MXPA99009940A - Agrobacterium mediated transformation of sorghum - Google Patents

Abstract

Methods and compositions for the efficient transformation of sorghum are provided. The method involves infection with Agrobacterium, particularly those comprising a super-binary vector. In this manner, any gene of interest can be introduced into the sorghum plant. The transformed gene will be flanked by at least one T-DNA border and present in the transformed sorghum in low copy number. Transformed sorghum, cells, tissues, plants and seed are also provided. The invention encompasses regenerated, fertile sorghum plants, transgenic seeeds produced therefrom, T1 and subsequent generations.

Description

'J and sorghum in substantial contribution to the increase in production in the United States. Currently, sorghum is ranked third among those produced in the United States and the preferred grain in areas of water availability due to its yield stability under drought conditions. * é-t z Sorghum "is plagued by diseases, especially in higher production environments, many of the X X" 2 *, - diseases are caused by highly pathogenic '* - #, - variables. In general, as the power of 'yield increases, so does the proportion of grain loss to occur. Until recently, the genetic improvement of sorghum * for agronomic and quality traits has been made by * traditional plant development methods and improved culture management practices. Advances in tissue culture and transformation technologies have given like,. * * * * * * - • 'result in the production of transgenic plants of all t% - "the main cereals, including sorghum To date, the key to this transformation are developments of microprojectile bombing devices for supplies of DNA in cells The bombardment of microprojectiles avoided two main restrictions of cereal transformation.These restrictions are the lack of a natural vector available - such as "Agrobacterium tumefaciens and makes it difficult to regenerate plants with" protoplasts "which are" used for transformation. He bombardment of particles can trigger '"*' '-. ~ dd-' '-" i' - * - ** - '_ JX' ¡~ "" - "'' ';:"' cells within the tissues or organs that have a high potential "morphogenic." However, the use of "bombardment of ... -. X". ', "t'f ~ -' **" - '. J. microproyecti? Es as a "de." transformation has :, its disadvantages *. Particularly, with the bombardment several copies J. -. of the gene can be "transferred" and are usually integrated into the target genome. These copies integrated by have generally been redisposed and mutated. In addition, * the event ... of transformation. may not be stable due to the point., of insertion or:, the means are not still an efficient process .,; "" (Casas det ^ al. (1993) Proc. To tl. Acad. 'Sci. USA' 90": 11212- 1T216). ' "'*";' '', i. '"" \ ~ - 15"Agrobacterium, a natural plant pathogen, has been widely used for the transformation of plants .... dicotyledonous. The Agr? Bacterium remains the most important vector . , 'widely used for the transformation of species 'dichotiiedons. Now, that the monocotyledonous plants are rarely natural hosts for Agrobacteri um, it has not "- '*" "" expected to be susceptible to the transfer of .. gene ... mediated by the bacteria. - '- "2 d: 2 Í? A advantage" of the transfer of. mediated gene. by . • s'X * "**" '"- * d •'" * - * "- - '- * $? * 7'" "-" '' J r ''; Agro¿ »acteriuip is that It offers the potential to regenerate 25 transgenic cells at relatively high frequencies without a significant reduction in the regrowth regimes, and the process of transferring DNA to the plant genome is defined, that is, DNA does not normally suffer any main rearrangement, and is usually integrated into the genome in individual or low numbers of copies t Transformation mediated by Agroba cterium involves the incubation of cells or tissues "with the bacteria, followed by the regeneration of transformed plant cells. through a callus stage. The inoculation of explants has proven to be the most effective means to create transgenic plants. Previous work with Agroba cteri um indicated that the bacterium can transfer T-DNA to a monocotyledonous host. However, there is clear evidence of integration of T-DNA only for asparagus, and even in this case, no transformed plants were produced. Due to the recalcitrant nature of monocotyledons to Agrobacterium infection, other methods, such as particle bombardment, were developed for the transformation of monocotyledons. More recently, the transformation of corn and rice using Agroba cterium has been reported. See, Ishida et al. (1996) "Na ture Biotechnology 14: 745- 750; EPA 0672752A1; EPA 0687730A1; and U.S. Patent No. 5,591,616. Among the indicated factors that affect the efficiency of the transformation including types and stages of infected corn tissues, the concentration of A. * T umefa hundreds, media compositions for tissue culture, selectable marker genes, vector types and strains of Agroba cterium, and maize genotype. The researchers concluded that the main obstacle in the transformation may have been the recovery of cells that acquired the T-DNA in their chromosomes. See, Ishida et al. , (1996) supra. Although the reports indicate that some genotypes of corn and rice can be transfected with Agroba cteri um, there is no report of sorghum transformation mediated by Agrobacteri um. While transgenic sorghum plants have been reported following the bombardment of microprojectile, transgenic plants were obtained only at very low frequencies. In addition, the inherent characteristics of sorghum cells make them a little irresponsible for transient expression. Houses e t al. (1993) Proc. Na ti. Acad. Sci. USA 90: 11212-11216. Therefore, there is a need for an efficient method for the transformation of sorghum, where a stable transformation of large inserts can be obtained. That is, there is a need for a method for the transformation of sorghum using Agrobacterium.

The present invention is designed for methods and compositions for the efficient transformation of sorghum. The method involves the use of bacteria belonging to the genus, Agrobacterium, particularly those that comprise a super-binary vector. In this way, any gene of interest can be introduced into the sorghum plant. The transferred gene can be flanked through at least one T-DNA limiter and presented in the transformed sorghum in a low copy number. _ Transformed sorghum cells, tissues, plants and seeds are also provided. Such transformed compositions are characterized by the presence of T-DNA limiters and a low copy number of the transferred gene. The invention encompasses regenerated, transgenic fertile sorghum plants, transgenic seeds produced therefrom, TI and subsequent generations. BRIEF DESCRIPTION D? THE DRAWINGS Figure 1 provides a diagram illustrating the construction of vector pPHP10525. La-- Figure 2 provides a vector plasmid map pPHPH264. Compositions and methods for the efficient transformation of sorghum are provided. Transformed sorghum plants are characterized by containing transferred nucleic acid such as a gene or genes transferred of interest flanked by at least one T-DNA limiter injected into the genome of sorghum plants. The plants are normal in morphology and fertility. In general, the transformed plants contain an individual copy of the transferred nucleic acid without any noticeable rearrangement. Alternatively, the transferred nucleic acid of interest is present in the sorghum transformed into low copy numbers. By low number of copies is meant that transformants comprising not more than five (5) copies of the transferred nucleic acid, preferably not more than three (3) of the transferred nucleic acid, more preferably less than three (3) copies of the transferred nucleic acid. The transferred nucleic acid will comprise at least one sequence of the T-DNA limiter. The. The methods of the invention are based on the use of gene transfer mediated by Agroba cteri um. The Agrobacterium-mediated gene transfer exploits the natural ability of Agroba cterium tumefa ciens to transfer DNA to plant chromosomes. Agrobacterium is a plant pathogen that transfers a group of genes encoded in a region called T-DNA from the Ti plasmid in plant cells at binding sites. The typical result of gene transfer is a tumorous growth called a crown gill, where the T-DNA is stably integrated into a host chromosome. The ability to cause crown gall disease can be removed by eliminating the genes in the T-DNA without losing the transfer and integration of the DNA. The DNA that will be transferred is bound to the limiting sequences that define the endpoints of an integrated T-DNA. Gene transfer through engineered Agrobacterium strains has become a routine for most dicotyledonous plants and for some monocotyledonous plants. However, there are no reports to date to produce transformed sorghum through transformation mediated by Agrobacterium. See, for example, Fraley et al. (1983) Proc. Na ti. Acad. Sci. USA 80: 4803; Watson et al. (1985) EMBO J 4: 211; Horsch et al. (1985) Science 227: 1229; Hernalsteens et al. (1984) EMBO J 3: 3039; Comai et al. (1984) Na t ure (London) 317: 741; Shah et al. (1986) Science 233: 418; Bytebier et al. (1987) Pro. Na ti. Acad. Sci. USA 54: 5345; Schafew et al. (1987) Na ture 327: 529; Potrykus, I. (1990) Biotechnol 8: 535; Grimsley et al. (1987) Na ture 325: 177; Gould et al. (1991) Plan t Physiol 95: 426; Ishida et al (1996) Na ture Biotecnhl ogy 14: 745; and U.S. Patent No. 5,591,616 and references cited therein - The Agroba cteri um strain used in the methods of the invention is modified to contain a gene or genes of interest, or a nucleic acid that is expressed in the transformed cells.The nucleic acid to be transferred is incorporated in the T region and is flanked by at least one T-DNA limiting sequence. A variety of Agrobacterium species is known in the art particularly for the transformation of dicotyledons, said Agrobacterium can be used in the methods of the invention See for example, Hooykaas, PJ (1989) Planz Mol. Biol. 13: 321; Smith et al. al. (1995) Crop Science 35: 301; Chilton, MO (1993) Proc. Nati. Acad. Sci. USA 90: 3119; Mollony et al., N: Monograph Theor Appl Genet NY, Springer verlag 19: 148, 1993; and Ishida et al * (1996) Nature Biotechnol.14: 745; Komari, T. et al. (1996) The Plant Journal 10: 165; incorporated here for reference. In the Ti plasmid, the T region is distinct from the vir region whose functions are responsible for the transfer of integration. Binary vector systems have been developed in which the foreign DNA that carries disarmed T-DNA manipulated and the functions of vir are present in separate plasmids. In this manner, a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid that replicates in E. coli. This plasmid is conjugatively transferred in a tri-parent mating in A. tumefaciens, which contains a virulence gene carrying a compatible plasmid. The vir functions are supplied 1 in trans to transfer to T-DNA in the plant genome. Such binary vectors are useful in the practice of the present invention. The preferred vectors of the invention are superbinary vectors. See, for example, U.S. Patent No. 5,591,616 and EPA 064662A1, incorporated herein by reference. Such a superbinary vector has been constructed containing a region of DNA that was originally from the virulence region of the Ti plasmid pTiBo542 (Jin et al., (1987) J. Bateriol 1 69: 4417 -4425 *), contained in Agroba um tumefaciens A281 supervirulent that exhibits an extremely high transformation efficiency. (Hood et al. (1984) Bio technol 2: 702-709; Hood et al. (1986) J. Bateriol. 168: 1283-1290; Komari et al. (1986) J. Ba c teriol 166: 88 -94; Jm et al. (1987) J. Bacteriol 169: 4417-4425; Komari T. (1989) Plant Science 50: 223-229; ATCC Accession No. 37394). Superbinary vectors are known in the art and include pTOK162 (Japanese Patent Application (Kokai) No. 4-222527, EP-A-504, 869, EP-A-604, 662, and U.S. Patent No. 5,591,616 incorporated herein for reference) and pTOK233 (Komari, T. (1990) Plan t Cell Reports 9: 303-306; and Ishida et al. (1996) Na ture Biotechnology 14: 745; incorporated herein by reference). Other superbinary vectors can be constructed through the methods set forth in the above references. The super binary vector pTPOK162 is capable of replication in both E. col i as in A. t umefaciens. In addition, the vector contains the VirB, virC and virG genes from the virulence region of pTiBo5422. The plasmid also contains an antibiotic resistance gene, a selectable marker gene and the nucleic acid of interest that will be transformed to the plant. The nucleic acid to be inserted into the sorghum genome is located between the two T-region limiting sequences. The superbinary vectors of the invention can be constructed having the characteristics described above for pTOK162. The T region of the superbinary vectors and other vectors for use in the invention is constructed to have restriction sites for the insertion of the genes that will be delivered. Alternatively, the DNA to be transformed can be inserted into the T-DNA region of the vector using homologous recombination in vivo. See, Herrera-Estrella et al. (1983) EMBO J. 2: 987-995; Horch et al. (1984) Science 223: 496-498). Such homologous recombination is based on the fact that the superbinary vector has a region homologous with the region of pBR322 or another similar plasmid. In this way, when the two plasmids are joined together, a desired gene is inserted into the superbinary vector - through genetic recombination through the homologous regions.

