WO2001023595A2 - Reduced gravity transformation process and product - Google Patents

Abstract

A transformation process is carried out in which the competence of a plant or animal cell is increased and/or induced by placing the cell in reduced gravity, and then the cell is transformed in reduced gravity. A transformation process is carried out in which a monocot plant cell is transformed in reduced gravity. In a process of increasing the frequency of transformation of cells, the cells are contacted with a chemical agent effective to increase the frequency of transformation of the cells, and then the cells are transformed in reduced gravity. In a process of providing an increased amount of transgenic gene product produced, a cell is provided having a gene which has been transformed into the cell by a transformation process. Then, gene product is produced from the gene in reduced gravity. A product is provided relating to a plant or animal cell having DNA transferred into the cell in reduced gravity. A cell has a chemical pathway enhanced by a gene product produced in reduced gravity from a gene transferred into the cell in either reduced gravity or atmospheric gravity. The transferred gene produces an increased amount of product or an altered product compared to the same gene product produced in atmospheric gravity.

Description

REDUCED GRAVITY TRANSFORMATION PROCESS AND PRODUCT

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of copending provisional applications

Serial No. 60/156,891, filed September 30, 1999, and Serial No. 60/159,278, filed October 13, 1999.

FIELD OF INVENTION This invention relates to a process of transforming cells under conditions of reduced gravity such as microgravity. In particular, the invention relates to enhancement of a transformation process that leads to increased production of transgenic plants and to increased accumulation of transgenic gene product by performing the process under conditions of reduced gravity.

BACKGROUND OF THE INVENTION The development of single gene transfer techniques for plant species is of great interest and value to plant breeders because it can be used for the rapid transfer of genes to plants. Numerous methods have been developed for transferring genes into plant tissues: Agrobacterium-mediated transfer (Murai et al., 1983; Fraley et al., 1983), direct DNA uptake (Pazkowski et al., 1984; Potrykus et al., 1985), microinjection (Crossway et al., 1986), high-velocity microprojectiles (Klein et al., 1987) and electroporation (Fromm et al., 1985; Fromm et al., 1986). A general problem with most of these gene transfer techniques is that the transformed tissues, either leaf pieces or cellular protoplast, must be subjected to some regeneration steps that are often time-consuming and tedious before a whole transformed plant can be obtained. Furthermore, tissue culture procedures, specifically those involving cell differentiation, can result in mobilization of transposable elements, rearrangement of the inserted DNA, or somatic mutations that cause the loss or alteration of genes or gene product accumulated by the expertise of many years of plant breeding.

Agrobacterium-mediated gene transfers are by far the most widely used gene transfer techniques and have been shown as well to transform monocotyledons, the grasses in particular, contrary to previous scientific opinion. Transformed seeds can be recovered following Agrobacteria infection using a variety of different techniques. These include wounding in the vicinity of apical shoot meristematic cells, axillary and adventitious shoot meristematic cells, scutella derived from immature embryos, microspores and protoplasts. By inoculating the seedling in one of these areas, transformed gametes are recovered. Following fertilization using either transformed eggs or pollen, transformed seed is recovered. This technique of Agrobacterium-mediated gene transfer can be accomplished without the need of any tissue culture intermediate steps. Additional confirmation of the transformation of mesocotyl cells of germinating seeds resulted when G4l8-τesistant Arabidopsis thaliana plants were produced by co-cultivating germinating seeds with

Agrobacterium containing a binary plasmid with a plant expressible neomycin phosphotransferase (NPT) II gene in its T-DNA region.

The development of gene transfer techniques for monocots and dicots is of commercial interest because it facilitates the development of new cultivars containing value-added traits and allows transfer of genes from wild species, for example, genes of pharmaceutical interest. Unfortunately, even though some dicotyledonous species such as soybean are susceptible to Agrobacterium infections, its use for transformation is limited due to low efficiency.

The development of a simple method for the transfer, stable integration, expression, and transmission of genetic material is of great interest and importance. U.S. Patent Nos. 5,169,770 and 5,376,543 to Chee et al. disclose an Agrobacterium-mediated process for transforming plant seeds and the use of the process for producing transformed plants, particularly dicotyledonous plants. There is no suggestion that the transformation process could be improved by performing the process in reduced gravity. In particular, the patents disclose the Agrobacterium-mediated transfer of the selectable, recombinant NPT II marker. There is no suggestion that expression levels of this gene would be enhanced in reduced gravity. The patents disclose injecting the Agrobacterium into the meristematic or mesocotyl cells. There is no suggestion to inoculate in reduced gravity with a phenolic compound such as acetosyringone to generate a chemotaxic gradient and to induce the expression of the vir region of the Ti plasmid thus mobilizing the transfer of T-DNA. U.S. Patent No. 6,020,539 to Goldman and Graves discloses an Agrobacterium-mediated process for transforming Gramineae. There is no suggestion to perform the process in reduced gravity. U.S. Patent No. 5,550,318 to Adams et al. discloses various methods for transforming monocots. Again, there is no suggestion to perform the methods in reduced gravity.

Tennessen, in American Institute of Physics Conference Proceedings, vol. 387(1), pp. 1019-1024 (1997), states that performing a gene transfer process in microgravity may increase suspension time of T-DNA in the nucleus, and thereby improve the probability of T-DNA recombination with the genomic DNA. However, the Tennessen paper makes no suggestion that microgravity can improve the competence of plant cells (the ability of the cells to receive T-DNA). Further, the paper relates solely to dicot plants, not monocot plants. U.S. Patent No. 4,954,442 to Gelvin et al. discloses using acetosyringone to aid in the induction of Agrobacterium virulence genes in a process for transforming plant cells. The acetosyringone is applied to the plant cells along with the Agrobacterium; there is no suggestion to pre-inject the plant cells with acetosyringone before exposing the cells to the Agrobacterium. Also, there is no suggestion to direct the Agrobacterium exposure to a specific site, such as the germ line cells. Further, there is no suggestion to perform the process in reduced gravity.

Rayl, in The Scientist, vol. 13(18), pp. 1, 8-10 (1999), reports that changes in the production of vitamin D₃ and erythropoietin occur in human cell cultures under conditions of microgravity. These changes do not occur following cell transformation by heterologous genes, but rather by modulations in genome-wide expression patterns.

