METHODS FOR EFFECTUATING MRNA-MEDIATED TRANSFER OF GENETIC INFORMATION INTO PLANTS AND PRODUCTS OF THE SAME

Abstract: Methods are disclosed for the beneficial genetic transformation of plants based on transfer of genetic information mediated by mRNA rather than DNA. Transgenic plants expressing proteins encoded by the mRNA are also disclosed.

Description: METHODS FOR EFFECTUATING mRNA-MEDIATED TRANSFER OF GENETIC INFORMATION INTO PLANTS AND PRODUCTS OF THE SAME Field of the Invention This invention relates to the fields of molecular biology and genetic engineering. More specifically, the invention provides methods of introducing foreign genetic material (mRNA) into plants. Incorporation by Reference Various scholarly articles and scientific publications are referenced in this patent application to describe the state of the art to which this invention pertains. Each of these publications is incorporated by reference herein in its entirety. Background of the Invention Methods of improving various crops are known. Some of these methods involve introducing foreign, i.e. nonendogenous, DNA into plant cells or tissues and analyzing the resulting plants for presence of exogenous nucleic acid and expression of the foreign gene. Although successful in certain instances, these methods are laborious, time consuming and variable in their reproducibility. There exists a need for the further development of methods for the beneficial genetic transformation of agricultural crops to increase protein yields or otherwise improve crops for human and/or animal consumption. Summary of the Invention In one of its aspects, this invention provides methods for producing transgenic plants by introduction therein of foreign mRNA, to impart to the resulting transgenic plants capability of synthesizing the foreign protein in subsequent generations. Based on the transfer of genetic information via mRNA molecules, as opposed to DNA molecules, in one of its aspects the invention provides a distinct improvement over methods presently employed in the art. Endogenous proteins of plants are beneficially augmented with valuable proteins from foreign sources utilizing the methods of this invention. In another of its aspects, this invention embraces a transgenic plant expressing beneficial exogenous proteins produced by obtaining a sample of mRNA encoding the exogenous protein, incubating and inoculating seed of the plant with the mRNA under conditions whereby the mRNA enters the seed, germinating the seed and growing the transgenic plant from the seed. Desirably the sample of mRNA is obtained from soybeans, most desirably from soybean cotyledons or sprouts. Alternatively, the sample of mRNA is obtained by transcribing an isolated DNA molecule that encodes the protein, such as a DNA molecule encoding a medically useful protein, preferably a human protein) such as γ-interferon, interleukin 1, interleukin 2 and human growth hormone. Further desirably, the plant is corn and most desirably corn strain 27-1 or 85089. In another of its aspects, this invention embraces a transgenic plant, preferably corn, expressing soy globulin protein, human interleukin 1, human interleukin 2, or human γ-interferon. Most desirably in this aspect of the invention the corn is strain 27-1; alternatively the corn may be strain 85089. In an exemplary practice of the invention, mRNA from soy cotyledon is isolated and purified and used for micro injection of com seed. The treated seeds are then germinated and resulting plants assayed for exogenous protein expression. Following microinjection and transformation, protein presence and content in propagated transgenic com plants is analyzed by protein detection techniques. The presence of soy DNA in the transformed com is confirmed by Southern blotting using soy globulin specific radiolabeled DNA probes. In alternative exemplary embodiments of the invention, mRNA may be isolated from soy sprouts. Additionally, other strains of com may be utilized, including com strain 85089. The mRNA may be microiηjected into recipient seeds once or twice or even more times. The methods described result in successful production of transgenic com plants expressing soy globulin protein. Other features and advantages of the invention will be understood from the detailed description and examples that follow. Detailed Description of the Preferred Embodiments and Best Mode Known for Practicing the Invention The following definitions are provided to facilitate understanding of the invention: mRNA: Messenger Ribonucleic Acid is a temporary complementary copy of the sense strand of protein coding DNA. In eukaryotes, this major intermediate of gene expression is transcribed from protein coding genes by RNA polymerase π. It is usually transcribed as a relatively long pre-mRNA which is then processed, still within the nucleus. Further