PROCESS FOR THE GENETIC MODIFICATION OF CEREALS WITH TRANSFORMATION VECTORS

Abstract: Novel transformation vectors may be used for the genetic modification of plant cells, plant tissue or plant organs. These transformation vectors are double-stranded DNA molecules which include a gene foreign to said plant cell, plant tissue or plant organ and associated with a selectable or identifiable trait when present therein and at least one DNA sequence substantially identical to a DNA sequence which is present in said plant cell, plant tissue or plant organ and capable or homologous recombination with said plant DNA sequence when the transformation vector is introduced into the plant cell, plant tissue or plant organ. Double-stranded DNA molecules which additionally include a gene associated with expression of a desired agronomic property or production of a desired product in plant cells, plant tissues, plant organs or in plants derived therefrom can be used to create plants having desirable properties or to produce commercially valuable products. These transformation vectors are especially useful for modifying the genetic make-up of cereals, including barley, wheat, rice and maize.

Description: PROCESS FOR THE GENETIC MODIFICATION OF CEREALS WITH TRANSFORMATION VECTORS FIELD OF THE INVENTION This invention relates to the use of genetic engineering techniques in the modification of plants, particularly cereals, such as wheat, rice, barley and maize. More particularly, it concerns the construction and use of transformation vectors which include at least one gene associated with the expression of a desired agronomic property or the production of a desired product, at least one gene associated with a selectable or identi- fiable plant trait and a DNA sequence derived from the chromosomal DNA of a plant. BACKGROUND OF THE INVENTION Considerable progress has been made in the application of genetic engineering techniques to produce commercially valuable products. In particular, natural and synthetic genes associated with the production of polypeptides such as insulin, somatostatin, and -thymosin have been cloned in bacteria and in yeast. In addition, there have been reports in the literature of successful cloning of foreign plant genes in bacteria and yeast and of successful transformation of eucaryotic cells " ith non- naturally occurring transformation vectors. However, there are no reports of successful transformation of photosynthetic plant cells with non-naturally occurring transformation vectors. Given the major importance of plants both for direct consumption and for the production of valuable products, it would be highly desirable to extend the techniques employed in microbial genetics to plant cells. However, there are major problems in doing so. These include: the lack of suitable transformation vectors; the difficulty of regenerating plants, particularly cereals such as wheat and maize, from cultured cells; and the fact that genetically modified plant cells must express foreign genetic information at specific developmental stages if they are to be useful. To overcome these difficulties, novel transformation vectors have been designed and constructed which have widespread application in the genetic modification of plant cells. More particularly, plant transformation experiments have been reported in which the transforming DNA was derived from the same species as the recipient plant into which it was introduced. Korohoda, J. and Strzalka, K., Z. Pflanzenphysiol. Bd. 94_:95-99 (1979); Soyfer, V.N. , et al., Environmental and Experimental Botany 18:105-111 (1978); Soyfer, V.N., et al., Molekulyarnaya Biologiya 12:637-645 (1978); Soyfer, V.N. , et al., Mutation Research _3£ : 303-310 (1976); Turbin, N.V. , et al.. Mutation Research r7 J 59-68 (1975); Leber, B. and Hemleben, V., Z. Pflanzenphysiol. Bd. 91:305-316 (1979); Soyfer, V.N., et al. , Theor. Appl. Genet. 58:225- 235 (1980); and Ondrej , M. , et al., Biol. Plant 21:127- 135 (1979) . DNA preparations reported in these exper¬ iments contained only DNA from the recipient species, and as such, were devoid of foreign genes. Moreover, these DNA preparations had no specific sequence or size homogeneity. Other plant transformation studies have been reported in which transforming DNA consisted only of non-homogeneous sequence foreign DNA devoid of DNA sequences substantially iden-tical to those of the recipient species. Johnson, C.B., et al., Nature New Biology 244:105-106 (1973); Bendich, A. and Filner, P., Mutation Research 13:199- 214 (1971); and Hess D. , Z. Pflanzenphysiol. Bd. 9_3:429- ft a - Q A Although the above-published reports suggest, and in some cases claim, transformation of plants, there does not presently exist any definitive physical confirmatio of the presence of transforming DNA molecules in the 5 cells, tissues or organs of recipient plants or their progeny. Moreover, refutations of specific reports maintain that claims for transformation have been based- upon misinterpretation and artifacts. Kleinhofs, A., et al., PNAS 21:2748-2752 (1975) and Behki, R.M. and 10 Lesley, S.M., In Vitro 15:851-856 (1979) . At present, no reports of plant transformation have involved the use of vectors consisting of specific combinations of foreign DNA and DNA sequences sub- 15. stantially identical to the DNA of the recipient species SUMMARY OF THE INVENTION Transformation vectors useful in the genetic modificatio 0 of photosynthetic plant cells, plant tissues and plant organs have been prepared. They are double-stranded DNA molecules which include a DNA sequence corresponding to a gene foreign to the plant cell, tissue or organ to be transformed and associated with a selectable or 5 identifiable trait when present therein and at least one DNA sequence substantially identical to a DNA sequen located in the plant cell, plant tissue or plant organ and capable of homologous recombination with the DNA present in the plant cell, plant tissue or plant organ 0 when the transformation vector is introduced therein. Preferably, the transformation vector also includes a DNA sequence containing a gene associated with expres¬ sion of a desired agronomic property or production of a 5 desired product. Examples of such genes include those associated with production of proteins possessing improv nutritional quality such as the zein genes of maize, the hordein genes of barley and the gliadin genes of wheat and those associated with resistance to environ¬ mental stress such as drought resistance, salt tolerance and herbicide resistance. This invention also concerns methods of preparing the transformation vectors, methods of introducing desired genes into plant cells, tissues or organs and the re- suiting plant cells, tissues or organs and plants derived therefrom. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a transformation vector which includes a foreign gene associated with expression of a selectable or identifiable plant trait and a DNA sequence substan¬ tially identical to a portion of a plant's DNA. Fig. 2 illustrates a transformation vector which includes a foreign gene. associated with expression of a desired property or production of a desired product and a DNA sequence substantially identical to a portion of a plant's DNA. Fig. 3 illustrates a transformation vector " as in Fig. 1 which additionally includes a DNA sequence associated with expression of a desired agronomic property or production of a desired product. Fig. 4 illustrates a transformation vector which includes a foreign gene associated with expression of a selectable or identifiable plant trait, a foreign DNA sequence associated with expression of a desired agronomic property or production of a desired product, and two DNA sequences each substantially identical to portions of a plant's DNA. The two DNA sequences are located at opposite ends of both the DNA sequence associated with the desire property or product and the DNA sequence containing the foreign gene associated with a selectable or identifiabl trait. Fig. 5 illustrates a transformation vector which include a foreign gene for a picillin resistance flanked at both ends by plant DNA expressed in wheat _in vitro. It also includes a DNA sequence containing codons for increased lysine levels flanked at both ends by wheat storage protein genes. Finally, it includes a sequence associated with replication and selection in E^ coli. Fig. 6 illustrates a transformation vector derived from the E^ coli plasmid pBR322. In this transformation vector, the selectable or identifiable trait is ampi¬ cillin resistance. The flanking DNA sequences are substantially identical to portions of maize chromosomal DNA expressed when the maize is in the germinating embryo state. The final DNA sequence is derived from a chicken and is associated with production of ovalbumin. DETAILED DESCRIPTION OF THE INVENTION Before proceeding, definitions of various terms used herein are set forth. TRANSFORMATION VECTOR - a double-stranded DNA molecule which is capable of mediating the introduction of genes into cells and includes at least one selectable or identifiable trait. PLANT - any photosynthetic member of the kingdom Planta which is characterized by a membrane-bound nucleus, genetic material organized into chromosomes, membrane- bound cytoplasmic organelles, and the ability to undergo meiosis. -6- PLANT CELL - the structural and physiological unit of plants, consisting of a protoplast and cell wall. PLANT TISSUE - a group of plant cells organized into a structural and functional unit. PLANT ORGAN - a distinct and visibly differentiated part of a plant such as root, stem, leaf or Gmb-yro-r ≤τu SELECTABLE OR IDENTIFIABLE TRAIT - a feature or quality encoded by one or more genes- present in a plant cell, plant tissue or plant organ which distinguishes the cell, tissue or organ from others lacking the feature or quality. PLANT CHROMOSOME - a DNA molecule present ' in the nucleus of a plant cell which is associated with proteins and RNA and undergoes reductive division during meiosis. HOMOLOGOUS RECOMBINATION - the exchange of nucleic acid sequences between two distinct DNA molecules based upon the interaction of substantially identical base sequences. CEREALS - organisms in the super ingdom - eucaryotes, kingdom - planta, subkingdom - embryophyta, phylum - tracheophyta, superclass - angiospermae, class - monocotyledonae, order - gramineae, including maize (corn) , wheat, rice and barley. SUBSTANTIALLY IDENTICAL DNA SEQUENCES - two DNA sequences which are sufficiently similar in nucleic acid base composition and sequence to facilitate homologous recom¬ bination with one another including different mutational forms of a gene or alleles. REPETITTVE SEQUENCES - DNA ' sequences which are present in more than 10 copies per haploid genome. PLASMIDS OR VIRUSES - extrachromosomal double-stranded DNA molecules capable of autonomous replication. ENVIRONMENTAL STRESS - any condition which results in altered metabolic activity, reduced growth, damage to cell, tissue or organ when imposed upon living cells and which is capable of causing cell death. Examples of environmental stresses are heat, drought, saline soils and pathogens. * INJECTION - the process of introducing molecules into cells, tissues and organs by positive pressure through a syringe. Macroinjection is accomplished without the aid of a microscope; microinjection is accomplished with the aid of a microscope. TRANSFORMATION - the process of introducing at least one gene into a cell which results in stable maintenance of the introduced gene. CO-TRANSFORMATION - the process of introducing at least two genes located on different molecules into the same cell which results in the stable maintenance of the introduced genes. FOREIGN GENE - a sequence of DNA encoding a specific product, products, or biological function which is obtained from a different species than that species into which the gene is introduced by a transformation vector. FOREIGN DNA - a sequence of DNA which is obtained from a different species than that species into which it is introduced on a transformation vector. Transformation vectors useful for the genetic modificatio of photosynthetic plant cells, plant tissues and plant organs may be prepared. They are double-stranded DNA molecules which include a gene foreign to the plant cell, tissue or organ to be altered and which are associated with the expression of a selectable or identi¬ fiable trait when the transformation vector is present in the plant cell, tissue or organ or in a plant derived therefrom. They also include at least one DNA sequence which is substantially identical to a portion of the DNA present in the plant cell, tissue or organ and which is capable of homologous recombination with the DNA present in the plant cell, tissue or organ after introduction of the transformation vector therein. Foreign DNA sequences which include a gene associated with a selectable or identifiable trait are available from a wide variety of sources including bacterial plasmids, plant viruses and plants. One group of such genes are those associated with resistance to antibiotics which are capable of inhibiting the growth of cereal cell cultures when present in the growth medium. These genes are generally Obtained from bacterial plasmids or bacterial transposons. Examples include genes for a picillin resistance which may be obtained from pBR322 or any Tn3-containing DNA sequence, genes for kanamycin or G418 resistance which can be obtained from Tn5 or any Tn5-containing DNA sequence and genes for chloramphenicol resistance. Another group of suitable foreign genes are those involved in the utilization of various substrates by plant cell cultures or the prevention of substrate inhibition. These genes may be obtained from organisms capable or incapable of utilizing a particular substrate. Examples include genes for galactose utilization, e.g. the gene for the enzyme UDP-4-galactose epi erase, which can be obtained