As will be apparent to one skilled in the art, now that method has been provided for the stable transformation of. sorghum, any nucleic acid of interest can be used in the methods of the invention. For example, a plant "of sorghum can be designed by genetic engineering to express genes for resistance to diseases and insects, genes that confer a nutritional value, genes" that confer male and / or male sterility, (antifungal, antibacterial or antiviral genes, Likewise, the method can be used to transfer any nucleic acid to control gene expression, for example, the nucleic acid to be transferred can encode an antisense oligonucleotide.The genes of interest are a reflection of the commercial markets and interests of those involved in grain development Grains and markets of interest change and as developing nations open up new world markets, new grains and technology will also emerge, in addition to an understanding of agronomic traits and traits such as yield and increase in eterosis, the choice of genes for the transformation t it will also change accordingly. Grains of special interest include corn, soybean, canola, sunflower, rapeseed, rice, tobacco, wheat, sorghum and alfalfa. General categories of genes of interest include, for example, those genes involved in the information, such as zinc extremities, those involved in communication, such as kinases, and those involved in the domestic aspect, such as heat shock proteins. More specific categories of transgenes, for example, include genes that code for important traits for agronomy, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Agronomically important traits such as oil, starch and protein content can be genetically altered in addition to using traditional harvesting methods. The modifications include an increased content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur and providing essential amino acids, and also the modification of starch. Modifications of hordothionine protein are described in WO 96/38563, WO 94/16078 and WO 96/38562 ~ and U.S. Patent No. 5, 703, 409_ issued December 30, 1997, the descriptions of which they are incorporated here for reference in their entirety. Another example is a seed protein rich in lysine and / or sulfur that encodes soy 2S albumin and the chymotrypsin inhibitor for barley Williamson et al. Eur. J. Bíochem. (1987) 165: 99-106, the descriptions of each are incorporated herein for reference. The derivatives of the following genes can be used through site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the lysine polypeptide with a high barley content (BHL) is derived from the barium chymotrypsin inhibitor, WO 98/20133 and PCT / US97 / 20441 filed on October 31, 1997, the descriptions of which are incorporated here for reference. Other proteins include proteins from plants rich in methionine, such as sunflower seed (Lilley, et al., Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, Applewhite, H. (ed.); American Oil Che ists Soc, Champaign, IL; (1989) 497-502; corn (Pedersen, et al., J. Biol. Chem. (1986) 261: 6279; Kirihara et al., Gene (1988) 71: 359; and rice (Musumura, et al., Plan, Mol. Biol., 1989). 12: 123. These references are incorporated here in their entirety Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors and transcription factors Insect resistance genes can encode pest resistance that they have a large production trawl such as the rootworm or the European corn ovipositor.For example, the genes of the microorsm Ba cillus th uringiensis encode toxic proteins that have been isolated, characterized and successfully used to reduce the infestation by ECB ( U.S. Patent No. 5,366,892 Foncerrada et al., Gene Encoding to Coleopteran-a ctive Toxin.) Other examples of genes useful in insect resistance include those that encode secondary metabolites and toxins from plants. "Genes that encode disease resistance treatments may include detoxification genes, such as against fumonosin or other toxins. Fumonisin resistance can be used to transform plant cells normally susceptible to Fusarium um or other toxin production fungi as described in U.S. Patent No. 5,792,931, issued August 11, 1998. Other examples are genes that confer viral resistance and antimicrobial peptides. the herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular, the sulfonylurea-type herbicides (eg, the acetolactate synthase gene ( AL?) Which contains mutations that lead to such resistance, in particular mutations S4 and / or Hra), genes that code for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or coarse (eg, the bar gene), or other genes known in the art. The gene encodes resistance to coarse herbicide, the npt ll gene encodes resistance to the antibiotics kanamycin and gentamicin, and the ALS gene encodes closulfuron herbicide resistance. Also the sterility genes can be encoded in an expression cassette and provide an alternative for physical depletion. Examples of genes used in such manners include male tissue preferred genes and genes with male sterility phenotypes, such as QM, described in U.S. Patent No. 5,583,210. Other genes include kinases and those that code toxic compounds for either male or female gametophysical development. Grain quality is reflected in traits such as the levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and cellulose levels. In corn, modified horotionin proteins, described in WO 96/38563, WO 94/16078, WO 96/38562 and U.S. Patent No. 5,703,409 issued December 30, 1997, provide descriptions of protein modifications for desired purposes. Commercial traits can also be encoded in a gene or genes that can increase, for example, starch for the production of ethanol, or provide protein expression. Another important commercial use of transformed plants is the production of polymers and bioplastics, such as those described in U.S. Patent No. 5,602,321, issued February 11, 1997. Genes such as B-ketothiolase, PHBase (polyhydroxybutyrate synthase) ) and acetoacetyl-CoA reductase (see Schubert et al., (1988) J. Bacteriol. 1 70) facilitates the expression of polyhydroxyalkanoates (PHA). For convenience, the nucleic acid that will be transferred may be contained within the expression cassette. "The expression cassette will comprise a region of transcriptional initiation linked to the nucleic acid or gene of interest, Such an expression cassette is provided with a plurality of restriction sites for the insertion of the gene or genes of interest that will be under the transcriptional regulation of the regulatory regions The transcriptional initiation region, the promoter may be native or homologous or foreign or heterologous to the host, or may be of natural sequence or synthetic sequence. Oddly, it is meant that the transcriptional initiation region is not found in the wild type host, into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to the transcription initiation region, which is heterologous to the coding sequence. The transcriptional cassette will include the transcription direction 5 '-3', a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region, functional in plants. The termination region may be native to the transcriptional initiation region, may be native to the DNA sequence of interest, or may be derived from another source. "Suitable termination regions are available from the Ti plasmid of A. tumefa ciens, such as the opalpine synthase termination regions and nopaline synthase, see, also, Guerineau et al. (1991) Mol. Gen. Gene t.262: 141-144; Proudfoot (1991) Cell 64: 611-614; Sanfacon et al. (1991) Genes Dev. 5: 141 -1 49; Mogen e t al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Bailas et al. (1989) Nucleic Acids Res. 17: 7391-7903; Joshi et al. (1987) Nucl ei c Acids Res. 15: 9627 -9639. Alternatively, the gene of interest can be provided on another expression cassette. When appropriate, the gene can be optimized for increased expression in the transformed plant. When mammalian, yeast or bacterial or dicotyledonous genes are used in the invention, these can be synthesized using preferred monocot or maize codons for improved expression. Methods for synthesizing preferred plant genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al., (1989) Nucleic Acids Res. 17: 477-498, -incorporated in the present for reference. The expression cassettes also usually contain 5 'leader sequences in the construction of expression cassette. Such leader sequences can act to improve translation. Translational leaders are n in the art and include: picornavirus leaders, eg, EMCV leader "(5 'non-coding region of encephalomyocarditis) (Elroy-Stein O., Fuerst, TR, and Moss, B. ( 1989) PNAS USA, 85: 6126-6130), leaders of .potivirus, for example, the leader TEV (Tobacco Attack Virus), (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); , 154: 9-20), and human immunoglobulin heavy chain protein (BiP), (Macejak, DG, and P. Sarnow (1991) Na ture, 353: 90-94, untranslated leader of the coating protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jogling, SA, and Gehrke L., (1987) Na ture, 325: 622-625, tobacco mosaic virus leader (TMV), (Gallie, DR et al. (1989) Biological molecule of RNA, pages 237-256, and leader of chlorotic spot virus virus (MCMV) (Lo mel, SA et al. (1991) Virology, 81: 382-385) See also "Della-Cioppa et al. (1987) Plan t Physiology, 84: 965-968 Other n methods for improving translation may also be used, for example, introns and the like. ** Expression cassettes may contain one or more of a gene or nucleic acid sequences that will be transferred and expressed in the plant transformed. In this manner, each nucleic acid sequence will operably be linked to 5 'and 3d regulatory sequences. Alternatively, multiple expression cassettes may be provided. In general, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are used for the selection of transformed cells or tissues. * Selectable marker genes include genes encoding antibiotic therapy, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) as well as genes that they confer insensitive resistance to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. (See DeBlock et al (1987) EMBO J., 5: 2513-2518; DeBlock et al. (1989) Plan t Physiol., 91: 691-704; Fromm et al. (1990) 8: 833-839) . For example, glyphosate or sulfonylurea herbicides have been used using genes encoding the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS). The ammonium resistance of glufosinate, bromoxynil, and 2,4-dichlorophenoxyacetate (2, -D) has been obtained using bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate monooxygenase, which detoxifies the respective herbicides. For the purposes of the present invention, the selected marker genes include, but are not limited to, genes encoding: neomycin phosphotransferase II (Fraley et al (1986) CRC Critical Reviews in Plant Science, 4: 1-25); cyanamide hydratase (Maier-Greiner et al., (1991) Proc.

