WO1994013787A1 - Procede pour favoriser la differenciation de cellules vegetales en culture - Google Patents
Procede pour favoriser la differenciation de cellules vegetales en culture Download PDFInfo
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- WO1994013787A1 WO1994013787A1 PCT/FR1993/001239 FR9301239W WO9413787A1 WO 1994013787 A1 WO1994013787 A1 WO 1994013787A1 FR 9301239 W FR9301239 W FR 9301239W WO 9413787 A1 WO9413787 A1 WO 9413787A1
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Classifications
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Definitions
- the present invention relates to a method for promoting the differentiation of cells in culture, in particular during the processes of tissue differentiation, embryogenesis, or organogenesis; it relates more particularly to a process intended for obtaining embryos from the culture of plant somatic cells using ⁇ lipid transfer proteins which will be referred to below as LTP, as well as these proteins themselves and their applications.
- LTP ⁇ lipid transfer proteins
- Somatic embryogenesis essentially comprises two stages: 1- the induction of the embryogenic potential by exogenous auxins, generally in high concentrations, which allow the appearance of proembryogenic aggregates (or masses) (PEM) which correspond to groups of 10 to 50 cells, in particular bas ⁇ stémmatique. 2- the transfer of these cells to media free of auxins which lead to the formation of somatic embryos from these PEMs, following the different stages of evolution of plant embryogenesis, namely: globules, heart and torpedo.
- PEM proembryogenic aggregates
- somatic embryos For some plant species, obtaining somatic embryos is very difficult, if not impossible. For example, with certain vine cultivars, the embryogenic capacities of somatic cells can be stopped, most of the time at the stage of proembryogenic aggregates formation, or else, the maturation of the embryos is blocked, in particular at the globular stage.
- EP 281375 proposed the addition of calmodu ne and calcium to a cell culture to promote the differentiation of roots and embryos.
- Application EP 55597 describes a method of stimulating the growth and embryogenesis of plants cultivated in vitro, by addition of arabino-galactan proteins. However, it has been shown (de V ⁇ es et al, 1989, Proceedings of the International Symposium of EUotechnology for Major Crops (ADEBIO ed.), Pages 22-23) that an extra-cellular glycoprotein of 52/54 kDa induces a cessation of embryonic development at the heart stage.
- the present invention is based on the discovery of proteins making it possible to ensure or stimulate cell differentiation, and in particular somatic embryogenesis, even under certain non-permissive conditions, in particular from somatic vine cells, said proteins also being able to have other applications.
- non-permissive conditions with respect to the realization of a given phenomenon, conditions known from the state of the art which partially or totally prevent the phenomenon from occurring.
- the present invention relates to a method for promoting the differentiation of cells in culture, in particular with a view to tissue differentiation, embryogenesis, or organogenesis, characterized in that '' is introduced into the culture medium in effective concentration to obtain the cell differentiation at least one lipid transfer protein or "LTP", or an LTP analog.
- LTP analog is meant any protein substance, such as protein, protein fragment or polypeptide, having at least 30% homology with an LTP or with an LTP fragment.
- the lipid transfer protein is an LTP obtainable from cells belonging to the same genus or the same species as cells in culture.
- the present invention relates to a process for obtaining plant somatic embryos from a culture of somatic cells in vitro, characterized in that the culture medium is added in a concentration effective to ensure the embryogenesis, at least one lipid transfer protein or LTP, or an analog of LTP.
- maturation will be used generally to designate the phenomena of development and germination of the somatic plant embryo, thus passing through the following stages of development: globule, heart, torpedo and seedling.
- the technology of in vitro cell culture of somatic cells is known and will of course have to be adapted to each of the cells in question, the physicochemical conditions, culture media and environment being adapted so as to allow the multiplication and the formation of aggregates. proembryogens, then embryogenesis.
- the corresponding media will be called "culture media for embryogenesis”.
- the LTPs usable according to the invention can be introduced at any time into the culture medium.
- Kader et al (Methods Enzymol., 1987, 148, 661-666) identified an LTP isolated from young corn plants.
- Nonspecific lipid transfer proteins could be characterized in the castor bean endosperm, spinach leaves, and barley grass.
- LTPs are also present in carrots and millets.
