MXPA00001464A - Genetically engineered duckweed - Google Patents

Abstract

Methods and compositions for the efficient transformation of duckweed are provided. Preferably, the methods involve transformation by either ballistic bombardment or Agrobacterium. In this manner, any gene or nucleic acid of interest can be introduced and expressed in duckweed plants. Transformed duckweed plants, cells, tissues are also provided. Transformed duckweed plant tissue culture and methods of producing recombinant proteins and peptides from transformed duckweed plants are also disclosed.

Description

LENTIL AQUATIC MAHtf > ULADA GENETICALLY This invention was made with governmental support under grant number R823570-01-1 from the United States Environmental Protection Agency. The government has certain rights over this invention.

FIELD OF THE INVENTION The present invention relates to methods and compositions for the transformation of aquatic lentil, particularly to transformation methods using ballistic bombardment and Agrobacterium.

BACKGROUND OF THE INVENTION Aquatic lentils are the only members of the monocot family, Lemnaceae. The four genera and 34 species are freshwater plants, free-floating and small, whose geographical scale covers the entire planet. Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institution ETH, Stiftung Rubel, Zurich (1986). Although they are the most morphologically reduced plants known, most aquatic lentil species have all the tissues and organs of much larger plants, including roots, stems, flowers, seeds and fronds. The species of aquatic lentil have been studied exhaustively, and there is a substantial literature regarding its ecology, systematics, life cycle, metabolism, susceptibility to diseases and pests, its reproductive biology, genetic structure and cellular biology. Hillman, Bot. Review 27, 221 (1961); Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institution ETH, Stiftung Rubel, Zurich (1986). The growth habit of aquatic lentils is ideal for microbial cullive methods. Pineapple proliferates rapidly through vegetative budding of new fronds, in a macroscopic form analogous to asexual propagation in yeasts. The aquatic lentil proliferates through vegetative budding from mehsiemaic cells. The Merisiemaic region is small and is found on the surface of the foliage. The merisiemálicas cells lie in two cavities, one on each side of the vein of the frond. The small region of the rib is also the site from which the root originates and the stem arises, which binds each frond to its mother leaf. The meristematic cavity is protected by a tissue cover. The fronds gel alíernalivameníe from those cavities. The periods of duplication vary with the species, and are brief as 20 to 24 hours. Landolt, Ber. Schweiz. Boí. Ges. 67, 271 (1957); Chang went to., Bull. Insí. Chem. Acad. Without. 24, 19 (1977); Daí o y Mudd., Plant Physiol. 65, 16 (1980); Venkaiaraman et al., Z. Pflanzenphysiol. 62, 316 (1970). The inlensive culíivo of the aquáíica lenleja results in the most alias rates of biomass accumulation per unit of time. Landolí and Kandeler, The family of Lemnaceae - A Monographic Síudy. Vol 2: Phyclochemislry, Physiology, Application, Bibliography, Veroffenllichungen des Geoboianischen Insíiíuíes ETH, Siflung Rubel, Zurích (1987)), varying the accumulation of dry weight from 6 to 10% of fresh weight (Tillberg et al., Physiol. Plañí. 46, 5 (1979), Landoll, Ber Schweiz, Bol. Ges. 67, 271 (1957), Stomp, unpublished data). It has been reported that the protein content of a number of aquatic lentil species grown under variable conditions ranges from 15 to 45% by dry weight (Chang et al, Bull, Inst. Chem. Acad. Sin. 24, 19 (1977) Chang and Chui, Z. Pflanzenphysiol., 89, 91 (1978), Poralh et al., Aquaíic Boíany 7, 272 (1979), Appenrolh et al., Biochem. Physiol. Pflanz., 177, 251 (1982)). Using these values, the level of prolein production per liter of medium in the aquatic lentil is of the same magnitude as the expression systems of genes in yeast. Hasfa now, the systematic optimization of the components of the medium and the culture conditions for the growth and coninido of maximum proleínas for specific strains of aquatic lentil has not been done. The sexual reproduction in the aquilic lentil is controlled by the components of the medium and the culíivo conditions, including foioperiod and density of culíivo. Floral induction is a procedure ^ Afeia colidiano laboratory in the case in some species. The plañías normally are auíopolinizan, and self-pollination can be achieved in the laboratory by gently stirring the culfivos. Through this method, inbred lines of Lemna gibba have been developed. Five spontaneous mutations have been idenified (Slovin and Cohen, Plañí Physiol., 86, 522 (1988)), and chemical and gamma-ray mulagenesis (using EMS or NMU) has been used to produce mulanides with defined characteres. The exogamy of L. gibba is tedious, but it can be carried out by manual pollination with conyrolate. The size of the genome of the lentils acálicas varies from 0.25-1.63 pg of DNA / 2C, varying the chromosome counts from 20 to 80, and averaging approximately 40 from the Lemnaceae (Landolí, Biosysiemaíic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Síudy. Geobalanischen Insituit ETH, Stiftung Rubel, Zurich (1986)). It is estimated that ploidy levels vary from 2-12 C.

Id .. The deníro genetic diversity of the Lemnaceae has been investigated using secondary producís, isozymes and DNA sequences. McClure and Alslon, Naíure 4916, 311 (1964); McClure and Alsíon, Amer. J. Bol. 53, 849 (1966); Vasseur el al., Pl. Syst. Evol. 177, 139 (1991); Crawford and Landolt, Syst. Bot. 10, 389 (1993). 20 Therefore, the characteristics described above make the lentil aquatic an ideal choice to develop an efficient and plane-based gene expression system. rft * f »^ - • * BRIEF DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for efficient transformation of the aquatic lens. The methods involve the use of ballistic bombardment, Agrobacterium or elecroporation to produce and expressively express a sequence of nucleolides of interest in aquaculture lentils. In this way, any gene or nucleic acid of interest can be intro- duced in the watery lens. Cells, tissues, plañís, and seeds of recycled lenses are supplied. In a first aspect, the present invention provides a method for transforming aquatic lentil with a nucleotide sequence of interest, wherein said nucleotide sequence comprises at least one expression cassette containing a gene that confers resistance to a selection agent. , the method comprising the steps of: a) providing an objelic tissue of aquatic lens, the cells of the aquatic lentil tissue including cell walls; and b) boosting the nucleotide sequence in the target tissue of the aquatic lens at a rate sufficient to perforate the cell walls and depositing the nucleotide sequence within a cell of the molecule to thereby produce an ransformed cell, wherein the sequence of nucleolides is carried by a microprojectile; and wherein the nucleotide sequence is driven into the tissue by driving the microprojectile therein.

As a second aspect, the present invention provides a method for transforming the aquatic lens with a nucleotide sequence of interest, the method comprising the steps of: a) inoculating an acidic leaflet with Agrobacterium comprising a vector comprising the nucleotide sequence , wherein the nucleotide sequence comprises at least one expression cassette containing a gene that confers resistance to a selection agent; and b) cocultivate the product with Agrobacterium to produce transformed tissue. As a third aspect, the present invention provides a method for transforming the aquatic lens by electroporation. As a fourth aspect, the present invention provides transformed aquatic lentil pads and transformed aquatic lentil tissue cultures produced by the methods described above. As a fifth aspect, the present invention provides a translucent aquatic leaflet, and methods for using aquatic leaflets transformed to produce a recombinant prolein or peptide. Aquatic legume offers an ideal plant-based gene expression system. A system of expression of genes of aquiline lenleja provides the basic technology that would be useful for a number of commercial and research applications. For research in molecular biology in plañías like a mud, a system of differentiated plants that can be manipulated with the convenience of the yeasts in the laboratory, provides a very fast system in which the physiological functions and the development of genesífajsteeos can be analyzed. Model plants such as the abacus and Arabidopsis are currently used for this purpose by molecular biologists of plañías. These plants require greenhouse or field facilities to grow (often difficult to obtain by plague molecular biologists). Aliemative systems of gene expression are based on cell counts or microbial cultures, where the effects of tissue-regulated gene expression and development are lost. Helerologous gene expression systems also require re-glycerin the gene of interest before insertion, a coszy, time-consuming procedure. A system of aquiline lenses overcomes these problems, and is much easier to develop and maintain in a laboratory. If it is desired to harvest the proteins or the expressed peptides (or the molecules produced by them), this can be achieved by any suitable technique known in the art, such as mechanical grinding or cell lysis. For the commercial production of valuable proteins, a system based on aquatic lentil has numerous advantages over existing tissue or microbial culturing systems. In the area of mammalian mammalian production, plañís sample post-translational processing, which is similar to that of mammalian cells, overcoming a major problem associated with the production of mammalian proteins in microbial cells. The aquatic lens is also produced much more economically than the cultures of mammalian cells. It has also been noted by others (Hiaít, Nature 334, 469 (1990)), that plant systems have the ability to assemble proteins from multiple subunits, a capacity that is frequently lacking in microbial systems. The production of idiopathic proteins by pneumonia also limits the risk of congenital infections, including animal viruses produced in mammalian cell culinae and in microbial systems. Clinical tests are a major problem in the production of therapeutic proteins. Unlike other suggested plant production systems, for example, soybean and tobacco, aquatic lentil can be cultured in fermentor / bioreactor containers, making it much easier to integrate the system into the existing industrial infraestructure of protein production . As a platform for manufacturing industrial enzymes and small molecules of lower cost, the lentil has the advantage that the production is rapidly scalable to almost any size, since it can be cultivated under field conditions using waste water rich in nuirienols. A genetically engineered aquatic legume system that grows in wastewater could produce a valuable product, while simultaneously cleaning the wastewater for additional use. Said system would transform a net capital loss (remediation of waste water from discharge) into a chemical or enzyme production system with a positive economic balance. The origin of the aquatic lexicon on chemical synthesis in field crops is that its G. &SS ^ IT production does not require arable land or irrigation water needed to increase food production for the world's growing population. These and other aspects of the present invention are described in more detail in the description of the invention given below.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a radiograph produced by Soulhem hybridization of untransformed aquatic lentil DNA and aquatic leg DNA transformed from D line with a radiolabelled 3.2 kb pBI121 fragment containing the GUS gene. Channels: 1) pBI121 DNA isolated and undigested. The expected main band is 12.8 kb. The minor molecular weight band represented perhaps the supercoiled plasmid. 2) pBI121 DNA digested with Hindl. This digestion linearizes the plasmid and shows the expected 12.8 kb band. The lower molecular weight band indicates incomplete digestion. 3) pBI121 DNA digested with Hind \\\ and EcoR1.

