Patent Application
20050155101
The present invention relates to an equipment for transforming plants, a system for transforming plants and a method for transforming plants, more particularly, it relates to the equipment, system and method suitable for transforming plant bodies according to an in planta method. Furthermore, the present invention relates to a method for selecting plants harboring a heterogeneous gene from a parent transformed plant body.
At present, transformation of higher plants is greatly noticed, as it leads to acceleration or efficiency of breeding and improvement of plants for increase in food production, and production of useful substances inherent in plants. Among them, mouse-ear cress, rice and tobacco and the like which are particularly investigated as a model plant of higher plants are enthusiastically transformed by a convenient in planta method, and a speed for investigating or developing them is being accelerated. Therefore, a method for experimenting, investigating and developing higher plants more exactly, conveniently, speedy and efficiently is demanded, which can provide a healthy and high quality plant in a qualitatively and quantitatively stable manner.
However, in a conventional transformation of a plant body by an in planta method, environmental management such as irrigation was required, because a plant body to be transformed was grown in a compost in a vessel such as a pot for growing a plant. In addition, in the conventional method, a size of one vessel was large relative to a number or a size of the plant to be used, thereby, a larger space and a larger environment-controlling facility were required in order to grow a number of plant bodies. Accordingly, in the conventional method, an experimental efficiency could be enhanced only at a relatively large institute. In addition, in the conventional method, an experiment in which a growth degree at transformation of the plant bodies to be used is severely uniformized could not be performed, because transformation of the plant bodies was conducted in a vessel unit. In addition, there are often plant bodies not suitable for immersing into a carrier solution. From above reasons, the experimental efficiency in the conventional method was low. Moreover, there were disadvantages in the conventional method that the compost is also immersed in a solution for transformation, that impurities are mixed in the solution, that the solution is infiltrated into the compost, and the like. In addition, in the case where a vacuum infiltration transformation of the in planta method is used, the number of plants which can be treated in one operation is limited because a volume of a vacuum chamber to be used is limited. Furthermore, there were problems in the conventional method that a special space is required for an acclimatization treatment after transformation, and that management of plant bodies until flowering and fructification is likely to go wrong.
In addition, hitherto, selection of plant bodies harboring a heterogeneous gene from a parent transformed plant body has been generally conducted by determining whether a seed has an ability to germinate or grow on a selection medium containing a drug for the presence or absence of a drug-resistant gene which has been introduced simultaneously with the heterogeneous gene. Thereafter, the grown plant is repotted to the compost. However, there are problems that in the case where multiple selections are conducted, cumbersome handling is required in which the plant seed or the plant body is transferred to different selective media at every selection, and that upon repotting the plant body from the selective medium to the compost, plant roots are cut. Moreover, a conventional selection method is undesirable, because it is also thought that a stress is burden on the plant body due to a rapid environmental change upon transferring it between a plurality of selective media or from under a selection condition to under a normal growth condition.
As described above, in the conventional transformation method of the plant body, wherein the plant body is grown in a compost, and in a conventional selection method of the plant harboring the heterogeneous gene from the parent transformed plant, a method for experimenting, investigating and developing higher plants more exactly, conveniently, speedy and efficiently could not be conducted.
The present inventors studied intensively in view of above problems and, as the result, found that above problems can be solved by growing a plant body to be transformed using a microporous body, and subjecting it as such to transformation according to an in planta method, or by germinating or growing a seed obtained from a transformed plant on the microporous body which has been immersed in a selective medium, which resulted in completion of the present invention.