Walther et al., in FEBS Lett., vol. 436, pp. 115-118 (1998), show that simulated microgravity conditions inhibit the genetic expression of interleukin-2 (IL-2) and its receptor (IL-2R) in activated T lymphocytes. The inhibition is related to changes in protein expression levels. There is no suggestion that the observed changes in IL-2 and IL-2R protein expression levels have any relation to the presence of or changes in DNA transformation events.

U.S. Patent No. 5,882,928 to Moses discloses the use of an improved in vitro fertilization method wherein immature mammalian oocytes are obtained very early in the mammal's menstrual cycle, they are cultured and matured, and then the oocytes are fertilized in vitro. There is no suggestion of culturing and maturing the oocytes in reduced gravity. There is also no suggestion of performing the in vitro fertilization process in reduced gravity. U.S. Patent No. 5,849,713 to Eisenbach discloses the use of an added sperm chemotactic factor, purifiable from human follicular fluid, either about 13 kDa or less than 1.3 kDa in molecular size and peptidic and hydrophilic in nature, in the process of fertilizing an ovum in vitro, wherein the sperm and ovum are incubated in a solution with the added chemotactic factor. There is no suggestion to incubate the sperm and ovum with the chemotactic factor in reduced gravity.

In addition, it has been shown that the use of Surfacten, a bovine pulmonary surfactant that is a commercially available naturally occurring phospholipid, improves the maturation rate of bovine embryos in vitro when added during the fertilization process, making the coculture medium approach the conditions found naturally in the bovine oviducts at or near the time of ovulation. There is no suggestion that this improved maturation of bovine embryos could be improved by treatment of the embryos under reduced gravity conditions.

Transformation experiments on E. coli bacteria in microgravity have been performed by Ciferri et al., Naturwissenschaften, vol. 73, pp. 418-421 (1986). Transformation processes of bacteria are very different from transformation of plants and animals.

Baumann et al., in Proceedings of the Fourth European Symposium on Life Sciences Research in Space, ESA SP 307 (1990), found a higher rate of protoplast fusion in microgravity versus atmospheric gravity. Protoplast fusion involves fusion of DNA.

SUMMARY OF THE INVENTION The present invention provides a transformation process in which the competence of a plant or animal cell is induced and/or increased by placing the cell in reduced gravity. Then, the cell is transformed in reduced gravity. The transformation in reduced gravity results in a significantly increased frequency of transformation compared to the same process performed in atmospheric gravity. Another embodiment of the invention relates to a transformation process in which a monocot plant cell is transformed in reduced gravity.

Another embodiment of the invention provides a process of increasing the frequency of transformation of cells by contacting the cells with a chemical agent effective to increase the frequency of transformation of the cells, and then transforming the cells in reduced gravity. In a preferred embodiment, the transformation in reduced gravity is performed in the presence of the chemical agent.

In another embodiment, the invention relates to a process of providing an increased amount of transgenic gene product by providing a cell having a gene which has been transferred into the cell by a transformation process, and then producing gene product from the gene in reduced gravity. The amount of gene product produced in reduced gravity is increased compared to the same gene product produced by the gene in atmospheric gravity.

In a further embodiment, the invention provides a product relating to a plant or animal cell having DNA transferred into the cell in reduced gravity. Another embodiment of the invention relates to a cell having a chemical pathway enhanced by a gene product produced in reduced gravity from a gene transferred into the cell in either reduced gravity or atmospheric gravity. The gene product produced in reduced gravity is provided in an increased amount, or is altered, compared to the same gene product produced in atmospheric gravity.

Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic plan view of soybeans which have been subjected to a reduced gravity transformation process in accordance with the invention, as described in Example I below. The soybeans are exposed to ultraviolet light at wavelength 365nm. Fig. 2 is a schematic plan view of control soybeans which have been subjected to a transformation process on Earth as described in Example II below.

The control soybeans are shown exposed to ultraviolet light.

Fig. 3 is a schematic plan view of soybeans which have been subjected to another reduced gravity transformation process in accordance with the invention, as described in Example III below. The soybeans are shown exposed to ultraviolet light.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION The transformation process of this invention is an improvement of all transformation techniques. This includes transformation of monocotyledonous plants and cells, dicotyledonous plants and cells, and animal cells. The transformation is performed under conditions of reduced gravity, and in a preferred embodiment with the use of a chemical agent in reduced gravity, to increase the frequency of transformation of the cells. This transformation process is also an improvement because heterologous, recombinant gene delivery results in an increase in the production of a gene product.

"Transformation" is used herein to mean genetic modification by the incorporation of exogenous DNA into plant or animal cells. "Exogenous DNA" is DNA not naturally found in the cell which is to be transformed. Exogenous DNA includes natural sequences from other sources and organisms, synthetic sequences including those with and without modified nucleotides, and DNA sequences that have not been genetically modified. The transformation process is applicable to any kind of cell that can receive transferred DNA, including both plant cells and animal cells. In a preferred Agrobacterium-mediated transformation process according to the invention, the process is applicable to any competent plant cell that can receive a gene covalently linked between the T-DNA border sequences that reside in Agrobacterium strains that are transfer competent (vir+). Some preferred plants for use in the invention are dicotyledons such as leguminous plants and other large seed dicots, e.g., peanuts, soybeans, common beans, squash, zucchini, peppers, melons, cucumbers and others. Some particularly preferred plants are dicotyledons of the family leguminoseae, such as Glycine max (the soybean) and Phaseolus vulgaris (the common bean). Other preferred plants for use in the invention are monocotyledons, such as plants of the family Gramineae and other grasses.