The digestion was incomplete, but produced the expected bands: 12.8 kb (which remains of the incomplete digestion), the band of approximately 9 kb, and a thin supercoiled band. The 3.2 kb band did not give visible hybridization in this exposure. 4) Aquatic lentil DNA not transformed with the equivalent of a DNA copy of double digested pBI121, giving the expected bands of 9 and 3.2 kb. 5) DNA of aquatic leia not transformed. 6) Undigested DNA from lentil line? aquatic transformed. 7) Hind digested DNA from the Iransformed aquatic lens line. 8) DNA digested with Hind \\\ and EcoR1 of line D of aquatic lynx Iransformed.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods for transforming the acrylic lentil. In preferred embodiments, the methods use ballistic bombardment or Agrobacterium to stably transform the cells of the lentil. Alternatively, the methods use electroporation to transform the water column. The methods and transformed plants of the present invention find use as a gene expression system based on pineapples that possess many of the properties of yeasts. Hasía where the inventors have knowledge, there are no previous reports of transferable transference of genes in the aquatic legume, nor of the regeneration of Iransformated lentil plants. In the present investigations, two strategies have been used for the production of transgenic aquatic lenses: 1) directly transferring and inserting introduced DNA into meristematic frond cells followed by asexual and self-propagating propagation to produce transgenic aquatic lentils (a pineapple system). plañía), and 2) transformation of undifferentiated callus cells, followed by selection of callus in proliferation, and regeneration -m of the frond (a callus system to piaUtta). Tissue culture systems limited for the production of callus of L. gibba and L. minor have been previously reported by Chang's group (Chang and Chui, Boí Bull, Academia Sinica 17, 106 (1976), Chang and Chui, Z. Pflanzenphysiol, Bd. 89.S, 91 (1978)) and Frick (Frick, J. Plant Physiology 137, 397 (1991)), respectively. The present investigations have significantly extended the work in this area, developing an organized callus system that regenerates fronds. Preferably, the present invention utilizes one of two systems for stable transformation to the aqualar lentil: ballistic transformation using microprojectile bombardment or Agrobacterium-mediated transformation. Although aquatic lentils would be expected to be refractory to transformation by Agrobacterium because they are monocroley plant, it has unexpectedly been found that the aquatic lentil can be transformed using Agrobacterium. Aquatic lentil plants transformed in accordance with the present invention can also be generated by electroporation. See, for example, Dekeyser et al., Plañí Cell 2, 591 (1990); D? Alluin et al., Plañí Cell 4, 1495 (1992); patent of E.U.A. No. 5,712,135 to D? Alluin al. An advantage of electroporation is that large pieces of DNA, including artificial chromosomes, can be transformed into the lentil by this method. Any suitable cell or cell type of acrylic lens can be transformed in accordance with the present invention. For example, nucleic acids can be introduced into aquatic lelitephae cells in tissue culture. Alternately, the small size and aquatic growth habit of the aquiline lentils allows nucleic acids to be introduced into aquatic lentil cells from intact embryos, fronds, roots, and other organized tissues, such as meristematic tissue. . As an additional ingredient, nucleic acids can be intro- duced into callus of the aquatic lens. It is preferred that the transformed aquatic legumes produced by the claimed methods exhibit normal morphology and be fertile by sexual reproduction. Preferably, the transformed proteins of the present invention contain a single copy of the transfered nucleic acid, and the transfered nucleic acid has no noiable rearrangements therein. Also preferred are water-based beads in which the transfered nucleic acid is present in low copy number (ie, no more than 5 copies, alternatively, no more than 3 copies, as an additional alternating, less than 3 copies of acid). nucleic per cell ransformed). The term "aquatic lens", as used herein, refers to members of the Lemnaceae family. 4 genera and 34 species of aquatic leia are known: Lemna genus (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L., miniscula, L. obscura, L. perpusilla, L. teñera, L. trisulca, L. turionifera, L. valdiviana); Spirodela genus (S. intermedia, S. polyrrhiza, S. punctata); genus Wolffia (Wa. angusta, Wa arrhiza, Wa. australina, Wa. borealis, Wa. 'brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. microscopic, Wa. neglecta) and genus Wolfíella (Wl. , Wl. Denticulata, Wl. Gladiata, Wl. Hyalina, Wi.longulata, Wl. Repunda, Wl. Rotunda and Wl. Neotropica). Any other genus or species of Lemnaceae, if any, is also an aspect of the present invention. Lemna gibba, Lemna minor and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being more preferred. The species of Lemna can be classified using the iaxonomic scheme described by Landolí, Biosyslemaíic Invesligaíion on fhe Family of Duckweeds: The family of Lemnaceae - A Monografh Sludy. Geobatanischen Insíiíui ETH, Síifíung Rubel, Zurich (1986)). As will be apparent to those skilled in the art, now that a method for the efficient transformation of the aqueous sheet has been provided, any nucleic acid of interest can be used in the methods of the invention. For example, an aquatic lentil plant can be manipulated to express disease and insect resistance genes, genes that confer numerical value, antimicrobial, antiviral or antiviral genes, and the like. Alternatively, leperalgic proteins and peptides (e.g., for veterinary or medical uses) or immunogens (e.g., for vaccination) may be expressed using the aquatic lens transformed in accordance with the present invention. Likewise, the method can be used to transfer any nucleic acid to control gene expression. For example, the nucleic acid that will be transfered can be encoded for an ampholyslide oligonucleotide. Alternatively, aquatic lentil can be transformed with one or more genes to reproduce enzymatic pathways for chemical synthesis (eg, for the synthesis of plastics) or other indusirial procedures (eg, queralinase). The nucleic acid can be aquatic lynx or another organism (ie, heterologous). In addition, nucleic acids of interest of prokaryotes or eukaryotes (eg, bacteria, fungi, yeasts, viruses, plains, mammals) can be obtained, or the nucleic acid sequence can be sinlylated in whole or in part. In particular preferred embodiments, the nucleic acid encodes a secreted prolein or peptide. Preferably, the transferred nucleic acid to be expressed in the transformed aquatic pellet encodes a hormone, growth factor or cilokin, more preferably, insulin, growth hormone (in particular, human growth hormone) and inermerone. In an amino acid form, it is also preferred that the nucleic acid expresses β-glucocerebrosidase. Also preferred are nucleic acids encoding peptides or proteins that can not be produced commercially in effective form by existing gene expression systems, due to cost or logistic resistances, or both. For example, some proteins can not be expressed in mammalian systems because proiein interferes with cell viability, cell proliferation, cell differentiation, or protein assembly in mammalian cells, proteins include, but are not limited to, proinin of reíinoblasloma, p53, angioslaíina and leplina. The present invention can be used advantageously to produce regulatory proteins in mammals; it is unlikely, given the large evolutionary dysency among mammals and higher plains, that these proteins will interfere with regulatory processes in the aquatic lens. It is also possible to use Iransgenic aqualic sheet to produce large quantities of proteins such as serum albumin (in particular, human serum albumin), hemoglobin and collagen, which challenge the production capacities of existing expression systems. Finally, as described in more detail below, superior plating systems can be manipulated to produce (i.e., synthesize, express, assemble) biologically active mullimeric proieins (e.g., monoclonal antibodies, hemoglobin, P450 oxidase and collagen, and similar), much more easily than mammalian systems can. Those skilled in the art will appreciate that the term "biologically active" includes mullimeric proteins in which the biological activity is altered, comparatively with the naive process (eg, suppressed or increased), in which the prolein has sufficient activity to be of interest for use in industrial or chemical procedures, or as an adjunct agent, vaccine or diagnostic reaction. An example of a method for producing biologically active multimeric proteins in the aqualelic lens uses an expression vector containing the genes qβ "encode for all subunits of the polypeptide, see, for example, During et al. (1990) Plant Molecular Biology 15: 281, van Engelen et al., (1994) Plant Molecular Biology 26: 1701. This vector is then introduced into the cells of the aqueous sheet using any known transformation method, such as a gene gun or transformation mediated by Agrobacterium. This method results in clonal cell lines expressing sludge the polypeptides necessary to assemble the multimeric protein.As a allemative method, independent vector constructions can be obtained that encode each subunit of the polypeptide.Each of these vectors is used to generate separate clonal lines of transgenic plans that express only one of the These polypeptides are then crossed to create progeny that express all the necessary polypeptides in a single plant. See Hiatl et al., (1989) Nature 342: 76; US patents Nos. 5,202,422 and 5,639,947 to Hiatt et al. A variation of this procedure is to obtain constructs of individual genes, to mix the DNA of these constructions, and then to add the mixture of DNA molecules in vegetale cells using ballistic bombardment or Agrobacterium mediated transformation, more preferably ballistic bombardment. As an additional variation, all or some of the vectors can code for more than one subunit of the multimeric protein (ie, so that there are fewer aquatic lentil clones that will be crossed with the number of subunits in the multimeric protein). By 7. In some cases, it may be desirable to produce less than all the subunits of a multimeric prolein, or even a subunit of individual prolein, in a water-based pineapple transformed, for example, for industrial or chemical procedures. or for diagnostic, therapeutic or vaccination purposes.