- the LTPs known from the vegetable sector are proteins generally having a molecular weight of approximately 10 kDa; non-specific LTPs and most of the specific LTPs are basic proteins, having an isoelectric point greater than 8.
- the inventors have been able to highlight the fact that the presence of auxin in the medium, which very often causes the appearance of non-permissive conditions, was not an obstacle to the formation of embryos if LTPs are added. .
- the LTP or the analog of LTP is added to the medium for the induction and / or multiplication of the aggregates of proembryogenic cells, generally containing an auxin. An increase in the embryogenic power of the strain is then observed and an increase in the number of proembryogenic aggregates formed.
- the process according to the present invention is also particularly advantageous for obtaining the formation of embryos and their maturation, especially in the case where they are transformed cells.
- the LTPs can be introduced into the maturation medium of the embryos, which is, according to the prior art, generally free of auxin, or even substantially free of auxin.
- the LTP or the analog of LTP is added to the maturation medium embryos.
- This maturation medium can be substantially free of auxin.
- the maturation medium contains an auxin, in particular in an amount necessary to ensure the viability of cell culture.
- auxins 2, -d ⁇ chloro phenoxyacetic acid (2.4 D), 1-naphthalene acetic acid (NAA), 2-naphthoxyacetic acid (NOA).
- 2.4 D 2, -d ⁇ chloro phenoxyacetic acid
- NAA 1-naphthalene acetic acid
- NOA 2-naphthoxyacetic acid
- LTPs in culture media for transformed cells, in particular vine cells, especially in the case where the cell strain has a disturbed somatic embryo genesis.
- these cells have been transformed by biolistics (by particle gun) or by appropriate cloning vectors, such as vectors derived from Agrobacté ⁇ um or other viral type systems.
- the multiplication step does not necessarily have to be, and maturation can take place directly.
- the LTP or the analog of LTP is present in the culture medium at a concentration of between 1 and 10 ⁇ g / ml of medium, preferably between 1 and 50 ⁇ g / ml.
- the abovementioned cell culture is a culture of vine cells.
- LTP or the analog of LTP comprises at least one amino acid sequence having at least 80% homology with one of the sequences shown in Figure 2.
- LTP or the analog of LTP comprises at least one amino acid sequence having practically 100% homology with one of the sequences represented in FIG. 2.
- LTP, or the analog of LTP has a molecular weight of about 9 kDa.
- the lipid transfer protein is an LTP obtainable from cells belonging to the same genus or the same species as cells in culture.
- LTP obtainable from cells belonging to the same genus or the same species as cells in culture.
- the invention also relates to a plant obtained by any of the aforementioned methods of the invention.
- PI, P2, P3 and P as well as the proteins or fragments of said proteins or polypeptides having at least 80% homology with at least one of these proteins, or the corresponding fragments of said proteins, in particular partially deleted proteins but which , preferably kept the lipid transfer capacities.
- the N-terminal sequence of these four proteins is indicated in the attached FIG. 1, the purity of these proteins was confirmed by the presence of a single type of N-terminal end for each sample.
- the corresponding proteins are proteins which are not N-glycosylated.
- the present invention relates to a protein substance consisting in particular of a protein, a protein fragment or a polypeptide, characterized in that said protein substance comprises an amino acid sequence, located preferably to its N-terminal extrunrun, having at least 80% homology with one of the sequences shown in Figure 1, and preferably having a transfer activity of lipids, in particular phospholipids.
- said protein substance comprises an amino acid sequence having practically 100% homology with one of the sequences represented in FIG. 1.
- Proteins P I and P2 have the same sequence on the first 40 amino acids from the N-terminus, while the amino acid compositions of proteins P3 and P4 are different. It appears from this observation that the proteins P I and P2 having the same
- the four proteins in question have a lipid transfer activity, as has been demonstrated by tests of in vitro transfer of phosphatidyl-choline t ⁇ tiée, between liposomes (donor membrane system) made of this phospholipid and mitochond ⁇ es constituting the membrane system recipient, according to the method described by Douady D. et ai. in the journal Biochim. Biophys. Acta (1982) vol. 710 p. 143-153.
- the present invention also relates to a DNA sequence coding for a tell protein as defined in the previous aspect.