That is, in the first aspect, the present invention provides an equipment for transforming plants which comprises:
According to the first aspect of the present invention, no environmental management such as irrigation is required for a long period of time because the plant body is grown by absorbing the aqueous nutrition supplied by the microporous body. In addition, according to the first aspect of the present invention, an area occupied by one plant body can be reduced, a large numbers of plant bodies can be efficiently grown and they can be efficiently subjected to transformation because soil is not required for growing or transforming the plant body. In addition, according to the first aspect of the present invention, an experiment in which only plant bodies of severely uniformized in their growth degree are selected and subjected them to transformation can be performed because the plant body can be independently grown in one unit. In addition, an exact experiment can be performed without contamination of any impurity into a solution for transformation, and the plant body can be easily handled, because no flowing medium such as soil is used in the experiment. In addition, in the conventional method, it is necessary that the carrier solution must be squeezed from the soil in light of biological containment, because the carrier solution is easily infiltrated into the soil upon transformation. Thereby, in the conventional method, an experimental or working efficiency is lowered and often a stress is burden to the plant body. On the contrary, in the present invention, microorganisms and the like in the carrier solution are hardly penetrated into the microporous body even in the case where the microporous body is contacted with the carrier solution upon transformation because the microporous body has been filled with the aqueous solution, thereby, it is not necessary that the carrier solution must be squeezed from the microporous body. Therefore, in the present invention, such the experimental or working efficiency can be enhanced, and the stress is not burden to the plant body. In addition, although the microporous body should be sterilized in a limited space such as in an autoclave and the like after use in light of biological containment and recycling or a disposition, the microporous body of the present invention can be efficiently treated even in such the case for reasons as described above.
In addition, in the second aspect, the present invention provides the equipment for transforming plants of the first aspect of the present invention, wherein the in plant method is a vacuum infiltration transformation.
According to the second aspect of the present invention, a transformation efficiency of the plant body can be further enhanced.
In addition, in the third aspect, the present invention provides a system for transforming plants which comprises:
According to the third aspect of the present invention, in addition to advantages of the first aspect of the present invention, a further accelerated and efficient experiment can be performed because a plurality of plant bodies can be subjected to transformation at the same time in parallel.
In the fourth aspect, the present invention provides the system for transforming plants of the third aspect of the present invention, wherein the in planta method is a vacuum infiltration transformation.
According to the fourth aspect of the present invention, the transformation efficiency of plant bodies in the third aspect of the present invention can be further enhanced.
In the fifth aspect, the present invention provides the system for transforming plants of the third or fourth aspect of the present invention, wherein the aqueous nutrition is contained in a holding means with contacting with the microporous bodies.
According to the fifth aspect of the present invention, the system may be comprised of only the microporous bodies and the holding means because the aqueous nutrition can be contained in the holding means, thereby, the system can be a more simple and convenient construction and the experiment can be performed more conveniently and efficiently and in a smaller space.
In the sixth aspect, the present invention provides the system for transforming plants of the third or fourth aspect of the present invention, which further comprises a storage tank for storing the aqueous nutrition and an aqueous nutrition-supplying means for connecting the microporous bodies with the aqueous nutrition in the storage tank, wherein the aqueous nutrition in the storage tank is supplied to the microporous bodies through the aqueous nutrition-supplying means.
According to the sixth aspect of the present invention, in addition to the advantages of the third and fourth aspects of the present invention, requirements such as a size of the microporous body and a volume of the aqueous nutrition may be loosen. Thereby, for example, the aqueous nutrition may be supplied from the storage tank through the aqueous nutrition-supplying means even in the case where the microporous body is short, the microporous body can be further miniaturized, and the experiment can be performed more conveniently.
In addition, in the seventh aspect, the present invention provides the system for transforming plants of any one of the third to sixth aspects of the present invention, wherein the microporous body has a cylindrical shape, and the plant seed is germinated and grown on an inner surface of the microporous body.
According to seventh aspect of the present invention, an area occupied by one microporous body can be further reduced, and a large numbers of the plant bodies can be efficiently grown in a smaller space, and can be efficiently subjected to the in planta method.
