gfp gene into the T-DNA of Agrobacterium;
  • Fig. 2 is an electrophoresis result for confirming that plasmid pGB for transformation is constructed
  • Fig. 3 is a photograph showing half a seed germinated for one day, including cotyledonary node, as a target plant tissue;
  • Fig. 4 is a photograph showing multiple shoots regenerated from cotyledonary node segments
  • Fig. 5 is a photograph showing the transformant selected in herbicide PPT (phosphinothricin)-containing medium, compared with a non-transformant
  • Fig. 6 is a Southern blot showing presence of the bar gene in the transformant genome following PCR amplification of the bar gene, to confirm that a target gene (bar gene) is introduced into the plant;
  • Fig. 7 is a Southern blot showing presence of the bar gene in the transformant genome
  • Fig. 8 is a Northern blot showing expression of the bar gene in the tranformant
  • Fig. 9 is a photograph of the tranformant growing in soil
  • Fig. 10 is a photograph showing resistance to herbicide of the tranformant, compared with a non-transformant
  • Fig. 11 is a photograph showing a second-generation progeny of the tranformant, growing in soil
  • Fig. 12 is a Southern blot showing presence of the bar gene and the gfp gene in the second generation tranformant genome, following PCR amplification of the bar and gfp genes;
  • Fig. 13 is a confocal fluorescence microphotograph showing expression of the gfp (green fluorescent protein) gene transformed into the transformant.
  • the present invention provides a method for producing a new breed of soybean by introducing a target gene into a soybean plant.
  • the target gene is derived from animals, plants or microorganisms. Examples of the target gene which may be used in soybean transformation include a gene for an enzyme conferring resistance to damages by harmful insects and herbicide, a gene for an enzyme enhancing crop yield, or improving qualities of protein and fatty acid of soybean seed, and the like.
  • the target gene may be any gene derived from microorganisms such as bacteria, fungi, yeasts, viruses, as well as plants and animals.
  • a target gene cassette according to the present invention is a useful gene construct capable of imparting desired genetic characteristics to plants. Therefore, a characteristic of the transformed plant depends on the target gene, which is inserted into the plant.
  • the DNA vector used for constructing the target gene cassette is containing an expression cassette consisted of a regulatory sequence such as a promoter and terminator, a transcription initiation region, a translation initiation codon, an amino acid coding region and a translation stop codon, and containing at least one origin of replication.
  • the gene construct used for transformation further contains a selectable gene, a marker gene, and a target gene, and the like.
  • a gene conferring resistance to herbicide can be used as a target gene, at the same time as a selectable gene.
  • genes, which may be used as a selectable gene include genes conferring resistance to, NPTII (neomycin phosphotransferase), HPT (hygromycin phosphotransferase), CAT (chloramphenicol acetyltransferase), nitrilase and gentamicin.
  • the genes provide resistance to biocides such as antibiotics, toxins and heavy metals.
  • genes which may be used as the marker genes include GUS ( ⁇ - glucuronidase) emitting indigo, CAT, luciferase emitting visible light, and gfp (green fluorescent protein) gene producing green fluorescent material, and the like.
  • a target plant tissue for transformation is generally selected from the group consisting of meristematic tissue such as a germ, a stem, a shoot tip and a cotyledonary node, and induced meristematic tissue such as callus.
  • the cotyledonary node of half a seed is used as a target plant tissue.
  • the cotyledonary node of half a seed which is germinated for one day, because it's cells are vigorously dividing and DNA synthesis is high, the possibility for transplantation of a target gene into the plant cells increases, upon inoculation of Agrobacterium (Binns et al, Ann Microbiol., 42: 575-606, 1988).
  • a recombinant vector is constructed to carry out transformation using the half a seed as a target plant tissue.
  • the bar gene conferring resistance to herbicide is used as a selectable gene
  • the gfp gene which causes green fluorescence, is used as a marker gene to select transformed plant in the beginning.
  • the bar and gfp genes are flanked by right and left borders of the T- DNA of Agrobacterium, respectively.
  • the bar gene expression cassette is composed of the NOS promoter, the bar coding region, and the NOS terminator, while the gfp cassette is composed of the 35S promoter, the gfp coding region, and the NOS terminator.
  • cultivar soybean is transformed with a herbicide-resistance gene.