from E_^ coli capable of utilizing galactose as a sole carbon source or from an E_^ coli F-prime plas id containing genes for the galactose utilization pathway. Other examples are genes for lactose utilizaton, including the gene for β-galactosidase and genes involved in preventing amino acid inhibition of plant cell culture growth. Examples of the latter are genes for asparto- kinase, ho oserine dehydrogenase and dihydropicolinic acid synthetase. In general, substrate utilization * genes are obtained by subcloning DNA fragments into * E * . coli and selecting for growth on an appropriate medium. In addition to the aforementioned foreign genes, genes associated with traits such as herbicide resistance, disease resistance or salt tolerance in one plant species may be employed as genes for selectable or identifiable traits in another. A transformation vector employing this principle is illustrated in Fig. 2. Various methods for preparing or obtaining foreign selectable or identifiable genes are known to those skilled '* in the art and may be employed in the practice of this invention, and it is not contemplated that the invention be limited to any one such method. DNA sequences substantially identical to DNA sequences present in plant cells, tissues or organs are obtainable by various methods including the following: (1) total plant DNA is cut by restriction endonucleases, ligated to plasmid DNA, and cloned in E^ coli. Plasmids con¬ taining plant DNA inserts are chosen following selection for insertional inactivation of an antibiotic resistance gene, size screening by agarose gel electrophoresis and DNA-DNA hybridization; (2) plant DNA is sheared, de¬ natured, allowed to partially reassociate and repeated sequences are separated from unique sequences by hydroxy- lapatite chromatography. The repeated sequence fraction is then cloned in E^ coli and plasmids are chosen as described in method (1) ; (3) DNA sequences containing genes expressed during specific developmental stages or in specific tissues are obtained by isolating messenger. RNA from the chosen stage or tissue, synthesizing DNA complementary to the messenger RNA and cloning the resulting DNA in Ξ^ coli. Plasmids chosen as described in method (1) are subjected to characterization by hybrid-selected ^n vitro translation of IΓLRNA or by frequency of occurrence of the sequence in the total given messenger RNA pool; and (4) sequences are obtained as described in method (3) followed by identification using DNA-DNA hybridization of the native gene sequence in a genomic DNA library prepared in Charon phages. Native genes isolated from gene libraries are charact¬ erized by restriction mapping and heteroduplex analysis to determine the number and location of intervening sequences. Structural gene sequences are then isolated by subcloning in E^ coli for use in vector construction. Transformation vectors of the type shown in Fig. 1 contain a foreign gene for a selectable or identifiable trait and a plant-derived DNA sequence substantially identical to DNA present in a plant cell, tissue or organ to be transformed. They are obtained by ligating the two sequences together or by ligating them to the same plasmid and cloning in Ξ^ coli using methods well known to those skilled in the art. Recombinant plasmids are identified by selection for insertional inactivation of resistance genes, colony hybridization, screening for insert size, selection for biological function of the foreign gene or any combination of these methods. Depending upon the method used, the resulting transformation vector may be linear or cir¬ cular. Although the plant-derived DNA portion of the trans¬ formation vector may vary widely, it is preferred that this DNA sequence be expressed during a specific devel-. opmental stage of the life cycle of the plant cell, tissue or organ or that this DNA sequence be a repetitiv sequence which is naturally present in the plant cell, tissue or organ. Also, it is preferred that this DNA sequence be substantially identical to a portion of the chromosomal DNA of the plant- cell, tissue or organ to be transformed. It is also preferred that the transformation vector contain two plant-derived DNA sequences located at flanking positions relative to the foreign gene for the selectable or identifiable trait. These sequences may be the same as or different from one another provided only that they be substantially identical to DNA sequenc located in the plant cell, tissue or organ to be trans¬ formed and be capable of homologous recombination with DNA present therein when the transformation vector is introduced into the plant cell, tissue or organ. In -order to introduce a desired agronomic property or permit production of a desired product in a plant cell, plant tissue, plant organ or in a plant derived therefro the transformation vector also includes a DNA sequence coding for the desired property or product. Of particula interest are non-selectable genes whose presence would be desirable in cereals. Suitable agronomic genes include storage protein genes such as the zein genes of maize, albumin and globulin genes of maize, gliadin genes of wheat, albumin and globulin genes of wheat, hordein genes of barley, animal protein genes such as chicken ovalbumin or collagen genes, non-cereal plant protein genes such as soybean storage protein genes, and synthetic genes. Also include are the following: (a) genes associated with disease resistance which cannot be selected for in vitro, including rust resistance in wheat, fungal smut resis¬ tance or stalk rot resistance in maize, powdery mildew resistance in barley, and rice blast resistance in rice; (b) genes associated with stress tolerance, in¬ cluding tolerance to heat, drought, salt, cold and minerals such as aluminum or boron; (c) genes associated with herbicide resistance, particularly systemic herb- icides that are not selectable in vitro; (d) genes associated with plant architecture, including genes associated with leaf shape, floral morphology, tiller number, and height and root length; and (e) genes associated with plant yield, vigor and the like. The DNA sequences containing these genes are obtained by combining specific plant genes with genes or gene fragments from a plant or a foreign source. For example, a gene for production of the corn storage protein, zein, having increased lysine content is obtained by first obtaining a purified zein gene from corn endosperm using the approach described i methods (3) and (4) hereinabove for obtaining plant DNA. This is followed- by insertion into the zein gene of a DNA sequence coding for increased lysine production such as a segment of the chicken ovalbu in gene or a synthetic DNA sequence. If this DNA sequence contains genes completely foreign to the recipient plant such as genes for herbicide resistance or disease resistance rather than foreign sequences serving to modify pre-existing genes such as zein with increased lysine content, the DNA sequence is constructed