Nati Acad. Sci. USA 88: 4250-4264); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) Bio / Technology, 11: 715-718); Tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Bio., 22: 901-912); dihydrodipicolinate synthase and desensitized aspartate kinase (Perl et al., (1993) Bio / Technology, 11: 715-718); gene ele bar (Toki et al. (1992) Plant Physiol, 100: 1503-1507 and Meagher et al., (1996) and Crop Sci., 36: 1367); tryptophan decarboxylase (Goddjin et al. (1993) Plant Mol. Biol., 222907-912); neomycin phosphotransferase (NEO) (Southern et al., (1982) J. Mol. Appl. Gen., 1: 321; hygromycin phosphotransferase (HPT or HYG) (Shimizu et al. (1986) Mol. Cell Biol., 5: 1074), dihydrofolate reductase (DHFR) (Kwok et al., (1986) PNAS USA 4552), phosphinothricin acetyltransferase (DeBlock et al., (1987) EMBO J. 6: 2513); 2, 2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al (1989) J. Cell. Biochem. 130: 330); acetohydroxy acid synthase (Anderson et al., U.S. Patent No. 4,761,373; Haughn et al. (1988) Mol. Gen. G net 221: 266); 5-enolpyruvyl-shikimate-phosphate synthase (AroA) (Comai et al (1985) Nature 317: 141); haloarylnitrilasse (Stalker et al., published in PCT applcn WO87 / 04181); carboxylase of A acetyl-coenzyme (Parker et al (1990) Plant Physiol. 92: 1220); dihydropteroate synthase (sul I) (Guerineau et al (1990) Plant Mol. Biol. 15: 121); 32 kD photosystem II polypeptide (psbA) (Hirschberg et al (193) Science, 222: 1346); etc. Also included are chloramphenicol resistance coding genes (Herrera-Estrella et al (1983) EMBO J., 2: 987-992); methotrexate (Herrera-Estrella et al. (1983) Nature, 303: 209-213; Meijer et al. (1991) Plant Mol Bio., 16: 801-820 (1991), hygromycin (Waldron et al. (1985) Plant Science, 108: 219-221 and Meijer et al (1991) Plantz Mol, Bio 16: 807-820), streptomycin (Jones et al (T987) Mol Gen. Genet., 2210: 86-91); Spectinomycin (Bretagne-Sagnard et al (1996) Transgenic Res., 5: 131-137); bleomycin (Hille et al. (1986) ~ Plant Mol. Biol., 1-111-116); sulfonamide (Guerineau et al. al. (1990) Plant Mol. Bio., 15: 127-136), bromoxynil (Stalker et al (1988) Science, 242: 419-423); 2, 4-D (Streber et al. (1989) Bio / Technology, 7: 811-816); glyphosate (Sha et al (1986) Science, 233: 478-481); phosphinothricin (DeBlock et al (1987) EMBO J., 5: 2513-2518); Spectinomycin (Bretagne-Sagnard and Chupeau (1996) Transgenic Research 5: 131-137). - The bar gene confers herbicidal resistance to glufosinate-type herbicides such as phosphinothricin (PPT) or bialaphos, and the like. As noted above, other selectable markers that can be used in vector construction, include, but are not limited to the pat gene, also for resistance to bialaphos and phosphinothricin, the ALS gene for imidazolinone resistance, the HPH gene or HYG for resistance to hygromycin, the EPSP synthase gene for glyphosate resistance, the H 1 gene for resistance to the He toxin, and other selective agents used routinely and known to a person skilled in the art. See generally, G.T. Yarranton (1992) Curr. Opin. Biotech 3: 506-511; Christopherson et al. (1992) Proc. Nati Acad. Sci. USA, 89: 6314-6318; Yao et al. (1992) Cell, 71: 63-72; Wd. Renzikoff (1992) Mol. Microbiol. , 6: 2419-2422; Barkley et al. (1980) The Operon, pp. 177-220; Hu et al. (1987) Cell, 48: 555-566; Brown et al. (1987) Cell, 49: 603-612; Figge et al. (1988) Cell, 52: 113-122; Ceuschle et al. (1989) Proc. Nati Acad. Aci. USA, 85: 5400-5404; Fuerst et al. (1989) Proc. Nati Acad. Sci. USA, 85: 2549-2553; Deuschle et al. (1990) Science, 248: 480-483; M. Goseen (1993) PhD Thesis, University of Heidelberg; Reines et al. (1993) Proc. Nati Acad. Sci. USA, 90: 1911-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Nati Acad. Sci. USA, 89: 3952-3956; Baim et al. (1991) Proc. Nati Acad. Sci. USA, 88: 5072-5076; Wyborski et al. (1991) Nuc. Acids Res., 19: 4647-4653; A. Hillenand-Wissman (1989) Topics in Mol. and Struc. Biol., 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. , 35: 1591-1595; Kelinschniclt et al. (1988) Biochemistry, 27: 1094-1104; Gatz et al. (1992) Plant J., 2: 391-404; A. L. Bonin (1993) PhD Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Nati Acad. Sci.- USA, 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. , 35: 913-919; Hlavka et al. (1985) Handbook of Exp. "" Pharmacology, 78; Gilí et al. (1988) Nature 334: 721-724. Such descriptions are incorporated herein for reference. The above list of selectable marker genes is not "limiting" Any selectable marker gene can be used in the present invention When appropriate, the selectable marker genes and other genes and nucleic acid of interest to be transferred can be synthesized for a optimal expression in sorghum, ie, the coding sequence of genes can be modified to improve expression in sorghum.Synthetic nucleic acid is designed to be expressed in transformed tissues and plants at a higher level. optimized selectable marker genes can result in superior transformation efficiency.Specific synthetic gene optimization methods are available in the art.The nuclsotide sequence can be optimized for sorghum or modified expression for optimal expression in monocotyledons. Preferred plant codons can be determined s from the highest frequency codons in the proteins expressed in sorghum. It is recognized that genes that have been optimized for expression in corn and other monocotyledons can be used in the methods of the invention. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perkal et al. (1991) Proc. Na ti. Acad. Sci. USA, 88: 3324-3328; and Murray et al. (1989) Nucleic Acids Research, 1 7: 477-498. U.S. Patent No. 5,380,831; U.S. Patent No. 5,436,391; and the like, incorporated herein for reference. It is further recognized that all or any part of the gene sequence can be optimized or synthesized. That is, fully optimized or partially optimized sequences can also be used. Additional sequence modifications are known to improve gene expression in a cellular host. These include sequence deletion encoding spurious polyadenylation signals, exon-intron cleavage signal signals, transposon-like repeats, and other well-characterized sequences, which can be dangerous for gene expression. The GC content of the sequence "can be adjusted to average levels for a given cell host, as calculated by the reference of known genes expressed in the host cell Where possible, the sequence is modified to avoid secondary mRNA structures of capillarity The methods of the invention are useful for transforming sorghum plant cells Such cells include callus which can be originated from any of the tissues of sorghum plants Preferably, the tissue used in the initiation callus is immature tissue "such as immature embryos, immature inflorescences, and the basal portion of young leaves. Alternatively, the callus can be originated from anthers, microspores, mature embryos and mainly from any sorghum tissue capable of forming callus and / or secondary embryos. A useful tissue to produce regenerable callus is the escutiform organ of mature sorghum embryos. Of particular interest, is the use of immature embryos. Such embryos can be isolated from immature palm seeds and treated for transformation. Alternatively, the embryos may be isolated and cultured for several days, generally from about 3 to about 10 days, preferably from about 5 to about 8 days., before inoculation with Agrobacterium. The method can also be used to transform "cell suspensions." Such cell suspensions can be formed from any sorghum tissue. * Immature embryos are an intact tissue that is capable of cell division to give rise to callus cells. which can then be differentiated "to produce tissues and organs of the whole plant." Immature embryos can be obtained from used reproductive organs of a mature sorghum plant Illustrative methods to isolate embryos, immature are described by Green and Phillips "(1976) Proc. Sci. 15: 411-421. See also Neuffer et al. in Maize for Biol ogi cal Research WF Sheridan (EP) University Press, University of North Dakota, Grand Forks, North Dakota, 1982, incorporated herein for "reference." Immature embryos are preferably aseptically isolated from the palm seed and developed and The immature embryos are preferably used at about two (2) days to about 20 days after pollination, more preferably from about 4 days to about 16 days after pollination and are kept in a sterile medium until they are used. more preferably from about 5 days to about 12 days after pollination. Generally, embryos exposed to Agrobacterium range in size from about 0.3 to about 4 mm, preferably from about 0.6"to about 0.3 mm, and more preferably from about 0.8 to about 1.5 mm. Agrobacterium of the invention can be divided into several stages.The basic stages include an infection stage (stage 1); a co-cultivation step (step 2), optional rest step (step 3), a step of selection (step 4); and a regeneration step (step 5). An optional pre-culture stage can be added before the infection stage. The preculture step includes culturing the immature embryos or other target tissue prior to the infection step in a suitable medium such as a N6, LSD1.5, or PHI-J medium (See Example 2). The pre-culture period may vary from about 1 to about 10, preferably from about 3 to about 7 days, more preferably from about 5 to about 6 days. Such a preculture stage was found to prevent the transformation of corn crop. See EP0672752A1.