A. Expression Cassettes In accordance with the present invention, the nucleic acid that will be transferred is synthesized in an expression cassette. The expression cassette comprises an initiation region of the transcription linked to the nucleic acid or gene of interest. Said expression cassette is provided with a plurality of resynchronization pins for the insertion of the gene or genes of interest (for example, a gene of interest, two genes of interest, etc.) which will be regulated by the transcription of the regulatory regions. . Preferably, the expression cassette codes for an individual gene of interest. In particular embodiments of the invention, the nucleic acid that will be transfected contains two or more expression cassettes, each of which encodes at least one gene of interest (preferably, a gene of interest). The region of initiation of the transcription (for example, a promoter) can be naive or homologous or inroduced or helerologous to the host, or it can be the nalural sequence or a synlelic sequence. For example, it is understood that the region of initiation of the transcription is not found in the host of lipo silveslre in which the region of initiation of transcription is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to an initiation region of the transcription that is helerologous to the coding sequence. Any suitable promoter known in the art can be used in accordance with the present invention (including promoters of bacteria, yeasts, fungi, insects, mammals and pineapples). Promoters of plañías are preferred, being more preferable the promoters of the aquáfica lenleja. Examples of promoters include, but are not limited to, the 35S promoter of cauliflower mosaic virus, the opine synthetase promoters (eg, nos, mas, oes, etc.), the ubiquitin promoter, the actin promoter , the promoter of the small subunit of ribulose bisphosphate (RubP) carboxylase, and the alcohol dehydrogenase promoter. The promoter of the small subunit of the RubP carboxylase of aquatic lentil is known in the art. Silverthrone eí al., (1990) Plant Mol. Biol. 15:49. Other promoters of viruses that infect plains, preferably aquatic lentils, are also suitable and include, but are not limited to, promoters isolated from Dasheen mosaic virus, Chlorella virus (for example, the adenine promoter melilfransferase). of the Chlorella virus; Miíra eí al., (1994) Plant Molecular Biology 26:85), tomato spotted marchiíez virus, labaco jingle virus, tobacco necrosis virus, ring-shaped labaco virus, ring spot virus package it, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, and the like. Finally, promoters can be chosen that give a desired level of regulation. For example, in some cases, it may be elusive to use a promoter that confers consi-facial expression (for example, the ubiquitin promoter, promoters of the RubP carboxylase gene family, and promoters of the acin gene family). In allemal form, in other situations, it may be advantageous to use promoters that are aclivated in response to specific environmental stimuli (for example, promoters of the heat shock gene, promoters of the drought-inducible gene, promoters of the gene inducible by pathogens, promoters of the gene induced by wounds, and promoters of the gene that can be induced by light / dark) or growth regulators (for example, promoters of genes induced by abscisic acid, auxins, cilocinins and gibberellic acid). As an additional ingredient, promoters can be chosen that give specific expression of proteins (for example, specific promoters of the root, leaf and flower). The franscription cassette includes, in the 5'-3 'direction of the transcription, a region of initiation of transcription and transcription, a nucleotide sequence of interest and a region of transcription termination and functional translation in plañías. Any suitable termination sequence known in the art can be used in accordance with the present invention. The termination region can be nalíva with the beginning region of the transcription, it can be native with the sequence of • 2 &; nucleotides of interest, or it can be derived from another source. Suitable termination regions are available from the plasmid Ti of A. tumefaciens, such as the termination regions of sinophacose oclopin and nopaline synarea. See also Guerineau et al., Mol. Gen. Genet. 262, 141 (1991); Proudfool, Cell 64, 671 (1991); Sanfacon et al., Genes Dev. 5, 141 (1991); Mogen et al., Plant Cell 2, 1261 (1990); Munroe et al., Gene 91, 151 (1990); Bailas al., Nucleic Acids Res. 17, 7891 (1989); and Joshi et al., Nucleic Acids Res. 15, 9627 (1987). Other examples of termination sequences are the termination sequence of the small subunit of the RubP carboxylase of the pea and the 35S termination sequence of the cauliflower mosaic virus. You will hear adequate termination sequences will be apparent to those skilled in the art. In an alignant form, the gene or genes of inferes can be provided in any other suitable expression cassette known in the art. When appropriate, the gene or genes can be optimized for increased expression in the transformed plan. When mammalian, yeast, bacterium, or dicoyledonous genes are used in the invention, they can be synthesized using preferred codons of monocyclic or aquatic lymph for improved expression. There are methods in the technique to synthesize favorite genes of plañías. See, for example, paiens of E.U.A. Nos. 5,380,831; 5,436,391; and Murray went to., Nucleic Acids. Res. 17, 477 (1989), incorporated herein by reference.

The expression cassettes may additionally contain 5 'leader sequences. These guide sequences can work to increase the extraction. Translation guides are known in the art and include: picomavirus guides, eg, EMCV guide (5 'non-coding region of encephalomyocarditis; Elroy-Síein al., Proc. Natl. Acad. Sci USA, 86, 6126 (1989), poivivirus guides, for example, TEV guide (tobacco etch virus, Allison et al., Virology, 154, 9 (1986)), human immunoglobulin heavy chain binding protein (BiP; Macajak and Sarnow, Nature 353, 90 (1991)), non-derived guidance of messenger RNA from the cover prolein of alfalfa mosaic virus (AMV RNA 4, Jobling and Gehrke, Nature 325, 622 (1987)); Guide to the virus of the mosaic of the mycobacterium (TMV, Gallie, MOLECULAR BIOLOGY OF RNA, 237-56 (1989)) and guide of the corn chlorotic mottle virus (MCMV, Lommel el al., Virology 81, 382 (1991)) See also Della-Cioppa et al., Plant Physiology 84, 965 (1987) Other methods known to increase translation, for example, can also be used. rones and the like The exogenous nucleic acid of interest may be operably further associated with a nucleic acid sequence encoding a transiloid peptide which directs the expression of the proiein or encoded peptide of interest to a particular cell compartment. Techniques are known in the art of transpiration peptides that direct the accumulation of proteins in cells of superior pineapples towards the chloroplast, mylochondrium, vacuole, nucleus and the endoplasmic relic (for its secretion outside the cell). Preferably, the transit peptide directs the expressed protein of the exogenous nucleic acid to the chloroplast or the endoplasmic relic. The transloid peptides that direct the proteins towards the endoplasmic relic are desirable to correct the processing of secreted proteins. It has been shown that the direction of proiein expression towards the chloroplasm (for example, using the translase peptide of the small subunit gene of RubP carboxylase), results in the accumulation of highly alkylated concentrations of recombinant proieins in this ORGANIZE IT An aquatic lentil nucleic acid encoding a RubP carboxylase transit peptide has already been cloned. Stiekma el al., (1983) Nucí. Acids Res. 11: 8051-61; see also patents of E.U.A. Nos. 5,717,084 and 5,728,925 to Herrera-Estrella et al. The transit peptide sequence of the small subunit of pea RubP carboxylase has been used to express and target mammalian genes in plants. See patents of E.U.A. Nos. 5,717,084 and 5,728,925 to Herrera-Estrella et al. In an allemal form, mammalian transit peptides can be used to direct the expression of the recombinant protein, for example, to the mitochondria and the endoplasmic reticulum. It has been shown that vegelale cells recognize mammalian transit plexuses, which are directed towards the endoplasmic reticulum; see US Patents Nos. 5,202,422 and 5,639,947 to Hiatt et al. The expression cassettes may contain more than one gene or nucleic acid sequence that will be transferable and expressed in the transformed cell. In this manner, each nucleic acid sequence will be operably linked to 5 'and 3' regulatory sequences. In an allernal way, multiple expression cassettes can be provided. In general, the expression cassette will comprise a selectable marker gene for the selection of the transformed cells. Selectable marker genes are used for the selection of transformed cells or tissues. Selectable marker genes include genes that encode antimicrobial resistance, such as those encoding neomycin phospholransferase II (NEO) and hygromycin phospholransferase (HPT), as well as genes that confer resistance to herbicidal compounds. The herbicide resistance genes generally code for a modified objective prolein insensitive to the herbicide, or for an enzyme that degrades or de -symites the herbicide in the plant before it can adhere. See, DeBlock et al., EMBO J. 6, 2513 (1987); DeBlock el al., Plant Physiol. 91, 691 (1989); Fromm el al., BioTechnology 8, 833 (1990); Gordon-Kamm et al., Plant Cell 2, 603 (1990). For example, resistance to sulfonylurea or glyphosate herbicides has been obtained, using genes that code for the muidenic objective enzymes, 5-enolpyruvylshikimafo-3-phosphate synthetase (EPSPS) and acelelacy synlelase (ALS). Resistance to glufosinalo of ammonium, boromoxinil and 2,4-dichlorophenoxyaceiolo (2,4-D) has been obtained by the use of genes bacíerianos that code for fosfinolricina accelillransferasa, a niírilasa, or a 2,4-díclorofenoxiacelaío monooxygenasa, which desloxifican the herbicides respecíivos.

For the purposes of this invention, selectable marker genes include, but are not limited to, genes encoding neomycin phosphransferase II (Fraley et al., CRC Critical Reviws in Plant Science 4, 1). (1986)); hydralyase cyanamide (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88, 4250 (1991)); aspartate kinase; dihydrodipicolinale sinielase (Perl et al., BioTechnology 11, 715 (1993)); gen bar (Toki et al., Plant Physiol., 100, 1503 (1992); Meagher et al., Crop Sci. 36, 1367 (1996)); tryptophan decarboxylase (Goddijn et al., Plant Mol. Biol. 22, 907 (1993)); Neomycin phosphotransferase (NEO; Soul em et al., J. Mol. Appl. Gen. 1, 327 (1982)); hygromycin phosphransferase (HPT or HYG; Shimizu et al., Mol.Cell. Biol. 6, 1074 (1986)); dihydrofolialo reductase (DHFR; Kwok et al., Proc. Natl. Acad. Sci. USA vol, 4552 (1986)); phosphinothricin acetyltransferase (DeBlock et al., EMBO J. 6, 2513 (1987)); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al., J. Cell. Biochem. 13D, 330 (1989)); acelohydroxy acid synilase (US Patent No. 4,761, 373 to Anderson et al., Haughn et al., Mol. Gen. Genet 221, 266 (1988); 5-enolpyruvyl-shikimalo-phosphato synthetase (aroA; Comai et al. , Nature 317, 741 (1985)), halogenaranylnitrilase (WO 87/04181 to Slalker et al.), Acetyl coenzyme A carboxylase (Parker ei al., Plant Physiol., 92, 1220 (1990)), dihydropleroate sintelase (sull; Guerineau et al., Plant Mol. Biol. 15, 127 (1990)) and 32 kDa foiosisiem II polypeptide (psbA; Hirschberg et al., Science 222, 1346 (1983)).