in such a way that the foreign sequence is joined to or flanked by a plant DNA sequence expressed during the desired developmental stage or in the desired tissue. Transformation vectors containing foreign genes associated with selectable or identifiable plant traits, genes associated with expression of a desired agronomic property or production of a desired product, and one or two plant-derived DNA sequences, are shown in Figs. 3 and 4, respectively. In the preferred embodiment shown in Fig. 4, the two sequences flank both the gene for a foreign selectable or identifiable trait and the agro¬ nomic gene. These transformation vectors are prepared using the methods described previously for transformation vectors which do not include the agronomic gene. They 'are characterized by analysis of protein products encoded by the genes contained- in the vectors and by DNA sequence and restriction endonuclease analysis. The transformation vectors of this invention may be introduced in plant cells, tissues or organs or into plants derived therefrom by various well known methods, including injection, both arcrdinjection and microin- jection, and by DNA-mediated transformation or cotrans- for ation. The introduction of the transformation vectors is carried out under suitable conditions permittin transformation, including homologous recombination between vector DNA and naturally-occurring ' plant DNA, to occur and permitting the subsequent expression of introduced genes. If the transformation vectors are introduced in plant cells or tissues, transformed cells or tissues may be selected or identified using the introduced trait. The transformed cells may -be grown in culture to produce desired products or plants having desired properties may be regenerated using methods known in the art. In the former case, product is recovered after cell growth using known methods. In the latter, regenerated plant- lets are transplanted to greenhouses when plants are grown using known methods. Thus, introduction of a transformation vector such as that shown in Fig. 1 which includes a gene associated with ampicillin resistance renders cereal embryos resis¬ tant to levels of ampicillin which would otherwise prevent plant growth. The transformed embryos are then grown until plan lets are produced. The plantlets are then transplanted and grown to produce a plant. * Addi¬ tional examples are shown in Figs. 5 and 6 which il¬ lustrate specific transformation vectors use ' ful in the genetic modification of cereals to produce desired properties. The transformation vectors may be introduced into growing plants or organs under suitable conditions such as the following. Vectors are injected into the free nuclear (milk-stage) endosperm of the karyopses using a syringe. Approximately 0.5 μl of solution per karyopsis is used. The solution typically has a vector DNA con¬ centration of about 1 mg/ml in a standard TRIS-EDTA/water solution. Microinjection into cells or tissues is accomplished using a micromanipulator manufactured by Leitz Manufacturing Corp. and borosilicate glass micro- syringes (5-10 μ in diameter at the orifice) . EXPERIMENTAL DETAILS METHODS AND MATERIALS I. ISOLATION OF PLANT DNA: The procedure is a modifi¬ cation of Kislev, N. and Rubenstein, I., Plant Physiol. 66:1140-1143 (1980). A. Isolation of nuclei: Plant tissue is ground in a Waring blender for one minute at low setting; one minute at fast. 50 grams tissue per 250 ml. MNlB + EtBr + PVP (Solutions are described more fully hereinafter.) Total tissue used was approximately 200 grams. Infuse under vacuum 10 minutes, place in beaker in large desiccator connected to water aspirator. Filter through three layers of cheesecloth; wash thoroughly with about 100 ml additional MNlB + EtBr + PVP. Pellet 1000g, 10 minutes in JA-10 at 4°C. Resuspend pellet in NSB + 0.15 percent Triton X-100 (1 ml per 5 grams original tissue) . Pellet 1000g, 10 min., 4°C. Resuspend by vortex and repellet. This step is repeated until the supernatant is essential clear. Resuspend in NSB (without Triton) . At this point, a sample was removed for nuclei count and the rest frozen at -80°C. Nuclei stained with aceto-carmin. Yield 9 X 10*> nuclei/ml; total volume 10 mis. B. Isolation of DNA from Plant Nuclei: To 10 mis of thawed nuclei the following was added: 6.8 ml 5 M NaCl, 16.6 mis PDM and pronase to 100 μg/ml from 1 mg/ml stock made in PDM. Incubate on ice for 15 minutes. Incubate in dark at 37°C for 1 hour. Draw up and down in 10 ml pipette and incubate 4 3/4 hours longer at 37°c in the dark. Centrifuge at 20,000g for 20 minutes at 0°C to pellet starch grains. Extract supernatant with salt saturated phenol for 15 minutes. The resulting phases are separated by a 15 minute spin at 9,000g at 16°C. Supernatant is extracted with 24:1 chloroform: isoamyl alcohol, buffer saturated. Phases are separated by 5 minute spin at 9,000g at 16°C. DNA spooled on glass rod by EtOH precipitation, vacuum dried (partially) and dissolved in 5 ml TE. The DNA is incubated with 80 μg/ml RNAase A on ice for 1 hour. Extracted with phenol, then extracted twice with chloroform. Extracted with ether until clear. Spooled by EtOH precipitation, vacuum dried and dissolved in 5 ml TE. r 16- Solutions for nuclei isolation and DNA purification MNlB: 4 mM n-octyl alcohol- 2 percent gum arabic (acacia) 0.4 M sucrose 2 mM CaCl2 20 M Tris-HCl, pH 7.4 400 μg/ml EtBr 2 mg/ml PVP NSB: 0.2 M sucrose 2 mM CaCl2 10 mM Tris-HCl, pH 7.4 0.15 percent Triton-X-100 Aceto-carmin : 0.5 percent w/v in 45 percent acetic acid PDM: 10 mM Tris-HCl, pH 8.6 0.5 percent SDS 10 mM EDTA 10 mM NaCl * Phenol: saturated with 1 M NaCl 10 mM Tris-HCl, pH 8.6 1 mM EDTA Chloroform:isoamvl alcohol :: 24:1 saturated with 10 mM NaCl 10 mM Tris-HCl II. ISOLATION OF PLASMID AND BACTERIAL DNA 1. Start from a single colony of E^ coli tested for the presence of the plasmid (Tet R , Amp 11 , Cam R , etc.). Grow 100 ml of LB broth culture to saturation in a 1- liter flask with vigorous agitation. Yield: -50 μg. (Preparation below is for one tube of an SW 50.1 rotor.) 2. Sediment Cells at 8000 rpm for 5 minutes at 5°C. 3. Resuspend cells in 1/4 volume 10 mM Tris (pH 8.5) and 1 mM Na3 EDTA and resediment. 4. Resuspend cells in 2 ml of 15 percent sucrose, 0.05 M Tris (pH 8.5), 0.5 M Na 2 EDTA, and 1 mg/ml of freshly prepared lysozyme and transfer to a 10-ml centrifuge tube that can be centrifuged to 20,000 rpm. Incubate at room temperature for 10-60 minutes. 5. Add 2 ml of Triton solution (0.1 percent Triton X- 100 [Sigma], 0.05 M Tris [pH 8.5], and 0.05 M Na 2 EDTA). Incubate at room temperature for 10-20 minutes. If lysis does not occur and the suspension does not become very viscous, incubate at 37°C for 30 minutes. 