According to the present invention, the plant cells are usually subjected to a preparation step prior to the transformation process. When the cells are plant seed cells, this usually involves initiating germination of the seed, explants like shoot meristems, microspores and or protoplasts. Germination can be initiated by imbibing the plant seed or by other means. The seed is exposed to hormones or sufficient moisture for a sufficient time and at a suitable temperature, to initiate germination of the seed. The preparation of the cells can be performed in either reduced gravity or atmospheric gravity. Prior to the transformation, certain cells can be targeted for transformation while other cells are left untargeted. For example, as described below, certain cells can be targeted by contacting the cells with a chemical agent effective to improve the frequency of transformation. In a plant seed, such cells may include those in an area of rapidly dividing cells that give rise to germ line cells. For instance, in a soybean seed the cells of the apical shoot meristem and mesocotyl regions may be targeted because they are responsible for ovule and pollen formation. Other cells which may be targeted are any cells that are competent to take up DNA. Competence may be a natural condition of certain cells, as with meristem or mesocotyl cells relative to Agrobacterium-mediated transformation. Competence may also be created as a result of environmental forces. For example, cells may be made competent by treatment with polyethylene glycol followed by Ca⁺² ion; by subjecting cells to electroporation; or, in the present instance, by subjecting cells to reduced gravity conditions. The transformation process of the invention can be used to transform either targeted or untargeted cells.

For a dicot such as a soybean plant, transformation may be carried out according to two major methodologies: organogenesis (shoot morphogenesis) and or embryogenesis. Organogenesis involves transformation of the shoot apical meristem region of the seed. The cells are first inoculated with Agrobacterium, for example, then co-cultivated with the bacterium to allow DNA transformation to occur. Regeneration of a mature plant from the transformed shoot meristem region is then carried out, and the mature plant is transferred to soil to allow production of transformed seeds. These transformed seeds can then be planted to produce future generations of transgenic plants, all containing the desired trait provided by the DNA incorporated into the plant's genetic material during transformation. Embryogenesis involves mature and immature embryos, protoplasts, microspores, or shoot meristem cells. The immature embryos are cultured to form a callus from which cell suspensions are initiated, or if desired meristem cells are cultured and callus formation is induced. Protoplasts from the cell suspension or callus are then transformed, by Agrobacterium for example, and then a mature plant is regenerated from a single transformed cell or from a mass of proembryogenic cells initiated during callus formation. Once transformed and regenerated, this method also allows for production of future generations of transgenic plants. Embryogenesis can also be done from microspores which are then subjected to Agrobacterium-mediated transformation, for example, followed by regeneration of a mature plant and then production of subsequent future generation transgenic plants.

For monocots, similar methodology choices for transformation exist. The organogenesis method is virtually the same for monocots such as maize as it is for dicots such as soybeans. In particular, Agrobacterium-mediated transformation of monocots also leads to successful transformation as with dicots, provided competent monocot cells are used. Embryogenesis with monocots varies slightly from that with dicots. Again, the method is applicable starting from immature embryos, microspores, or shoot meristem cells. The only real difference arises with the use of immature embryos. The immature monocot embryos are again cultured to promote callus formation, as with dicots, but the type II formed from monocots is used for transformation studies. From this callus, a cell suspension is made, and then transformation is undertaken, for example by using Agrobacterium, and a mature plant is again regenerated from a single transformed cell or a mass of proembryonic cells initiated during callus formation.

Whether organogenesis or embryogenesis is the methodology of choice, for both monocots and dicots, the DNA delivery method for the actual DNA transformation step may vary greatly. DNA may be introduced into the desired cell by Agrobacterium infection, electroporation, high velocity microprojectile bombardment, liposomes, or other methods. All methods are applicable to the transformation of dicots or monocots in reduced gravity.

In a preferred animal cell transformation process, the transformation is in vitro fertilization between a male and female sex cell. Preferred animal sex cells include human egg and sperm, and bovine egg and sperm, although any animal species whose sex cells are capable of being successfully fertilized in vitro could be used. U.S. Patent No. 5,882,928 to Moses describes a process for transformation (in vitro fertilization) of human sex cells. The transformation of the cell is performed in reduced gravity conditions.

For purposes of this invention, "reduced gravity" means any gravity less than about 0.9 G, preferably less than about 0.5 G, and more preferably less than about 0.1 G. Most preferably, the transformation process is conducted in conditions of microgravity, which means, for purposes of the invention, a gravity less than about 0.001 G. In contrast, "atmospheric gravity" or "positive gravity" means the gravity normally experienced on Earth (i.e., 1 G). Any suitable means for achieving reduced gravity or microgravity conditions can be used for performing the transformation process. In one preferred embodiment, the transformation process is performed under reduced gravity or microgravity conditions in space, e.g., aboard the Space Shuttle, the Space Station, a sounding rocket, or a satellite. In another preferred embodiment, the transformation process is performed under reduced gravity conditions simulated on Earth using a machine or other device suitable for this purpose.

It has been found that by placing the cells in reduced gravity prior to the transformation process, the competence of the cells is significantly increased. "Competence" means the ability of the cells to receive and incorporate the exogenous DNA. It is believed that the state and architecture of the cell is changed in reduced gravity to increase its ability to receive and incorporate the DNA. As with the natural competence of meristem and mesocotyl cells to transformation using Agrobacterium, reduced gravity conditions induce and/or increase the competency of cells for effective transformation events.

Because of the increased competence of the cells, the transformation process performed on the cells in reduced gravity according to the invention results in a significantly increased frequency of transformation compared to the same process performed in atmospheric gravity. For example, it has been found that the frequency of transformation of soybean seed cells performed in microgravity is at least about ten times greater than the frequency of the same process performed in atmospheric gravity. Based on this experiment, one would expect a frequency of transformation at least about five times greater when performed in reduced gravity compared to atmospheric gravity.

This transformation process can be any type of process capable of transferring DNA into a cell. Numerous different types of transformation processes are well known to persons skilled in the art. Some examples include the use of bacterial vectors, the use of viral vectors (e.g., geminivirus vector), the use of microspores, microprojectiles, electroporation, microinjection, particle bombardment, and protoplast fusion.

Also, different tissues can be used in the transformation process. For example, soybean tissue culture methods can be used in the process. Vens et al., in Journal of Biotechnology, vol. 47, pp. 203-214 (1996), describe a semi-automatic cell culture device suitable for experiments under microgravity.

In a preferred embodiment, the cell is a plant cell, and the transformation process is an Agrobacterium-mediated transformation process. The Agrobacterium strain can be any type suitable for containing a transferable gene in a vector. A preferred Agrobacterium strain is Agrobacterium tumefaciens, but other strains such as Agrobacterium rhizogenes and Agrobacterium rubi can also be used.