  • a sterilized seed is germinated for 16 to 24 hours and then its half a seed is used as a target plant tissue (see
  • a target plant tissue is wounded with a bundle of needles, preferably a bundle of 30 needles.
  • the shoots are generally induced in cells of a target plant tissue without inducing callus. Therefore, the transformation with Agrobacterium can be easily carried out only in the case where Agr'obacterium directly accesses to the cells of a target plant tissue. To do this, it is very important to wound the cells of the target plant tissue, in which shoots are induced.
  • the transformation efficiency is enhanced more than 8 times versus transformation efficiency without wounding (see Table 2). Furthermore, the transformation efficiency with a bundle of 30 needles is remarkably higher than that of multiple wounding with one needle (see Table 3).
  • the efficiencies for transformation also vary according to breed of plant. As shown in Table 5 below, when the herbicide-resistance gene was introduced into cultivar Jungery, Danyup and Paldal, respectively, with Agrobacterium, the transformation efficiency of cultivar Jungery was 15 times and 7 times higher than Paldal and Danyup, respectively.
  • cultivar Jungery can be more effectively used in transformation of plant.
  • the present inventors performed PCR amplification, Southern blotting and Northern blotting. As a result, the inventors confirmed that bar gene, the target gene, is stably expressed in the transformant (see Fig. 6, Fig. 7, Fig. 8). Further, to confirm that the transformant so produced has resistance to herbicide, the inventors applied 100 ppm of herbicide on leaves of the transformant and then compared with a non-transformant. As shown in Fig.10, as for the non-transformant, its leaves gradually wilted and died, while the transformant grew vigorously. Further, as shown in Fig.
  • second-generation plants can be obtained by germinating seeds obtained from the transformant.
  • the bar gene of the second-generation plant was amplified by PCR, and then Southern blotting was carried out to detect the presence of the herbicide-resistance gene. As a result, it can be seen that the bar gene was stably introduced into the second-generation transformant (see Fig.12).
  • a useful target gene-inserted transformant can be produced using the transformation method of the present invention.
  • plants which may be used in the transformation include cultivar soybean; food crops such as rice, wheat, barley, corn, soybean, potato, red bean, oats and sorghum; vegetables such as Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; special crops such as ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rape; fruits trees such as apple, pear, jujube, peach, kiwi fruit (Actinidia argutd), grape, tangerine, persimmon, plum, apricot, and banana; ornamental plants such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and forage crops such as ryegrass,
  • Example 1 Construction of herbicide-resistance bar gene-inserted recombinant plasmid for transformation
  • Recombinant plasmid pGB was constructed to use in transformation of soybean (see Fig. 1).
  • the recombinant plasmid pGB was constructed by inserting herbicide-resistance bar gene as a selectable gene and green fluorescent protein gfp gene as a marker gene into T-DNA plasmid pGA643 for plant transformation.
  • the expression cassette of the bar gene for conferring resistance to herbicide PPT is composed of the NOS (nopaline synthase) promoter, the bar coding region, and the NOS terminator.
  • the expression cassette of the gfp gene is composed of the 35S promoter, the gfp coding region and the NOS terminator.
  • the recombinant plasmid pGB was digested with restriction enzymes, Clal and EcoRI, or Kpnl, and then the digested fragments were separated by agarose gel electrophoresis (see Fig. 2). Finally, the sequence data of the recombinant plasmid pGB was obtained by sequencing.
  • Example 2 Selection of target plant tissue for transformation and transformation using the same
  • Target plant tissue was selected to introduce recombinant plasmid thereinto and then transformed with Agrobacterium.
  • Cultivar Jungery which is transformed at high efficiency, was used.
  • Mature soybean seeds were immersed in 70% ethanol for 1 minute, and then in 20% Clorox solution (5.25%o sodium hypochlorate) for 12 minutes to disinfect their surfaces and then washed four times with sterilized distilled water.
  • Clorox solution 5.25%o sodium hypochlorate
  • the mature seeds so treated were immersed in sterilized distilled water for 16 to 24 hours so as to easily wound them, and germinated at room temperature. Because a whole seed is not easy to manipulate or wound, half a seed was used. A whole seed was divided into two equal parts in direction of cotyledonary node using a knife. As a result, the half a seed with bud was used as the target plant tissue of the present invention (see Fig. 3).