In addition, in the eighth aspect, the present invention provides the system for transforming plants of any one of the third to seventh aspects of the present invention, wherein the plants are selected from the group consisting of a useful tree such as bishop's flower (Ammi majus), onion (Allium cepa), garlic (Allium sativum), celery (Apium graveolens), asparagus (Asparagus officinalis), sugar beet (Beta vulgaris), cauliflower (Brassica oleracea var. botrytis), brusseles sprout (Brassica oleracea var. gemmifera), cabbage (Brassica oleracea var. capitata), rape (Brassica napus), caraway (Carum carvi), chrysanthemum (Chrysanthemum morifolium), spotted hemlock (Conium maculatum), coptis Rhizome (Coptis japonica), chicory (Cichorium intybus), summer squash (Curcurbita pepo), thorn apple (Datura meteloides), carrot (Daucus carota), carnation (Dianthus caryophyllus), buckwheat (Fagopyrum esculentum), fennel (Foeniculum vulgare), strawberry (Fragaria chiloensis), soybean (Glycine max), hyacinth (Hyacinthus orientalis), sweet potato (Ipomoea batatas), lettuce (Lactuca sativa), birds-foot trefoil (Lotus corniculatus, Lotus japonicus), tomato (Lycopersicon esculentum), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), rice (Oryza sativa), parsley (Petroselinum hortense), pea (Pisum sativum), rose (Rosa hybrida), egg plant (Solanum melongena), potato (Solanum tuberosum), wheat (Triticum aestivum), maize (Zea mays), sugar beat (Beta vulgaris), cotton (Gossypium indicum), rape (Brassica campestris), flax (Linum usitatissimum), sugarcane (Saccharum officinarum), papaya (Carica papaya), Squash (Cucurbita moschata), cucumber (Cucumis sativus), watermelon (Citrullus vulgaris), melon (Cucumis melo), Winter Squash (Cucurbita maxima ) and the like; a foliage plant such as snapdragon (Antirrhinum majus), mouse-ear cress (Arabidopsis thaliana), croton (Codiaeum variegatum), cyclamen (Cyclamen persicum), poinsettia (Euphorbia pulcherrima), barberton daisy (Gerbera jamesonii), sunflower (Helianthus annuus), fish geranium (Pelargonium hortorum), petunia (Petunia hybrida), African violet (Saintpaulia ionatha), dandelion (Taraxacum officinale), torenia (Torenia fournieri), Dutch clover (Trifolium repens), cymbidium (Cymbidium) and the like; a woody plant such as beat tree (Azadirachta indica), orange (Citrus), common coffee (Coffea arabica), ribbon gum (Eucalyptus), para rubber tree (Hevea brasiliensis), holly (Ilex aquifolium), trifoliate orange (Poncirus trifoliata), almond (Prunus amygdalus), carolina poplar (Populus canadensis), oriental arborvitae (Biota orientalis), Japanese ceder (Cryptomeria japonica), Norway spruce (Picea abies), pine genus (Pinus), grapevine (Vitis vinifera), apple (Malus pumila), apricot (Prunus armeniaca), persimmon (Diospyros kaki), fig (Ficus carica), chestnut (Castanea crenata), Lombardy poplar (Populus nigra), Eleuthero (Acanthopanax senticosus) and the like.
According to the eighth aspect of the present invention, an experiment can be performed more rapidly and efficiently for plant bodies on which investigation utilizing transformation is enthusiastically performed at present.
In addition, in the ninth aspect, the present invention provides a method for transforming plants which comprises steps of:
According to the ninth aspect of the present invention, there can be provided a method for transforming plants having advantages as described for the first aspect of the present invention.
In addition, in the tenth aspect, the present invention provides the method for transforming plants of the ninth aspect of the present invention, wherein the in planta method is a vacuum infiltration transformation.
According to the tenth aspect of the present invention, there can be provided a method for transforming plants having advantages as described for the second aspect of the present invention.
In addition, in the eleventh aspect, the present invention provides a method for transforming plants which comprises steps of:
According to the eleventh aspect of the present invention, there can be provided a method for transforming plants having advantages as described for the third aspect of the present invention.
In addition, in the twelfth aspect, the present invention provides the method for transforming plants of eleventh aspect of the present invention, wherein the in planta method is a vacuum infiltration transformation.
According to the twelfth aspect of the present invention, there can be provided a method for transforming plants having advantages as described for the fourth aspect of the present invention.
In addition, in the thirteenth aspect, the present invention provides the method for transforming plants of the eleventh or twelfth aspect of the present invention, which further comprises a step of selecting only microporous bodies having the plant bodies which have grown to a stage suitable for transformation to hold them in the holding means before immersing the plant bodies in the carrier solution, and subjecting the plant bodies to transformation.
According to the thirteenth aspect of the present invention, only plant bodies exactly uniformized in their growth stage can be subjected to the transformation experiment, thereby, a more exact experiment can be conducted.