6. Spin at 20,000 rpm for 1 hour in a JA-21 rotor at 5 d C. 7. Decant supernatant by pouring into a graduated test tube until the very viscous material above the pellet is reached. Usually this is less than the last half inch of clarified supernatant. Sometimes the supernatant is relatively nonviscous down to the pellet; in this case, take it all. 8. Adjust volume to 4.0 ml. Add 3.7 g of solid CsCl and 0.4 ml of 10 mg/ml of ethidium bromide (Sigma). Refractive index should be between 1.390 and 1.396 or a density of 1.59 g/ml. The density is the most reliable and can be determined easily by weighing a known volume. This mixture will just fill one SW 50.1 rotor tube. 9. Centrifuge at 35,000 rpm for 48 hours at 20°C. 10. View bands by illuminating the tube with long- wavelength UV. The lower band contains the covalently closed circular DNA. Collect the DNA by puncturing the side of the tube with a 20- to 21-gauge syringe needle. Relaxed DNA: p = 1.55 g/ml Native superhelical DNA: p = 1.59 g/ml For larger culture volumes, use a fixed-angle or vertical rotor.- Rotor 50 Ti 60 Ti 60 Ti 60 Ti 60 Ti Volume of culture (liters) 0.1 0.8 1.5 3 6 Number of rotor tubes 1 1 2 4 8 Lysozyme solution (ml) 4.5 13.5 27 54 108 Triton solution (ml) 4.8 14.5 29 58 116 Adjusted supernatant (ml) 8.7 27.5 55 110 220 CsCl (g) 8.3 26.13 52. ,26 104.5 209 Ethidium bromide solution (ml) 0.9 2.75 5. .5 11 22 III. RESTRICTION ENDONUCLEASE CLEAVAGE OF DNA Restriction endonucleases were obtained from New England BioLabs. The reaction conditions for each enzyme were essentially as described by the suppliers. Restriction enzymes were added to a concentration of approximately one unit/μg of DNA and incubated from 1 to 3 hours at 37°C. The reaction buffer , for Eco RI contained 50 mM NaCl, 100 mM tris-HCl, pH 7.4, 6 mM MgCl 2 , and 6 mM 2- mercaptoethanol. The buffer for Hae III contained 6 mM tris-HCl, pH 7.4, 6 mM MgCl 2 , and 6 mM 2-mercaptoethanol. The buffer for Hind III, Bam HI and PstI contained 50 mM NaCl, 6 mM tris-HCl, pH 7.4, 6 mM MgCl 2 . and 6 mM 2-mercaptoethanol. The reactions were stopped by heating the solution at 65°C for 5 minutes. -19- IV. ALKALINE PHOSPHATASE (BAP) TREATMENT OF BAM HI CUT pBR322 The method was substantially as described in Ullrich, A., et al., Science 196:1313 (1977). BAP from Millipore was dialyzed against 100 mM Tris- HCl, pH 9.0 for 6 hours with two buffer changes - enzyme concentration is approximately 0.2 unit/ml. Reaction Conditions and Mixture DNA (5 μg) 20 μl - directly from Bam HI reaction 100 mM Tris, pH 9.0 33 μl BAP 50 μl Incubate at 37°C for 1 hour; Phenol extract, re-extract interface; ether extract; ethanol precipitate; and dry. V. USE OF T4 LIGASE The method was a modification of that described in Weiss, B., et al. , J. Biol. Che . 2_43.:4543-4555 (1968) as described hereinbelow. Into a tube in which DNA was dried after BAP treatment, the following was added: Reaction Mixture H 2 0 61.7 μl Bam HI cut Barley DNA 10.0 μl 10X ATP 10.0 μl 10X buffer 10.0 μl ligase 8.3 μl (from N.E. BioLabs, final cone, 250 U/μg DNA) reaction carried out at -14°C -20- VI. TRANSFORMATION WITH PLASMID DNA Transformation was carried out using the method of Mandel, M. and Higa, A., J. Mol. Biol. 5_3:159 (1970). 1. Dilute overnight LB culture of HB101, RD102 = HBlOl/λ, or BNN45 1:100 into fresh LB broth [BNN45 1:100 into fresh LB broth] (40 ml in 250-ml flask) at 37°C. This amount of culture is sufficient for 20 transformations. 2.. Collect cells at Agoo = °- 6 ^ 2 ~ 3 hours) and sediment at ! 5000 rpm for 5 minutes at 2°C. 3. Resuspend in 20 ml of 50 mM CaCl 2 for 5-60 minutes at 0°'C. 4. Sediment cells at 5000 rpm for 5 minutes at 2°C and resuspend in 2 ml of 50 mM CaCl 2 for 5-60 minutes at 0°C. 5. Add 0.1 ml of cells to DNA in 0.1 ml of 0.1 M Tris (0.09 M Tris-HCl, 0.01 M Tris base) at pH 7.2 Tris for 10 minutes at 0°C. 6. Heat treat: Use 2 minutes at 37°C. 10 minutes at room temperature, 2 minutes at 37°C plus 10 minutes at room temperature, 2 minutes at 45 β C plus 10 minutes at room temperature, and 2 minutes at 45 β C -are all equal in efficiency (unlike with λ) . 7. Add 1 ml of LB broth and incubate at 37°C for 20 minutes. 8. Add 2.5 ml of LB soft agar (with no drugs) at 47°C and pour on LB plates containing 10 μg/ml of tetracycline or 50 μg/ml of ampicillin (fresh) . Tetracycline plates can be stored at 10°C for a few weeks before use. Ampicillin plates can be stored at 10°C for a few days before use. -21- VII. SLAB GEL ELECTROPHORES S DNA samples were analyzed by electrophoresis in slab gel of either agarose or polyacrylamide, depending on the expected size of the DNA molecules. Generally, circular DNA and linear DNA molecules greater than 1200 base pairs in length were separated on 1 percent to 1.4 percent agarose slab gels (vertical or horizontal). DNA samples containing molecules smaller, than 1200 base pairs in length were separated on 5 percent to 8 percent vertical polyacrylamide slab gels. Vertical garose and poly¬ acrylamide gels were 20 cm long and 1.5 mm thick; horizontal agarose gels were approximately 3 mm thick and of varying lengths. Electrophoresis 'buffer in all cases contained 40 mM tris base, 5 mM sodium acetate, and 1 mM EDTA, adjusted to pH 7.6 with acetic acid. Polyacrylamid gels were prepared from a 30 percent stock acrylamide solution (29:1: :acrylamide:bisacrylamide) using 65 mg ammonium persulfate and 50 ul TEMED per 100 mis of gel. Samples were mixed with bromphenol blue and either sucros or glycerol before layering on the gel. Electrophoresis was carried out at 150 volts or less for one to four, hour at room temperature without buffer circulation. After electrophoresis, the gels were stained with 0.4 μg/ml ethidium bromide in water for 30 minutes and the DNA band * visualized with a short wavelength ultraviolet light. Gels were photographed with a Polaroid camera equipped with an orange filter using * Polaroid Type 107 film. In some experiments, gels were prepared with 2 ug/ml ethidiu bromide in both the gel and the electrode buffer. In these cases, staining was not necessary after electro¬ phoresis, although contrast was generally poor unless the gel was destained by soaking in water for 1 hour. -22- VIII. POLYSOME ISOLATION FROM WHEAT AND CORN TISSUE CULTURES ISOLATION OF POLYRIBOSOMES Cultures are A188 corn and "Chinese Spring" wheat (48.6g FR. Wt.); grind with mortar and teflon pestle (2 ml extraction buffer/lg tissue - on ice; pellet 500g, 5 min. , JA-17 (low speed spin); pellet 30,000g, 10 in., JA-17 (medium speed spin) ; layer supernatant on cushion buffer (10 ml supernatant/5 ml buffer); pellet * 130,000g, 2 hours, TI 70; and freeze pellet at -70°C. PURIFICATION OF TOTAL POLYRIBOSOMAL RNA (rR A and mRNA) Resuspend pellet in 50 mM NaHOAc (0.15 ml/g cells); add SDS to 1.0 percent; extract with phenol/chloroform; pre¬ cipitate with ethanol; pellet - dry; redissolve in Milli- H 2 0 (sterile) 1-2 ml; dilute sample for spectrophotometer 1/100. A 260 = 0.875. When 35 μg/ml, A 260 = 1.0 .". yield RN iprj- j = 6125 μg BUFFERS FOR POLYSOME ISOLATION Extraction Buffer - CELLS ENDOSPERM FOR 100 ml MgCl 2 50 mM 50 mM 5 ml KC1 50 mM 50 mM 5 ml Triton X-100 1.0% - 1 ml Sucrose 0.2 M 0.2 M 10 ml Tris-HCl 200 mM 20 mM 20 ml β-Mercaptoethanol 5.0 mM 5.0 mM 35 λ EGTA 25 mM 25 mM 5.0 ml pH 8.0 7.6 -23- Cushion Buffer CELLS * ENDOSPERM FOR 100 MgCl 2 10 mM 10 mM 1.0 ml KC1 40 mM 40 mM 4.0 ml Tr is-HCl 40 mM 40 mM 4.0 ml Sucrose 1.5 M 1.5 M 75.0 m pH 8.0 7.6 IX. QLIGO dT CELLULOSE CHROMATOGRAPHY OF RNA [SEPARATION OF mRNA FROM RNA] 2.5g OdT cellulose in 50 ml binding buffer - define 3X; equilibrate in sterile plastic column with binding buffe at room temperature; read A 2 60 RNA, add NaCl to 0.25 M a elute with binding buffer; load sample and wash with binding buffer until A 2 gn<0.05; elute mRNA with elution buffer; wash column with binding buffer minus SDS; pool mRNA fractions and make to 0.25 M NaCl; reapply pooled fraction to column and wash with binding buffer - SDS until 2 5 Q <0.05; elute mRNA with elution buffer; and precipitate with ethanol in NaOHAc. Final product is 14. micrograms of cell culture mRNA derived from 48.6 grams starting tissue. 