In the infection stage, the cells that will be transformed are isolated and exposed to Agrohacterium. If the target cells are immature embryos, the embryos are isolated and the cells are contacted with a suspension of Agro acterium. As noted above, Agroba cterium has been modified to contain a gene or nucleic acid of interest. The nucleic acid is inserted into the T-DNA region of the vector. In the invention, general molecular tecues are provided, for example, by Sambrook et al. (eds) Molecular Cloning: A Labora tory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The Agrobacterium containing the plasmid of interest is preferably kept in master plates of Agrobacterium with freezing at about -80 ° C. As used herein, the term "Agroj ac erium capable of transferring at least one gene" refers to Agroba czerium that contains the gene or nucleic acid of interest, generally in a plasmid that is suitable for mediating the events required for transfer the gene to the cells that will be infected. The master plates can be used to inoculate agar plates to obtain Agrobacterium which is then resuspended in a medium to be used in the infection process. Alternatively, bacteria from the master plate can be used to inoculate broth cultures that are developed for logarithmic phase before transformation. The concentration of Agrobacterium used in the infection stage and the co-cultivation stage can affect the frequency of transformation. Also, very high concentrations of Agrobacterium can damage tissue that will be transformed, such as immature embryos, and result in a reduced callus response. In this manner, the concentration of Agrobacterium useful in the methods of the invention may vary depending on the strain of Agrobacterium used, the tissue to be transformed, the genotype of sorghum to be transformed and the like. To perfect the transformation protocol for a particular sorghum line or tissue, the tissue that will be transferred, (immature embryos, for example), can be incubated with various concentrations of Agrobacterium. Also, the level of expression of the marker gene and the transformation efficiency can be analyzed for several concentrations of Agrobacterium. Although the concentration of Agrobacterium may vary, generally a concentration range of about 1 x 10O cfu / ml to about 1 x 10 10 will be used, preferably in the range of about 1 xlO 3 cfu / ml to about 1.5 x 10A cfu / ml and even more preferably at about 0.5 x IO 9 cfu / ml at about 1.0 x 109 cfu / ml will be used. The tissue that will be transformed is usually added to the suspension of Agrobacterium in a liquid contact phase containing a concentration of Agrobacterium to optimize the transformation efficiencies. The contact phase facilitates maximum contact of the cells / tissue that will be transformed with the suspension of Agrobacterium. The cells are contacted with the suspension of Agrobacterium um for a period of at least three (3) minutes to about 15 minutes, preferably from about 4 minutes to about 10 minutes and more preferably from about 5 minutes to about 8 minutes. . The liquid contact phase of the infection step is presented in a liquid solution including the major inorganic salts and vitamins of the N6 medium referred to herein as "N6 salts" (Chu CC Proc. Symp. Plant Tissue Culture, Science Press Peking, pp. 43-50, 1987). As used herein, the medium containing "N6 salts" includes a medium containing about 400-500 mg / l of ammonium sulfate and preferably about 463.0 mg / l of ammonium sulfate; about 1.0-2.0 mg / l of boric acid and preferably about 1.6 mg / l of boric acid; about 100-140 mg / l of anhydrous calcium chloride and preferably about 125 mg / l of anhydrous calcium chloride; about 20-50 mg / l Na2-EDTA and preferably about 37.25 mg / l Na2-EDTA; about 20-40 mg / l of ferrous sulphate.7H20 and preferably about 27.8 mg / l_ of ferrous sulphate.7H20; about 80-100 mg / l of magnesium sulfate and preferably about 90.37 mg / l of magnesium sulfate; approximately 1.5-7 mg / l of magnesium sulfate. H2O and preferably about 3.33 mg / l of magnesium sulfate. H20; about 0.4-1.6 mg / l of potassium iodide and preferably about 0.8 mg / l of potassium iodide; about 1,500-3,500 mg / l of potassium nitrate and preferably about 2,830 mg / l of potassium nitrate; about 200-600 mg / l of monobasic potassium phosphate and preferably about 400 mg / l of monobasic potassium phosphate; and about 1.0-2.5 mg / L of zinc sulfate.7H20 and preferably about 1.5 mg / L of zinc sulfate.7H20. Other equivalent liquid suspensions are known in the art and can be used. See, for example, Ishida et al. (1996) Na ture Biotechnology 14: 745-750; EPA 0672752A1; EPA 0687730A1; and U.S. Patent No. 5,591,616. For example, media containing MS salts can also be used in the infection stage. The MS salts include approximately 1,650.0 mg / l of ammonium nitrate, approximately 6.2 mg / l of boric acid, approximately 332.2 mg / l of anhydrous calcium chloride, approximately 0.025 mg / l of cobalt chloride .6H20, approximately 0.025 mg / l. l of cupric sulfate. "5H20, approximately 37.26 mg / l Na2 EDTA, approximately 27.8 mg / l of ferrous sulfate .7H20, approximately 180.7 mg / l of magnesium sulfate, approximately 16.9" mg / l of manganese sulfate. H20, approximately 0.25 mg / l of molybdic acid (sodium salt) .2H20, approximately 0.83 mg / l of potassium iodide, approximately 1,900.0 mg / l of potassium nitrate, approximately 170.0 mg / l of potassium phosphate monobasic and approximately 8.6 mg / l of zinc sulfate.7H2 ?. In addition, other means such as LS and those established in the examples can be used, the macro and micro salts in the MS medium are identical to the macro and micro salts in the LS medium, but the two media differ in the composition of some of the vitamins and other components (Skirvin RM, In: Cloning Agricultural Plan ts Via In Vi tro Techniques, B. Conger, ed., CRC Press, Knoxville, Tenn., pp. 51-140, 1981). In addition, media in the infection stage generally exclude AgN03. AgNOi is generally included in the co-cultivation, resting (when used) and selection stage when the N6 medium is used.