Also included are fossils encoding for resistance to chloramphenicol (Herrera-Eslreila et al., EMBO J. 2, 987 (1983)); meíotrexato (Herrera-Estrella et al., Nature 303, 209 (1983); Meijer et al., Plant Mol. Biol. 16, 807 (1991); hygromycin (Waldron et al., Plant Mol. Biol. 5, 103 ( 1985), Zhijian et al., Plant Science 108, 219 (1995), Meijer et al., Plant Mol. Bio 16, 807 (1991)), streptomycin (Jones et al., Mol. Gen. Genet. 86 (1987)), spectinomycin (Bretagne-Sagnard et al., Transgenic Res. 5, 131 (1996)), bleomycin (Hille et al., Plant Mol. Biol. 7, 171 (1986)), sulfonamide (Guerineau et al. al., Plant Mol. Bio., 15, 127 (1990), bromoxynil (Stalker et al., Science 242, 419 (1988)), 2,4-D (Streber et al., Bio / Technology 7, 811 (1989). )), phosphinothricin (DeBlock et al., EMBO J. 6, 2513 (1987)), spectinomycin (Breiagne-Sagnard and Chupeau, Transgenic Research 5, 131 (1996)): The bar gene confers resistance to glycosylated glyphosate herbicides such as phosphinocyrrhine (PPT) or bialaphos, and the like, as mentioned earlier, other selectable markers that can be used in the s vector constructs include, but are not limited to, the pat gene, also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, the HPH gene or HYG for hygromycin resistance, the EPSP synlelase gene for resistance to glyphosate, the Hm gene? for resistance to the He toxin, and other selective agents used in the past and known to those skilled in the art. See generally, Yarranton, Curr. Opin. Biotech 3, 506 (1992); Chisíopherson ei al., Proc. Natl. Acad. Sci. USA 89, 6314 (1992); Yao et al., Cell 71, 63 (1992); Reznikoff, Mol. Microbiol. 6, 2419 (1992); BARKLEY ET AL., Ttti OPERON 177-220 (1980); Hu et al., Cell 48, 555 (1987); Brown et al., Cell 49, 603 (1987); Figge et al., Cell 52, 713 (1988); Deuschle et al., Proc. Natl. Acad. Sci. USA 86, 5400 (1989); Fuerst et al., Proc. Natl. Acad. Sci. USA 86, 2549 (1989); Deuschle et al., Science 248, 480 (1990); Labow el al., Mol. Cell. Biol. 10, 3343 (1990); Zambretti et al., Proc. Natl. Acad. Sci: USA 89, 3952 (1992); Baim et al., Proc. Natl. Acad. Sci. USA 88, 5072 (1991); Wyborski et al., Nuc. Acids Res. 19, 4647 (1991); Hillenand-Wissman, Topics in Mol. And Struc. Biol. 10, 143 (1989); Degenkolb et al., Antimicrob. Agents Chemother. 35, 1591 (1991); Kleinschnidt et al., Biochemistry 27, 1094 (1988); Galz et al., Plant J. 2, 397 (1992); Gossen el al., Proc. Natl. Acad. Sci. USA 89, 5547 (1992); Oliva et al., Antimicrob. Agents Chemother. 36, 913 (1992); HLAVKA ET AL., HANDBOOK OF EXPERIMENTAL PHARMACOLOGY 78 (1985); and Gilí et al., Nature 334, 721 (1988), said descriptions being incorporated herein by reference. The previous list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention. When appropriate, selectable marker genes and other genes and nucleic acids of interest that will be transformed can be syn- ellized for the expression of the optic in the aquatic lens. That is, the coding sequence of the genes can be modified to increase expression in aquatic lentil. The synthetic nucleic acid is designed £ 7 to be expressed in the tissues and pfiUfas transformed to a higher level. The use of optimized selectable marker genes can result in higher transformation efficiency. Methods for the optimization of synthetic genes are available in the art. The nucleotide sequence may be opiimized for expression in the aqueous lens, or alternatively it may be modified for optimal expression in monocolleys. Preferred codons of pineapples can be determined from the codons of frequency further down in the proteins expressed in the aquatic lentil. HE recognizes that genes that have been opimimized for expression in the aquatic and monocotyledonous lens can be used in the methods of the invention. See, for example, EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak et al., Proc. Natl. Acad. Sci. USA 88, 3324 (1991) and Murray et al., Nuc. Acids Res. 17, 477 (1989), and the like, incorporated in the present as reference. It is further recognized that the entire gene sequence or any part thereof can be optimized or sini-elized. In • you will hear words, you can also use fully optimized or partially oplimized sequences. You know you will hear sequence modifications that incremen gene expression in a cellular host. These include the elimination of sequences encoding false polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to the ** < £ «« gene expression. The e * O content of the sequence can be adjusted to average levels for a given cell host, as calculated in relation to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted secondary messenger messenger RNA strains.

B. Corns and Target Tissues The methods of the invention are useful for transforming cells of the aqueous sheet, preferably merismatic and frond cells. Said cells also include callus, which can originate from any tissue of aquatic lentil plants. Preferably, the tissue used for the start of the callus is merislemic tissue. Alternatively, the callus can originate from any other frond cell, or mainly from any other vessel of the aquicic sheet capable of forming callus. Alternatively, any tissue capable of undergoing subsequent clonal propagation, either by organogenesis or embryogenesis, can be used to transform the aqueous lens in accordance with the present invention. The term "organogenesis", as used herein, means a process by which fronds and roots are sequentially developed from meristematic centers. The term "embryogenesis", as used herein, means a process by which fronds and roots are developed in concert (not sequentially), either from somatic cells or gametes.

The method can also be used to transform cell suspensions. Said suspensions of cells can be formed from any tissue of the aquatic lens. Aquatic lentils form different types of callus: (a) a semi-organized compact callus (designated as type I); (b) a friable, white and undifferentiated callus (designated as type II); and (c) a differentiated green callus (designated as íll lll). In tissue culture, calluses can only regenerate plañías in two forms: by embryos and by formation of shoots (in the aquatic lentil, the frond is the outbreak). Methods for the induction of callus are known in the art, and the particular conditions to be used can be optimized for each species of aquatic lentil and for the desired callus type, as demonstrated in the following examples. Preferably, type I or III callus, most preferably calli of I, are used to transform the aquatic lentil according to the present invention. Calluses can be induced by culturing tissues of the aqualar lentil in medium containing plant growth regulators, ie, cytokinins and auxins. Preferred auxins for the induction of callus from tissue of aquatic lentil include 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalenacetic acid (NAA). Preferred auxin concentrations are 1-30 μM, more preferably 5-20 μM, even more preferably 5-10 μM. The preferred cyclokinin is benzyladenine (BA) or fidiazuron (TDZ). Preferred concentrations of cyclokinin are 0.1-10 μM, more preferably 0.5-5 μM, still more preferably 0.5-1 μM. In other more preferred embodiments, callus is induced by culturing the water column in medium containing BA or TDZ and 2,4-D or NAA. In general, low concentrations of auxin or "weak" auxin (eg, indolelic acid), promote the proliferation of fronds rather than callus formation, and aliases "strong" auxin or auxin concentrations (eg, 2, 4-D), promote callus formation. The preferred basal media for callus formation include N6 medium (Chu et al., Scienia Sinica 18, 659 (1975)) and medium Murashige and Skoog (Murashige and Skoog, Physiol. Plant, 15, 473 (1962)), Murashige and Skoog medium being more preferred. In general, the frequency of callus induction is variable. In these species, the callus may not be visible for two to three weeks in summer, and may last 4 to 8 weeks of culíivo before the corns are of sufficient size for their transformation. Preferably, the induction of callus is carried out for a period of 1 to 10 weeks, more preferably 2 to 8 weeks, even more preferably 3 to 5 weeks. For callus growth, the preferred means are as for the induction of callus, but the concentration of auxin is reduced.