500 ml Binding Buffer 50 ml 0.5 M NaCl 10 ml 0.1 M Tris (pH=7.5) 25 ml 0.5% SDS 1 ml 1 mM EDTA -24- 500 ml Elution Buffer 0.01 Tris (pH=7.5) 1 mM EDTA X. TERMINAL TRANSFΞRASE TAILING The method is a modification of Roychodhury, R. , et al., Nucl. Acid Res. 3_ :863 (1976). Stock Buffers 1. TT buff 10X 1.4 M Sodium cacodylate pH 6.9 0.3 M Tris-HCl pH 6.9 (each made up concentrated and pH adjusted separately, then mixed in correct proportions) 2. Cobalt Chloride - 10X 10 mM 3. dNTP 2.5 mM stock - 0.125 mM final concentration 4. gelatin-1 mg/ml autoclaved 5. S-mercaptoethanol-5 mM 6. enzyme - PL Biochemicals spec. act. 26,000 U/mg cone. 15,000 U/ml Calculation: No. of residues added/3' end CPM/pmole 3'end pmoles NTP/pmole 3'end = CPM/pmole NTP IfUR- ' -25- To tail 1 μg linear ρBR322 using dGTP: * dry - -32P-dGTP * (~1 μCi) buffer 5 H 2 0 17.5 CoCl 2 5 DNA 10 dGTP (2. ,5 mM) 2.5 gelatin 5 incubate 15 minutes at 37°C 8 -mercaptoethanol 1 enzyme 4 Incubate 5 minutes at 37°C. Stop reaction by freezing in-t-S-r-f- ce w with EtOH/dry ice; phenol extract; extract in-te-r-phaae-; <£ ~ ~M chromatograph on G150 column, EtOH precipitate. XI. TRANSFER OF DNA TO NITROCELLULOSE Transfer from Agarose Gels A. Agarose Gel 1. Electrophoretically separate DNAs using a horizontal 14.5 X 13.5 X 0.8-cm (150 ml) gel electrophoresis apparatus. Use 0.7 percent agarose and Tris-acetate buffer. 2. Each 0.4-cm slot can be loaded with up to 5 μg of restriction-endonuclease-cleaved DNA. Use about 1 μg of cleaved bacterial DNA. 3. Ethidium bromide (0.5 μg/ml) is included in the electrophoresis buffer, and the gel is photo¬ graphed under shortwave UV light. B. Breakage of Large DNA in Gel by Depurination 1. Place the gel (14.5 X 13.5 X 0.8 cm [150 ml]) in a tray and add 250 ml of 0.25 M HC1 at room temperature. 2. Rock occasionally for 15 minutes, decant the acid, and repeat steps 1 and 2. 3. Rinse with water briefly and proceed immediately to step C. C. Alkaline Denaturation Add 250 ml of 0.5 M NaOH and 1.5 M NaCl and gently agitate for 1-5 minutes. 2. Decant alkali and repeat step C-l. D. Neutralization 1. For transfer to nitrocellulose, neutralize with 500 ml of 0.5 M Tris-HCl (pH 7.5) (60g Tris base and 30-ml concentration of HC1 per liter) and 1.5 M NaCl (90g/l) at room temperature for 30 minutes with gentle agitation. E. Transfer to Paper 1. Prepare a stack of 12 sheets of Whatman 3MM paper (57 X 46 cm, cut or folded and torn into quarter sheets) on a sheet of plastic wrap or i a tray and saturate with buffer. Two sheets ar wet and laid down at a time; the bubbles are rolled out with a rod or pipette. Buffer: Use 20X SSPE (see XII. hereinafter) for transfer to nitrocellulose. « 2. Place the gel on the 3MM paper ' . 3. A nitrocellulose filter sheet (S&S B85 or HAWP Millipore) cut to the size of the gel, is place on top of the gel. 4. Five sheets of 3MM paper (the same size as the filter) wet with water are placed on top of the nitrocellulose filter without bubbles. 5. A stack of about 6 cm of paper towels cut to size are placed on top of the 3MM paper. The stack is uniformly compressed with a 1-kg weigh on a thick sheet of plexiglass. 6. Allow the transfer to occur for 2 hours or longer. 7. The filter is turned over with the shrunken gel attached, and the gel lanes and edges are marked with a soft-lead pencil. 8. The nitrocellulose filter is rinsed in 2X SSPE for 10 minutes and dried in a vacuum oven at 80°C for 2 hours. 9. Hybridization is conducted as described in XII. REFERENCES: Southern, Ξ.M. , J. Mol. Biol. 98_:503 (1975). Wahl, G.M. , et al., Proc. Natl. Acad. Sci. £ : 3683 (1979) XII. Hybridization to DNA or RNA on Solid Support 1. Use about 10 6 to 5 X 10 6 dpm of probe (nick-translated 32 P) dissolved in TE (10 mM Tris [pH 7.5] and 1 mM Na3 EDTA) in a polypropylene tube. 2. a. Heat at 95°C for 10 minutes to denature DNA. or b. Add 1/10 volume of 1 M NaOH, wait 10 minutes, add 1/10 volume of 1.8 M Tris-HCl and 0.2 M Tris base. 3. Place DNA filters in a heat-sealable plastic bag. 4. Pretreat filter by placing in a heat-sealable bag and adding 4 ml/100-cm 2 filter of 50 percent v/v for- mamide (MCB) , 5X SSPE, 5X BFP (IX BFP = 0.02 percent w/v of bovine serum albumin, Ficoll [m.w. 400,000] and polyvinyl pyrrolidone) , 1 percent glycine, and 100 μg/ml of denatured, sonicated carrier DNA (salmon sperm) . Incubate for at least 1 hour at 42°C. Remov prehybridization solution. Prepare 4 ml/100-cm 2 filter of a solution containing 50 percent v/v for- mamide, 5X SSPE, IX -BFP, 100 μg/ml of denatured, sonicated carrier DNA (salmon sperm) , 10 percent w/v sodium dextran sulfate 500 (a 50 percent w/v stock solution can be prepared), and 0.3 percent SDS. Add half of solution to bag with the filter and mix. To remaining half, add the denatured probe, mix well, and add to bag. The sodium dextran sulfate may be omitted. 5. Add -10 6 dpm denatured DNA probe. Be careful not to get any 32 P on the sealing area. Heat-seal the bag. 6. Place sealed bag(s) into a second bag and heat-seal. wet paper towel placed in the second bag will help prevent drying of the filters. 7. a-b. Hybridize for 3-48 hours at 42°C for 50 percent formamide reaction, or c. Hybridize for 3-24 hours at 65°C for aqueous reaction. 8. Remove radioactive hybridization mix from bag. The probe may be used for additional hybridizations. 9. Cut open bag and remove filter. 10. Wash three to four times for 5-15 minutes in 250 ml of 2X SSPE + 0.2 percent SDS at 45°C with agitation. 11. Dry, cover in plastic wrap, and expose to X-ray film. The filter .can be identified and oriented by in¬ cluding within the plastic wrap a piece of paper marked with radioactive ink. (See XIII hereinafter.) 100X BFP = 2 percent w/v bovine serum albumin, Ficoll, and polyvinyl pyrrolidone. -30- 1 liter of 20X SSPE SSPE = 0.18 M NaCl Na 2 EDTA 7.4 g 20 mM 10 mM (Na 1-5 )P0 NaOH (50%) 8.8 mM 0.16 M 1 mM Na 2 EDTA NaH 2 P0 4 .H 2 0 27.6 g 0.2 M pH 7.0 NaCl 210 g 3.6 M XIII. Autoradiography of 3 on solid Support 1. Wrap filter in plastic wrap to avoid contamination of screens and holders. 