- the present invention also relates to the different uses of protein substances as defined above when describing the fourth aspect of the invention, in particular in compositions comprising them alone or in combination with other active principles, in particular lipid compounds. These compositions can be used, for example as additive for cell cultures, but can also be used for other applications in the biological field in which their lipid transfer activity and / or on tissue differentiation can prove to be valuable.
- the present invention also relates to a probe consisting of at least one oligonucleotide, characterized in that the base sequence of said oligonucleotide is deduced from a part of the amino acid sequence of a protein substance such as previously defined, in particular of one of the proteins PI to P4.
- oligonucleotide is deduced from an amino acid sequence which comprises at least 6 or 7 amino acids, preferably from position 3 of the N-terminal end of the protein substance , especially protein.
- the probe can also consist of a mixture of oligonucleotides as defined above.
- Figure 1 Sequences of PI, P2, P3 and P4 proteins
- Figure 2 Compared sequences of LTPs from different species
- Figure 3 Effect of the addition of the FI fraction (PI, P2 and P3 proteins) on the maturation of the embryos present of different concentrations of auxin and at two different cell densities, compared to a control culture.
- Figure 4 Effect of the addition of the FI fraction on differentiation at the preglobular stage, in the presence of different concentrations of auxin and at two different cell densities
- FIG. 5 Effect of the addition at different concentrations of the F2 fraction (P4 protein) on the maturation of embryos.
- a suspension of vine cells derived from the 41 B line (Vitis vinifera cv. Chasselas x Vitis berlandie ⁇ ) is used. The undifferentiated growth of these cells is maintained by weekly subcultures in the presence of auxin, 2-naphthoxy acetic acid (NOA) at the concentration of 5 ⁇ M, as described previously by Coutos-Thevenot, et al. . in Plant Cell Tissue Organ Culture 29, 125-133 (1992).
- auxin 2-naphthoxy acetic acid
- the filtered cells are washed in a medium free of auxins and inoculated at a rate of 1 ⁇ l in a GMo culture medium according to the protocol described in the same document.
- the medium is harvested by first separating the PEMs or the embryos with a particulate glass filter No. 2 and No. 4 respectively (Pyrex France).
- the conditioned medium is clarified by passing it under vacuum through a fiberglass membrane (GF / C hatman) and concentrated 100 to 200 times using an AMICON ultrafiltration system with an AMICON YM3 membrane (cutoff threshold 3 kDa).
- the protein content is determined by the Lowry method.
- the proteins are purified using an HPLC system (Waters Massachussets USA) using a UV detector M 440 under the following conditions: 1) the medium harvested after 1 5 days of incubation and concentrated 100 times, is loaded onto a cation exchange column (model SP5PW from Waters Massachussets USA) previously equilibrated with a 25mM buffer of sodium phosphate pH 6.5 ( speed 0.8 ml / minute). About 4mg of protein is placed on the column. The basic proteins are removed in 30 minutes by a linear gradient of aqueous sodium chloride solutions 0 to 0.3 M. The fractions corresponding to the different peaks observed on the elution profile are collected and analyzed by SDS-PAGE electrophoresis. Two fractions having a molecular weight of approximately 10 kDa are obtained, which are then subjected to a second purification step. The IF fraction represents 15.3% by weight of the initial basic proteins, and the F2 fraction represents 10.2%
- the elution profile obtained for the first major peak (fraction F1) of step 1 provides 3 secondary peaks corresponding to the proteins PI, P2, and P3.
- the P4 protein is recovered in the elution of the second major peak (fraction F2) of step 1.
- For 100 ⁇ g of the fraction F1, 42.5 ⁇ g of PI, 6.54 ⁇ g of P2 and 0.33 ⁇ g of P3 are collected after this chromatographic treatment. From 100 ⁇ g of fraction F2, 46 ⁇ g of P4 is collected.
- the molecular weights of the proteins PI, P2, P3 and P4 were determined in a conventional manner by mass spectrometry. The results are shown in Table I.
- the N-terminal sequence on 40 amino acids was analyzed using a 470A sequencer in the liquid gas phase (ABI, Foster City, California).
- the amino acids phenylhydantoin are analyzed on a 120A analyzer.