C. Transformation of aquatic lentil by ballistic bombardment One embodiment of the invention is a method for transforming aquatic lentil with a nucleotide sequence of interest, wherein the nucleoid sequence contains less an expression cassette that has a gene that confers resistance to a selection agent. The nucleolide sequence is carried by a microprojectile. As far as the inventors are aware, there are no previous reports of aquatic lens production stably transformed by means of ballistic transformation. In accordance with preferred embodiments of the present invention, the ballistic transformation method comprises the steps of: (a) providing an aquatic lens fabric as a target; (b) boosting the microprojectile that possesses the nucleoid sequence in the body of the aquatic lens at a rate sufficient to perforate the walls of the tissue cells of the tissue, and deposit the sequence of denuclear nucleoids of a cell of the tissue to provide the nucleus. way an ransformated entity. In particular embodiments of the invention, the method further includes the step of culturing the transformed yeast with a selection agent, as described below. In a further alternative embodiment, the selection step is followed by the step of regenerating aquaculture lentils Iransformed from the transformed tissue. As mentioned below, the technique could be carried out with the nucleotide sequence as a precipitate (wet or dehydrated by freezing) alone, instead of the aqueous solution containing the nucleoid sequence. Any ballistic device for transforming cells can be used to implement the presented invention. Examples of apparatuses are described by Sandfojfd et al. (Particulate Science and Technology 5, 27 (1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356. Said apparatuses have been used to transform maize cells (Klein et al., Proc. Nall, Acad. Sci. USA 85, 4305 (1988) ), soy tripe (Chrisíou et al., Plant Physiol. 87, 671 (1988)), McCabe et al., BioTechnology 6, 923 (1988), yeast mitochondria (Johnson et al., Science 240, 1538 (1988). )), and chloroplases of Chlamydomonas (Boynton et al., Science 240, 1534 (1988)). In the investigations presented here, a commercially available helium gene gun (PDS-1000 / He) manufactured by DuPonl was used. Alternatively, an apparatus configured as described by Klein et al. (Naíure 70, 327 (1987)). This apparatus comprises a bombardment chamber, which is divided into two compartments separated by an adjustable allura stop plate. An acceleration tube is mounted on top of the bombing chamber. A macroprojectile is driven below the acceleration tube on the stop plate by a powder charge. The stop plate has a hole formed in it, which has a smaller diameter than the microprojectile. The macroproyeclil owns the microprojectiles, and the macroproyecíil is directed and shot in the hole. When the macroproject is stopped by the stop plate, the microprojectiles are propelled through the hole. The target tissue is placed in the bombardment chamber, so that a microprojectile driven through the hole penetrates the cell walls of the cells in the target tissue and deposits the nucleotide sequence of interest carried therein into the cells of the objective tissue. . The bombardment chamber is partially evacuated before use to prevent the almospherical trap from unduly replenishing the microprojectiles. The chamber is only partially evacuated, so that the target vessel is not dewatered during the bombardment. A vacuum is adequate around 400 to about 800 milliliters of mercury. In alternative modalities, the ballistic transformation is carried out without the use of microprojects. For example, an aqueous solution that conengates the sequence of nucleolides of interest as a precipitate can be cross-linked by the macroprojectile (for example, by placing the aqueous solution directly on the contact exíremo of the macroprojectile plate without a microprojectile, where it is retained by surface tension ), and the single solution driven into the objective tissue of the plant (for example, driving the macroprojectile down the acceleration tube in the same way as described earlier). Other methods include placing the nucleic acid precipitate ("wet" precipitate) or a nucleotide precipitate dehydrated by freezing directly on the contact end of the macroprojectile plate without a microprojectile. In the absence of a microprojectile, it is thought that the nucleotide sequence must be driven into the target tissue at a rate greater than that required if it is carried by a microprojectile, or make the sequence of • ** i &z ^ 1 nucleolides travel a shorter distance to the target tissue (or both procedures). It is more preferred to carry the nucleotide sequence on a microprojectile. The microprojectile can be formed of any material having sufficient density and cohesiveness to be propelled through the cell wall, given the speed of the particle and the dissipation that the particle must travel. Non-limiting examples of materials that can be used to obtain microprojects include metal, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (eg, example, graphite, diamond). Accelerated melanic particles are also preferred. Non-limiting examples of suitable materials include fungus, gold and iridium. The particles should be of a sufficiently small size to avoid excessive disintegration of the cells with which they bind in the void in the target area, and large enough to provide inertia required to penetrate the cell of interest in the objective tissue. Particles that vary in diameters of about half are suitable. (micrometer to approximately three micrometers) The particles do not need to be spherical, since surface irregularities on the particles can increase their DNA carrying capacity 20 The nucleolide sequence can be immobilized on the particle by precipitation. used will fluctuate, depending on real factors such as the particle acceleration procedure used, as is well known in the art.

Carrier particles can optionally be coated with encapsulating agents such as polylysine to improve the stability of the nucleotide sequence immobilized thereon, as described in EP 0 270 356 (column 8). After performing the ballistic bombardment of the target object, the transforms can be selected, and transformed aquilike lenses can be regenerated as described later in section E.

D. Transformation mediated by A robacterum 10 In one embodiment of the present invention, aquatic lentil is transformed using Agrobacterium tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium tumefaciens. The transference of genes mediated by Agrobacterium exploits the natural capacity of A. tumefaciens and A. rhizogenes to transfer DNA into chromosomes of plañías. Agrobacterium is a fiopainogen that transferes a series of genes encoded in a region called DNA T of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, in vegetale cells. The typical result of the transfer of the Ti plasmid is a tumorous growth called crown gall, in which the T-DNA is integrated stably in the chromosome of a host. The integration of plasmid Ri into the chromosomal DNA of the host results in a condition called "hairy root". The ability to cause disease in the host plant can be removed by deletion of the genes in the DNA T without ? ^ * s ^ .s ^ ^ a- - > m loss of transference and integration of DNA. The DNA that will be transferred is adhered to edge sequences that define the terminal ends of an integrated T-DNA. The transfer of genes by genetically manipulated Agrobacterium strains is now common for many dicotyledonous crop plants. However, considerable difficulty has been experienced in using Agrobacterium to transform monocolongae plantains, in particular, cereals. Hasla, where the inventors know, until the date there are no reports of aquatic lentil production that has been transformed by means of Agrobacterium-mediated transformation. Although the following discussion will focus on using A. tumefaciens to achieve gene transfer in aquatic legume, those skilled in the art will appreciate that this discussion applies equally well to A. rhizogenes. The transformation using A. rhizogenes has been developed analogously to that of A. tumefaciens, and has been successfully used to transform, for example, alfalfa, Solanum nigrum L., and tulipero (US Patent No. 5,777,200 to Ryals al. .). As described in the patent of E.U.A. No. 5,773,693 to Burgess et al., It is preferable to use a disarmed strain of A. tumefaciens (as described below); however, A. rhizogenes of ipo silvesíre can be used. An illusive strain of A. rhizogenes is strain 15834.

The strain of Agrobacterium used in the methods of the present invention is modified to contain a gene or genes of interest, or a nucleic acid that will be expressed in the transformed cells. The nucleic acid that will be transferred is incorporated into the T region, and is flanked by DNA T border sequences. Several strains of Agrobacterium are known in the art, particularly for the transformation of dicotyledonous pineapples. Said Agrobacterium can be used in the methods of the invention; see, for example, Hooyskaas, Plañí Mol. Biol. 13, 327 (1989); Smilh ef al., Crop Science 35, 301 (1995); Chillón, Proc. Nati Acad. Sci. USA 90, 3119 (1993); Mollony et al., Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al., Nalure Bioiechnol. 14, 745 (1996); and Komari et al., The Plañí Journal 10, 165 (1996), whose descriptions are incorporated herein by reference. In addition to the T region, the Ti (or Ri) plasmid contains a vir region. The vir region is important for efficient transformation, and seems to be specific to the species. Systems of binary vectors have been developed in which the manipulated disassembled DNA T that possesses the introduced DNA and the vir 's functions are present in separate plasmids. In this manner, a modified T-DNA region comprising introduced DNA (the nucleic acid to be transferred) is consiruido in a small plasmid which replicates in E. coli. This plasmid is transferable in conjugated form in an ireparential pairing or by eleclroporation in A. tumefaciens which contains a plasmid compatible with virulence gene sequences. The vir functions are supplied in the trans position to transfer the T-DNA into the genome of the pineapple. Said binary vectors are useful in the practice of the present invention. In preferred embodiments of the invention, vectors derived from C58 are used to transform A. tumefaciens. Alternatively, in other modalities, superbinary vectors are used. See, for example, US patent. No. 5,591, 615 and EP 0 604 662, incorporated herein by reference. Said superbinary vector has been consíruido, and contains a region of DNA that originates from the hypervirulence region of the Ti plasmid, pTBB542 (Jin et al., J. Bacleriol. 169, 4417 (1987) contained in the strain A281 supervirulenla of A. tumefaciens which exhibits highly transformed transformation efficiency (Hood et al., Biotechnol., 2, 702 (1984), Hood et al., J. Bacteriol., 168, 1283 (1986), Komari et al., J. Bacteriol. 166, 88 (1986), Jin et al., J. Bacíeriol., 169, 4417 (1987), Komari, Plañí Science 60, 223 (1987), ATCC accession No. 37394. Examples of supernormal vectors known to the experts in the art include pTOK162 (Japanese patent application (Kokai) No. 4-222527, EP 504,869, EP 604,662, and US patent. Do not. ,591, 616, incorporated herein by reference) and pTOK233 (Komari, Plant Cell Reports 9, 303 (1990), Ishida et al., Nalure Biotechnology 14, 745 (1996), incorporated herein by reference). Other superbinary vectors can be constructed by the methods described in the above references. The superbinary vector pTOK162 is capable of replicating itself in E. coli and in A. tumefaciens. In addition, the vector contains the virG, virC and virG genes of the virulence region of pTtBo542. The plasmid also contains an antibody resistance gene, a selectable marker gene and the nucleic acid of interest that will be transformed in the pineapple. The nucleic acid to be inserted into the genome of the aqueous lens is located between the two border sequences of the T region. Superbinary vectors of the invention having the characteristics described above for pTOK162 can be constructed. The T region of the supernatant vectors and other vectors for use in the invention is intended to have reslurry sites for the insertion of the genes that will be delivered. Alternatively, the DNA that will be transformed can be inserted into the T-DNA region of the vector using homologous recombination in vivo. See, Herrera-Eslrella et al., EMBO J. 2, 987 (1983); Horch et al., Science 223, 496 (1984). Said homologous recombination depends on the fact that the superbinary vector has a region homologous to the region of pBR322 or other similar plasmids. In this way, when the plasmids are assembled, a desired gene is inserted into the superbinary vector by genetic recombination through the homologous regions. Any suitable vector for transforming aquatic lentil can be used in accordance with the present invention. For example, the heterologous nucleic acid sequences of interest and the flanking T-DNA can be carried by a binary vector lacking the vir region. The w'res region then provided in a disarmed Ti plasmid or in a second S ^^. Áíß-i-Sc? binary plasmid As an alternative alternative, the helicer nucleic acid sequence and the DNA T border sequences can be placed at the T-DNA site on the Ti plasmid through a double recombination event whereby the new T-DNA replaces the T-DNA. of the original Ti plasmid. The vir region can be provided by the Ti plasmid or in a binary plasmid. As a further alternative, the helerologous nucleic acid sequence and the flanking T-DNA can be integrated into the bacterial chromosome as described in the U.S. patent. No. 4,940,838 to Schilperoort et al., And the vir region can then be provided in a Ti plasmid or in a plasmid binary It can be considered that the Agrobacterium-mediated Iransformation process of the present invention comprises several steps. The basic steps include an infection step and a co-cultivation step. In some modalities, these steps are followed by a selection step, and you will hear modalities for a selection and a regeneration step. An optional pre-culture step may be included before the • infection step. The step of previous cultivation consists in cultivating the callus, frond or other target tissue before the infection step on an adequate medium. The previous cultivation period can vary from approximately 1 to 21 days, from preference of 7 to 14 days. It was found that said previous culino step prevents the transformation of corn crops; see, for example, EP 0 672 752.