Wild type virulent and/or auxotrophic strains of Agrobacterium tumefaciens are known to infect plants and to elicit a neoplastic response in these plants. The tumor-inducing agent in the bacterium is a plasmid that functions by transferring some of its DNA into its host plant's cells where it is integrated into the chromosomes of the host plant's cells. This plasmid is called the Ti plasmid, and the virulence of the various strains of Agrobacterium tumefaciens is determined in part by the vir region of the Ti plasmid which is responsible for mobilization and transfer of the T-DNA. The T-DNA section is delineated by two 23-base-pair repeats designated right border and left border, respectively. Any genetic information placed between these two border sequences may be mobilized and delivered to a susceptible host. At a minimum, the T-DNA includes the right border sequence. Once incorporated into a chromosome, the T-DNA genes behave like normal dominant plant genes. They are stably maintained, expressed and sexually transmitted by transformed plants, and they are inherited in normal Mendelian fashion. A gene foreign to the Agrobacterium tumefaciens and to the host plant which is inserted into the T-DNA by genetic manipulation will also be transferred to the host plant's cells by the Ti plasmid. Thus, the Ti plasmid can be used as a vector for the genetic engineering of plant cells. The plasmid can be a single plasmid or a binary system that has been adapted for transfer in either the cis- or trans- configuration.

The gene can be any exogenous gene. Preferably, the transformation process transfers a gene into the cell that can express a value-added molecule. The value-added molecule can be a protein or a non-protein. Examples of genes in plants that express value-added proteins include genes relating to insect resistance, herbicide resistance, enhanced nutritional quality, or ability to produce a useful substance such as a medicinal drug (e.g., an antibiotic, an antibody product such as a hepatitis B vaccine, or a drug or drug intermediate useful in the treatment of arthritis, heart disease, high blood pressure, or other conditions or diseases). For example, seeds having the transformed gene can be used to grow plants on Earth, and the useful substance extracted and isolated from the plants. Alternatively, the useful substance could be isolated from the seeds.

The reduced gravity plant transformation process of this invention is simple and rapid, and it avoids the use of any tissue culture techniques. Transformed plants can be directly obtained from the transformed plant cells.

In another aspect, the invention relates to a process of increasing the frequency of transformation of cells by first contacting the cells with a chemical agent effective to improve the frequency of transformation. In one embodiment, the chemical agent is an exogenous chemical which is exposed to the cells during the transformation process. Alternatively, the chemical agent can be produced by the cells, possibly as a result of a stimulus during the process. For example, plant cells may be wounded to induce the production of the chemical agent. For animal cells, the chemical agent may be isolated from follicular fluid. The chemical agent can be any chemical that improves the transformation process.

In a preferred plant cell transformation process according to the invention, the chemical agent is a phenolic compound as secreted by wounded plant cells or added as an exogenous agent to plant cells. Acetosyringone is an example of such a chemical agent. The acetosyringone serves two purposes: (1) exogenous application of acetosyringone to plant cells chemotaxically attracts the bacteria to the treated cells; and (2) acetosyringone activates the Ti plasmid's virulence (v/ ) gene that is involved in DNA transfer from the bacterium to the plant cell's chromosome. In an Agrobacterium-mediated transformation process, preferably the Agrobacterium strain is primed by exposing the strain to the chemical agent before exposing the plant cells to the Agrobacterium strain.

In a preferred in vitro fertilization-type animal cell transformation process according to the invention, the chemical agent preferably would work by chemotaxically and chemokinetically modulating the sperm towards the egg, and/or by improving the maturation rate of the fertilized embryo by making the coculture medium approach conditions found in the oviducts of the animal. Preferably, the chemical agent is a compound isolated from follicular fluid, such as the hydrophilic peptide factors isolated from human follicular fluid having a molecular size of about 13 kDa or less than 1.3 kDa (e.g., as disclosed in U.S. Patent No. 5,849,713 to Eisenbach). These factors improve the transformation process in two ways:

1) by chemotaxically inducing human sperm to swim towards the human egg; and

2) by acting chemokinetically to increase sperm motility.

Another preferred chemical agent for animal cell transformations is a surfactant which would improve in vitro fertilization by improving the maturation rate of the fertilized embryo. Surfacten, a commercially available and naturally occurring phospholipid, is an example of such a chemical agent. Surfacten would be added to the coculture medium in the presence of the sperm and egg during the in vitro fertilization process. Surfacten acts to improve the maturation of bovine embryos in vitro by creating coculture conditions which approach those in the bovine oviducts. For either plant or animal cells, the contact of the cells with the chemical agent can be performed either in reduced gravity or atmospheric gravity.

After the cells have been contacted with the chemical agent, the cells are transformed in reduced gravity. This aspect of the invention is applicable to any type of transformation process that may be performed in reduced gravity. The different types of transformation processes are well-known to persons skilled in the art. In one preferred embodiment of the invention, the chemical agent is used in a process in which the transformation is conducted on plant cells by use of a motile bacterium containing a transferable gene in a vector, such as the Agrobacterium- mediated process described above. By use of the chemical agent in a plant cell transformation process performed in reduced gravity, the frequency of transformation is significantly increased compared with the same process performed in atmospheric gravity. Examples of preferred plant cells are the cells of plant seeds, immature embryos, e.g., in an embryonic suspension culture, microspores and/or protoplasts. The plant cells can be cells of dicot plants (e.g., soybeans or common beans), or cells of monocot plants (e.g., corn or grasses). In another preferred embodiment of the invention, the chemical agent is used in a process in which the cells involved in transformation are male and female sex cells of an animal, and the transformation process is an in vitro fertilization process. For in vitro fertilization processes with animal cells, a chemical agent can also be added at the same time as fertilization of the ova, to improve embryo maturation. Examples of preferred animal cells are human egg and sperm cells, or bovine egg and sperm cells. In a further aspect, the invention relates to a process of providing an increased amount of transgenic gene product. A cell is provided having a gene which has been transferred into the cell by a transformation process. Any type of transformation process can be used for transferring the gene. Gene product is produced from the gene in reduced gravity. It has been found that the amount of transgenic gene product produced in plants in reduced gravity is increased compared to the same gene product produced in atmospheric gravity. The reason(s) for the increased amount of gene product are not known. However, possible mechanisms include: an increase in transcription (e.g., due to an increased binding of transcription factors to the DNA to be transcribed, or due to an increased rate of transcription in reduced gravity, possibly by easier and/or faster unwinding of the double-stranded DNA as it is "read"); an increase in RNA stability (possibly through faster/easier folding into more stable secondary and tertiary structures); an increase in RNA processing (such as could occur if processing enzymes were also expressed at an increased level, or bind and release their target RNAs faster and more efficiently in reduced gravity); more efficient translation (possibly by more efficient rRNA recognition of the mRNA sequence to be translated, or more efficient movement of the ribosome along the mRNA as it is translated, or possibly by faster and more efficient acylation of the correct amino acid to the corresponding tRNA, along with faster/more efficient recognition of the required tRNA molecule by its corresponding tRNA synthetase); and a change in post- translational modification and/or rate of protein turnover (i.e., either through an increase or decrease in rate of the modifications, or a change in the types of modifications carried out in the cell. For example, the active/inactive form of the enzyme may be stabilized/ destabilized by preferential phosphorylation in reduced gravity relative to the form that is normally phosphorylated in atmospheric gravity. Also, reduced gravity may affect the half-life and ubiquitine-mediated decay of a protein). Not only is an increased amount of transgenic gene product produced in plants in reduced gravity, but the gene product produced is complete and functional, i.e., it is made correctly.