In the co-cultivation stage, the cells that will be transferred are co-cultured with Agrobacterium. For immature embryos, co-cultivation with Agroba cteri um occurs in solid medium. The embryos are placed csn down on the solid medium and the medium can include AgN03 at a range of approximately 0.85 to 8.5 mg / l, although 0.01 to 200 mg / l can also be used. The embryos are co-cultured with Agrobacterium um of about * 1-30 days, preferably about 2-20 days and more preferably about 3-10 days. Following the co-cultivation stage, the transformed cells can be subjected to a resting stage.As noted above, the resting stage is optional.If no resting stage is used, an extended co-cultivation step can to be used to provide a culture period before the addition of a selective agent.For the resting stage, the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase is performed in the absence of any selective pressure to allow preferential inhibition and callus development of the transformed cells containing the heterologous nucleic acid. "Une" "antibiotic is added to inhibit the development of Agrobacterium um Such antibiotics are known in the art to inhibit Agrobacterium and which include cefotaxime, timetine, vancomycin, carbenicillin and the like. _ Antibiotic concentrations will vary according to With its standard for each antibiotic, for example, carbenicillin concentrations will vary from the range of about 50 mg / L to 250 mg / L of • carbenicillin in a solid medium, preferably approximately 75 mg / L to 200 mg / L, preferably about 100-125 mg / l Those skilled in the art of transforming monocotyledonous sulfate 7.H20 will recognize that the concentration of antibiotic can be optimized for a particular transformation protocol "without undue experimentation. The resting phase cultures are preferably allowed to stand in the dark, at 28 ° C for about 1 to about 15 days, preferably about 3 to about 10 days, and more preferably about 5 to about 8 days *. Any of the means known in the art will be used for the rest stage.Following the co-cultivation stage, or following the rest stage, where it is used, the transformed cells "are exposed to selective pressure to select those cells that have received and which are expressing the heterologous nucleic acid polypeptide introduced by Agrobacterium. When the cells are embryos, the embryos are transfected into plaques as a solid medium that includes both an antibiotic to inhibit the growth of Agrobacterium and a selection agent. The agent used to select the transformants will select the preferential development of explants. containing a selectable labeled insert placed within the superbinary vector and supplied by Agrobacterium. Generally, any of the means known in the art suitable for sorghum cultivation can be used in the selection step, such media containing N6 salts or "MS salts." During selection, the embryos are cultured until the formation of Normally, the calluses developed in the selection medium are allowed to grow to a size of approximately 1.5 to approximately 2 cm in diameter. "After the callus have reached the appropriate size, the calluses are cultured in a regeneration medium in darkness for several weeks, generally about 1 to 3 weeks, to allow the somatic embryos to mature. Preferred regeneration means include media containing MS salts, such as PHI-E and PHI-F media as provided in the examples. The calluses are then grown in a root medium in a light / dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art and preferred methods are Kamo et al. (Bot. Ga z 1 46 (3) -. 321 -334, 1985), West et al. (The Plan t Cell 5: 1361-1369, 1993) and Duncan et al. (Plan ta 1 65: 322-332, 1985). The small seedlings are then transfected into tubes containing a medium formed from roots and allowed to grow and grow more roots for about another week. The plants are then transplanted to a soil mix in containers in a greenhouse. Now that it has been shown that sorghum can be transformed using Agrobacterium, alterations to the general method described here can be used to increase the efficiency of transforming selected innate lines, which can exhibit some recalcitrance to the transformation. affecting the efficiency of transformation include the types and stages of infected tissues, the concentration of A. tumefacins, the composition of the means for tissue culture, the selectable marker genes, the duration of any of the steps described above, the vectors and the strains of Agroba cterium, and the genotype of sorghum, so these factors can be varied to determine which is the optimal transformation protocol for any line of sorghum, it is recognized that not every genotype will react the same as the conditions of transformation and may require a slightly different modification of the protocol. However, by altering each of the variables, an optimal protocol can be derived for any line of sorghum. Although any line of sorghum or variety can be used in the transformation methods of the invention, examples of sorghum lines include, but are not limited to, public lines such as CS3541, M91051, SRN39, Shanqui red, IS8260, IS4225, Tx430, P898012, P954035, PP290 (Casas et al., Supra) and the commercially important innate owner pioneer lines such as PH860, PH987, PHB180, PHB123 and PHB82. Other modifications may also be used including the provision of a second stage of infection to increase infection by Agrobacterium. Also, the vectors and methods of the invention can be used in combination with the bombardment of particles to produce transformed sorghum plants. Bombardment of particles can be used to increase healing in tissues that are transformed by Agroba cteri um (Bidney et al (1990) Plan t Mol. Biol. 18: 301-313; EP0486233, incorporated herein by reference). Methods for particle bombardment are well known in the art. See, for example, Sanford et al., U.S. Patent 4,945,050; McCabe et al. (1988) Biotechnol ogy, 6: 923-926). Also see, Weissinger et al. (1988) Annual Rev. Gene t. 22: 421-411; Datta et al. (1990) Biotechnol ogy, 8: 736-740; Klein et al. (1988) Proc. Na ti. Acad. Scí. USA, 85: 4305-4309; Klein et al. (1988) Biotechnology, 6: 559-563 (Corn); Klein et al. (1988) Plant Physiol, 91: 440-444; Fromm et al., (1990) Biotechnology, 8: 833-839; and Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment". In: Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods; Springer-Verlag, Berlin (1995); Hooydaas-Van Slogteren & Hooykaas (1984) Na ture (London), 311: 763-764; Bytebier et al., (1987) Proc. Na ti. Acad. Sci. USA, 84: 5345-5349; all of these are incorporated for reference herein. After the cells are cured by the bombardment of miroproyectiles, the cells are inoculated with a solution of Agrobacterium. The additional infection stage and particle bombardment can be useful to transform those sorghum genotypes which are particularly recalcitrant to Agrobacterium infection. The following examples are presented by way of illustration and not by way of limitation. "EXPERIMENTAL DATA Example 1: Construction of vectors and strains of Agroba cteri um PHP10525: All vectors were constructed using standard molecular biology techniques (Sambrook et al. eds.) supra.) A reporter gene and a selectable marker gene for expression and selection were inserted between T-DNA limiters of a superbinary vector.The report gene included the ß-glucuronidase gene * gene (GUS) (Jefferson, RA et al (1986) Proc. Na ti, Acad. Sci (USA) 83: 8447-8451) in whose coding region the second intron of the ST-LSl potato gene was inserted (Vancanneyt, et al. (1990) Mol. Gen. Genet 220: 245-250), to produce the intron-GUS, in order to avoid the expression of the gene in Agrobacterium (see Ohta, S. et al. (1990) "Cell Physiol Plan, 31 (6): 805-813." Referring to Figure 1 (a), the 2 kb fragment of the promoter region of the ubiquitin maize gene Ubi-1 (Christensen et al. (1992) Plan t Mol. Biol. 18: 675-689) with the 5 'Ba Hl site of the GUS site A fragment containing 2 bases at 310 of the terminator of the potato proteinase inhibitor (pinll) gene (An et al. (1989) Plant Cell 1: 115-122) was ligated at a shaved end downstream of the GUS coding sequence to create the GUS expression cassette The 3 'end of the terminator carries a Notl restriction site.