In the infection step, the delatas that will be transformed are exposed to Agrobacterium. The cells are then contacted with Agrobacterium, typically in a liquid medium. As described above, Agrobacterium 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. General molecular biology techniques used in the invention are well known to those skilled in the art; see, for example, SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (1989). The Agrobacterium containing the plasmid of interest is preferably maintained in Agrobacterium master plates with frozen supply material at about -80 ° C. Master plates can be used to inoculate agar plates to obtain Agrobacterium which is then resuspended in the medium to be used in the infection process. Alternatively, bacteria from the master plate can be used to inoculate broth cultures that are brought to a logarithmic phase before their transformation. The concentration of Agrobacterium used in the infection step and in the co-culture step can affect the frequency of the transformation. In the same way, concentrations very similar to Agrobacterium can damage the tissue that will be transformed, 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 Agrobact & rium used, the substance that is being transformed, the species of aquatic lentil that is being transformed, and the like. In order to optimize the Iransformation protocol for a particular aquatic legume species or tissue, the tissue to be transformed can be incubated with several concentrations of Agrobacterium. In the same way, the level of expression of the marker gene and the efficiency of transformation for several concentrations of Agrobacterium can be evaluated. Although the Agrobacterium concentration may vary, a concentration scale of about 1 x 10 3 cfu / ml to about 1 x 10 10 cfu / ml, preferably about 1 x 10 3 cfu / ml to about 1 x 109 cfu / ml, and even more preferably from about 1 x 10 8 cfu / ml to about 1 x 109 cfu / ml. The tissue to be transformed is generally added to the Agrobacterium suspension in a liquid contact phase containing a concentration of Agrobacterium to optimize the efficiencies of Iransformation. The concoction phase facilitates the maximum condensation of the tissue that will be transformed with the Agrobacterium suspension. As usual, the infection is allowed to proceed for 1 to 30 minutes, preferably 1 to 20 minutes, more preferably 2 to 10 minutes, even more preferably 3 to 5 minutes before the co-culling step. Experts in the technique will appreciate that conditions can be optimized to achieve the highest level of infection and transformation by Agrobacterium. For example, in preferred embodiments of the invention, the cells are subjected to osmotic pressure (eg, 0.6 mmol mannol) during the infection and co-culfivo steps. Additionally, to increase the efficiency of Iransformation, the tissue can be grown in medium containing an auxin, such as IAA, to promote cell proliferation (ie, it is thought that Agrobacterium is integrated into the genome during mitosis). As you will hear alternatives, it is possible to use lesion of the uterus, pressure with vacuum or culture in medium that contains aceyosyringone to promote the efficiency of Iransformation.

In the co-culture step, the cells that will be transformed are cocultivated with Agrobacterium. Typically, the co-culture is carried out on a solid medium. Any suitable medium can be used, as a means of Schenk and Hildebrandí (Schenk and Hildebrandí, Can. J. Bol. 50, 199 (1972)) containing 1% sucrose and 0.6% agar. The optimal co-culture time varies with the particular tissue. The fronds are cocullivated with Agrobacterium for about 2 to 7 days, preferably 2 to 5 days, more preferably 3 to 5 days, and most preferably 4 days. In coníraste, corns are cocullivados with Agrobacterium duraníe 0.5 to 4 days, more preferably 1 to 3 days, most preferably 2 days. The co-culture can be carried out in darkness or under soft lighting conditions to increase the efficiency of transformation. In addition, as previously described for the inoculation step, co-cultivation can be carried out in medium that conies IAA or acetyringone to promote the efficiency of Transformation. Finally, the step of co-culture can be carried out in the presence of cytokines, which acyuate to increase cell proliferation. After the passage of co-cultivation, the transformed tissue may be subjected to an optional step of rest and desconamination. For the rest / desconamination step, the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase can be carried out in the absence of any selective pressure to allow the recovery and proliferation of the transformed cells containing the nucleic acid helerologist. An antibiotic is added to inhibit the growth of Agrobacterium. Such antibiotics which are known in the art and which are known to inhibit Agrobacterium include cefoximax, imelin, vancomycin, carbenicillin, and the like. For example, carbenicillin concentrations will fluconate from about 50 ml / l to about 250 ml / l of carbenicillin in solid media, preferably from about 75 mg / l to about 200 mg / l, more preferably to about 100-125 mg / l. l. Those skilled in the art of monocot transformation will recognize that the concentration of antibiotics can be optimized for a particular transformation protocol without undue experimentation. Preferably, the resting phase cultures are allowed to stand in the dark or in dim light, preferably in dim light. Any of the means known in the art can be used for the rest interval. The resting / deconditioning step can be carried out as necessary to inhibit the growth of Agrobacterium, and to increase the number of cells transformed in order to practice the selection. Typically, the resting / deconfiguration step can be carried out for 1 to 6 weeks, preferably 2 to 4 weeks, more preferably 2 to 3 weeks, before carrying out the selection step. In more preferred embodiments, the selection period begins within 3 weeks after co-culture. Some strains of Agrobacterium are more resistant to antibiotics than others. For less resistant strains, decontamination is typically carried out by adding fresh decontamination media to the corns every 5 days or more. For stronger strains, a stronger antibiotic (eg, vancomycin) can be added to calluses every third day. After the co-culture step, or rest / de-lamination step, the transforms can be selected and the regenerated water samples can be regenerated as described later in section E.

E. Selection of transformants and regeneration of transformed aquatic lentil plants The tissue or callus of aquatic lentil is transformed in accordance with the present invention, for example, by ballistic bombardment or Agrobacterium-mediated transformation, each of which is described in FIG. more detail in sections C and D, respectively. AfterZ ^ "^ * 4 of the transformation step, the transformed tissue is exposed to selective pressure to select those cells that have received and are expressing the polypeptide of the helerologous nucleic acid inlroduced by the expression cassette.The agent used to select the transformants will select for preferential growth of cells containing at least one selectable marker insert located within the expression cassette, and provided by ballistic bombardment or by Agrobacterium The conditions under which the selection of transformants (of any type of tissue or callus) This procedure is generally the most critical aspect of the methods described here.The transformation procedure subjects the cells to eslrés, and the selection procedure can be toxic even for the transforms, typically in response to this problem. , the transformed tissue is initially submitted Weak selection, using low concentrations of the selection agent, and dim light (eg, 1-5 μmoles / m2 sec.), with a gradual increase in the selection gradient applied by increasing the concentration of the selection agent and / or incrementing the luminous intensity. The selection pressure can be removed for a period of time, and it can be reapplied if it is observed that the product is under stress. In addition, the transformed fabric may be given a period of "rest" as described above in section D, before any selection pressure is applied. The selection medium generally contains a simple carbohydrate, such as sucrose of 1 to 3%, so that the cells can not carry out the M7 «- photosynthesis. In addition, the selection is carried out initially under conditions of dim lighting, or even in complete darkness, to maintain the melabolic activity of the cells at a relatively low level. Those skilled in the art will appreciate that the specific conditions under which the selection is carried out can be optimized for each species or strain of the aquatic leech, and for each type of tissue that is being transformed without undue experimentation. There is no particular time limit for the selection step. In general, the selection will be carried out for a sufficient time to destroy the non-transformants, and to allow the transformed cells to proliferate at a rate similar to the cells not transformed to generate a sufficient callus mass before the regeneration step. In this way, the selection period will be longer with cells that proliferate at a more lens speed. Aquatic type I callus, for example, proliferate relatively more leniently, and selection can be carried out for 8 to 10 weeks before regeneration. Metodes are known in the art to regenerate certain plañías from transformed cells and calluses; see, for example, Kamo et al., Bot. Gaz. 146, 327 (1985); Wesl e al al., The Plant Cell 5, 1361 (1993); and Duncan e al., Plañía 165, 322 (1985). Several refinements are recommended to these methods to regenerate aquatic lenses. The regeneration of fronds consecutive to transformation and selection, can be carried out more reliably with callus of type I and II. The regeneration of type I calluses, for example, can be idenified by green centers (sites where the fronds are slashing) that appear on the surface of the pale yellow callus. Typically, the regeneration of the aquatic lentil does not occur under the same conditions of the medium that supports the proliferation of calluses. 5 A poor solid medium (for example, agar-water or Schenk medium and Hildebrandt medium concentration containing 0.5% sucrose and 0.8% agar) is preferred. However, it is usually necessary to intermittently cultivate the calli in regeneration of the aquatic lens for brief periods on a medium of complete concentration to maintain the balance of nullies in the cells in regeneration. In some cases, with strains or regeneration species, this procedure may have to be repeated several times before the fronds are regenerated. Typically, regrowth regulators are not added to the regeneration of fronds (since they inhibit the organization of the fronds); however, cytokines such as BA and adenine sulfate, can increase the regeneration of fronds in the case of some species. Callus cultures do not lose their ability to regenerate fronds during prolonged periods of callus culture. During the regeneration process, any known method The technique in the art can be used to verify that plants in regeneration are, in fact, transformed with the transfered nucleic acid of interest. For example, hislochemical staining, ELISA test, Soufhern hybridization, Northern hybridization, Western hybridization, PCR and the like can be used for 'isi- :.; ^ 7ift7 go S ^ € sß; 7; *, j delect the transferred protein or nucleic acids in the callus tissue and regenerating plants. Now that it has been demonstrated that the aquilic lenticel can be transformed using ballistic bombardment and Agrobacterium, alterations to the general methods described herein can be used to increase the efficiency or to transform strains that may exhibit some obscenity to the transformation. Factors that affect the efficiency of transformation include aquatic lentil species, infected tissue, tissue media composition, selectable marker genes, the duration of any of the steps described above, types of vectors, and the light / dark conditions. Specifically for Agrobacterium mediated transformation, the concentration and strain of A. tumefaciens or A. rhizogenes should also be considered. Therefore, these and other factors can be varied to determine what is an optimal transformation protocol for any species or strain of a particular watercourse. It is recognized that not any species and strain will react in the same way to the transforming conditions, and that they may require a slightly different modification of the protocols described herein. However, by altering each of the variables, an optimal protocol can be derived for any aquatic lentil line. The following examples are offered by way of illustration and not by way of limitation. As used in the examples, "hr." means hour, "sec" means second, "g" means gram, "mg" means milligram, "I" means liter, "ml" means milliliter, "rr? oles" means millimoles "mM" means millimolar, "μM" means micromolar, "m" means methyl, "mm" means millimeter, "BA" means benzyladenine, "2,4-D" means 2,4-dichlorophenoxyacetic acid, "NAA" means naphnelenaclic acid, and "IAA" means indoleacetic acid.