In addition, in the fourteenth aspect, the present invention provides a method for selecting plants harboring a heterogeneous gene from a parent transformed plant body, which comprises steps of:
According to the fourteenth aspect of the present invention, the plant body harboring the heterogeneous gene can be conveniently subjected to a plurality of selections by merely changing the aqueous nutrition for selection into which the microporous body is immersed. In addition, a root of a grown plant body is not cut because there is no necessity that the plant body is repotted to the compost. In addition, in the case where subjecting to different selections, a stress due to a rapid environmental change is not burden on the plant seed or the plant body because the aqueous nutrition for selection is gradually changed in the microporous body. Thereby, the plant body harboring the heterogeneous gene from the parent transformed plant body and the plant seed obtained from the plant body can be selected in more exactly, conveniently, speedy and efficiently.
Furthermore, in the fifteenth aspect, the present invention provides a method for selecting plants harboring a heterogeneous gene from a parent transformed plant body which comprises conducting, at least once, steps of:
According to the fifteenth aspect of the present invention, in addition to advantages as described for the fourteenth aspect of the present invention, the plant harboring the heterogeneous gene from the parent transformed plant body and expressing it as phenotype can be selected more exactly.
The term “plant seed” herein means a plant seed in a state which has not been germinated yet. In addition, the term “plant body ” herein means a plant from a plant after germination to a plant grown to the suitable stage for transformation according to the in plant method. And, in the case where it is simply referred to as “plant” herein, it means that both of such the plant seed and plant body are included. In addition, the term “transformed plant body” herein means a plant body to which a heterogeneous gene is introduced by the method of the present invention or other method for transformation well known in the art, and the term “heterogeneous gene” means any extraneous gene for being intentionally introduced into the plant, including a gene participating in plant morphogenesis, a gene of particular enzyme, a gene participating in production of a useful material, a gene relating to disease resistance and the like, in addition to a marker gene of a drug resistance for screening the transformed plant.
The term “carrier solution” herein means a suspension or a homogenate of a carrier such as bacteria and virus which carries a plasmid vector comprising a particular heterogeneous gene, with which the plant body can be transformed according to the in plant method. Examples of the carrier solution include, for example, a suspension of Agrobacterium tumefaciens, Agrobacterium rhizogenes and the like, and an additive suitable for transformation of the plant body by the in planta method, for example, a buffer, an osmoregulating agent, a pH-adjusting agent, a surface active agent, a plant growth-adjusting agent and the like can be contained therein. In addition, for the method for transforming, the transformation efficiency can be further enhanced by utilizing a vacuum infiltration transformation among the in planta method, and even in that case, similar additives can be contained in the carrier solution and similar carriers can be used.
In addition, in the case where the terms “upper end” and “lower end” are used herein, they mean a side of the microporous body to which the plant seed is placed and an opposite side thereto, respectively.
Next, embodiments of the present invention will be illustrated with referring to
Firstly, a microporous body (1) having a surface on which a plant seed is germinated and grown is shown in
That is, it is not always restricted by this theory, it seems that a movement of the aqueous nutrition is caused based on a difference in suction at interfaces between the aqueous nutrition and the microporous body and between the microporous body and the plant body. The aqueous nutrition retained by the microporous body is dried depending on a portion of the aqueous nutrition utilized by the plant body on the microporous body, thereby, a force due to the capillary action is restored depending on the dried portion of the aqueous nutrition in the microporous body. As the result, the microporous body absorbs and retains the aqueous nutrition again, and only a necessary amount of the aqueous nutrition is always supplied to the plant. Therefore, according to the present invention, even a plant susceptible to dry or high wet environment can be stably and conveniently germinated and grown.
In addition, a holding means (2) which can removably hold a plurality of microporous bodies (1) is shown in
Next, as shown in
In addition, in another embodiment, as shown in
In addition, in another embodiment, in the system for transforming plants shown in
Then, a series of steps of seeding the plant seed to germinate, selecting the plant body grown to the stage suitable for transformation, and subjecting the plant body to transformation by the in planta method according to the embodiments as described above will be illustrated below.