  • the half a seed was further germinated in the germination medium for 10 days. Then, the cotyledonary nodes of the seed were separated and cultured in 1 ppm of PPT-containing germination medium for 3 weeks to selectively induce multiple shoots (see Fig. 4). To select independent transformants, individual shoots were cut and the cuts were cultured and re-selected in 2 ppm of PPT-containing root inducing medium (MS salt solution (1/2 Murashige and Skoog), vitamin solution (Gamborg B5)) for 2 weeks. Non-transformed shoots wilted and died, while transformed shoots rooted (see Fig. 5). Shoots with roots were transferred to soil after acclimation in the manner of gradually opening the lid of their container. As shown in
  • Cotyledonary nodes of half a seed were wounded to 1-2 mm depth using a bundle of 30 needles in order to wound effectively and then Agrobacterium were inoculated thereinto.
  • Table 2 the transformation efficiency was 21% and was enhanced more than 8 times versus transformation efficiency without wounding.
  • inoculation of Agrobacterium with no wounding transformed shoots were obtained once, while as for inoculation of Agrobacterium with a bundle of needles, transformed shoots continued to be regenerated at least three times from the inoculated cotyledonary nodes. Accordingly, inoculation of Agrobacterium with a bundle of needles makes it possible to wound target cells of the inoculated cotyledonary nodes. As a result, efficiency for transformation of soybean increases and thus stably transformed soybean plants are regenerated in large quantities.
  • Example 4 Comparison of transformation efficiency according to degree of growth of target plant tissue
  • Target tissue Number of segments resistant shoots efficiency (%) cotyledonary node
  • telomeres a target gene that was amplified by PCR using NOS promoter- and NOS terminator-specific primers having SEQ ID NO. 1 and SEQ ID NO. 2, respectively, and then agarose gel electrophoresis was conducted.
  • Southern blotting was conducted using a bar gene-specific probe having SEQ ID NO. 3, no signal appeared in the non-transformant. While, as for the transformant, a 1.2 kb band was visualized. Based on the fact, it can be seen that the bar gene was transformed into a plant tissue (see Fig. 6). Furthermore, to confirm that a target gene is stably inserted into genomic
  • DNA of the transformant about 5 ⁇ g of genomic DNA was digested with Sacll restriction enzyme, separated by 0.8% agarose gel electrophoresis, and transferred to a nylon membrane.
  • Sacll restriction enzyme Sacll restriction enzyme
  • a Southern blotting using bar gene-specific probe a 0.6 kb fragment derived from a portion of the bar coding region, and 7 kb and 23 kb fragments derived from the expression cassette of the bar gene were visualized (see Fig.
  • a target gene was inserted into the genome of the transformed soybean plant. Furthermore, whole RNA was isolated from the transformed soybean plant, and a Northern blotting was performed. As a result, it could be seen that the inserted foreign gene was stably expressed in the transformant. As a result of the Northern blotting using bar gene-specific probe, no signal appeared in the non-transformant, while bar genes were stably expressed in the transformant (see Fig. 8).
  • Example 7 Test for acclimation and herbicide-resistance of transformant Seedlings selected in media were transplanted into soil containers. When the seedlings reached the lids of the containers, they were acclimated by opening the lids.
  • Second-generation plants Seeds obtained from soybean tranformants were germinated in soil to obtain second-generation (Tl) plants (see Fig. 11). To confirm that the second-generation plants had inserted foreign gene, genomic DNA was isolated and then PCR amplification and Southern blotting were performed. Specifically, a foreign gene was amplified by PCR using NOS promoter- and NOS terminator-specific primers, and then Southern blotting was carried out using a bar gene-specific probe. As a result, no signal appeared in the non-transformant, while strong signals appeared in transformant 2. In addition, in Southern blotting of gfp gene, strong signals appeared in all the transformants (see Fig. 12).
  • the present invention provides a useful method for transforming plants, in particular soybeans, by introduction of a target gene into the plants with Agrobacterium. Specifically, half a seed germinated for one day as a target tissue is suitably wounded and a herbicide-resistance gene is introduced thereinto. Then, transformed soybean plants are selected in herbicide- containing media. Accordingly, using the transformation method of the present invention, a new breed of transformed soybean, which is commercially valuable, can be developed and productivity of soybean can be remarkably enhanced.