EXAMPLES Cultivation of tissues This section presents experiments that belong to methods to obtain corns of the aquatic lentil. Numerous examples use Lemna gibba G3 as the aquatic lentil strain, the strain used to optimize the cultivation parameters: (1) formulation of the basal medium, (2) type and concentration of the growth regulators leaves plant, and (3) ) transfer program. As the knowledge of callus formation increased, it was applied to aquatic lentic species. The aquatic lentils form three types of callus: (a) a semi-organized compact callus (designated as type I); (b) a friable, white, undifferentiated callus (designated as type II); and (c) a differentiated green callus (designated as íll lll). The cultivation of tissues, the callus can only regenerate plants in two ways: by embryos and through the formation of shoots (in the aquilic leaf, the frond is the outbreak). The data presented below demonstrate that M can regenerate transformed aqueous sheeting from all the known ways of frond regeneration from callus.

EXAMPLE 1 18 combinations of an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), and a cytokinin, benzyladenine (BA), were tested to determine their effects on the induction of callus in a species of aquatic lentil, G3 of Lemna gibba. 0 Lentil fronds were developed in liquid medium of Hoagland (Hoagland and Snyder, Proc. Amer. Soc. Hort. Sc. 30, 288 (1933)) containing sucrose at 3%, for 2 weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experimentation. For the induction of callus, 18 portions of 100 ml of Murashige and Skoog basal medium (Murashige and Skoog, Physiol., Plant 15, 473 (1962)) containing 3% sucrose, 0.15% Gelriie and Baclo-agar were prepared. Difco at 0.4% with 2,4-D concentrations of 10, 20 and 50 μM and BA concentrations of 0.01, 0.1, 1.0, 2.0 and 10.0 μM. All media were adjusted to pH 5.8, autoclaved at 121 ° C for 20 minutes, cooled, and each 100 ml were poured into 4 100 mm x 15 mm Petri dishes. A very large experimental factorial design of high concentrations of 2,4-D x six concentrations of BA was used (18 isolations in ? ~ -ss. ^ s * ^ ^ * "total) with 4 replicas, with one Petri dish per replica and 5 fronds per Petri dish For the induction of the calluses, 5 individual fronds of aquatic lentil with the abaxial side down were placed on Each plate of medium The 72 plates were incubated at 23 ° C for 27 days under a foioperiod of 16.5 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. Aquatic legume tissue was transferred to fresh media of the same type, and incubation was continued for another 35 days under the same conditions of light and culture under illumination.10 The results, measured as the frequency of callus induction (number of fronds) that form any callus / number of fronds), showed many types of callus proliferation after 62 days of incubation: (1) A white-yellow compact callus was identified and designated as "I-type I." a low frequency of fronds, approximately 5%, this type of callus proliferated. (2) A friable white callus was identified and designated as "type II". Between 20 and 40% of the fronds • This type of callus proliferated. (3) A green callus that varied in its degree of cellular organization was identified and designated as "type III". This type of callus was produced by more than 50% of all the fronds proliferated during the incubation time. The large callus types showed proliferation in all the 18 combinations of 2,4-D and BA with variable frequencies. The proliferation of callus was the most vigorous in a wide scale of concentrations of 2,4-D of 20-50 μ and tf concentrations of BA enlre 0.01 and 0.1 μM.

EXAMPLE 2 40 concentrations of an auxin, 2,4-dichlorophenoxyacelic acid (2,4-D), and a cilocinine, benzyladenine (BA) were tested to better optimize the concentrations of auxin and cytokine for the induction of callus. from the fronds of the G3 • 10 Lemna gibba. Aquatic lentil fronds were developed in Hoagland liquid medium containing sucrose at 3%, for 2 weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the 15 experimentation. For the induction of callus, 40 100-ml portions of Murashige and Skoog medium containing 3% sucrose, Gelriie at 0.15% and Difco Bacto-agar at 0.4% were prepared with 2,4-D concentrations of 20, 30 , 40, 50, 60, 70, 80 and 100 μM and BA concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0 μM. All media were adjusted to pH 5.8, autoclaved at 20-121 ° C for 20 min., Cooled, and each 100 ml poured into 4 100 mm x 15 mm Peiri boxes. An experimental factorial design of eight concentrations of 2,4-D x five concentrations of BA was used (40 isolates in * «SE 5 * 4 total) with 4 replicas, with a box of ^ ftf per replica and 5 fronds per Petri dish. For the induction of the callus, 5 individual fronds of aquatic lentil with the abaxial side down on each medium plate were placed. The plates were incubated at 23 ° C for 27 days under a photoperiod of 16 5 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 27 days, the acid of the aqueous sheet was transferred to fresh media of the same type, and the incubation was continued for another 35 days under the same conditions of temperature and culture under illumination. • 10 The results obtained after 63 days of incubation showed that fresh callus had proliferated: calluses of (1) type I, (2) type II and (3) type III. The regression analysis (quadratic response surface) of the numerical frequency data (# of fronds that form any callus / # loal of fronds), revealed differences in the answer of the frond for the induction of calluses of the different types. The frequencies of the callus types II and III were affected significantly by the concentrations of (f 2,4-D and BA, however, the frequency of type I callus was significantly affected only by the concentration of 2,4-D.) No specific concentration of 2,4-D or BA occurred for induction opium calluses on a wide scale of both growth regulators. Approximately 50% of the fronds produced callus of type III, approximately 25% produced calluses of type II, and less than 5% produced calluses of type I. ro5 EXAMPLE 3 We tested 40 combinations of an auxin, dicamba, and a cytokinin, benzyladenine (BA), to compare the efficacy of relactate dicamba conira 2,4-D for the induction of callus in a species of aquatic lens, Lemna G3 gibba Aquatic lentil fronds were developed in liquid Hoagland medium containing sucrose at 3%, lasting 2 weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with an intensity light of approximately 40 μmoles / m2 sec. before the experiment. For the induction of callus, 40 100 ml portions of Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite and 0.4% Difco Agar-agar with dicamba concentrations of 10, 20, 30, 40 were prepared. 50, 60, 80 and 100 μM, and BA concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0 μM. All media were adjusted to pH 5.8, subjected to auíoclave at 121 ° C for 20 minutes, cooled, and each 100 ml were poured into 4 boxes • Peep 100 mm x 15 mm. An experimental factorial design of eight concentrations of dicamba x five concentrations of BA was used (40 years ago). in loíal) with 4 replicas, with a Pelri box per replica and 5 fronds per box of Pefri. For the induction of the callus, 5 individual fronds of aquatic lentil with the abaxial side down on each medium plate were placed. Plates were incubated at 23 ° C for 27 days under a photoperiod of 16 m hours of light / 8 hours of darkness * and with a luminous intensity of approximately 40 μmoles / m2 sec. After 28 days, the aquatic lentil tissue was transferred to fresh media of the same type, and the incubation was continued for another 45 days under the same conditions of temperature and culture under illumination. After 73 days of incubation, callus proliferation blots were observed: callus of (1) íip I, (2) íip II and (3) ííp III. In general, callus proliferation was poor and occurred at dicamba concentrations of 10 and 20 μM; above 30 μM, callus proliferation did not occur. The calluses of • 10 types II and III proliferated in response to dicamba; and the proliferation of type I calluses was rare.

