Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1999-06-04
2003-09-30
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S420000, C435S430100, C435S410000, C800S295000, C800S298000
Reexamination Certificate
active
06627441
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from Canadian Patent Application No. 2,240,135 filed Jun. 5, 1998.
FIELD OF THE INVENTION
This invention relates to a development treatment for somatic embryos, particularly conifer embryos, including water stressing and growth regulator treatment, preferably including the use of a relatively high molecular weight non-permeating osmoticum and abscisic acid or equivalents, characterized by an increase in the concentration of the growth regulator or the intensity of the water stressing during the course of the growth regulator treatment.
BACKGROUND OF THE INVENTION
Somatic embryogenesis offers the potential to produce clonally large numbers of plants of many species at low cost. Somatic embryos, develop without the surrounding nutritive tissues and protective seed coat found in zygotic embryos, so considerable research has been devoted to causing somatic embryos to functionally mimic seeds with regard to efficient storage and handling qualities. The development of techniques for somatic embryogenesis in conifers has greatly improved the ability to culture conifer tissues in vitro and now offers the means to propagate clonally commercially valuable conifers of a number of species. However, there is still room in the technology for improvement of the quality and vigour of plants resulting from somatic embryos, including those from all species of conifers.
It has been suggested to use abscisic acid (ABA) or osmoticum or both for enhancing storage levels in plant cells. For example, it was shown that somatic embryos of Theobroma cacao could be induced to accumulate fatty acids approaching the composition of commercial cocoa butter by using a high sucrose concentration in the culture medium (Pence et al. 1981; Physiol. Plant. 53:378-384). Modifying the culture conditions by osmoticum concentration and/or ABA content similarly improved lipid accumulation in
Brassica napus
L. somatic- (Avjioglu and Knox 1989; Ann. Bot. 63:409-420) and microspore-derived (Taylor et al. 1990; Planta 181: 18-26) embryos as well as somatic embryos of carrot (Dutta and Appelqvist 1989; Plant Sci. 64: 167-177) and celery. Also, the level of storage lipids in
P. abies
somatic embryos was improved by optimizing the ABA level to between 10-20 &mgr;M, but the somatic embryos contained about 4% of the lipid level obtained by zygotic embryos (Feirer et al. 1989; Plant Cell Rep. 8:207-209).
Japanese laid-open patent publication No. 1-218520, issued on Aug. 31, 1989, describes a process for producing plant body regenerative tissue. The process includes a step of cultivating a plant body regenerative tissue in a medium containing ABA and having an osmotic pressure of 180 to 2500 mmol/kg. In-order to control the osmotic pressure within the specific range, a non-toxic substance such as sugar, alcohol, an amino acid or glycol is added.
Water stress plays an important role in maintaining embryos in a maturation state (Kermode 1990, Crit. Res. Plant Sci. 9, 155-194). Kermode suggests that low water content rather than ABA prevents precocious germination during later stages of development. This is important because precocious germination of embryos during development in seeds would be lethal during desiccation.
A conventional way to water stress plant cells grown in vitro is to increase the osmotic concentration of the culture medium through the use of plasmolysing osmotica. For example, increased concentrations of plasmolysing osmotica such as sucrose have been used to promote somatic embryo maturation of many plant species. Sucrose at levels of 3 to 6% was found to improve somatic embryo development of many conifers (Attree and Fowke 1993). It seems that high concentrations generally led to repressed embryo development. Mannitol had a similar effect on maturation of conifer somatic embryos (Roberts 1991; Physiol. Plant. 83; 247-254). Low levels of mannitol (2-6%) led to a doubling of the number of mature embryos recovered at the end of the maturation period; however, the treatment could only be applied as a short pulse (one week) as prolonged maturation treatment with mannitol became detrimental to further embryo maturation.
Poor embryo response using sucrose and mannitol or other simple sugars and salts may be due to the absorption of such plasmolysing, osmotica by the symplast of plant cells. Such absorption facilitates adjustment of tissue osmotic potential (osmotic recovery) without lowering the tissue water content. Additionally, direct or indirect metabolic effects on specific plant metabolites can occur, due to utilization of the solute by the embryo or its toxic effects.
Alternatives to plasmolysing osmotica are non-plasmolysing osmotic stresses as well as other forms of non-plasmolysing stresses which have the same effect as drought conditions. Such stresses can be induced using a controlled environmental relative humidity (r.h.) or, for example, by non-permeating high molecular weight compounds such as polyethylene glycol (PEG) or dextran. These compounds are usually available in a wide range of molecular weights. For example, PEG is available in molecular weights ranging from 200 to 35,000. However, only those with a molecular weight above 1000 would normally be considered to be non-permeating. This is because the large molecular size of these solutes excludes their passage through plant cell walls, so preventing entry into cells and consequently preventing plasmolysis, while still removing water (Carpita et al, 1979). Consequently, their non-plasmolysing effect reduces tissue water content in a manner similar to the effects of water stress observed in cells of plants subjected to drought conditions. The effect is constant and cell turgor can only be restored by cells actively increasing their cellular solute concentrations. PEG has been most commonly used to apply water stress to whole plants, to osmotically prime whole seeds to synchronise germination and improve seedling vigour.
Embryo drying occurs naturally in most seeds, and has a role to play in the developmental transition between maturation and germination. Thus, desiccation leads to enhanced germination of both zygotic and somatic embryos. Desiccation of whole somatic embryos is also an alternative method of germplasm storage. Somatic embryos produced continuously year-round could therefore be dried and stored until the appropriate planting season, or shipped to new locations.
A number of prior patents and publications describe methods for the desiccation of somatic embryos. In U.S. Pat. No. 4,615,141 issued on Oct. 7, 1986, Janick and Kitto describe a method for stimulating desiccation. tolerance to asexual plant embryos which are then desiccated. In this method, the embryos are removed from medium containing auxin and cytokinin to a hormone-free development medium. During subsequent development, the somatic embryos are pre-treated by increasing the sucrose concentration of the development medium from normal levels to high levels, or by applying ABA. The hydrated embryos are then encapsulated in a hydrated coating material. The coating material dries to form a thin, non-toxic film enclosing one or more embryos, protecting the embryos during storage but readily redissolving in an aqueous solution. The use of ABA and increased sucrose during embryo development is suggested to improve subsequent survival of the encapsulated embryos during desiccation. Once the embryos have been encapsulated, they are dried at a temperature ranging from 20 to 30° C. for a period of at least five hours.
In U.S. Pat. No. 4,777,762 Oct. 18, 1988, Redenbaugh et al. describe a method for producing desiccated analogs of botanic seeds which are created by providing ABA during the development phase then removing a portion of the water by slow or fast drying so that the plant tissue is no longer saturated with water. The method also involves encapsulating meristematic tissue in a hydrated gel or polymer and removing water by slow or fast drying. The formation of somatic embryos is induced and the embryo
Campell Bruce R.
Childs Lisa C.
Grunberg Anne Marie
Lum Brian J.
Michael Best & Friedrich
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