post-transcriptional modifications are made to most eukaryotic mRNA's to add a 5' cap structure and a 3' poly A tail. Transgenic Plants: Plants which have been engineered via recombinant nucleic acid techniques to express exogenous proteins encoded by other plants or animals. Southern Hybridization Analysis: A technique developed by Edward Southern in 1975 in which denatured DNA is transferred from agarose gels in which fragments have been separated by electrophoresis to a nitrocellulose or a nylon membrane laid over the gel, before hybridization with a complementary nucleic acid probe. This step is required as hybridization to the gel itself is very inefficient. The technique is ubiquitous in molecular genetics and its numerous applications usually revolve around the identification of a particular DNA sequence within a mixture of restriction fragments, for example to determine the presence, position, and number of copies of a gene in the genome. It is also an integral technique used in DNA typing. Western Blotting: A method for detecting one or more specific proteins in a complex protein mixture such as a cell extract, which is named by analogy with Southern hybridization to detect DNA sequences and Northern blotting to detect RNA's. The procedure involves fractionating the protein mixture by denaturing SDS- PAGE gel electrophoresis and transferring and immobilizing the mixture onto a solid membrane of either nitrocellulose or nylon by electro-blotting. The loaded membrane is then incubated with an antibody raised against the protein of interest. The antibody-antigen complex so formed on the membrane can then be detected by a procedure which involves the application of a secondary antibody, raised against the first antibody, and to which an enzyme has been covalently linked. The insoluble reaction product generated by enzyme action can then be used to indicate the position of the target protein on the membrane. Ouchterlony Double Agar Immunodiffusion Assay: An assay in which antigen and antibody are placed in wells cut in an agar gel which then diffuse towards one another and precipitate to form an opaque line in the region where they meet in optimal proportions. A preparation containing several antigens often gives rise to multiple lines. The immunological relationship between two antigens can then be assessed by setting up precipitation reactions in adjacent wells: The lines formed by each antigen may be completely confluent, indicating immunological identity. They may show a spur, as in the case of partially related antigens, or they may cross, indicative of unrelated antigens. Methods of the invention entail isolation and purification of mRNA from a selected source. Following isolation, mRNA is micro injected separately into plant seeds (e.g., com kernels), which are thereafter planted and allowed to grow to maturity. Following transformation, protein content in the resulting generations of plants is analyzed. The presence of the foreign protein in the treated plant is demonstrated via immunological and biochemical methods. Experimental data, which is set forth in detail hereinbelow, show that micro injection into com kernels of mRNA from plants or animals results in the generation of transgenic com plants which synthesize the protein encoded by the selected mRNA. Without intending to be limited by any particular explanation of this phenomenon, it appears that the mRNA is reverse transcribed and incorporated into the com genome. Thus, for example, soy mRNA-produced globulin was expressed in succeeding generations of com. The presence of soy globulin nucleic acid sequences was demonstrated by Southern blotting of the transformed com DNA with complementary 32 P-labeled soy globulin probes. The method of the invention has the following steps, resulting in the production of transgenic com in which the transgene is stably inherited by subsequent generations: 1. Isolate or otherwise obtain the desired mRNA. 2. Obtain seeds of the plant species desired to be genetically modified. 3. Prepare the seeds by imbibing them with water. 4. Microinject the mRNA into the seeds. 5. Plant the seeds and allow them to grow to maturity. 