For the selectable marker, a Cauliflower 35S Mosaic Virus promoter with a duplicated enhancer region was created. (2X35S; bases -421 to -90 and -421 to +2 by Gardner et al (1981) Nucí Acids Res. 9: 2871-2888) with a flanking Notl 5 'site and a PstI site of 3 ' A fragment of PstI / SalI containing the tobacco virus leader 79_bp (Galliet et al (1987) Nucí Acids. Res. 15: 3257-3273) was inserted downstream of the promoter followed by the SalI / BamHI fragment containing the first intron of the corn alcohol dehydrogenase gene ADH1-S (Dennis et al., (1984) Nucí Acids Res. 12: 3983-3990). The BAR coding sequence (Thompson et al (1987) EMBO J. 5: 2519-2523) was cloned into the BamHI site with the pinll terminator ligated downstream to create the BAR expression cassette. The pinll terminator was flanked by a Salí 3 'site. The plasmid, pPHP8904 (Figure lb) was constructed by inserting the GUS expression cassette as a HindIII / NotI fragment and the BAR expression cassette as a NotI / SacI fragment between the right-to-left T-DNA limiters at the psBll sites and HindIII and Sacl. The GUS cassette was inserted near the edge of T-DNA. Plasmid psBll was obtained from Japan Tobacco Inc. (Tokyo, Japan). The construction of pSBll from psB21 and the construction of psB21 from starting vectors as described in Komari et al. (1996) Plan t J. 10: 165-174. The T-DNA of pPHP8D04 was integrated into the superbinary plasmid psBl (Saito et al., EP 672 752 Al) through homologous recombination between the two plasmids (Figure 1, psBl x pPHP8904). Plasmid pSBl was also obtained from Japan Tobacco, Inc. The strain of E. col i HB101 containing pPHP8904 bound with the Agroba cteri um strain LBA4404 harboring pSBl to create the cointegrated plasmid in Agrobacteriiim, designed as LBA4404 (pPHP10525) as shown in Figure 1, using the method of Ditta et al. (1980) Proc. Na ti. Acad. Sci. USA 77: 7347-7351. LBA4404 (pPHP10525) was selected based on the resistance of the transformed Agroba cterium to spectinomycin and verified as a recombinant by **** a restriction digestion of the plasmid. PHP11264: PHP11264 was basically built as described above. One difference observed is the selectable marker gene. For the selectable marker, a corn ubiquitin promoter (UBI) (Christensen et al. (1992) Plant Mol. Biol. 18: 675-689) was used, with 5 'added of the BAR gene. The UBI intron was inserted downstream of the promoter. The BAR coding sequence was cloned from the UBI intron stream (Thompson et al (1984) Nuc.Aids Res. 12: 3983-3990). A fragment containing phase 2 to 310 of the terminator of the potato proteinase inhibitor (pinll) gene (An et al. (1989) Plan t Cell 1: 115-122) was ligated downstream of the BAR coding sequence to create the expression cassette of BAR. See Figure 2. Example 2: Transformation of a drought resistant sorghum crop P898012. Preparation of the Agrobacterium suspension: Individual colonies of species were selected and traced sequentially at least three times to ensure the purity of the Agrobacterium strain. From the final scored plates, a single colony was selected and used to start a liquid culture. After the development of stationary phase, the liquid culture was used to make glycerol supply materials and minipreparation of plasmid DNA. Sambrook et al., Supra. The resulting plasmid DNA was digested with SalI to verify the co-integrated LBA4404 (pPHPl0525). The glycerol supply materials were stored at -80 ° C and used as a source for master plates. The Agroba cterium was grated from an aliquot frozen at -80 ° C on a plate containing the PHI-L medium and cultured at 28 ° C in the dark for 3 days. The PHI-L medium comprised 25 ml / 1 of the Supply Solution A, 25 ml / 1 of the Supply B, 450.0 ml / 1 of the Supply C, and spectinomycin (Sigma Chemicals) added at a concentration of 50 mg / l in sterile ddHO (supply solution A; K2HP04 60.0 g / 1, "NaH2P04 20.0 g / 1, pH adjustment at 7.0 w / KOH and autoclave, supply solution B: NH4C1 20.0 g / 1, MgSO4.7H20 6.0 g / 1, KCl 3.0 g / 1, CaCl2 0.20 g / 1, FeS04.7H20 50.0 mg / l, autoclave, supply solution C: glucose 5.56 g / 1, agar 16.67 g / 1 (* A-7049, Sigma Chemicals, St. Louis, MO) and autoclave). The plate "can be stored at 4 ° C and was usually used for about 1 month. Several colonies were collected from the master plate and scratched on the plate containing the PHI-M medium [yeast extract (Difco) 5.0 g / 1 / peptone (Difco) 10.0 g / 1; I was born 5.0 g / 1; agar (Difco) 15.0 g / 1; pH 6.8, containing 50 mg / L of spectinomycin] and incubated at 28 ° C in the dark for 2 days. - Five ml of PHl- [100 ml / 1 of a 10X solution of macronutrients N6 (463.0 mg / l (463.0 mg / l (NH2) 2S04, 400.0 mg / l KH2P04, 125.33 mg / l CaCl2, 90.37 mg / l MgSO4, and 2,830.0 mg / l KN03), 2.44 mg / l boric acid, 37.1 mg / l Na2-EDTA.2_H20, 27.88 mg / l FeS04.7H20, 7.33 mg / l MnS0, .H20, 0.77 mg / l Kl, 0.6 mg / l ZnS04.7H20, 0.15 mg / l Na2Mo02.2H20, 1.68 g / 1 KN03, 0.8 mg / l glycine, 3.2 mg / l nicotinic acid, 3.2 mg / l pyridoxine, HCl, 3.4 mg / l Thiamine HCl, 0.6 g / 1 Myo-inositol, 0.8 mg / l 2, 4-D, 1.2 mg / l Dicamba (Sigma), 1.98 g / 1 L-proline, 0 3 g / 1 casein hydrolyzate, 68.5 g / 1 sucrose and 36.0 g / 1 glucose, pH 5.2] or PHI-I [MS salts (GIBCO BRL) 4.3 g / 1, nicotinic acid (Sigma) 0.5 mg / l, pyridoxine, HCl (Sigma) 0.5 mg / l; thiamine HCl 1.0 mg / l; myo-inositol (; Sigma) 0.10 g / 1; casamino vitamin test acids (Difco Lab) 1.0 g / 1; 2, 4-D 1.5 mg / l; sucrose 68.50 g / 1, glucose 36.0 g / 1, adjust pH to 5.2 w / KOH and sterilize by filtering] and 5 μl of 100 mM _ (3 ', 5' - Tell me toxi-4 '-hydroxyacetofonone, Aldrich, Chemicals) were added to a 14 ml Falcon tube in a cover. Approximately 3 complete loops (5 mm loop size) Agroba cterium were collected from a plate and suspended in the tube, then the tube was stirred to make a uniform suspension. One ml of the suspension was transferred to a spectrophotometer tube and the OD of the suspension was adjusted to 0.72 at 550 nm by adding either more Agrobaterium or more of the same suspension medium. The concentration of Agrobacterium was approximately 1 x 109 cfu / ml. The final suspension of Agroba cterium was formed in aliquots in 2 ml microcentrifuge tubes, each containing 1 ml of the suspension. The suspensions were then used as quickly as possible. Isolation of the embryo, infection and co-cultivation: Immature embryos of sorghum were isolated with a size of approximately 0.8 to 1.5 mm of sterilized immature palm seeds from the sorghum line P898012, a drought-resistant sorghum culture obtained from Purdue University . The immature palm seeds were removed from a sorghum head and placed in a glass vessel with autoclave. These immature palm seeds were immersed in 50% bleach and 0.1% Tween 20 in this vessel and the vessel was shaken very well to allow the solution to cover all the palm kernels and reach any interior part of the vessel. Vacuum was applied for 10 minutes and rinsed with autoclaved distilled distilled water, twice on a cover and these palm seeds were kept in sterile water until they were used. The immature embryos were isolated from sterilized palm seeds using a sterile spatula (Baxter Scientific Products S1565). The isolated embryos were cultured on a PHI-J medium without acetosyringone in the dark at approximately 25 ° C for 5 days and these "pre-cultured embryos were inoculated with a suspension of 109 cfu / ml Agrobacterium.The Agrobacterium was suspended in a medium PHI-G: Approximately 1 ml of the Agroba cterium suspension was added to approximately 100 isolated embryos in a sterile tube and the tube was vortexed for 30 seconds.The tube was allowed to stand for 5 minutes on the cover. cterium and the embryos were emptied into a Petri dish containing the PHI-J medium [Sales MS 4.3 g / 1; nicotinic acid 0.50 mg / l; nicotinic acid 0.50 mg / l; pyridoxine HCl 0.50 mg / l; thiamine. HCl 1.0 mg / l; myo-inositol 100.0 mg / l; 2, 4-D 1.5 mg / l; sucrose 20.0 g / 1; glucose 10. "0 g / 1; L-proline 0.70 g / 1; MES (Sigma) 0.50 g / 1; 8.0 g / 1 agar (Sigma A-7049, purified) and 100 μM acetosyringone" with a final pH of 5.8. The PHI-B medium was also used. The PHI-B medium: [CHU (N6) basal salts (Sigma C-1416) 4.0 g / 1; Mixture of vitamins Eriksson (1000X, Sigma-1511) 1.0 ml / 1; thiamine. HCl 0.5 mg / l; 2.4-D 1.5 mg / l; L-proline 0.69 g / 1; silver nitrate 0.85 mg / l; gelrite (Sigma) 3.0 g / 1; sucrose 30.0 g / 1; acetosyringone 100 μM; pH 5.8]. Any of the embryos left in the tube were transferred to the plate using a sterile spatula. The suspension of Agroba cterium was removed and the embryos were placed with the lateral axis down on the medium.The plate was sealed with a Parafilm tape or a product of Pylon Vegetative Combination Tape called "EG" .CUT "and available in sections of 18 mm x 50 m; Kyowa Ltd., Japan), and incubated in the dark at approximately 25 ° C for 5 more days of co-cultivation Rest or Rest Stage: No resting or resting step was used Selection and Regeneration Steps: For selection, all the embryos were then transferred to new plates containing PHI-J medium without glucose and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of phosphinothricin (PP) as a selection medium, placing approximately 20 embryos on each plate The plates were sealed as described above and incubated in the dark at 25 ° C for the first two weeks of selection.The embryos were then transferred to a fresh selection medium at the end of the two weeks. After a period of another 2 weeks of culture, these embryos were subcultured in the same medium of PHI-J, except that the PPT concentration was increased from 5 mg / L to 10 mg / L. were transferred to a fresh selection medium containing 10 mg / l of PPT at 3-week intervals continuing at 25 ° C in the dark. The tissue was subcultured by transferring to fresh selection medium for a total of about 3.5 months to obtain callus resistant to herbicides. For regeneration, the callus was then cultured in a PHI-E medium [MS salts 4.3 g / 1; myo-inositol 0.1 g / 1; nicotinic acid 0.5 mg / l; thiamine. HCl 0.1 mg / l; Pyridoxine HCl 0.5 mg / l; Glycine 2.0 mg / l; Zeatin 0.5 mg / l; sucrose 60.0 g / 1; Agar (Sigma, A-7049) 8.0 g / 1; indoleacetic acid (IAA, Sigma) 1.0 mg / l; abscisic acid (ABA, Sigma) 0.1 μM; PPT 10 mg / l; carbenicillin 100 mg / l adjusted to pH 5.6] in the dark at 28 ° C for 1-3 weeks to allow maturation of all somatic embryos. The callus was then cultured in PHI-F medium [MS salts 4.3 g / 1; myo-inositol 0.1 g / 1; Thiamine. HCl 01. mg / l; Pyridoxine HCl 0.5 mg / l; Glycine 2.0 mg / l; nicotinic acid 0.5 mg / l; sucrose 40.0 gl; gelrite 1.5 g / 1; pH 5.6] at 25 ° C under a daylight program of 16 hours of light (270 uE m-2 sec.-1) and 8 hours of darkness until buds and roots developed. Each small seedling was then transferred to a 25 x 150 mm tube containing the PHI-F medium and developed under the same conditions for approximately another week.The plants were transplanted into containers with a mixture of soil in a greenhouse. GUS + events in the callus stage or regenerated plant stage Confirmation of stable transformation: 25 T0 plants were regenerated from the callus and grown in the greenhouse Five plants were selected at random for the PCR assay for the presence of the BAR and GUS genes The PCR result confirmed that the 5 plants were stably transformed with Agroba cteri um The 25 plants, as well as the untransformed control plants, were painted with a 1 'solution of the Liberty hcide in their leaves to verify resistance to the hcide due to the expression of the BAR gene All the 25 regenerated plants were resistant to Liberty, while the The control plants exhibited sensitivity to Liberty. Example 3: Optimization of the transformation process for particular sorghum lines.