Plant cells that have been subject to a transformation process in microgravity exhibit the following characteristics when compared with a control group transformed in atmospheric gravity: (1) increased frequency of transformation at target cells such as are found in the meristematic or mesocotyl regions; (2) enhanced accumulation of heterologous gene products in cells of the meristematic or mesocotyl regions; (3) increased frequency of transformation across the surface of exposed cotyledons that includes the cotyledonary node; and (4) enhanced accumulation of heterologous gene products in non-targeted cells. The transgenic gene product provided in an increased amount can be any type of gene product, such as a protein. In some embodiments, the gene product is operative (e.g., glycosylation/non-glycosylation co-factors), while in other embodiments, the gene product is non-operative (e.g., soybean seedcoat peroxidase).

In another aspect, the invention is a product relating to a cell having DNA transferred into the cell in reduced gravity. The product can be a gene product produced by the transferred DNA, such as a protein, a catalytic RNA, a tRNA, or an rRNA. Alternatively, the product can be a multicellular product including the cell having the transferred DNA, such as a plant seed, an organ or an organelle. The product of the invention can also be a product that is regenerated from the cell, such as a whole plant regenerated from a plant seed including one or more of the cells, and plant progeny derived from a regenerated plant. In yet another aspect, the invention relates to a cell having a chemical pathway enhanced by a gene transferred into the cell in reduced gravity. The transferred gene produces an increased amount of product or an altered product compared to the same gene transferred in atmospheric gravity. The increased product or altered product enhances the pathway of the cell. For example, the gene product is involved in a rate limiting step of the chemical pathway, and the amount of gene product is increased such that the step is no longer rate limiting, and the pathway can proceed at an increased rate. As another example, the transferred gene produces an altered product that allows a previously inactive chemical pathway to be turned on. In a particular example, the cell is a plant cell having a pathway for producing betaglucan, and the gene product is an isomerase that causes the plant cell to produce 1,3 -betaglucan instead of 1,6-betaglucan. Some non-limiting examples of gene products produced according to the invention include enzymes, lipids, isoflavones, flavones, terpenes, and terpenoids.

Example I Microgravity Soybean Transformation Experiment

A soybean transformation experiment was performed in both microgravity according to the invention (described in Example I) and atmospheric gravity as a control (described in Example II, below). On Earth, one thousand Glycine max (soybean) seeds, cv. Athow, were surface sterilized with 15% Clorox for 10 minutes, followed by 4-5 rinses with distilled water, and then placed on moistened germination paper in a temperature controlled oven at 26°C and allowed to imbibe for 24 hours. The seed coats were removed and the decoated seeds were divided into halves. The mesocotyl region of the imbibed seeds, with the plumule still attached, was injected with 20μM acetosyringone solution with a 29-gauge needle at three different points in the mesocotyl region. The seeds were wrapped in germination paper, 50 per roll, and sealed in a bag. The bag was placed into an Astroculture Box (ACB) designed to contain the experiment. Virulent Agrobacterium tumefaciens were grown in nutrient media to an optical density of 0.33A (600 ran) and placed into a separate sealed bag, which was placed into the ACB. The Agrobacterium tumefaciens contained a Ti plasmid that included an smRS-GFP marker gene covalently linked to the T-DNA, and a vir gene that mobilizes the transfer of the T-DNA. The smRS-GFP gene encodes the solubilized form of the red-shifted green fluorescent protein, thereby creating fluorescence in a cell into which it is transferred. (The Agrobacterium tumefaciens is available from Indiana Crop Improvement Assoc, Lafayette, IN.) A bacterial primer solution of 2 μM of acetosyringone dissolved in dimethylsulfoxide and water was added into a syringe and placed in the ACB. The ACB was sealed, placed in a SPACEHAB locker, and loaded into the space shuttle Discovery.

The transformation experiment was performed in the microgravity of space aboard the space shuttle, beginning four days after the seeds were injected with acetosyringone. The bacterial primer solution in the syringe was injected into the media containing the Agrobacterium tumefaciens, and incubated for 2 hours. This mixture was then injected into the bag containing the acetosyringone-injected soybeans. The seeds and bacterial solution were coincubated for 22 hours. During the coincubation, if transformation occurred, the bacteria transferred T-DNA to the plant cells. Transfer was mediated by the vir gene products encoded on the plasmid. Once the vir genes were activated, cleavage of the T-DNA commenced. Proteins involved with nuclear localization signals transported the T-DNA into the cell where it was integrated into the plant's chromosomal DNA. At this point the plant's cells were capable of the production of the heterogenous protein encoded by the transgenes. In one aspect of the experiment, by targeting the tissue responsible for ovule and pollen formation, the genes of interest could be carried to the next generation. After the coincubation of the seeds and bacterial solution, the solution was withdrawn from the bag.