6. Screen the grown plants and select those that contain the protein encoded by the desired mRNA. For the first step, isolating or otherwise obtaining the desired mRNA, any mRNA for which expression in plants is desired is suitable for use in the present invention. Exemplified herein are two broad categories of mRNA: (1) mRNA encoding major structural proteins of plants, e.g., soy globulins; and (2) mRNA encoding proteins having useful biological activity; e.g., human growth hormone (HGH) and human γ-interferon. Other mRNAs that have been introduced into plants by the methods of the invention include the following: into com variety 85079: soy globulin, human growth hormone, human interleukins 1, β and 2, human γ-interferon; into com variety 27-1: soy globulin; into com variety 340: human interleukin 2; into American sweet com: soy globulin and human γ-interferon; into rice: soy globulin into wheat: soy globulin and (tentatively) chicken myosin. Other mRNAs contemplated for use in the present invention include, but are not limited to, human albumin, human urokinase plasminogen activator, hiruden (anticoagulant from leech) and human thymosin-4. In sum, the invention contemplates use of any plant or animal mRNA. mRNA is isolated according to standard methods, e.g., as disclosed by Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory (1989) or Ausubel et al. (eds) Current Protocols in Molecular Biology. John Wiley & Sons (2001). In a preferred embodiment wherein the desired mRNA is a major structural or storage protein (e.g., soy globulin), a suitable quantity of the mRNA may be obtained by isolating a total mRNA component from a tissue that expresses the protein, e.g., soy cotyledons or sprouts. In an embodiment wherein the desired mRNA is not a major component, the mRNA may be produced by in vitro transcription of an isolated DNA molecule that encodes the protein, e.g., γ-interferon- encoding DNA from human cells. In vitro transcription systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland. Next, seeds of plants for which genetic modification is desired are obtained. These may be seeds of any plant species, without limitation. Preferably, they are seeds of agronomically important crops and are amenable to microinjection techniques. In a preferred embodiment, they are seeds from com or wheat. In another embodiment, they are seeds from rice. In other embodiments, they may be from other agriculturally important plants such as soybean, barley, alfalfa, sorghum and rye, or they may be seeds of vegetable or fruit crops, including but not limited to tomato, carrot, cucumber, pea, broccoli, cauliflower, cabbage, spinach, and the like. In co , which is a highly preferred plant for use in the invention, any variety is suitable. Particularly suitable are varieties 27-1 and 85089. Seeds are soaked in water for a suitable time, e.g., 2-3 days for com, such that they are fully imbibed and amenable to microinjection. Microinjection is accomplished by standard techniques, through the use of commercially available micro injection systems. In com, the microinjection needle is placed as close to the central axis of the seed as possible. A suitable amount of mRNA to be injected into com is 1 μg per μl distilled water. The seed may be injected more than once before it is planted, and it may be injected with more than one specific mRNA. Following injection, the seeds are planted and grown, and suitable parts of the grown plants are analyzed for the presence of the protein encoded by the injected mRNA. Such analyses are well known to those of skill in the art. They range from protein isolation (e.g., actually purifying the foreign protein and subjecting it to amino acid composition or sequencing analysis) or detection techniques (e.g., immunological techniques such as Western blotting, immunopreciptiation or Ouchterlony diffusion) to mRNA or DNA detection (e.g., dot blots, northern blots, Southern blots using specific DNA probes). The following examples are provided to illustrate a preferred practice of the invention. They are not intended to limit the invention in any way. EXAMPLE 1 Stable Transformation of Com with mRNA Encoding Soybean Globulin Genetic transformation of plants based on transfer of information encoded by mRNA, rather than DNA, has been demonstrated in com. mRNA was extracted from the cotyledon and sprout of the soybean and both mRNAs were separately introduced into com kernels which were then planted. Materials and Methods Isolation of mRNA: The cotyledon and/or sprouts (shoot and root axis) of the germinating soybean were used for the extraction of mRNA according to published procedures (Niu, M. C, L. C. Niu, C. Ma, Z. P. Lin and Y. L. Zhang (1980) Scientia Sinica, 23: 119-122.). Eighty percent of the poly A mRNA in soy encodes soy globulin protein (1 IS and 7S). Microinjection of Com Seeds: Com seeds from strains 27-1 and 85089 were treated separately and analyzed as follows: 200 com seeds, i.e. kernels as a batch were washed twice using tap water. They were then soaked in double distilled water at 4NC for 48 to 72 hours. During this time the water was changed 2 or 3 times. The imbibed seeds were then microinjected with soy mRNA, (isolated from either soy sprout or soy cotyledon) using a commercially available micro injector which is a graduated glass tube of 100 microliter capacity with a stainless steel needle attached. The needle was inserted into the seed as close to the central axis as possible. The amount injected was 1 microgram of soy mRNA