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Abstract

La présente invention concerne un procédé permettant de transformer des plantes par manipulation génétique, et plus particulièrement, un procédé permettant de transformer des plantes avec une agrobactérie contenant un gène bar présentant une résistance à un herbicide, par une méthode de lésion efficace sur un tissu cible approprié. Le procédé de transformation décrit dans cette invention permet d'obtenir une race de plante améliorée par rapport à une race de plante classique.
PCT/KR2002/000232 2001-02-16 2002-02-14 Procede efficace permettant de mettre au point des plantes transgeniques par manipulation genetique WO2002066599A2 (fr)

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AU2002234979A AU2002234979A1 (en) 2001-02-16 2002-02-14 Efficient method for the development of transgenic plants by gene manipulation

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KR10-2001-0007818A KR100402513B1 (ko) 2001-02-16 2001-02-16 유전자 조작을 통한 식물체의 효과적인 형질전환 방법

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Cited By (7)

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WO2008112628A3 (fr) * 2007-03-09 2008-12-24 Monsanto Technology Llc Préparation et utilisation d'explants d'embryons végétaux pour la transformation
WO2010065534A3 (fr) * 2008-12-01 2010-10-14 Ceres, Inc. Procédés de transformation de miscanthus
US7935529B2 (en) 2003-08-05 2011-05-03 Monsanto Technology Llc Method for excision of plant embryos for transformation
US7937890B2 (en) 2003-08-05 2011-05-10 Monsanto Technology Llc Method and apparatus for substantially isolating plant tissues
EP2734035A4 (fr) * 2011-07-22 2015-03-25 Basf Plant Science Co Gmbh Procédé de transformation de plante
CN105132457A (zh) * 2015-10-19 2015-12-09 宁夏农林科学院 一种快速遗传转化苜蓿的方法
CN106636196A (zh) * 2017-02-21 2017-05-10 辽宁省农业科学院大连生物技术研究所 一种优化的、高效率的农杆菌介导的花生转化方法

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DE69429012T2 (de) * 1993-12-09 2002-08-22 THE TEXAS A & M UNIVERSITY SYSTEM, COLLEGE STATION Transformation von musa-arten durch verwendung von agrobacterium tumefaciens
US5693512A (en) * 1996-03-01 1997-12-02 The Ohio State Research Foundation Method for transforming plant tissue by sonication
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US7935529B2 (en) 2003-08-05 2011-05-03 Monsanto Technology Llc Method for excision of plant embryos for transformation
US7937890B2 (en) 2003-08-05 2011-05-10 Monsanto Technology Llc Method and apparatus for substantially isolating plant tissues
US10433503B2 (en) 2003-08-05 2019-10-08 Monsanto Technology Llc Method and apparatus for substantially isolating plant tissues
US9790512B2 (en) 2007-03-09 2017-10-17 Monsanto Technology Llc Preparation and use of plant embryo explants for transformation
WO2008112628A3 (fr) * 2007-03-09 2008-12-24 Monsanto Technology Llc Préparation et utilisation d'explants d'embryons végétaux pour la transformation
US8466345B2 (en) 2007-03-09 2013-06-18 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US8872000B2 (en) 2007-03-09 2014-10-28 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US11485980B2 (en) 2007-03-09 2022-11-01 Monsanto Technology, Llc Method of meristem excision and transformation
US12195736B2 (en) 2007-03-09 2025-01-14 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US11807858B2 (en) 2007-03-09 2023-11-07 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US8044260B2 (en) 2007-03-09 2011-10-25 Monsanto Technology Llc Method of meristem excision and transformation
US9714428B2 (en) 2007-03-09 2017-07-25 Monsato Technology Llc Methods for plant transformation using spectinomycin selection
US11542514B2 (en) 2007-03-09 2023-01-03 Monsanto Technology, Llc Apparatus for the preparation and use of plant embryo explants for transformation
US9885053B2 (en) 2007-03-09 2018-02-06 Monsanto Technology Llc Method of meristem excision and transformation
US8357834B2 (en) 2007-03-09 2013-01-22 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US8030544B2 (en) 2007-03-09 2011-10-04 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
US10920235B2 (en) 2007-03-09 2021-02-16 Monsanto Technology Llc Apparatus for the preparation and use of plant embryo explants for transformation
US10584345B2 (en) 2007-03-09 2020-03-10 Monsanto Technology Llc Preparation and use of plant embryo explants for transformation
US10717983B2 (en) 2007-03-09 2020-07-21 Monsanto Technology Llc Method of meristem excision and transformation
US10907167B2 (en) 2007-03-09 2021-02-02 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
WO2010065534A3 (fr) * 2008-12-01 2010-10-14 Ceres, Inc. Procédés de transformation de miscanthus
US10557141B2 (en) 2011-07-22 2020-02-11 Basf Plant Science Company Gmbh Plant transformation method
EP2734035A4 (fr) * 2011-07-22 2015-03-25 Basf Plant Science Co Gmbh Procédé de transformation de plante
CN105132457B (zh) * 2015-10-19 2018-08-03 宁夏农林科学院 一种快速遗传转化苜蓿的方法
CN105132457A (zh) * 2015-10-19 2015-12-09 宁夏农林科学院 一种快速遗传转化苜蓿的方法
CN106636196A (zh) * 2017-02-21 2017-05-10 辽宁省农业科学院大连生物技术研究所 一种优化的、高效率的农杆菌介导的花生转化方法

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