The experiment was in orbit aboard the space shuttle for seven additional days. Within 10 hours of the space shuttle's landing on Earth, the locker containing the ACB was removed from the space shuttle and the seeds were removed from the ACB. The seeds were unwrapped and washed. Within 48 hours, the seeds were scored for frequency of transformation by utilizing the visual marker (the smRS- GFP marker gene). When successful transformation occurred in a cell, a red fluorescence could be visualized under 365-nm ultraviolet light in a darkened room, indicating that the smRS-GFP gene had been transferred into the cell. Between 8% and 9% of the seeds incubated in microgravity gave rise to seedlings that displayed visual red fluorescence appearing at the injection site and certain other areas throughout the seed. The fluorescence appeared to be concentrated at the injection site. These data demonstrate the successful ability to integrate transferred genes into plant cells in a microgravity environment.

Fig. 1 shows some examples of soybean seeds 10 which were subjected to the reduced gravity transformation process described in Example I. The soybeans are shown exposed to ultraviolet light. The areas of fluorescence 12 in the seeds 10 are indicated as dotted areas. As shown in the drawing, the soybean seeds include a cotyledon 14 having a mesocotyl region 16. The mesocotyl region was the target region of cells injected with the acetosyringone. One of the illustrated soybean seeds 10' displays no fluorescence, indicating that transformation did not occur. The other soybean seeds 10 display fluorescence in the mesocotyl region 16, indicating that transformation of cells occurred in this region. The soybean seeds also display fluorescence in a region 18 spaced from the mesocotyl region along the edge and/or at the end of the soybean seed. This indicates that the cells of the soybean seeds were transformed both in the target region 16 and in a non-target region 18 of the soybean. It is believed that the non-target region was injured during separation of the cotyledons, causing the soybean seed to produce endogenous acetosyringone in this region.

It should be noted that when the transformation process was successful in the soybean seeds, the seeds always displayed fluorescence at least in the targeted mesocotyl region. This indicates the effectiveness of the present method in targeting a particular region of cells for transformation. Another finding was that the fluorescent product of the smRS-GFP gene was produced in the transformed cells in significantly greater amounts than when the same process was performed in atmospheric gravity. The reduced gravity process resulted in significantly enhanced accumulation of the gene product in both targeted and non-targeted cells. This was easily observed because the amount of fluorescence in a cell is directly related to the amount of fluorescent protein produced by the gene. The soybeans seeds that were transformed in reduced gravity, as shown in Fig. 1, exhibited significantly more fluorescence than soybean seeds transformed on Earth, as described in Example II and shown in Fig. 2.

Example II Atmospheric Gravity Soybean Transformation Experiment

The control soybean transformation experiment was performed under atmospheric gravity conditions on Earth. The virulent Agrobacterium tumefaciens strain containing the plasmid with the smRS-GFP marker gene was used in the experiment. The experimental times and conditions were the same as in the microgravity experiment described in Example I. Five hundred Glycine max seeds, cv. Athow, were surface sterilized with 15% Clorox for 10 minutes, followed by 4-5 rinses with distilled water. The seeds were wrapped in germination paper, 50 per roll. The paper was moistened with water and placed in warm room at 26°C, and the seeds were allowed to imbibe for 24 hours. The seed coats were removed and the decoated seeds were divided into halves. The mesocotyl region of the imbibed seeds, with the plumule still attached, was injected with a 20μM acetosyringone solution at three different points in the mesocotyl region. The seeds were wrapped in germination paper, 50 per roll, and sealed in a bag. The germination paper was sealed within bags and kept at 26°C for four days. The virulent Agrobacterium tumefaciens was incubated with the bacterial primer solution for two hours, then the bacteria was coincubated with the seeds for 22 hours. The bacterial solution was withdrawn from the bag, and the seeds were incubated for an additional seven days. The seeds were inspected for visual fluorescence under 365-nm ultraviolet light. The data of the plant cells transformed in atmospheric gravity was compared with the microgravity transformed plant cells from Example I. The results showed that 0.8% of the Glycine max seedlings incubated in atmospheric gravity displayed visual fluorescence, compared with 8% to 9% for the process performed in microgravity. These data demonstrate that an increased frequency of transformation occurred in microgravity compared to atmospheric gravity.

Fig. 2 shows some examples of soybean seeds 20 which were subjected to the transformation process on Earth as described in Example II. A lower percentage of the soybean seeds showed fluorescence 22 in Example II compared to Example I, indicating that the frequency of transformation was less on Earth than in reduced gravity. In Fig. 2, one of the soybean seeds 20 shows fluorescence while the other of the soybean seeds 20' do not show fluorescence. It should be noted that in the soybean seed 20 showing fluorescence, the amount of fluorescence is significantly reduced compared to the fluorescence shown in the soybean seeds 10 in Fig. 1. This indicates that the amount of gene product produced in the transformed cells was less on Earth than in reduced gravity.

Example III

Microgravity Soybean Transformation - Second Experiment

A second soybean transformation experiment was performed in microgravity aboard the space shuttle, similar to the experiment described in Example I. The second experiment was very successful in transforming the soybean seeds. However, the seeds died during the days following their removal from the space shuttle after return to Earth, for reason(s) that are not completely understood. Some examples of soybean seeds 30 from the second microgravity experiment are shown in Fig. 3. Almost all areas of the soybean seeds showed significant fluorescence 32. By examining the soybean seeds under conventional lighting as well as ultraviolet light, it was determined that in most of the soybean seeds 30, the only areas that did not show fluorescence were areas 34 of dead tissue. Thus, except for the dead cells of the soybean seeds, almost all the cells of the seeds showed evidence of successful transformation. One of the soybeans seeds 30" in Fig. 3 has speckles or spots of fluorescence 32 at scattered locations all over the seed. This indicates that multiple successful transformations of individual cells occurred all over the seed. It also suggests that the fluorescence over almost all areas of the soybean seeds 30 was the result of multiple successful transformations of individual cells, rather than the result of protein produced by the transformed cells migrating to other cells. It is believed that the fluorescence of the soybean seed 30" would eventually cover almost the entire seed, similar to the other seeds 30, if the growth of the seed 30" had continued.