in 1 microliter of double distilled water. Over 90% of the injected seeds germinated and grew into com plants. Protein Extraction from the Com Kernels: Groups of six kernels were soaked overnight in double distilled water at 4°C. After removing the outer skin they were dehydrated by acetone soakings, then de- fatted by ether soakings and then ground into a fine powder. Protein was extracted with 3 ml of extraction fluid (10% glycerine, 5% 2-mercaptoethanol, 2.3% SDS, 8 M/L urea, 0.02% of bromophenol blue in 0.625 M/L Tris-HCl, pH 6.8). The sediment was removed by centrifugation (4000 m for 10 minutes.). The supernatant was boiled 3 mins. and centrifuged (5000 φm for 5 minutes.). The supernatant was then stored at -20°C for future analysis. SDS-PAGE: Using the discontinuous SDS-PAGE procedure (Laemmli, U. K. (1970) Nature, 227: 681-685), the extracted protein was separated into a series of bands and stained with Coomassie brilliant blue R250. Ouchterlony Double Agar Immunodiffusion Test: The plate used for the test was made of 1.2% agar. Rabbit anti-soy protein serum was used to detect the presence or absence of soy protein in the mRNA-treated kernels and untreated control samples. Detection of Soy Protein by Western Blotting Technique: The additional band obtained from the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) test was transferred to a nitrocellulose membrane (Vaessen, R. T. M. J., J. Kreke and G. S. P. Groot (1981) FEBS letter 124: 193-196). The membrane was washed with the following buffer: (0.02 M/L Tris buffered saline (TBS), pH 7.4, and 0.05% Tween 20 (TTBS)) and sealed in a dark chamber containing 4% Bovine se m albumin (BSA) in TTBS at 30NC for two hours. After washing with TTBS fluid the soy protein was detected by the ProtoBlot Western Blot AP System according to Promega's technical manual. Detection of Soy DNA by Molecular Southern Hybridization: DNA was extracted from the soy mRNA treated and control com samples. The DNAs were electrophoresed on gels and transferred to a nitrocellulose membrane. Commercial reverse transcriptase was used to catalyze the transcription of soy mRNA into cDNA (Szkukalek, A., and F. Solymosy (1994) Plant Science 99:183-187). This cDNA was nick translated into 32P-labelled cDNA. Southern blotting analysis was carried out on the treated and control com DNAs with 32P-labeled cDNA probes complementary to soy mRNA. Appearance of hybridization bands demonstrates presence of the soy globulin DNA sequences in the treated com. In control samples, the hybridization band is absent. A typical gel would comprise a lane for a soy DNA control; for DNA isolated from uninjected, control com samples; and for DNA isolated from soy mRNA treated com from different fields. Results Protein extraction, followed by biochemical analyses revealed that these com kernels express soy globulin. Additionally, the presence of soy niRNA-induced soy globulin protein was demonstrated in subsequent generations of com by Western blotting. DNA isolated from com treated with soy globulin mRNA was subjected to Southern hybridization analysis with 32P-labeled cDNA probes specific for soy globulin RNA. This revealed presence of soy globulin nucleotide sequences in the treated com. These results demonstrate the feasibility of creating valuable transgenic plants via introduction of mRNAs encoding agronomically important proteins. Genetic transformation of economically important plant species is highly desirable. To date, strategy for gene transfer in plants often involves introduction of foreign DNA into protoplasts or callus to enable its introduction into the nuclear genome. However, regeneration of certain plant species from protoplasts so transformed has been difficult to achieve. This example describes the transforming capacity of soy globulin mRNA in com. The results achieved indicate that delivery of genetic information by mRNA molecules is useful for the stable transformation of monocot species. The method of the invention using mRNA molecules represents a more reliable means of introducing new genes into plants than that provided by the transfer of genetic information using heterologous DNA. Furthermore, mRNA induced genetic changes are stable, whereas genetic changes induced by DNA are not. Two strains of high yield com, 85089 and 27-1, were utilized for transformation in these experiments: Total RNA was isolated from powdered soybeans, either cotyledon or sprouts, by the cold phenol extraction procedure and purified by oligo DT chromatography. The mRNA thus isolated was adjusted to 1 micro gram per microliter; 1 microliter of the resulting solution was injected separately into kernels of imbibed com. The injected seeds from the two different strains were sown in rows separated by a passageway, in a garden measuring 10 x 12 meters. The planting of strain 85089 was