Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
2000-01-26
2003-04-01
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S405000, C435S252100, C435S822000
Reexamination Certificate
active
06540997
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of preparation and storage of viable Agrobacterium cultures.
BACKGROUND OF THE INVENTION
The preservation of dried microorganisms has present application in such areas as pharmaceuticals and agriculture. Furthermore, research of such organisms is greatly benefited by technology that allows for their long-term preservation. Also, the potential benefits of such preserved microorganisms is great, since dried microorganisms may be stored for long periods of time while retaining significant viability. Freeze-drying is, by far, the most common method employed to prepare dried microorganisms. However, freeze-dried bacteria generally require long-term storage at refrigerated or frozen temperatures to maintain optimum viability. Additionally, freeze-drying requires expensive, specialized equipment. Freeze-drying is also not the optimum method for preparing at least some types of bacterial cultures for storage.
P. Louis et al.,
Survival of Escherichia Coli During Drying and Storage in the Presence of Compatible Solutes,
41 Appl. Microbiol. Biotechnol. at 684 (1994) discloses the use of compatible solutes, small, highly water-soluble organic molecules, and air-drying for preservation of other gram-negative bacterial species, including
Escherichia coli.
These air-dried cultures of
Escherichia coli
are quite stable for several months with out requiring freezing to preserve bacterial cell viability.
Agrobacterium, a genus of gram-negative bacteria, is commonly found in soil and some strains can be plant pathogens. One species of Agrobacterium is
Agrobacterium tumefaciens,
which is the causative agent of crown gall tumors in plants. Another species,
Agrobacterium rhizogenes
is the causative agent of hairy root disease in plants. Both Agrobacterium species,
tumefaciens
and
rhizogenes,
can potentially cause tumor-like growth dysfunction in plants by transferring foreign bacterial genes into plant cells. The ability of Agrobacterium to transfer foreign DNA into plant cells has made Agrobacterium strains very useful vectors for foreign gene transfer into plants in the process known as plant genetic engineering. Additionally, infective virulence of Agrobacterium may be significantly enhanced by treating the Agrobacterium cultures with phenolic compounds, which may be produced by plants in response to wounding, and acidic environmental conditions, such as those found inside some plant tissues. M. Limami et. al.,
Natural Genetic Transformation by Agrobacterium rhizogenes,
118 Plant Physiol. at 543 (1998), J. Porter,
Host Range and Implications of Plant Infection by Agrobacterium Rhizogenes,
10 Critical Reviews In Plant Sciences, vol. 4, at (1991), H. Matthews et. al.,
The Promotion of Agrobacterium Mediated Transformation in Atropa Belladona L. by Acetosyringone,
136 J. Plant Physiol. at 404 (1990), S. Turk et. al.,
Localization of the VirA Domain Involved in Acetosyringone-mediated Vir Gene Induction in Agrobacterium Tumefaciens,
25 Plant Molecular Biol. at 899 (1994).
There is a great need for a preparation of Agrobacterium that can be conveniently stored at room temperature for an extended period of time since it has great potential utility in agricultural biotechnology research. Such cultures can be transported and stored without the need for refrigeration or freezing. Such handling efficiency would make the use of Agrobacterium under actual field conditions much more practical. There is also a need for a room temperature-stable preparation of Agrobacterium for the preparation of plant biotechnology educational kits. These kits could be displayed in conventional retail settings with extended shelf life. Additionally, there is a need for a process for enhancing Agrobacterium virulence, and to incorporate the process into a method for preserving Agrobacterium preparations. The process would have enhanced utility for routine genetic transformation of plants.
SUMMARY OF THE INVENTION
It is an object of the present invention to meet the above-described needs and others. Specifically, it is an object of the present invention to provide a virulent preparation of Agrobacterium, and a method for making the same. The method may include the step of growing Agrobacterium either in a liquid media, i.e., nutrient broth, or on solid bacteriological media, i.e., nutrient agar, to a stationary growth phase. The method may also include the step of harvesting the grown bacteria by centrifugation.
The method includes the step of suspending bacteria in an Agrobacterium desiccation medium. The Agrobacterium desiccation medium may be an aqueous solution that includes an osmoprotectant that may be approximately 625 millimolar sucrose, an acidulant that may be 50 millimolar sodium dihydrogen phosphate (NaH
2
PO
4
), and a phenolic compound that may be 100 micromolar ethyl vanillin. Other natural or synthetic phenolic compounds may be used with or in place of the micromolar ethyl vanillin such as, sinapyl alcohol, coniferyl alcohol, sinapic acid, sinapic acid methyl ester, sinapinic acid, syringic acid, syringaldehyde, p-hydroxybenzoic acid, vanillalacetone, vanillin, ferulic acid, ferulic acid methyl ester, 5-hydroxyferulic acid methyl ester, flavonoids, lignins, lignans, and acetosyringone. In a preferred embodiment of the invention, the phenolic compound is micromolar ethyl vanillin.
Approximately one gram weight, or one milliliter volume, of bacterial cells per 2 milliliters of the Agrobacterium desiccation medium may be used to prepare the bacterial suspension. After the bacterial cells have been suspended in the aqueous solution, the cell suspension may be dried immediately or stored refrigerated until processing is desired.
The method includes the step of dehydrating the bacteria, which may be performed by slowly air drying the bacterial suspension at approximately 20 to 25° C. and less than 30% relative humidity. When the bacterial suspension is completely dry, it constitutes a shelf-stable, virulent culture of Agrobacterium.
The bacterial suspension may be conveniently applied to a compatible supporting matrix (i.e. autoclaved toothpicks or cellulose powder) prior to the dehydrating step to facilitate ease of handling or packaging. Additionally, the dried bacterial suspension may be ground and diluted with compatible dry or liquid additives to produce a variety of useful forms of the culture.
One use of these cultures is, for example, educational kits to demonstrate the natural genetic engineering of plants. It is beneficial to incorporate into these educational kits cultures of Agrobacterium that remain viable upon storage at room temperatures since these educational kits may then be stored and displayed for sale with other ordinary merchandise in a retail store setting rather than requiring special handling such as refrigerated shipping and storage.
It is a further object of the present invention to provide a virulent preparation of Agrobacterium cells, which includes Agrobacterium cells, an acidulant, and a phenolic compound. The phenolic compound is preferably ethyl vanillin. However, the phenolic compound can be at least one of the following compounds: ethyl vanillin, sinapyl alcohol, coniferyl alcohol, sinapic acid, sinapic acid methyl ester, sinapinic acid, syringic acid, syringaldehyde, p-hydroxybenzoic acid, vanillalacetone, vanillin, ferulic acid, ferulic acid methyl ester, 5-hydroxyferulic acid methyl ester, flavonoids, lignins, lignans, and acetosyringone.
The virulent preparation of Agrobacterium according to the invention can include sodium dihydrogen phosphate as the acidulant.
It is a primary objective that the preparation is shelf stable at ambient temperature for a plurality of months. The preparation as described above accomplishes this objective.
At least one dry excipient material can be included in the virulent preparation of Agrobacterium. A food coloring agent can also be added. At least one flow agent may be included in the preparation, as well as a plant hormone and/or a bacterial growth promoter.
Benson David K.
Naff David M.
Ware Deborah K.
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