- the PI to P4 sequences were compared to the LTP protein sequences of carrot (c) spinach (millet (Mi) and corn (M)).
- the four protein fractions and a purified corn LTP used as a control are tested for their lipid transfer activity, by the technique described by Douady, D., Grosbois, M., Guerbette, F. and Kader, 3.C. (1982) Biochim. Biophys. Acta 710, 143-153. Proteins are incubated for 30 minutes at 30 ° C with purified mitochond ⁇ es from corn and with a preparation of radioactive liposomes. The labeling is carried out on the one hand with t ⁇ tiated phosphatidylcholine and on the other hand with a liquid non-transferable by these LTPs: cholesteryl (1 - 14C) oleate.
- the mitochondria are resuspended in 1% of Triton X 100 and the activity ratio (in cpm) between 3H and 14C is measured to determine the transfer activity which is expressed as a percentage of t ⁇ tiated phosphatidyichohne transferred after correction of contaminating radioactivity due to cholesteryl oleate not exchanged.
- Proteins can be classified into two groups based on their ability to transfer phospholipids (see Table II). The PI and P2 proteins exhibit high specific activities, while
- the culture medium used for the development of somatic embryos is a modified liquid culture medium MS, 0 comprising in particular 18 g / 1 of maitose and 4.6 g / 1 of glycerol replacing sucrose and 1 g / 1 of hydrolyzate casein.
- This medium is used at a pH of 5.8 adjusted by the addition of 0.1 N sodium hydroxide; either such that, or NOA (naphthoxyacetic acid) is added at concentrations of 0.5 ⁇ M or 5 ⁇ M.
- the culture medium is autoclaved for 20 min at 120 ° C.
- the undifferentiated cell cultures, originating from the cell culture maintained according to the protocol of Example 1, are successively filtered through nylon filters having pores with a size of 500 ⁇ m, then of 200 ⁇ m, so as to not preserve for culture inoculation only ⁇ cell aggregates of size between these two values.
- the undifferentiated cell aggregates retained on the second filter are then washed 3 times with modified MS medium, then suspended in 30 ml of this medium.
- the cell density expressed in ⁇ l of cell volume per ml of medium is determined after sedimentation (1 ⁇ g) of the cells in a graduated conical tube.
- modified culture medium MS then containing 0, 0.5 or 5 ⁇ M NOA
- 80 ml of modified culture medium MS are then inoculated with a suspension of cellular aggregates at a density of 0, 1 or 1 ⁇ l / ml, in a 250 ml Erlenmeyer flask.
- the volumes of culture medium can be reduced to approximately 1 ml, in 24-well microtiter dishes (Falcon) but the cell density must be respected.
- the culture mediums are then supplemented to To of the experiment by increasing doses of the protein fraction F I isolated according to Example 1 and containing essentially the LTP P I, P2 and P3.
- the respective doses are 2 and 10 ⁇ g / ml; a control without LTP is also cultivated.
- the development curves of the proembryogenic structures are established using arbitrary units: 0 for undifferentiated PEM, 4 for embryos at the globular stage, the values 1, 2 and 3 corresponding to intermediate stages of proglobular embryos in the cell mass.
- the first globules appear only after 21 days. This time for the appearance of the first globules is shortened to 1 day when the middle place is supplemented with 2 ⁇ g / ml of LTP, and to 6 days when the medium is supplemented with 10 ⁇ g / ml of LTP. The same effect is observed for a density of 1 ⁇ i / ml, with a slight delay.
- auxin With an initial cell density of 1 ⁇ l / ml, auxin is rapidly metabolized and there is a beginning of differentiation even in the absence of LTP. The differentiation is accelerated by the complementation in fraction FI.
- Figure 3 shows the progression of embryogenesis by counting the proportions of embryos at different stages of their development in a medium containing no auxin, compared to the total number of embryos.
- A are represented the results for an initial cell density of 0.1 ⁇ l / ml; e in B for an initial density of 1 ⁇ l / ml. (A) at 0.1 ⁇ l PCV / ml.