2. Place wrapped filter in bottom of 8 X 10-inch X-ray film holder being sure no plastic wrap protrudes from holder. 3. An intensifying screen is attached to the lid of the film holder (Dupont Cronex Lightning-Plus ZC; 224-156 without blockers, 8 X 10 in.). 4. In a dark room, place one sheet of 8 X 10-inch Kodak X-Omat R ' film or Dupont Cronex 4 film in the film holder. The Cronex 4 film is about one-quarter to one-half as fast as the X-Omat R film, but it is of higher resolution. 5. . Close and lock film holder before turning on lights. Wrap holder in aluminum foil and place in -70°C freezer. 6. Expose for several hours to several days 7. To develop film, -remove holder from freezer and bring to room temperature before removing aluminum foil. This is to prevent condensation on film and damage to intensifying screen. 8. In a dark room, remove aluminum foil, open holder, and remove film. 9. Develop in X-ray film processor, or_ 5-minute Kodak liquid X-ray developer 1-minute stop bath (3 percent acetic acid) 10-minute Kodak rapid fixer 15-minute running water hang to dry XIV. cDNA SYNTHESIS AND CLONING The methods used are those described in Buell, G.N., et al. j. Biol. Chem. 25_3:2471-2482 (1978) and Wickens, M.P., et al., J. Biol. Chem. 253:2483-2495. XV. MEDIA USED FOR CEREAL TISSUE CULTURE (IPX) Total Volume = 4 Liters A. NH4NO3 .66.0 g. KNO3 76.0 g. MgS0 .7H 2 0 14.8 g. KH 2 P0 4 6.8 g. Ferric versenate 1.6 g. L.-Asparagine 600 mg, B. CaCl 2 .2H 2 0 (stock @ 15 g/100 ml.) 116 ml MICRONUTRIENTS (stock solution) 40 ml H3BO3 620 mg/100 ml. MnS0 4 .H 2 0- 1690.0 mg/100 ml ZnS0 4 .7H 2 0 m 860.0 mg/100 ml Na 2 Mo0 .2H 0 25.0 mg/100 ml. CuS0 4 .5H 2 0 2.5 mg/100 ml. CoCl 2 .6H 2 0 2.5 mg/100 ml. D. Cereal vitamin #1 (Thiamine @ .0125 g/250 ml.)-- 40 ml. E. KI (stock solution @ 75 mg/100 ml.) - 40 ml. Dissolve all ingredients completely and bring volume to 4 liters in a graduated cylinder. Dispense into 100 ml. aliquots, using plastic whirl-pack bags. Freeze promptly for long term storage. XVI. PROCEDURE FOR ESTABLISHING CEREAL TISSUE CULTURES Immature embryos (—1.0-1.5 mm in length) are isolated, asceptically from sterilized seeds. Embryos are placed polar axis down, (scutellum up) on culture initiation medium (basal medium and 1 mg/1 2,4-D) and incubated at 28 β C (in presence or absence of light) . XVII. SELECTION Selection is accomplished by transferring cultures to basal media supplemented with 1 μg/ral 2,4-D and 50 μg/ml - ampicillin. These cultures are transferred every 102- - ~ τ» weeks to media adjusted with 2,4-D and ampicillin as judged by the viability of the cultures. Surviving cultures are regenerated by step-down transfer to hormone- free media. Regenerants are grown to maturity in the greenhouse, where self or cross pollinations are made and phenotypic data is recorded. DNA can be isolated from leaves of these plants and used for DNA/DNA hybridization analysis. XVIII. PLANT STRAINS Strains used are maize: IPRI M62 - a derivative of A188X "Hayse White"; A188; and wheat: _^ Aestivum cv. "Chinese Spring". -33- PROCESS SCHEMA: CONSTRUCTION OF pZVD7 and pTVDl etiolated plant seedlings purified pBR322 purifi 1ed nuclei Hind I III digest purifie id -nuclear DNA bacter Iial alkaline treatment pBR322 transforma Ition of E. coli HBlOl identifica Ition of recombinant plasmid-containing colonies by selection and screening isolation iof plasmid DNA " R£ -34- CONSTRUCTION OF pZVD and pTVD SERIES isolation of cell culture mRNA purified pBR322 ψ synthesis of double-stranded cENA res ϊtriction digestion terminal transferase tail addition term Iinal transferase taili inant plasπid-containing colonies by selection and screening analysis of cDNA clones hybridization of insertion of (restriction) specific clones to selection ge (hybridization) gene library into specifi (hybrid-selected translation) clones analysis of gene structure isolat 4ion of structural components for vector construction -35- VECTOR INSERTION, TRANSFORMANT SELECTION AND ANALYSIS OF TRANSFORMED REGENERANTS recipient plant (vegetative) pollination in planta injection Soyfer, V.M., et al. 1976 Mutation Res. 36:303-310 culture initiation- -Gosch, G. , et al. 1979 ol al. 1980 •selection ■Science 209 :1414-1422 regeneration confirmation ization- -Southern, E.M. 1975 J. Mol. Biol. 98:503 see b oc em ca and genetic analysis Day 1 ca. 400 M62 karyopses (free nuclear stage endosperm, milk stage) injected with 0.5 μl - Tris-EdTA +-§4?- (ca. 1 mg/ml) . f Day 5 M62 embryos (1.0-1.75 mm) plated onto C1D for culture initiation. Thirty-one non-injected . controls plated. Day 11 -ptt?- and control cultures transferred to C0.75D + 50 μg/ml ampicillin Day 17 All cultures transfered to either C0.75D + 50 μg/ml ampicillin or CQ.75D + 100 μg/ml ampicillin Day 33 All cultures transferred to either C0.1D + 100 μg/ml ampicillin or C1D + 100 μg/ml ampi¬ cillin * Fresh weight of each embryo-derived culture recorded. Day 39 62 plants regenerated from -pM?—inj cted, ampi- £! cillin-selected culture by transferring cultures to basal media minus hormone. -37- Fresh weights ( g.) =>ZV07 -ff-M*?-injected non-injected controls 147 521 6 64 158 540 9. ao 175 540 21 85 176 562 21 104 177 570 22 129 207 575 22 191 225 725 23 235 232 740 23 274 260 760 30 285 285 765 32 285 295 835 32 353 401 910 37 398 408 975 38 800 500 1035 40 842 510 1340 40 515 44 57 n - 31 n - 31 X = 518.2 X = 149.1 s = 300.3 s = 210.9 CHARACTERIZATION OF TRANSFORMED PLANTS Initial DNA/DNA hyb idization has been conducted on regenerated plants, confirming the presence in these plants of DNA sequences substantially identical to DNA sequences of the transformation vector. Numerous phenotypic abberations have been observed-in regenerated plants including: 1. abnormal leaf origination (opposite as opposed to alternate) 2. abnormal leaf pigmentation (albino sections, transluscent striations) 3. Extreme leaf curling and deformation 4. Abnormal silk morphology PRELIMINARY CYTOGENETIC CHA ACT- ERIZATION OF REGENERATED PLANTS CYTOGENETIC CHARACTERIZATION OF BIVALENT ASSOCIATIONS WITH NUCLEOLI IN pZVD7-12 (EXPERIMENTAL) AND M62 (CONTROL) PACHΗ±NE DIAKENESIS fZVOl-YZ normal 2 biv.* abnormal normal 2 biv.* abnormal '£ΑJ M -±mm 163 34 2 43 32 21 M62 34 0 0 56 0 0 * associated with nucleolus