A series of steps of plant transformation shown in
Furthermore, the transformed plant body can be confirmed by any method known to those skilled in the genetic engineering art, but preferably it can be confirmed by integrating a certain drug resistant marker in a carrier in advance, and then selecting a seed obtained from the plant body for an ability to grow on a medium containing the drug. Thereafter, an expression of a target heterogeneous gene aimed at introducing into the plant (for example, morphology of the plant, enzyme activity, production of particular material, disease resistance or the like) can be finally confirmed by mating the selected plants, collecting a seed therefrom, germinating and growing the seed, and selecting them.
The microporous body used in the present invention is not particularly limited as far as the aqueous nutrition which is contact with a portion thereof can be supplied and retained throughout the interior thereof through communicating pores therein, but the microporous body having a water-absorbing ability being capable of retaining usually 0.05-0.5 (wt/wt), preferably 0.05-0.3, and more preferably 0.1-0.2-fold amount of water at 20° C., and having communicating pores having a pore diameter of usually 0.2-900 μm, preferably 0.2-80 μm, more preferably 0.2-9 μm, most preferably 0.2-3 μm at a pore rate (vol/vol) of usually 0.05-1, preferably 0.1-0.4, more preferably 0.2-0.3 relative to the microporous body is preferred.
Examples of such the microporous body include those obtained by kneading, forming and firing non-metal inorganic solid raw materials such as No.10 clay, porcelain No.2 clay (Shiroyama Cerapot Kabushiki-Kaisya) and Murakami clay (produced in Niigata Prefecture in Japan) according to the conventional method, as well as open-cell type plastic foam materials such as polyvinyl alcohol foam, polyurethane foam, polystyrene foam, vinyl chloride resin foam, polyethylene foam, polypropylene foam, phenol resin foam, urea resin foam and the like. In particular, in the case where non-metal inorganic solid raw materials are made into the microporous body which easily absorbs and releases water, it is preferable that those raw materials are fired while containing, for example, petalite or alumina at 50-60% by weight. Generally, the petalite as described above preferably contains usually 70-90% by weight, preferably 75-85% by weight, more preferably 75-80% by weight of SiO2, usually 10-20% by weight, preferably 12-18% by weight, more preferably 15-17% by weight of Al2O3, usually 2-5% by weight, preferably 3-4.5% by weight, more preferably 3.5-4.2% by weight of LiO2, usually 0.1-0.5% by weight, preferably 0.2-0.5% by weight, more preferably 0.3-0.45% by weight of K2O, and usually 0.5-2% by weight, preferably 0.7-1.8% by weight, more preferably 0.8-1.6% by weight of inevitable impurities. In addition, non-metal inorganic raw materials may contain powdery inorganic foam. Further, the microporous body used in the present invention may be comprised of a non-metal inorganic material, the strength of which is not substantially lowered even when it absorbs water.
As a method of forming a non-metal inorganic solid raw material into the microporous body, there are forming methods which are known in the art such as slip casting forming, extruding forming, press forming and potter's wheel forming. In particular, from a viewpoint of large scale production and reduction in the cost, extruding forming is preferable. In addition, drying after forming can be performed using the conventional methods and conditions known in the art. Subsequent firing of a formed body is not particularly limited as far as firing is performed by the conventional conditions and methods. For example, oxidative firing by which a desired pore is easily obtained can be selected. A firing temperature is 1000° C. to 2000° C., preferably 1100° C. to 1500° C., more preferably 1150° C. to 1300° C., and most preferably approximately 1200° C. When a temperature for firing a non-metal inorganic solid raw material is below 1000° C., a sulfur component easily remains and, on the other hand, when the temperature is higher than 2000° C., the microporous body having the desired water-absorbing property can not be obtained.
On the other hand, as a method of molding a microporous body made of an open-cell type plastic foam, for example, there are melt foaming molding, solid phase foaming molding, casting foaming molding and the like.
Main steps in melt foaming molding comprise melting and kneading, molding of an unfoamed sheet, heat foaming or extrusion foaming, cooling, cutting and processing. In solid phase foaming molding, a polymer is foamed in the solid phase or in the state near the solid phase. In addition, in casting foaming molding, a liquid raw material (monomer or oligomer) is cast and foamed while reacting it in the air. In order to foam an open-cell type plastic foam, a foaming agent is generally used.
