Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Patent
1995-08-08
1999-06-22
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
435410, 435420, 435421, 435430, 4354301, 435431, C12N 500, C12N 502
Patent
active
059142707
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
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. The invention relates, more specifically, to a method for obtaining embryos by culturing plant somatic cells and using lipid transfer proteins (LTPs) and to these proteins and their uses.
BACKGROUND OF THE INVENTION
The principle of obtaining embryos by culturing somatic cells in vitro is well known, on paper. In particular, see D. J. Gray and J. A. Mortensen, Plant Cell, Tissue and Organ Culture (1987) vol. 9 pp. 73-80. Regarding vine somatic embryos in particular, see U.S. Pat. No. 4,532,733. While somatic embryogenesis may be feasible for certain species of plants, this method is, by contrast, very difficult, if not impossible, to implement in the case of other species.
In order to understand the problems which are posed, it is necessary to recall briefly the principles of culturing somatic cells which are intended to form embryos.
Somatic embryogenesis essentially comprises two steps: general at high concentrations, which give rise to the appearance of proembryogenic aggregates (or masses) (PEM), which correspond to groups of from 10 to 50 cells which are, in particular, of a meristematic nature. to the formation of somatic embryos from these PEM, following the different developmental stages of plant embryogenesis, namely: globules, heart and torpedo.
In the case of certain plant species, it proves to be very difficult, if not impossible, to obtain somatic embryos. For example, in the case of certain vine cultivars, the embryogenic capacities of the somatic cells can either be halted, usually at the stage of proembryogenic aggregate formation, or else maturation of the embryos is blocked, in particular at the globular stage.
This is all the more unfortunate since there exists, in particular in the case of the vine, a very great need for obtaining somatic embryos, in vitro, on a large scale. Thus, the in vivo methods for producing embryos are often inadequate and undesirable since they lead, as a general rule, to genetic recombinations which involve the loss of the novel genetic features of the vine variety or the stock-vine whose multiplication is desired.
European patent application EP 281375 proposed adding calmodulin and calcium to a cell culture in order to promote differentiation of roots and embryos.
European patent application EP 455597 describes a method for stimulating the growth and embryogenesis of plants cultivated in vitro, by addition of arabino-galactan proteins. However, it has been demonstrated (de Vries et al., 1989, Proceedings of the International Symposium of Biotechnology for Major Crops (A.D.E.B.I.O. ed.), pages 22-23) that an extracellular glycoprotein of 52/54 kDa induces arrest in embryonic development at the heart stage.
SUMMARY OF THE INVENTION
The present invention is based on identifying proteins that ensure or stimulate cell differentiation, in particular somatic embryogenesis, even under certain nonpermissive conditions, in particular from vine somatic cells. It is also possible for the proteins to have other uses.
Within the meaning of the preceding paragraph, "nonpermissive conditions" are understood to mean, in relation to achieving a given phenomenon, conditions which are known from the state of the art partially or totally to prevent the phenomenon from being achieved.
This is why, in accordance with a first aspect, the present invention relates to a method for promoting the differentiation of cells in culture. In particular, the present invention relates to a method for promoting tissue differentiation, embryogenesis or organogenesis. The method is characterized in that at least one lipid transfer protein or "LTP", or one LTP analog, is introduced into the culture medium in a concentration which is effective for obtaining differentiation of the cells. An LTP analog is understood to mean any proteinaceous substance, such as a protein, protein fragme
REFERENCES:
patent: 4532733 (1985-08-01), Krul
patent: 4714679 (1987-12-01), Krul
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 pp. 137-145.
P. Strek et al., Cell-Specific Expression Of The Carrot EP2 Lipid Transfer Protein Gene, The Plant Cell, vol. 3, No. 9, Sep. 1991, pp. 907-921.
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, Apr. 1989, pp. 1560-1564.
L. Sossountzov et al., Spatial And Temporal Expression Of A Maize Lipid Transfer Protein Gene, The Plant Cell, vol. 3, No. 9, Sep. 1991, pp. 923-933.
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, Feb. 1992, pp. 749-757.
P. Coustos-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, pp. 125-133.
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, Nov. 1993, pp. 885-889.
Boulay Michel Paul Henri
Coutos-Thevenot Pierre Marie Louis
Deloire Alain Jean
Guern Jean Rene Denis
Jouenne Thierry Georges
Lankford , Jr. Leon B.
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