Claims: -39- What is claimed is: 1. A transformation vector useful for genetic modifi¬ cation of a photosynthetic plant cell, plant tissue or plant organ comprises a double-stranded DNA molecule whic includes a gene foreign to said plant cell, plant tissue or plant organ and associated with a selectable or identi fiable trait when present therein and at least one DNA sequence substantially identical to a DNA sequence presen in said plant cell, plant tissue or plant organ and capable of homologous recombination with said plant DNA sequence when said transformation vector is introduced into said plant cell, plant tissue or plant organ. 2. A transformation vector in accordance with Claim 1 which additionally includes a gene asociated with ex¬ pression of a desired property or production of a desired product in said plant cell, plant tissue or plant organ or in a plant derived therefrom. 3. A transformation vector in accordance with Claim 1 wherein -said DNA sequence is expressed during a specific developmental stage of said plant cell, plant tissue or plant organ. 4. A transformation- vector in accordance with Claim 1 wherein said DNA sequence comprises repetitive sequences derived from said plant cell, plant tissue or plant organ. 5. A transformation vector in accordance with Claim 1 wherein said DNA sequence is substantially identical to a DNA sequence located in the chromosomal DNA of said plant cell, plant tissue or " lant organ. 6. A transformation vector in accordance with Claim 1 which includes two DNA sequences, each substantially identical to a DNA sequence located in said plant cell, plant tissue or plant organ, said DNA sequences being either the same as or different from one another and being capable of homologous recombination with said plant DNA sequences when said transformation vector is introduced into said plant cell, plant tissue or plant organ. 7. A transformation vector.in accordance with Claim 6 which additionally includes a gene associated with expression of a desired property or production of a desired product in said plant cell, plant tissue or plant organ or in a plant derived therefrom. 8. A transformation vector in accordance with Claim 6 wherein said DNA sequences are expressed during a specific developmental stage of said plant cell, plant tissue or plant organ. 9. A transformation vector in accordance with Claim 6 wherein said DNA sequences comprise repetitive sequences derived from said plant cell, plant tissue or plant organ. 10. A transformation vector in accordance with Claim 6 . wherein said DNA sequences are substantially identical to DNA sequences present in the chromosomal DNA of said plant cell, plant tissue or plant organ. 11. A transformation vector in accordance with Claim 1 wherein said foreign gene associated with a selectable or identifiable trait is- derived from a plasmid. 12. A transformation vector in accordance with Claim 1 wherein said foreign gene associated with a selectable or identifiable trait is derived from a plant virus. 13. A transformaton vector in accordance with Claim 1 wherein said foreign gene associated with a selectable or identifiable trait is derived from a plant. 14. A transformation vector in accordance with Claim 1 wherein said selectable or identifiable trait is ability to grow on a medium on which growth of said plant cell, plant tissue or plant organ is otherwise not possible. 15. A transformation vector in accordance with Claim 14 wherein said growth medium contains an antibiotic. 16. A transformation vector in accordance with Claim 1 wherein said plant is a cereal. 17. A transformation vector in accordance with Claim 16 wherein said cereal is barley, wheat, rice, triticale or maize. 18. A transformation vector in accordance with Claim 1 wherein said double-stranded DNA molecule is linear. 19. A transformation vector in accordance with Claim 1 Wherein said double-stranded DNA molecule is circular. 20. A transformation vector in accordance with Claim 1 wherein said plant organ is an embryo. 21. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is associated with production of proteins possessing improved nutritional quality in a plant derived from said plant cell, plant tissue or plant organ". 22. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is derived from barley and associated with production of hordein in a plant derived from said plant cell, plant tissue or plant organ. 23. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is derived from wheat and associated with production of gliadin in a plant derived "from said plant cell, plant tissue or plant organ. 24. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is associated with resistance to environmental stress in a plant derived from said plant cell, plant tissue or plant organ. 25. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is associated with resistance to elevated salt concentrations in water or soil which contacts a plant derived from said plant cell, plant tissue or plant organ. 26. A transformation vector in acordance with Claim 2 or Claim 7 wherein said additional gene is associated with drought resistance in a plant derived from said plant cell, plant tissue, or plant organ. 27. A transformation vector in accordance with Claim 2 or Claim 7 wherein said additional gene is associated with resistance to a herbicide in a plant derived from said plant cell, plant tissue or plant organ. 28. A method of preparing the transformation vector of Claim 1 which comprises preparing a double-stranded DNA molecule which includes a gene foreign to a plant cell, plant tissue or plant organ and associated with expression -43- of a selectable or identifiable trait when present therein, separately recovering from a plant cell, plant tissue or plant organ a double-stranded DNA sequence which is substantially identically to a DNA sequence present in the plant cell, plant tissue or plant organ to be trans¬ formed and joining the DNA molecule so prepared with the. foreign gene to form the transformation vector. 29. A method of introducing a gene associated with a desired property or product into a plant which comprises introducing into a plant cell, plant tissue or plant organ capable of cultivation and iri vitro regeneration to yield said plant a transformation vector in accordance with Claim 2 or Claim 7 under suitable conditions permitting subsequent expression of said gene in said plant and growing said plant cell, plant tissue or plant organ to yield said plant. 30. A method in accordance with Claim 29 wherein said introduction comprises injection. 31. A method in accordance with Claim 29 wherein said introduction comprises transformation. 32. A plant prepared in accordance with the method of Claim 29. 33. A photosynthetic plant which includes a foreign gene associated with expression of a desired property or pro- duction of a desired product. -44- 34. A transformation vector useful for genetic modifi¬ cation of a photosynthetic plant cell, plant tissue or plant organ comprises a double-stranded DNA molecule which includes a gene foreign to said plant cell, plant tissue or plant organ and associated with expression of a desired property or production of a desired product and at least one DNA sequence substantially identical to a DNA sequence present in said plant cell, plant tissue or plant organ and capable of homologous recombination with.said plant DNA sequence when said transformation vector is introduced into said plant cell, plant tissue or plant organ.

Inventors: ERWIN ROBERT L, HUBBARD ERNEST T, ERWIN, ROBERT, L., HUBBARD, ERNEST, T.
Applicants: INT PLANT RESEARCH INST, INTERNATIONAL PLANT RESEARCH INSTITUTE
Application year: 1982
Main classification: A01H1
IPC codes: A01H1, C12N15