- FIG. 5 shows the results obtained after 21 days of culture, in the presence of NOA at the concentration of 5 ⁇ M for an initial cell density of 0.1 ⁇ l / ml, and in the presence of different concentrations of the fraction F2: 0.1 , 2 and 3 ⁇ g / ml.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP6513885A JPH08504329A (ja) | 1992-12-14 | 1993-12-14 | 培養の植物細胞の分化を促進する方法 |
US08/448,481 US5914270A (en) | 1992-12-14 | 1993-12-14 | Method for promoting the differentiation of plant cells in culture |
EP94902805A EP0673414A1 (fr) | 1992-12-14 | 1993-12-14 | Procede pour favoriser la differenciation de cellules vegetales en culture |
AU57022/94A AU684484B2 (en) | 1992-12-14 | 1993-12-14 | Method for enhancing the differentiation of plant cells in culture |
Applications Claiming Priority (2)
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FR92/15044 | 1992-12-14 | ||
FR9215044A FR2699190B1 (fr) | 1992-12-14 | 1992-12-14 | Procédé pour favoriser la différenciation de cellules, substances protéiques possédant une activité de transfert de lipides, compositions les contenant, séquences d'ADN et sondes d'oligonucléotides correspondantes. |
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WO1994013787A1 true WO1994013787A1 (fr) | 1994-06-23 |
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PCT/FR1993/001239 WO1994013787A1 (fr) | 1992-12-14 | 1993-12-14 | Procede pour favoriser la differenciation de cellules vegetales en culture |
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US (1) | US5914270A (fr) |
EP (1) | EP0673414A1 (fr) |
JP (1) | JPH08504329A (fr) |
AU (1) | AU684484B2 (fr) |
CA (1) | CA2151587A1 (fr) |
FR (1) | FR2699190B1 (fr) |
NZ (1) | NZ258928A (fr) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999059398A3 (fr) * | 1998-05-15 | 2000-01-20 | Univ Florida | Systeme de regeneration de la vigne et son utilisation |
WO2000070054A1 (fr) * | 1999-05-14 | 2000-11-23 | University Of Florida | Plants de vigne resistant aux agents pathogenes |
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US6174818B1 (en) * | 1999-11-19 | 2001-01-16 | Taiwan Semiconductor Manufacturing Company | Method of patterning narrow gate electrode |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2537157A1 (fr) * | 1982-12-03 | 1984-06-08 | Rhode Island Education | Procede de propagation in vitro de la vigne par regulation de la croissance d'embryon |
EP0455597A1 (fr) * | 1990-04-30 | 1991-11-06 | Sandoz Ltd. | Méthode de stimulation de croissance, de division ou d'embryogenèse de plantes en culture in vitro |
WO1992020801A1 (fr) * | 1991-05-24 | 1992-11-26 | Universidad Politecnica De Madrid | Nouveaux peptides antipathogenes et compositions les contenant |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US4714679A (en) * | 1982-12-03 | 1987-12-22 | The Board Of Governors For Higher Education, State Of Rhode Island And Providence Plantations | In vitro propagation of grape |
-
1992
- 1992-12-14 FR FR9215044A patent/FR2699190B1/fr not_active Expired - Fee Related
-
1993
- 1993-12-14 NZ NZ258928A patent/NZ258928A/en unknown
- 1993-12-14 WO PCT/FR1993/001239 patent/WO1994013787A1/fr not_active Application Discontinuation
- 1993-12-14 US US08/448,481 patent/US5914270A/en not_active Expired - Fee Related
- 1993-12-14 JP JP6513885A patent/JPH08504329A/ja active Pending
- 1993-12-14 CA CA002151587A patent/CA2151587A1/fr not_active Abandoned
- 1993-12-14 EP EP94902805A patent/EP0673414A1/fr not_active Withdrawn
- 1993-12-14 AU AU57022/94A patent/AU684484B2/en not_active Ceased