done in late April and strain 27-1 in late May. The difference in planting dates was done to avoid cross-hybridization. Uninjected com kernels (control) were sown in a field several miles away. Over 90% of the uninjected and soy mRNA-injected seeds germinated and grew into plants. All were harvested in October. The methods of the present invention were performed using com strains, 27-1 and 85089. mRNA used for microinjection was prepared from at least two sources in the soy plant, e.g. the cotyledons and the sprouts. Microinjected com seeds were planted and the com kernels of the first generation were harvested. SDS-PAGE of isolated com protein from the first generation revealed an additional band at approximately 67 KD. Ouchterlony double agar immunodiffusion assays using rabbit anti-soy protein semm demonstrated that the additional band was of soy origin and due to the presence of soy protein globulin. The kernels of the first generation were then planted in order to see if the soy protein globulin was transmitted to the next generation. Extracts from the kernels of the first generation of com were analyzed for detection of soy protein as follows: SDS-PAGE: The com kernels of one ear of com constituted one sample. A total of 134 samples were analyzed from the mRNA- treated group and 10 from the control. Of the 134 samples in the mRNA-treated group, 29 exhibited an additional band, and the balance did not. An additional band was observed at 67 KD in the treated com extracts. No new band was observed in control extracts isolated from untreated com. The data obtained from SDS-PAGE analysis of 134 samples of mRNA treated com are summarized in Table 1. When soy cotyledon mRNA is microinjected once into com strains 85089 and 27-1, 18.75% of the 85089 com plants demonstrate an additional band, whereas 23.68% of the 27-1 com plants demonstrate an additional band. Hence, strain 27-1 is more efficiently transformed than strain 85089. This is further supported by data obtained when each strain, 27-1 and 85089, is microinjected twice with mRNA isolated from soy cotyledon. In these experiments, 26.47% of the 27-1 com plants exhibited an additional band, as compared to 20% of the 85089 com plants, as set forth in Table 1. Soy sprout mRNA was microinjected once or twice into com strain 85089. The data indicate that a second injection of soy sprout mRNA into com strain 85089 did not result in an increase in the number of plants exhibiting an additional band, as shown in Table 1 below. Comparing results obtained following microinjection of com strain 85089 with mRNA from the cotyledon or the sprout shows that mRNA isolated from soy cotyledon is more efficient in generating transgenic com plants than mRNA isolated from the soy sprout, as set forth in Table 1, below: The Ouchterlony Double Agar Diffusion Test: Based on the results from the SDS-PAGE, com kemel-extracted protein with and without the additional band, and from the control kernels, were subjected to the double agar diffusion test with rabbit anti- soy protein serum. All of the com protein with the additional band reacted to the anti- serum as shown by the precipitation line, but varied in intensity. See Table 2, below: No precipitation line was found in the control, nor in the com proteins obtained from samples not expressing the additional band. Extracts from the kernels of the second generation of com were analyzed as 5 follows: The Western Blotting Technique: The kernels of the first generation were planted and harvested as the second generation. The kernel extracts from this second generation were subjected to the Western blotting technique. The results were that two bands appeared between 60-70 KD. No band was observed in the control. The demonstration of soy protein in the kernels of the second generation of com illustrates 10 that the soy mRNA transferred genetic information into the com genome, such that soy globulin protein is expressed in the next generation. Southern Blotting: Finding the soy protein in the second and succeeding generations of com indicates that the mRNA encoded genetic information has been reverse transcribed and incoφorated into the com genome. This is presumably due to 15 the presence of reverse transcriptase in the com seed. Southern blotting demonstrated presence of soy globulin encoding DNA in the transformed com. The presence of the hybridizing band provides evidence of reverse transcription of the injected soy mRNA. No soy derived nucleic acid sequences were detected in DNA obtained from untreated com. To summarize, identification of soy protein in the com was carried out in three steps. The first method employed was SDS-PAGE. An additional band from the com extract grown from the mRNA-treated kernels was observed. This band was absent in extracts isolated from untreated com. Second, the extract of kernels