Hypothetical Example Simulated Microgravity Soybean Transformation Experiment

A soybean transformation experiment could be performed in simulated microgravity conditions using a Random Positioning Machine ("RPM"), also known as a three-dimensional clinostat, as described in Walther et. al, and first developed by Hoson at the Osaka City University. Such a device involves placement of the samples as close as possible to the center of a first frame, which is then rotated within a second frame. Each frame is driven by a separate motor and the rotation of each frame is controlled by computer software to generate random, autonomous rotation with respect to the other. Rotation velocity of the frames is typically about 60° per second. Such conditions easily and inexpensively simulate the conditions of microgravity, usually obtained only in space. Additional information about such a device is provided in Hoson, T. et. al., (1992) Bot. Mag., vol. 105, pp. 53-70; and Hoson, T., et. al., (1997), Planta, vol. 203, pp. S187-S197. In this simulated microgravity experiment, Glycine MAX (soybean) seeds, cv. Athow, would be surfaced sterilized with 15% Clorox for 10 minutes, followed by 4-5 rinses with distilled water, and then placed on moistened germination paper in a temperature controlled oven at 26°C and allowed to imbibe for 24 hours. The seed coats would be removed and the decoated seeds divided into halves. The mesocotyl region of the imbibed seeds, with the plumule still attached, would be injected with 20 μM acetosyringone solution, with a 29-guage needle at three different points in the mesocotyl region. The seeds would be wrapped in germination paper, 50 per roll, and sealed in a bag. The bag would be placed into an RPM or other suitable microgravity simulator. The bacteria would be primed by growing virulent Agrobacterium tumefaciens in nutrient media and then injecting this media with acetosyringone dissolved in dimethylsulfoxide, to obtain a final concentration of 2 μM acetosyringone in the bacterial solution. The bacteria would be incubated for 2 hours, also under simulated microgravity conditions using an RPM or similar device, and then injected into the bag containing the acetosyringone-injected soybeans. The soybean/bacterial solution would then be incubated for an additional 22 hours under simulated microgravity conditions. After 22 hours, the bacterial solution would be removed from the soybeans using a syringe, and the soybeans would then be subjected to simulated microgravity conditions for an additional 7-10 days, mimicking space-flight conditions. It is also possible to tailor the microgravity time to any desired length, since this method of simulated microgravity transformation is not limited by the time-table of the space shuttle flights. After subjection to simulated microgravity conditions, the seeds would be removed from the RPM or similar device and placed once again in atmospheric gravity conditions, and scored for efficiency of transformed tissue by observance of the presence or absence of fluorescence from the smRS-GFP marker gene present in the transforming DNA sequence. In this example, it would also be possible to return the transformed seeds to atmospheric gravity at a gradual rate, thus lessening the potentially negative impact of a rapid return to atmospheric gravity conditions on the transformed seeds. Hypothetical Example Reduced Gravity Transformation of Monocots

It is expected that reduced gravity will have a great effect on transformation efficiency in monocots, such as rice, wheat, sorghum, maize, barley and grasses.

For example, microspores of cereals could be used to transform with

Agrobacterium mediated transformation. Auxotrophic strains could be used to eliminate wash steps etc. performed under reduced gravity.

The principle and mode of operation of this invention have been described in its preferred embodiments. However, it should be noted that this invention may be practiced otherwise than as specifically illustrated and described without departing from its scope.

Claims

What is claimed is:

1. A transformation process comprising increasing and or inducing the competence of a plant or animal cell by placing the cell in reduced gravity, and then transforming the cell in reduced gravity.

2. A process according to claim 1, wherein the cell is a microspore.

3. A process according to claim 1, wherein the cell is contained in a shoot apex.

4. A process according to claim 1, wherein the cell is a protoplast.

5. A process according to claim 1, wherein the cell is derived from a callus or an embryogenic cell suspension.

6. A process according to claim 1, wherein the cell is an egg.

7. A process according to claim 1, wherein the cell is a plant cell.

8. A process according to claim 7, wherein the transformation process comprises exposing the plant cell to an Agrobacterium that contains a transferable gene in a vector.

9. A process according to claim 7, wherein the transformation process comprises exposing the plant cell to an Agrobacterium that contains a transferable gene in a vector.

10. A process according to claim 1, wherein the cell is transformed by a process relating to biolistics, electroporation, liposomes, or sonication.

11. A process according to claim 1, wherein the cell is targeted.

12. A process according to claim 11, wherein the targeted cell is any dividing cell of a plant seed.

13. A process according to claim 12, wherein the targeted cell is a meristematic or mesocotyl cell of a plant seed.

14. A process according to claim 1, wherein the transformation process performed in reduced gravity has a frequency of transformation at least about five times greater than the same process performed in atmospheric gravity.

15. A process according to claim 1, comprising the additional step of regenerating the cell in atmospheric gravity.

16. A process according to claim 1, comprising the additional step of regenerating a cultivar from the transformed cell or its descendent.

17. A process according to claim 16, wherein the transformation process transfers to the cultivar a gene that encodes a value-added molecule.

18. A process according to claim 17, wherein the value-added molecule is a protein.

19. A process according to claim 1, wherein the cell is an animal cell.

20. A process according to claim 19, wherein the transformation process is an in vitro fertilization process.

21. A transformation process comprising transforming a monocot plant cell in reduced gravity.

22. A process according to claim 21, wherein the cell is a microspore.

23. A process according to claim 21, wherein the cell is contained in a shoot apex.

24. A process according to claim 21, wherein the cell is a protoplast.

25. A process according to claim 21, wherein the cell is derived from a callus or an embryogenic cell suspension.

26. A process according to claim 21, wherein the cell is an egg.

27. A process according to claim 21, wherein the transformation process performed in reduced gravity has a frequency of transformation at least about five times greater than the same process performed in atmospheric gravity.

28. A process according to claim 21, wherein the transformation process comprises exposing the monocot plant cell to an Agrobacterium that contains a transferable gene in a vector.

29. A process according to claim 21, comprising the additional step of regenerating a cultivar from the transformed cell, and wherein the transformation process transfers to the cultivar a gene that encodes a value-added molecule.