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2537157A1 (fr) * | 1982-12-03 | 1984-06-08 | Rhode Island Education | Procede de propagation in vitro de la vigne par regulation de la croissance d'embryon |
US4532733A (en) * | 1982-12-03 | 1985-08-06 | The Board Of Governors For Higher Education, State Of Rhode Island | In vitro propagation of grape |
EP0455597A1 (fr) * | 1990-04-30 | 1991-11-06 | Sandoz Ltd. | Méthode de stimulation de croissance, de division ou d'embryogenèse de plantes en culture in vitro |
WO1992020801A1 (fr) * | 1991-05-24 | 1992-11-26 | Universidad Politecnica De Madrid | Nouveaux peptides antipathogenes et compositions les contenant |
Non-Patent Citations (7)
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L. SOSSOUNTZOV ET AL.: "SPATIAL AND TEMPORAL EXPRESSION OF A MAIZE LIPID TRANSFER PROTEIN GENE.", THE PLANT CELL, vol. 3, no. 9, September 1991 (1991-09-01), ROCKVILLE, MD US, pages 923 - 933 * |
M. GROSBOIS ET AL.: "CHANGES IN LEVEL AND ACTIVITY OF PHOSPHOLIPID TRANSFER PROTEIN DURING MATURATION AND GERMINATION OF MAIZE SEEDS.", PLANT PHYSIOLOGY, vol. 90, no. 4, August 1989 (1989-08-01), ROCKVILLE, MD US, pages 1560 - 1564 * |
P. COUTOS-THEVENOT ET AL.: "EXTRACELLULAR PROTEIN PATTERNS OF GRAPEVINE CELL SUSPENSIONS IN EMBRYOGENIC AND NON-EMBRYOGENIC SITUATIONS.", PLANT SCIENCE, vol. 86, no. 2, 1992, LIMERICK, IE, pages 137 - 145 * |
P. COUTOS-THEVENOT ET AL.: "FOUR 9-kDa PROTEINS EXCRETED BY SOMATIC EMBRYOS OF GRAPEVINE ARE ISOFORMS OF LIPID-TRANSFER PROTEINS.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 217, no. 3, November 1993 (1993-11-01), BERLIN, DE, pages 885 - 889 * |
P. COUTOS-THEVENOT ET AL.: "SOMATIC EMBRYOGENESIS FROM GRAPEVINE CELLS. I - IMPROVEMENT OF EMBRYO DEVELOPMENT BY CHANGES IN CULTURE CONDITIONS.", PLANT CELL, TISSUE AND ORGAN CULTURE, vol. 29, no. 2, 1992, THE HAGUE, NL, pages 125 - 133 * |
P. STERK ET AL.: "CELL-SPECIFIC EXPRESSION OF THE CARROT EP2 LIPID TRANSFER PROTEIN GENE.", THE PLANT CELL, vol. 3, no. 9, September 1991 (1991-09-01), ROCKVILLE, MD US, pages 907 - 921 * |
S. TORRES-SCHUMANN ET AL.: "A PROBABLE LIPID TRANSFER PROTEIN GENE IS INDUCED BY NaCL IN STEMS OF TOMATO PLANTS.", PLANT MOLECULAR BIOLOGY, vol. 18, no. 4, February 1992 (1992-02-01), DORDRECHT NL, pages 749 - 757 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999059398A3 (fr) * | 1998-05-15 | 2000-01-20 | Univ Florida | Systeme de regeneration de la vigne et son utilisation |
AU750133B2 (en) * | 1998-05-15 | 2002-07-11 | University Of Florida | Regeneration system for grape and uses thereof |
US6455312B1 (en) | 1998-05-15 | 2002-09-24 | University Of Florida | Regeneration system for grape and uses thereof |
US7326826B2 (en) | 1998-05-15 | 2008-02-05 | University Of Florida Research Foundation, Inc. | Selection of fungal resistant grape somatic embryos |
WO2000070054A1 (fr) * | 1999-05-14 | 2000-11-23 | University Of Florida | Plants de vigne resistant aux agents pathogenes |
US6995015B1 (en) | 1999-05-14 | 2006-02-07 | University Of Florida | Pathogen-resistant grape plants |
Also Published As
Publication number | Publication date |
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EP0673414A1 (fr) | 1995-09-27 |
FR2699190B1 (fr) | 1995-03-03 |
JPH08504329A (ja) | 1996-05-14 |
CA2151587A1 (fr) | 1994-06-23 |
US5914270A (en) | 1999-06-22 |
FR2699190A1 (fr) | 1994-06-17 |
AU684484B2 (en) | 1997-12-18 |
NZ258928A (en) | 1996-07-26 |
AU5702294A (en) | 1994-07-04 |
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