exhibiting the additional band were analyzed by an Ouchterlony double agar immunodiffusion test using rabbit anti-soy protein semm. A precipitation line was found between the anti-serum and the extract from the kernels exhibiting an additional band by SDS-PAGE (Table 2). No line appeared between the anti-serum and the control extract. Third, in order to see if the soy protein globulin was expressed in the next generation of com, the kernels of the first generation were planted again. The com grown from these kernels was harvested and the protein extracts analyzed by Western blotting. The results of this experiment showed the appearance of two bands believed to represent the two globulins of the soy protein, 7S and 1 IS (1). Finally, the presence of soy globulin encoding nucleic acids in mRNA treated com was demonstrated by Southern hybridization assays using 32P-labeled soy specific probes. EXAMPLE 2 Identification of Human Growth Hormone and Human Gamma-Interferon in Transgenic Com Kernels The method used for the identification of foreign protein in the transgenic com described in this example is the procedure of Yu and Niu published in the book The Role of RNA in Development and Reproduction, pp. 893-903, 1981 (incoφorated by reference) (editors: Niu and Chuang, China Press and distributed by Nostrand and Reinhard Co., New York, Cincinnati, Toronto, London and Melbourne). mRNA encoding Human growth hormone (HGH) and γ-interferon protein were obtained. These were injected into com kernels according to the methods set forth in Example 1. The injected com kernels were then planted and allowed to grow (referred to herein as the "first generation"). Kernels from the first generation were themselves planted and allowed to grow into mature plants ("second generation"). For the first generation, groups of six kernels were extracted by the same procedure used for the extraction of soy globulin from soy mRNA-treated com, as described in Example 1. For the second generation, groups of six kernels from each of six cobs (one cob from each of six com plants) were used. All extracts were centrifuged. The aqueous layers were subjected to SDS-PAGE analysis. SDS-PAGE analysis revealed that kernels produced by plants treated with HGH mRNA demonstrated an extra band, presumably the HGH, as compared with untreated plants. SDS-PAGE analysis further revealed that kernels produced by plants treated with γ-interferon demonstrated an extra band at approximately equivalent molecular weight as that observed for the HGH-treated plants. It is believed that the locations of both protein bands are close because the molecular weight of HGH and γ-interferon are close. The extra bands from each gel plate were cut out and eluted from the gel. To each eluate from both the γ-interferon gel plate and the HGH gel plate was added the rabbit antisemm against γ-interferon or the rabbit antiserum against HGH, respectively. There were two resulting precipitates: one from the γ-interferon gel plate and the other from the HGH gel plate. As controls, purified γ-interferon and purified HGH, respectively, were immunoprecipitated with the same respective rabbit antisera. The various immunoprecipitates are referred to as follows: Extra band from γ-interferon mRNA treated kernels: g-AB + g-protein Immunoprecipitate with purified γ-interferon: g-AB + g-AG Extra band from HGH mRNA treated kernels HGH-AB + HGH- protein Immunoprecipitate with purified HGH: HGH-AB + HGH- AG Both of the precipitates from the g-AB + g-AG and from the g-AB + g-protein (which was eluted from the extra band) were subjected to SDS-PAGE. It was seen that both precipitates dissociated to two bands. Those bands, which lined up horizontally close to the level of the 17 kDa molecular weight marker, are proteins of similar molecular weight (about 15-17 kDa). These bands were eluted. One eluate is the purified protein and the other is the γ-interferon from the com kernels. Yu and Niu in 1981 analyzed the composition of the protein digested by trypsin with ion exchange chromatography. They found that peptide maps of the two proteins (experimental and control) are similar in chemical composition. Instead, here I analyzed the amino acid composition of the two proteins (purified antigen and the extracted γ-interferon from kernels) and found that the respective amino acid compositions were practically the same. Both precipitates from HGH-AB + HGH-AG and from HGH-AB + HGH- protein were processed in the same way as above. The amino acid compositions of the two proteins (the antigen and the extracted HGH) were found to be practically the same. While the preferred embodiments of this invention have been described and specifically exemplified above, the invention is not limited to such embodiments. Modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims and equivalents thereto.