In addition, the microporous body may be any shape depending upon a kind of a subject plant and a state upon use, but preferably it may be a shape such as cylindrical shape, plate shape, pillar shape, barrel shape having a bottom, pillar shape having a honeycomb cross section, and barrel shape having a polygonal cross section (for example, hexagonal or tetragonal barrel).
Next, the holding means used in the present invention may be made of any material and has any shape as far as it can hold the microporous body by fitting or pressing to immerse the plant body in the carrier solution and transform it by the in planta method as described above, but preferably it is made of expanded polystyrene, expanded polyethylene or expanded polyurethane of a plate shape having pores or tapered recessions as described above. In addition, the holding means is preferably made of a material having an elastic property such that the microporous body can be removably held repetitively. In addition, the holding means preferably has a specific density such that it floats on the carrier solution while the plant body is immersed in the carrier solution by inverting it as shown in
However, in the system for transforming plants of the present invention, it is also intended that the holding mean for holding the microporous body is moved to above the carrier solution, inverted there, and put down to be immersed in the carrier solution by another transporting means which is not shown in Figures.
In addition, in an embodiment, the holding means used in the present invention may be one having a partial cylindrical recession (40) to which the microporous body can be attached from a side direction thereof as shown in
In another embodiment, the holding means used in the present invention may be one which can suspend a plurality of microporous bodies thorough caps equipped with a hook (13) or a magnet (14) as shown in
In addition, in another embodiment, the holding means used in the present invention may be one of a plate shape having a surface on which a recessed portion (15) having a diameter approximately same as an outer diameter of the microporous body is provided (50). In this embodiment, a plurality of microporous bodies can be held by fitting the lower end of the microporous body into this recessed portion. In addition, the plant bodies on a plurality of microporous bodies can be immersed in the carrier solution approximately at the same time by suspending the holding means by which a plurality of microporous bodies are held with a hook which is provided on an opposite side of the holding means.
In addition, in another embodiment, the holding means used in the present invention is comprised of a cap (18) having a microporous body-fitting portion (16) and a pin (17), and a plate on which a recessed portion (20) having a guide groove (19) is projected, wherein the microporous body can be stably held by guiding the pin to the groove to fit it with the cap. In this embodiment, the holding means which holds a plurality of microporous bodies can be suspended with a hook, which is not shown in Figures, provided on the opposite side of the holding means. In light of a distribution or transportation system, preferably the hook is removable.
In addition, in another embodiment, the holding means used in the present invention may be one by which side portions of a plurality of microporous bodies are fixedly supported (60). In this embodiment, a wave-shape pawl is provided in the holding means, which can fixedly support the microporous bodies such that they do not slip down from the holding means. In addition, in the holding means of this embodiment, a wing portion (21) is provided such that a labor for supporting microporous bodies with the holding means can be relieved and the microporous bodies fixedly supported by the holding means can be mounted on a side wall of the storage tank upon immersing the plant bodies in the carrier solution.
In addition, in the present invention, in the case where the plant body is immersed in the carrier solution containing the gene with which the plant body is transformed, the microporous body on which the plant body is germinated and grown in the embodiment as described above may be removed from the holding means, and it may be subjected to transformation by the in planta method by mounting on a slope (22) of a carrier solution tank (70) as shown in
In addition, in the present invention, the plant body grown on the microporous body to the stage suitable for transformation by the in planta method may be surrounded with a sleeve (24) as shown in
The sleeve may have any shape and may be made of any material as far as it can surround the plant body, but preferably it is comprised of a transparent material such that the condition of the plant body can be observed, and more preferably it is comprised of a plastic film such as a cellophane film. In addition, the sleeve may be fixed to the holding means in addition to an upper portion of the microporous body as shown in