Using the methods detailed above in Examples 1 and 2, the transformation protocols can be optimized for any genotype of sorghum. To demonstrate, you can test the following established protocol groups for particular parameters for a transformation efficiency effect. It is recognized that other protocols and other proven sorghum lines can be formulated. The following is an illustration of the stages to optimize the transformation. Materials: Agrobacterium strain: LBA4404 (PHP11264) (UBI-UBI intron-Bar-PinlI) (Figure 2). Lines of sorghum: P898012 PH391 (a selected pioneer species). Treatments: The following table illustrates a number of treatments that will indicate which factors are important for the efficient transformation of the two illustrative sorghum lines.

Table 1 Treatment Conditions Detailed procedure of these experiments: The following is a brief description of the treatments established in Table 1. Generally, the treatment protocols will be carried out for each of the sorghum lines and will follow the details given in the Examples 1 and 2 with the following differences. Treatment -1: Fresh immature sorghum embryos of 0.8 to 1.5 mm were isolated from sterilized immature palm seeds. The isolated embryos were inoculated with 109 cfu / ml of a suspension of Agrobacterium um (Agrobacterium um suspended in PHI-I medium) and co-cultivated with Agrobacterium in PHI-J medium plus 10 mg / l of ascorbic acid at 25 ° C. ° C in the dark for about 3 days. After co-cultivation, the embryos were transferred to PHI-J medium plus 10 mg / l of ascorbic acid without glucose and acetosyringone for approximately 4 days for a resting or rest period. After the resting or resting stage, all the embryos were then transferred to new plates containing the PHI-J medium plus 10 mg / l ascorbic acid without glucose and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or _ 1.5 mg / l of Bialafos as a means of selection. After a culture period of 2 weeks, the embryos were "subcultured in the same medium of PHI-J except that PPT was increased from 5 mg / l to 10 mg / l or Bialafos was increased from 1.5 mg / l to 3 mg / l. The selection procedure was similar to that used in Example 2, except that both PPT and Bialafos could be used as the selective agents.After recovering growth and development, the callus was moved to PHI-E. then they were grown on a PHI-F medium under a daylight program during 16 hours of light (270 uE m ~ 2segd1) and 8 hours of darkness until buds and roots developed. Each small seedling was then transferred to a 25x150 mm tube containing the PHI-F medium and developed under the same conditions for approximately another week. The plants were transplanted into containers with a mixture of soil in a greenhouse. The stable transformation was confirmed by DNA analysis and herbicide resistance. Treatment -2: Immature embryos of fresh sorghum from 0.8 to 1.5 mm of sterilized immature palm seeds were isolated. The isolated embryos were inoculated with a suspension of 109 cfu / ml Agrobacterium. (Agroba cterium suspended in a PHI-I medium) and co-cultivated with Agrobacterium um in PHI-J medium plus 10 mg / l ascorbic acid at 25 ° C in the dark for approximately 7 days. No resting or resting stage was applied. After cultivation, all embryos were transferred to new plates containing the PHI-J medium plus 10 mg / l of ascorbic acid without glucose and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or 1.5 mg of Bialafos as a means of selection. The selection procedure was the same as that used in Treatment 1. After recovering the callus with good development, the callus was moved to PHI-F. The calluses were then cultured in a PHI-F medium under a daylight program for 16 hours of light (270 uE m_2sec) and 8 hours of darkness until buds and roots developed. Each small seedling was then transferred to a 25x150 mm tube containing the PHI-F medium and developed under the same conditions for another week. The plants were transplanted into containers with a mixture of soil in a greenhouse. The stable transformation was confirmed through DNA analysis and herbicidal resistance. Treatment -3: Mature embryos of fresh sorghum were isolated 0.8 to 1.5 mm from sterilized immature palm seeds. The isolated embryos were inoculated with a suspension of 106 to 108 cfu / ml Agroba cteri um. { Agroba c terium suspended in the PHI-I medium) and were co-cultured in Agrobacterium um in a medium of PHI-J plus 10 mg / l of ascorbic acid at 25 ° C in the dark for approximately 3 days. The embryos were then subjected to a second stage of infection. The embryos were inoculated with a suspension of 108 to 109 cfu / ml of Agrobacterium um (Agrobacterium suspended in PHI-I medium) and cultured with Agrobacterium in PHI-J medium plus 10 mg / l of ascorbic acid for 4 days. more at 25 ° C in the dark. No resting or resting stage was applied and the selection started immediately after co-cultivation. All embryos were then transferred to new plates containing the PHI-J medium plus 10 mg / l of ascorbic acid without glucose, and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or 1.5 mg / l of Bialafos as a means of selection. The selection procedure was the same as that used in Treatment 1. After recovering the callus with good development, the callus was moved to PHI-E. The calluses were then cultured in a medium of PHI-F under a daylight program for 16 hours of light (270 uE pf2 sec "1) and 8 hours of darkness until buds and roots developed. transferred to a 25x150 mm tube containing the PHI-F medium and developed under the same conditions for approximately another week.The plants that were transplanted to containers with a mixture of soil in a greenhouse.The stable transformation was confirmed through analysis of DNA and herbicide resistance Treatment -4: Fresh immature sorghum embryos of 0.8 to 1.5 mm of sterilized immature palm seeds were isolated.The isolated embryos were cultured in a medium of PHI-J plus 10 mg / l ascorbic acid at 25 ° C for approximately 7 days Pre-cultured embryos were inoculated with a suspension of 109 cfu / ml of Agrobacterium (Agrobacterium suspended in PHI-I medium) and cultured in the medium PHI-J plus 10 mg / l ascorbic acid at 25 ° C in the dark for approximately 7 days. No resting or resting stage was applied and the selection started immediately after co-cultivation. All embryos were then transferred to new plates containing the PHI-J medium plus 10 mg / l of ascorbic acid without glucose and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or 1.5 mg / l of Bialafos , as a means of selection. The selection procedure was the same as that used in Treatment 1. After a well-developed callus was recovered, the callus was moved to PHI-E. The calluses were then cultured on a PHI-F medium under a light program. day for 16 hours ele light (270 uE pds-egd1) and 8 hours of darkness until buds and roots developed. Each small seedling was then transferred to a 25 x 150 mm tube containing PHI-F medium and developed under the same conditions for approximately another week. The plants were transpired to containers with a mixture of soil in a greenhouse. The stable transformation was confirmed through DNA analysis and herbicide resistance.