Claims: I claim: 1. A method for producing transgenic plants which express an exogenous protein, comprising: a) obtaining a sample of mRNA encoding said exogenous protein; b) incubating seed of said plant with said mRNA under conditions whereby said mRNA enters said seed; c) germinating said seed; and d) growing said transgenic plant from said seed. 2. The method of claim 1, wherein said exogenous protein is soy globulin. 3. The method of claim 1, wherein said exogenous protein is selected from the group consisting of human growth hormone, human γ-interferon, human interleukin 1, human interleukin β and human interleukin 2. 4. The method of claim 1, wherein said seed is com seed. 5. The method of claim 1, wherein said exogenous protein is detected with methods selected from the group consisting of Western blotting, double agar immunodiffusion, and Sodium dodecyl sulfate polyacrylamide gel electrophoresis. 6. The method of claim 1, wherein said mRNA is introduced into said seeds by microinjection. 7. The method of claim 2, wherein said mRNA is isolated from soy cotyledons or sprouts. 8. The method of claim 3, wherein said mRNA is produced by transcription of an isolated DNA molecule. 9. A transgenic plant expressing exogenous proteins, produced by a method comprising: a) obtaining a sample of mRNA encoding said exogenous protein; b) incubating seed of said plant with said mRNA under conditions whereby said mRNA enters said seed; c) germinating said seed; and d) growing said transgenic plant from said seed. 10. A transgenic plant as claimed in claim 9, wherein said exogenous protein is soy globulin. 11. A transgenic plant as claimed in claim 9, wherein said exogenous protein is selected from the group consisting of human growth hormone, human γ-interferon, human interleukin 1, human interleukin β and human interleukin 2.. 12. A transgenic plant as claimed in claim 9, wherein said seed is com seed. 13. Seeds obtained from the transgenic plant of claim 9. 14. The seeds of claim 13, which are com kernels. 15. A method of producing transgenic com plants expressing soy globulin, comprising: a) obtaining soy globulin encoding mRNA; b) incubating com seed with said mRNA under conditions whereby said mRNA enters said com seed; c) germinating said com seed treated as in step b; d) growing a plant from said germinated seed; and e) detecting said soy globulin in said transgenic com plant. 16. The method of claim 15, wherein the com is variety 27-1 or variety 85089. 17. A transgenic com plant produced by the method of claim 14. 18. A transgenic com plant expressing soy globulin protein. 19. The plant of claim 18 in which the plant is com variety 27-1 or 85089. 20. Transgenic com kernels expressing soy globulin protein.

Inventors: NIU MAN CHIANG, NIU, MAN, CHIANG
Applicants: MULTIGENESIS CORP, NIU MAN CHIANG, MULTIGENIS CORPORATION, NIU, MAN, CHIANG
Application year: 2001
Main classification: A01H5
IPC codes: C12N15