Examples of the plant as a subject of the system for transforming plants of the present invention are not particularly limited as far as it can be transformed by the in planta method or the vacuum infiltration transformation, but preferably include bishop's flower (Ammi majus), onion (Allium cepa), garlic (Allium sativum), celery (Apium graveolens), asparagus (Asparagus officinalis), sugar beet (Beta vulgaris), cauliflower (Brassica oleracea var. botrytis), brusseles sprout (Brassica oleracea var. gemmifera), cabbage (Brassica oleracea var. capitata), rape (Brassica napus), caraway (Carum carvi), chrysanthemum (Chrysanthemum morifolium), spotted hemlock (Conium maculatum), coptis Rhizome (Coptis japonica), chicory (Cichorium intybus), summer squash (Curcurbita pepo), thorn apple (Datura meteloides), carrot (Daucus carota), carnation (Dianthus caryophyllus), buckwheat (Fagopyrum esculentum), fennel (Foeniculum vulgare), strawberry (Fragaria chiloensis), soybean (Glycine max), hyacinth (Hyacinthus orientalis), sweet potato (Ipomoea batatas), lettuce (Lactuca sativa), birds-foot trefoil (Lotus corniculatus, Lotus japonicus), tomato (Lycopersicon esculentum), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), rice (Oryza sativa), parsley (Petroselinum hortense), pea (Pisum sativum), rose (Rosa hybrida), egg plant (Solanum melongena), potato (Solanum tuberosum), wheat (Triticum aestivum), maize (Zea mays), sugar beat (Beta vulgaris), cotton (Gossypium indicum), rape (Brassica campestris), flax (Linum usitatissimum), sugarcane (Saccharum officinarum), papaya (Carica papaya), Squash (Cucurbita moschata), cucumber (Cucumis sativus) watermelon (Citrullus vulgaris), melon (Cucumis melo), Winter Squash (Cucurbita maxima) and the like; a foliage plant such as snapdragon (Antirrhinum majus), mouse-ear cress (Arabidopsis thaliana), croton (Codiaeum variegatum), cyclamen (Cyclamen persicum), poinsettia (Euphorbia pulcherrima), barberton daisy (Gerbera jamesonii), sunflower (Helianthus annuus), fish geranium (Pelargonium hortorum), petunia (Petunia hybrida), African violet (Saintpaulia ionatha), dandelion (Taraxacum officinale), torenia (Torenia fournieri), Dutch clover (Trifolium repens), cymbidium (Cymbidium) and the like; a woody plant such as beat tree (Azadirachta indica), orange (Citrus), common coffee (Coffea arabica), ribbon gum (Eucalyptus), para rubber tree (Hevea brasiliensis), holly (Ilex aquifolium), trifoliate orange (Poncirus trifoliata), almond (Prunus amygdalus), carolina poplar (Populus canadensis), oriental arborvitae (Biota orientalis), Japanese ceder (Cryptomeria japonica), Norway spruce (Picea abies), pine genus (Pinus), grapevine (Vitis vinifera), apple (Malus pumila), apricot (Prunus armeniaca), persimmon (Diospyros kaki), fig (Ficus carica), chestnut (Castanea crenata), Lombardy poplar (Populus nigra), Eleuthero (Acanthopanax senticosus) and the like.
In addition, the aqueous nutrition used in the present invention is an aqueous solution which contains an inorganic element such as nitrate nitrogen, ammonium nitrogen, phosphorus, potassium, calcium, magnesium, iron and manganese, copper, zinc, molybdenum, boron and the like; all kinds of vitamins such as thiamin, pyridoxine, nicotinic acid, biotin, folic acid and the like; a natural material such as coconut milk, casein hydrolysate, yeast extract and the like; an organic nitrogen source such as glutamic acid, aspartic acid, alanine and the like; an agent for controlling plant growth such as auxin, cytokine, gibberellin and the like; a carbon source such as dextrose, sucrose, fructose, maltose and the like; an antibiotic such as kanamycin, hygromycin and the like; and an agrochemical such as basta and the like.
In addition, the in planta method and the vacuum infiltration transformation can be performed, for example, as described in Bechtold N, Ellis J, Pelletier (1993) “In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.” C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199. or “A Model Plant Laboratory Manual” ed. M. Iwabuchi, K. Okada, and I. Shimamoto, Springer-Verlag Tokyo, Apr. 13, 2000. Briefly, in the vacuum infiltration transformation, for example, a shape of the plant body grown on the microporous body of the present invention is conditioned such that it becomes suitable for transformation, and then the plant body is immersed in the carrier solution for transformation. The plant body is placed in a vacuum chamber while immersing in the carrier solution. The plant body is placed under a decompression condition for the predetermined period (for example, in the case where a subject plant is mouse-ear cress, it may be placed at approximately 400 mm Hg (approximately 50 kPa) for 4-12 minutes), and thereafter, the decompression condition is gradually released. After the treatment, the plant body is subjected to a suitable acclimatization condition to grow, and the transformant is selected on a medium containing an antibiotic according to the conventional method, and optionally a plant seed may be obtained from the transformant.