Treatment -5: Immature embryos of fresh sorghum 0.8 to 1.5 mm were isolated from sterilized immature palm seeds. The isolated embryos were cultured in a PHI-J medium at 10 mg / l ascorbic acid at 25 ° C for about 7 days. The pre-cultured embryos were bombarded with gold or tungsten particles at 200 μg of Hg to increase healing. The bombarded embryos were inoculated with a suspension of 109 cfu / ml of Agrobacterium um (Agrobacterium suspended in the PHI-I medium) immediately after the bombardment and were combined in a PHI-J medium plus 10 mg / l ascorbic acid. at 25 ° C in the • darkness for about 7 days. No resting stage was applied and the selection started immediately after co-cultivation. All * the embryos were transferred to new plates containing the PHI-J medium plus 10 mg / l of ascorbic acid without glucose and acetosyringone, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or 1.5 mg / l of Bialafos as a means of selection. The selection procedure was the same as that used in Treatment 1. After recovering the well-developed callus, the callus was moved to PHI-E. The calluses were then cultured in a PHI-F medium under a daylight program of 16 hours of light (270 uE pf2sec_i) and 8 hours of darkness that developed buds and roots. Each small seedling was transferred to a 25 x 150 mm tube containing the PHI-F medium and developed under the same conditions for approximately another week. The plants were transported to containers with soil mix in a greenhouse, stable transformation was confirmed through DNA analysis and herbicide resistance Treatment -6: To provide a negative control, immature embryos of fresh sorghum were isolated. 0.8 to 1.5 mm of sterilized immature palm seeds No infection or co-cultivation with Agroba cterium was used The embryos were cultured in PHI-J 2 plus 10 mg / l of ascorbic acid without glucose and acetosyringone at 25 ° C in the darkness, but adding 100 mg / l of carbenicillin and 5 mg / l of PPT or 1.5 mg / l of Bialafos, no callus growth was expected.Analysis of Results: The "regenerated T0 plants were analyzed through Southern methods. or PCR to confirm stable transformation. In addition, the plants were painted with the Liberty herbicide to verify the expression of the BAR gene. The generation of IT was analyzed to confirm the transmission of the transgene. The following comparison determines the specific nature of this technology. The results of the treatments will provide a guide to the factors that are important for the transformation of the sorghum lines to be tested. Similar methods can be used to efficiently transform any line of sorghum. A comparison of the data to * from: Treatments 1 and 2 will indicate the importance of the rest or rest phase in the transformation; Treatments 2 and 3 indicate if a second infection increases the transformation; Treatments 2 _ and 4 indicate the importance of pre-culture of isolated embryos; and Treatments 4 and 5 indicate whether the bombardment can improve the transformation efficiency with Agrobacterium. Using the methods of the invention, any line of sorghum can be stably transformed. The transformed plants of the invention are an improvement over the transformed sorghum plants produced by bombardment. In contrast to the plants produced by bombing, the plants of the invention contain a transferred nucleic acid which does not undergo any major rearrangement. In addition, the transferred nucleic acid is integrated into the genome at low copy numbers, usually in an individual copy. Example 4: Transformation results P898012 was used as a model line to test the treatment conditions set forth in Example 3. The embryos were harvested, cultured and transformed as described in Treatments 1-6, respectively. The transformation frequency for each treatment is established in Table 2. Table 2 Transformation Results "? 'G = embryos and greenhouse The data showed that for P89012, stable transgenic events were easily produced with treatments -1, treatment -2 and treatment -4. In treatment -1, 21 stable events of 1,392 embryos were generated in 12 experiments and the average transformation frequency was 1.5%, varying from 0.6-6.6% In treatment -2, 19 events of 1,399 embryos were generated in 13 experiments and the average frequency was 1.4%, varying from 0.8-4.3 In treatment -4, eight events of 641 embryos were generated in 6 experiments and the average frequency was 1.2%, varying from 0.4-2.7%, however, in experimental practice, treatment 2 is preferred since it is a simplified procedure, the embryos could not have been moved in different ways, in the treatment -4, a 3-day preculture period was tested ", 4 days, 5 days and 7 days. The transformation frequencies were of the same scale in these different days culture treatments. The data from these different pre-cultures of days were combined together to represent the results of treatment 4. An event of 442 embryos occurred in treatment 5. The bombardment (treatment -5) actually reduced the transformation frequency of 1.2% (treatment -4) to 0.2% (treatment -5). The preculture for 5 days and 7 days was tested in this treatment and the data were combined together since no obvious difference could be observed in these two pre-culture conditions. "_ No event occurred in infection control without Agrobacterium (treatment 6) Example 5: Additional optimizations Using P898012, additional optimization of the transformation conditions was tested Some conditions such as the use of an antioxidant and The source of sorghum embryos was tested. "The results indicate that an antioxidant and the source of embryos (conditions of development of the sorghum plant) played an important role in the transformation efficiency of sorghum. "Antioxidant: Antioxidants have been used in Agrobacterium-mediated transformations in at least one plant in the technique.The results indicated that the antioxidants were very" critical for the Agrobacterium-mediated transformation of grapes (Vi tis vinifera L.) (Avihai Peri et al. al. (1996) Na ture Biotechnology 14: 624-628). To test the role of antioxylators in sorghum, the protocol of treatment 2 was followed with the addition of 1% PVPP (polyvinylpolypyrrolidone) in the co-cultivation medium (710P).

The results are given in Table 3. Table 3 Example 6: Transformation of PH391: The preferred conditions of Example 4 were repeated using the sorghum developed in the PH391 field. PH391 is a line chosen in the sorghum cultivation program. The transformation methodologies developed using line model P898012 were also tested in PH391 to verify the common application of these methodologies in the general transformation of sorghum. The results are presented in Table 4.

Table 4 All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference, to the same degree as if each publication or individual patent application was specifically and individually indicated to be incorporated for reference. Although the above invention has been described in detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (43)

1. A method for transforming sorghum with a nucleotide sequence of interest, the method is characterized in that it comprises the steps of: contacting tissue from a sorghum plant with

Agrobacterium comprising a vector comprising the nucleotide sequence, wherein the nucleotide sequence comprises at least one expression cassette comprising a gene that confers resistance to a selection agent; co-culturing the tissue with Agrobacterium in a concentration of approximately lxlO3 cfu / ml to approximately lx1010 cfu / ml; culturing the tissue in a medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium and the selection agent; regenerate transformed sorghum plants.

2. The method according to claim 1, characterized in that the tissue is immature embryos.

3. The method according to claim 2, characterized in that it further comprises the step of cultivating embryos for several days before the co-cultivation step.

4. The method according to claim 1, characterized in that the vector is a superbinary vector.

5. The method according to claim 4, characterized in that the concentration of Agrobacterium ranges from about 1 x 103 cfu / ml to about 1.5 x 109 cfü / ml.

6. The method of compliance with the claim

1, characterized in that the contact passage is presented in a liquid suspension.

The method according to claim 1, characterized in that the co-cultivation step is presented in a solid medium.

The method according to claim 1, characterized in that it also comprises a resting stage after the co-cultivation step.

The method according to claim 8, characterized in that the rest or rest step comprises culturing the tissue in a medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium.

The method according to claim 1, characterized in that the antibiotic is selected from the group consisting of cefotaxime, timetine, vancomycin and carbenicillin. 11. "The method according to claim 10, characterized in that the antibiotic is carbenicillin 12.

The method according to claim 11, characterized in that the concentration of carbenicillin in the selection step is approximately 50 mg / la approximately 200. mg / l 13.

The method according to claim 1, characterized in that the gene that confers resistance to a selection agent is selected from the group consisting of bar, pat, ALS, HPH, HYG, EPSP, and Hml.

The method according to claim 13, characterized in that the gene that confers resistance to a selection agent is bar

15. The method according to claim

1, characterized in that the expression cassette comprises a second gene of interest.

16. A method for transforming sorghum with a nucleotide sequence, the method is characterized in that it comprises the steps of: contacting a tissue of a sorghum plant with an Agrobacterium comprising a vector comprising the nucleotide sequence, wherein the nucleotide sequence comprises at least one expression cassette comprising a gene that confers resistance to a selection agent; co-culture the tissue with Agrobacterium; culturing the tissue in a medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium and the selection agent "regenerating transformed sorghum plants.

17. The method of compliance with the claim

16, characterized in that the tissue is immature embryos.

18. The method of compliance with the claim

17, characterized in that it further comprises the step of cultivating embryos for several days before the co-cultivation step. The method according to claim 16, characterized in that the superbinary vector is selected from PHP11264 and "PHP10525."

20. The method according to claim 19, characterized in that the concentration of Agroba cterium ranges from approximately 1 x 103 cfu / ml to approximately 1.5 x 109 cfu / ml.

21. The method according to claim 16, characterized in that the contact passage is presented in a liquid suspension 22.

The method according to claim 16, characterized in that the co-cultivation step is presented in a medium solid. -231 The method of compliance with the claim

16, characterized in that it also comprises a resting stage after the co-cultivation step.

24. The method according to claim 23, characterized in that the rest or rest step comprises culturing the tissue in a medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium.

25. The method according to claim 16, characterized in that the antibiotic is selected from the group consisting of cefotaxime, tymmethine, vancomycin and carbenicillin.

26. The method according to claim 25, characterized in that the antibiotic is carbenicillin. * =

27. The method according to claim 26, characterized in that the concentration of carbenicillin in the selection step is from about 50 mg / l to about 200 mg / l.

28. The method according to claim 16, characterized in that the gene that confers resistance to a selection agent is selected from the group consisting of bar, pat, ALS, HPH, HYG, EPSP, and Hml.

29. The method according to claim 28, characterized in that the gene which confers resistance to a selection agent is bar.

30. The method of compliance with the claim

16, characterized in that the expression cassette comprises a second gene of interest.

31. A transformed sorghum plant, such plant comprises less than 5 copies of nucleic acid of interest flanked by at least "one T-DNA limitation sequence incorporated into its genome.

32. The sorghum plant according to claim 31, characterized in that the plant comprises less than 3 copies of the nucleic acid sequence.

33. The sorghum plant according to claim 31, characterized in that the nucleic acid comprises at least one expression cassette comprising a gene that confers resistance to a selection agent. 3 .

The sorghum plant according to claim 33, characterized in that the selection agent is selected from the group consisting of bar, pat, ALS, HPH, HYG, EPSP, and Hml.

35. The sorghum plant according to claim 34, characterized in that the gene that confers resistance to a selection agent is bar.

36. The sorghum plant according to claim 33, characterized in that the nucleic acid comprises a second expression cassette comprising a gene of interest.

37. The transformed seed of the plant according to claim 31.

38. The transformed seed of the plant according to claim 32.

39. The transformed seed of the plant according to claim 36.

40. A transformed plant cell, the cell is characterized in that it comprises less than 5 copies of a nucleic acid of interest flanked by at least one T-DNA limitation sequence incorporated. in its genome, where the plant is sorghum.

41. The plant cell according to claim 40, characterized in that the nucleic acid comprises at least one expression cassette comprising a gene that confers resistance to a selection agent.

42. A tissue of transformed plant, the tissue is characterized in that it comprises less than 5 copies of a nucleic acid of interest flanked by at least one T-DNA limitation sequence incorporated in the genome of cells of such tissue, wherein the plant e's sorghum

43. The plant tissue according to claim 42, characterized in that the nucleic acid comprises at least one expression cassette comprising a gene that confers resistance to a selection agent.