30. A process of increasing the frequency of transformation of cells comprising:

(a) contacting the cells with a chemical agent effective to increase the frequency of transformation of the cells; and (b) in reduced gravity, transforming the cells.

31. A process according to claim 30, wherein the cells are microspores.

32. A process according to claim 30, wherein the cells are contained in a shoot apex.

33. A process according to claim 30, wherein the cells are protoplasts.

34. A process according to claim 30, wherein the cells are derived from a callus or an embryogenic cell suspension.

35. A process according to claim 30, wherein the cells are eggs.

36. A process according to claim 30 wherein the cells are transformed in the presence of the chemical agent.

37. A process according to claim 30, wherein the chemical agent is exogenous to the cells.

38. A process according to claim 30, wherein the chemical agent is produced by the cells.

39. A process according to claim 30, wherein the cells are plant cells.

40. A process according to claim 30, wherein the chemical agent is acetosyringone.

41. A process according to claim 30, wherein the plant cells are meristematic or mesocotyl cells of a plant seed.

42. A process according to claim 30, wherein the plant cells are plant embryonic cells.

43. A process according to claim 30, wherein the transformation is conducted by use of a bacterium containing a transferable gene in a vector, and wherein the chemical agent increases the frequency of transformation by chemotaxically attracting the bacterium or by activating a gene of the bacterium involved in the transformation.

44. A process according to claim 43, comprising the additional step of contacting the bacterium with the chemical agent before contacting the cells with the bacterium.

45. A process according to claim 30, wherein the cells are animal cells.

46. A process according to claim 45, wherein the chemical agent is selected from surface cell receptors, proteins, peptides and hormones.

47. A process according to claim 45, wherein the transformation process is an in vitro fertilization process.

48. A process according to claim 47, wherein the transformation process is conducted in the presence of a chemical agent which increases the frequency of transformation by improving maturation of the in vitro ova and/or fertilized embryo.

49. A process according to claim 47, wherein the transformation process is conducted in the presence of a chemical agent which increases the frequency of transformation by either chemotaxically attracting sperm to an egg, or chemokinetically increasing the swim speed of sperm towards the egg.

50. A process according to claim 49, wherein the chemical agent is isolated from human follicular fluid, and is a hydrophilic peptidic factor having a molecular size of about 13 kDa or less than 1.3 kDa.

51. A process of providing an increased amount of transgenic gene product comprising:

(a) providing a cell having a gene which has been transferred into the cell by a transformation process; and (b) in reduced gravity, producing gene product from the gene, the amount of gene product produced in reduced gravity being increased compared to the same gene product produced by the gene in atmospheric gravity.

52. A process according to claim 51, wherein the cell is a microspore.

53. A process according to claim 51, wherein the cell is contained in a shoot apex.

54. A process according to claim 51, wherein the cell is a protoplast.

55. A process according to claim 51, wherein the cell is derived from a callus or an embryogenic cell suspension.

56. A process according to claim 51, wherein the cell is an egg.

57. A process according to claim 51, wherein the gene product is a protein.

58. A process according to claim 51, wherein the gene product is functional.

59. A process according to claim 51, wherein the increase in gene product is caused at least in part by an increase in transcription.

60. A process according to claim 51, wherein the increase in gene product is caused by an increase in mRNA stability.

61. A process according to claim 51, wherein the increase in gene product is caused at least in part by an increase in RNA processing.

62. A process according to claim 51, wherein the increase in gene product is caused at least in part by more efficient translation.

63. A process according to claim 51, wherein the increase in gene product is caused at least in part by a change in post-translational modification.

64. A process according to claim 51, wherein the increase in gene product is caused at least in part by a change in the rate of protein turnover.

65. A gene product produced by the process of claim 51.

66. A product relating to a plant or animal cell having DNA transferred into the cell in reduced gravity.

67. A process according to claim 66, wherein the cell is a microspore.

68. A process according to claim 66, wherein the cell is contained in a shoot apex.

69. A process according to claim 66, wherein the cell is a protoplast.

70. A process according to claim 66, wherein the cell is derived from a callus or an embryogenic cell suspension.

71. A process according to claim 66, wherein the cell is an egg.

72. A product according to claim 66, wherein the product is a protein produced by the DNA.

73. A product according to claim 66, wherein the product is a catalytic RNA produced by the DNA.

74. A product according to claim 66, wherein the product is an anti-sense

RNA produced by the DNA.

75. A product according to claim 66, wherein the product is an anti-gene DNA produced by the DNA.

76. A product according to claim 66, wherein the product is a tRNA produced by the DNA.

77. A product according to claim 66, wherein the product is an rRNA produced by the DNA.

78. A product according to claim 66, wherein the product is an organ or an organelle.

79. A product according to claim 66, wherein the product is a plant seed.

80. A product according to claim 66, wherein the product is a plant.

81. A product according to claim 66, wherein the product is a plant progeny.

82. A cell having a chemical pathway enhanced by a gene product produced in reduced gravity from a gene transferred into the cell in either reduced gravity or atmospheric gravity, the gene product produced in reduced gravity being provided in an increased amount, or being altered, compared to the same gene product produced in atmospheric gravity, the increased gene product or altered gene product enhancing the pathway of the cell.

83. A cell according to claim 82, wherein the cell is a microspore.

84. A process according to claim 82, wherein the cell is contained in a shoot apex.

85. A process according to claim 82, wherein the cell is a protoplast.

86. A process according to claim 82, wherein the cell is derived from a callus or an embryogenic cell suspension.

87. A process according to claim 82, wherein the cell is an egg.

88. A cell according to claim 82, wherein the gene is transferred into the cell in reduced gravity.

89. A cell according to claim 82, wherein the cell is a plant cell having a pathway for producing betaglucan, and wherein the gene product is an isomerase that causes the plant cell to produce 1,3 -betaglucan instead of 1,6-betaglucan.

90. A cell according to claim 82, wherein the gene produces an increased amount of product to enable the pathway to proceed at an increased rate.

91. A cell according to claim 82, wherein the gene produces an altered product that enables the pathway to be turned on.

92. A cell according to claim 82, wherein the gene product is selected from the group consisting of enzymes, lipids, isoflavones, flavones, terpenes, and terpenoids.