Transformation of Mouse-Ear Cress (Arabidopsis thaliana)
A cylindrical microporous body for early cultivation made from Murakami clay (collected in Niigata Prefecture, Japan) (outer diameter 14 mm, inner diameter 9 mm, height 45 mm) was immersed in the first aqueous fertilizer (0.1% of Hyponex), and a mouse-ear cress seed was placed on an upper end surface of a wet microporous body.
After four weeks cultivation under continuous lumination at 3000 lux at 23° C., a grown plant body was repotted to a cylindrical microporous body for expanded cultivation made from the same material (outer diameter 14 mm, inner diameter 9 mm, height 85 mm) immersed in the second aqueous fertilizer (NaH2PO4.2H2O 1.5 mM, Na2HPO4.12H2O 0.25 mM, MgSO4.7H2O 1.5 mM, Ca(NO3)2.4H2O 2 mM, KNO3 3 mM, Na2.EDTA 67 μM, FeSO4.7H2O 8.6 μM, MnSO4.4H2O 10.3 μM, H3BO3 30 μM, ZnSO4.7H2O 1.0 μM, CuSO4.5H2O 1.0 μM, (NH4)6.Mo7O24.4H2O 0.024 μM, CoCl2.6H2O 0.13 μM) such that a root of the plant body was contacted with a wet surface of the microporous body and, thereafter, cultivated under a continuous rumination at 3000 lux at 23° C. for four weeks again.
On the other hand, Agrobacterium tumefacience EHA105 harboring a binary vector pBI121 was incubated in a LB medium containing 10 mg/l of kanamycin at 28° C. for 48 hours. After incubation, a culture was centrifuged, and the resulting bacterial pellet was resuspended in an infiltration medium (IM) of the volume twice of an initial medium (IM=½ Gamborg's B-5 medium containing 0.1% Hyponex 5-10-5, 1% sucrose, 0.02% Silwet L-77 and 0.044 μM 6-benzylaminopurine).
The mouse-ear cress plant bodies grown on ten microporous bodies as described above were inverted together, and then immersed in 300 ml of IM medium containing Agrobacterium which was placed in 500 ml volume beaker. Such the mouse-ear cress plant bodies immersed in the beaker were subjected to decompression in a vacuum chamber for 5 minutes. Infiltrated plant bodies were grown for six weeks under the condition as described above, and approximately 10000 seeds were harvested therefrom.
Among the harvested seeds, approximately 5000 seeds were placed on a selective medium (½ MS medium containing 100 mg/l kanamycin), and transformed plant bodies which had survived even after 2 weeks were selected.
As the result, transformed plant bodies which correspond to 0.14%, 0.10% and 0.12% of seeds were obtained (triplicate results). On the other hand, it is reported that, in a mouse-ear cress experiment of soil cultivation, 70 transformed plant bodies were obtained from 60000 seeds (0.12% transformation efficiency, Bechtold et al., cited above). In light of this, it is found that the plant cultivated on the microporous body of the present invention has a similar transformation efficiency to that of a plant cultivated in the soil.
However, as described above, a larger number of plant bodies can be cultivated in a limited area such as in an artificial weather conditioner by the microporous body of the present invention than in a conventional soil, because an area necessary for cultivating one plant body on the microporous body of the present invention can be reduced than in the conventional soil cultivation (in the case of mouse-ear cress; 54 plant bodies/30×40 cm in the soil cultivation, 63 plant bodies/46×2 cm in the microporous body cultivation). Therefore, an effect greater than an actual transformation efficiency is exhibited because a transformation treatment can be conducted to a larger number of plant bodies.
According to the present invention, there can be provided an equipment for transforming plants, a system for transforming plants, and a method for transforming plants, which can be utilized in more exact, convenient, speedy and efficient experiment, investigation and development, and which can supply a healthy and high quality plant in a qualitatively and quantitatively stable manner.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP03/04681 | 4/14/2003 | WO |
