Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Medium – per se – for culture – maintenance – regeneration – etc.
Reexamination Certificate
1998-05-28
2001-04-17
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Medium, per se, for culture, maintenance, regeneration, etc.
C435S410000, C435S420000, C047S05810R, C504S101000
Reexamination Certificate
active
06218184
ABSTRACT:
FIELD OF INVENTION
This invention relates to a new plant tissue culture medium which can be used successively from propagation to acclimatization to the formation of the mature plantlet, and to a method for production of said medium. The invention further relates to a culture method in which this medium is used in a light-transmissible container which is at least partially equipped with a porous gas permeable film.
PRIOR ART
Cell culture and meristem culture of highier plants is a multistage process, starting from a primary culture and successive sub-cultures to form multiple axillary shoots or plantlets by such methods as “enhanced axillary branching”, “protocorm-like body formation”, or “shoot primordium formation”, followed by the culturing of mature plantlets. Culturing of mature plantlets comprises the growth of shoots from “protocorm-like bodies”, “shoot primordium” or the like, followed by differentiation of adventitious roots. In this stage, the culture medium (support material) is a critical factor in the growth and development of the root structure of the plant.
Agar is the most commonly used medium for this type of plant tissue culture. However, an agar medium cannot be aerated, this root induction is poor. Root development is better with an agar medium specifically formulated for such purpose, but the adventitious roots formed therein cannot function effectively, and when the plantlets thereof are transplanted directly into soil, the exposed foliage soon withers.
In one method in use to resolve this problem, after removal from the agar medium the plantlets are first acclimatized in perlite, vermiculite, or similar media for a period before transplanting them into soil either outdoors or in greenhouses. However, this acclimatization normally requires an extended period of one to three months, during which rooting is poor causing the plantlets to wither or die, or the plantlets remain vitrificated and cannot develop into normal plants. The method has many disadvantages in tens of time and labor and yield.
Alternatively, rockwool, or perlite and vermiculite have been used in place of agar. However, rockwool itself does not decompose when the plantlets are transplanted directly into the soil, and the root structure can be damaged upon attempting to remove it, thereby reducing the rooting ratio. Also, root growth is retarded because rockwool as a culture substrate is too hard. On the other hand, the granular structure of perlite and vermiculite make it difficult for the plants to be affixed to the medium, and this poor contact results in uneven growth rates among the plants. In addition, perlite and vermiculite are difficult to work with when planting since the granules easily stick to the tips of the forceps.
Moreover, glass, polycarbonate, or other non-porous containers are generally used in the aforementioned culture methods, but because of the poor gas permeability of these materials, the plantlets are spindly with poor root structure and cannot be transferred outdoors directly. As a result, the culture process had to be divided into two separate stages, a propagation and root induction stage, and an acclimatization stage.
These conventional plant tissue culture methods as described present many problems in acquiring mature plantlets. Root development is poor, as is primary growth, and the yield is reduced because of the poor rooting ratio. As well, the necessity of dividing the culture process into the aforementioned two stages increases both the time and labor. This invention was developed in order to resolve these problematic points.
That is, an objective of this invention was to provide a culture medium which would improve the efficiency of propagating and acclimatizing tissue cultured plants, and a method of producing said medium.
Another objective of this invention was related to a novel culture method using this culture medium.
This invention is characterized by a novel plant tissue culture medium comprised in part of vermiculite with an average particle diameter between 0.1-10 mm and cellulose fibers of an average fiber length between 0.01-5 mm; in addition, it provides a culture method in which said culture medium is used in a light-transmissible container equipped at least in part with a porous gas permeable film, preferably having an air permeability between 1-50 sec/100 ml as measured in accordance with JIS standard P8117.
The use of this culture medium formed from a specific type of vermiculite and cellulose fibers enables the culturing process as a whole to be performed easily and efficiently. Root development and plant growth are good, and healthy plants can be produced in a single culturing without the need for an acclimatization stage. Plantlets can also be transplanted directly without damaging the roots. The inventors also observed that when this medium is used in combination with a light-transmissible container equipped with a gas permeable film, the aforementioned efficacy is noticeably improved, and this invention was therein completed.
DISCLOSURE OF INVENTION
This invention provides a composite culture medium formed from vermiculite and cellulose fibers. There is no specific restriction on the type of vermiculite, but a baked vermiculite with an average particle diameter between 0.1-10 mm, preferably between 1-5 mm, is best. Pretreated vermiculite of various types can be used. Pretreatment processes include heating, cooling, refining, expansion, pulverization, pelletization, impregnation, coating, or other chemical or physical processing.
The cellulose fibers should have an average fiber length between 0.01-5 mm, preferably between 0.02-4 mm. There is no specific restriction on the type of cellulose, and cotton lint, cotton linter, softwood cellulose, hardwood cellulose, bast fiber cellulose, hemp cellulose, regenerated cellulose, bacteria-derived cellulose, or the like, or any mixture thereof can be used. As well, pretreated cellulose of various types can be used. Pretreatment processes include heating, cooling, refining, conversion to amorphous cellulose, swelling, depolymerization, chemical derivatization, cross-linking, conversion of cellulose crystalline type, regeneration from dissolved cellulose, pulverization, pelletization, impregnation, coating, or other chemical or physical processing.
The admixture ratio of vermiculite to cellulose fiber will vary with the method of molding, but a ratio between 50:50-95:5 parts by weight is preferable, more preferably in a 65:35-90:10 ratio. Cellulose ratio above or below these values are not desirable. At a ratio of less than 5% by weight, intertwining of the vermiculite and cellulose fibers is poor making the admixture difficult to mold. Conversely, if the cellulose content exceeds 50% by weight, the resultant molded product is too hard for embedding the plantlets.
The vermiculite and cellulose fibers can be molded, for example, by either of two methods, wet molding or dry compression molding. In wet molding, the vermiculite and cellulose fibers are admixed in a liquid, preferably water, and the slurry so formed is poured into a mold. The liquid is then removed and the mold is dried. In dry compression molding, an admixture of dry components is compressed.
For wet molding, the cellulose fibers should preferably have an average fiber length between 0.1-4 mm, more preferably from 0.5-3 mm. The vermiculite should preferably have an average particle diameter between 0.1-10 mm, more preferably from 0.5-5 mm. If the cellulose fibers are too short, the mold becomes too hard when dried, and is still too hard upon the addition of a nutrient liquid medium, making it difficult to embed the plantlets. Conversely, if the cellulose fibers are too long, the vermiculite and cellulose fibers cannot entwine, making it difficult to form an uniform admixture and molded product. In either case, the final product is not desirable as a culture medium.
Similarly, extremes in vermiculite particle size are not desirable. If the particles are too fine, the nutrient liquid medium cannot be effectively retained by the subst
Hasegawa Osamu
Tadokoro Fumihiko
Takahashi Nobumitsu
Arent Fox Kintner & Plotkin & Kahn, PLLC
Lankford , Jr. Leon B.
Nisshinbo Industries Inc.
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