EXAMPLE 4 Two concentrations of 2,4-D were used in combination with BA to determine if the growth of the calluses could be maintained in lines of • Callos are stable from the fres types observed with G3 from Lemna gibba. Leaflets of the aquiline lele in liquid medium were developed by Schenk and Hildebrandt (Schenk and Hildebrandt, Can J. Bot. 50, 199 (1972)) containing sucrose at 1% for two weeks at 23 ° C under a pholoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experimentation. For the induction of callus, 400 ml of Murashige and Skoog medium were prepared with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar and 0.01 μM BA, with 2,4-D concentrations of 10. and 40 μM. All media were adjusted to pH 5.8, autoclaved at 121 ° C for 20 minutes, cooled, and each 200 ml portion was poured into 8 boxes of 100 mm x 15 mm Peiri. A randomized experimental block design of two treatments with four replicas was used, with a Pelri box per replica and 5 fronds per Peiri box. For the induction of callus, 5 individual aquatic lentil fronds were placed with the abaxial side down on each plate • 10 medium. The plates were incubated at 23 ° C for 27 days under a foioperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 4 weeks, the aquatic tissue was transferred to fresh media of the same type, and the incubation was continued for another 4 weeks under the same conditions temperature and culture under lighting. After 8 weeks of incubation, fres types were observed • of callus proliferation: calluses of (1) type I, (2) type II and (3) type III. The callus fresipos were transferred to fresh medium of identical composition, and the incubation was continued under identical culino conditions with cualro. subcultures per week. After two more months of culture, the calli of types I and III on 2,4-D at 10 μM and BA at 0.01 μM, were viable culíivos of callus in healthy proliferation. Type II callus did not proliferate. Although the proliferation of calluses could be maintained on a subculture program of 68 weeks, a decrease in calybs was observed during the third and fourth weeks of the subculiivous period.

EXAMPLE 5 The subculture program was tested to maintain the proliferation of callus with Lemna gibba G3. Leaf fronds of the aquatic leaf were developed in Schenk and Hildebrandí liquid medium containing sucrose at 1% for two weeks at 23 ° C under a furoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experiment. For the induction of callus, 500 ml of Murashige and Skoog medium were prepared with 3% sucrose, 0.15% Gelrile and 0.4% Difco agar-agar, with concentrations of 2,4-D at 30 μM and BA at 0.02. μM. All media were adjusted to pH 5.8, subjected to auíoclave at 121 ° C for 30 minutes, cooled, and poured into 20 Pelri boxes of 100 mm x 15 mm. A two-year randomized experimental block design with four replicas was used, with five Peiri boxes per replica and five fronds per Petri dish. For the induction of callus, 5 individual aquatic lentil fronds were placed with the abaxial side down on each medium plate. The plates were incubated at 23 ° C for 2 weeks under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 2 weeks, the 69 Z tissue of the aquatic grass in half of the plates (10 plates) was transferred to fresh medium of the same composition, and the incubation was continued under the same conditions to those of the non-transferred tissue. After 4 weeks, the tissue was evaluated for callus proliferation. Three types of callus proliferated: calluses of type I, type II and type III. No difference was observed in the proliferation or type of callus between the tissue of the aquatic lentil transferred at 2 weeks, comparatively with the tissue of the aquatic lentil incubated for 4 weeks without transfer. The calluses of types I and III were subcultured far from the fronds • 10 originals, and were cultured in the middle between Murashige and Skoog with 3% sucrose, 0.15% Gelrile, 0.4% Difco Bacillus agar, 20 μM 2.4-D and 0.01 μM BA. Proliferating callus was subcultivated coniinely in fresh medium of the same composition at 2 week intervals. The longer intervals between the transference resulted in a decrease abrupt in the health of calluses between 2 and 3 weeks. The proliferation of calluses continued without loss of vigor when a program of • subculture for two weeks.

EXAMPLE 6 20 Two different basal media, Murashige and Skoog and Niisch and Nilsch (Science 163, 85 (1969)), were tested to compare their relative efficacy for the induction of G3 tripe of Lemna gibba. They were developed fronds of aquatic lentils in liquid water of Schenk and Hildebrandt containing 1% sucrose for two weeks at 23 ° C under a foioperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experimentation. For the induction of callus, 500 ml of Murashige and Skoog medium and Niisch and Nilsch medium were prepared with 3% sucrose, 0.15% Gelrile and 0.4% Difco Bacto-agar, with concentrations of 2,4-D 30 μM and BA at 0.01 μM. All media were adjusted to pH 5.8, autoclaved at 121 ° C for 30 minutes, cooled, and poured into 20 100 mm x 15 mm Petri dishes. • 10 A randomized experimental block design with two replications with two replicas was used, with five Pelri boxes per replica and 5 fronds per Petri dish. For the induction of callus, 5 individual lentil leaflets were placed with the abaxial side down on each medium plate. The plates were incubated at 23 ° C for 2 weeks under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 2 weeks, the • Aquatic lentil tissue was transferred to fresh medium of the same composition, and incubation was continued under the same conditions. After 4 weeks, the tissue on all the plates was evaluated for the proliferation of calluses. The fronds cultivated in the middle of Nitsch and Niísch could not proliferate significant amounts of calluses. The color of the aquatic lens in this medium was pale and had begun to turn yellow.

The fronds of the aquatic lentil cultivated in the middle of Murashige and Skoog proliferated the three usual types of callus: callus of type I, type II and type III. Type I and III calluses were subcultured away from the original fronds, and were cultured in Murashige and Skoog medium with 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacto-agar, 2.4-D at 10 μM and BA at 0.01 μM. Proliferating callus was continuously subcultured in fresh medium of the same composition at 2 week intervals. The longer intervals between the transfer resulted in an abrupt decrease in the health of the corns between 2 and 3 weeks. The proliferation of • 10 corns coníinuó without loss of vigor.

EXAMPLE 7 Different baseline media, Murashige 15 and Skoog, Schenk and Hildebrandl and Gamborg B5 (Gamborg et al., In Vitro 12, 473 (1976)), were tested to compare their relative efficacy for callus induction and • G3 growth of Lemna gibba. Leaflets of the aquatic leaflet were developed in Schenk and Hildebrandl liquid medium containing sucrose at 1% for two weeks at 20-23 ° C under a photoperiod of 16 hours of light 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experiment. For the induction of callus, 500 ml of the three media were prepared with 3% sucrose, 0.15% Gelrite and 0.4% Difco agar-agar, with concentrations of 2,4-D at 30 μM and BA at 0.02 μM. All media were adjusted to pH 5.8, autoclaved at 121 ° C for 30 min., they were cooled, and poured into 20 Pelri boxes of 100 mm x 15 mm. A randomized experimental block design of fres fralamienlos with two replicas was used, with five boxes of Pelri per replica and 5 fronds per box of Pefri. For the induction of callus, 5 individual leaf lenses were placed with the abaxial side down on each medium plate. The plates were incubated at 23 ° C for 2 weeks under a furoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 2 weeks, the leech of the aquatic lentil was transferred to fresh medium of the same composition, and the incubation was continued under the same conditions. After 4 weeks, the tissue in all the plates was evaluated for callus proliferation. Fronds grown in Gamborg medium B5 were pale, and yellow senescent fronds were present. No noticeable proliferation of calluses had occurred. The fronds grown in the middle of Schenk and Hildebrandt were dark green, and aberrant fronds proliferated, and no appreciable proliferation of calluses had occurred. The aquatic lentil fronds cultivated in the middle of Murashige and Skoog proliferated the three common types of callus: callus of type I, II and III. The callus of íipos I and III were subcultured away from the original fronds, and they were grown in culture in Murashige and Skoog medium with 3% sucrose, 0.15% Gelriie, 0.4% Difco Bac-agar, 2,4-D at 10 μM and BA at 0.01 μM. Proliferating callus was continually secreted in fresh medium of the same composition at 2 week intervals. The longer intervals between the transference resulted in an abrupt decrease in the health of the corns between 2 and 3 weeks. The proliferation of callus continued without loss of vigor.

EXAMPLE 8 Basal media were used: Murashige and Skoog (MS), Schenk and Hildebrandí (SH), Nilsch and Nifsch (NN) and Gamborg B5 (B5), to compare their efficacy to support the proliferation of G3 type II callus. Lemna gibba in liquid medium. Lentils of aquatic lentil were developed in Schenk and Hildebrandt liquid medium containing sucrose at 1% for two weeks at 23 ° C under a furoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. before the experimentation. For the induction of callus, 500 ml of Murashige and Skoog medium were prepared with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar, with concentrations of 2,4-D at 30 μM and BA at 0.02. μM. All media were adjusted to pH 5.8, subjected to auíoclave at 121 ° C for 30 min., Cooled, and poured into 20 100 mm x 15 mm Petri dishes. For the induction of callus, 5 individual aquatic lentil fronds were placed with the abaxial side down on each plate. medium. The 20 plates were incubated at 23 ° C for 2 weeks under a furoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. After 2 weeks, the aquatic lentil tissue was transferred to fresh medium of the same composition, and the incubation was continued under the same conditions. After 4 weeks, the callus tissue of type II was used to inoculate liquid medium for callus culture in suspension. For the storage of suspended calli, 100 ml of each of the basal media, MS, SH, NN and B5, were prepared with 3% sucrose, 10 μM 2,4-D and 0.01 μM BA. The media were adjusted to pH 5.8, 25 ml aliquots were placed in 125 ml flasks, and the 16 flasks of the media were autoclaved at 121 ° C for 18 minutes. After cooling, each flask was inoculated with 1 to 2 small pieces of friable type II white callus. The mairas were wrapped with aluminum foil and incubated at 23 ° C for 2 weeks, with agitation at 100 rpm, in the dark. After two weeks, the mairas were evaluated for callus proliferation. A slight amount of growth was observed with Murashíge and Skoog medium and with Nilsch and Niísch medium. The flasks were incubated for 2 weeks without media change, and no further callus proliferation was observed.

LO 9 Threinol and two aquatic lentil strains of 15 species, widely representative of the genetic diversity of the Lemnaceae, were used to determine the degree to which the methods and means for the induction of callus developed with Lemna gibba G3 were extrapolated through the whole family. Table I gives a list of the strains tested. Lentils of aquatic lentil were developed in Schenk and Hildebrandt liquid medium containing sucrose at 1% for two weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmol / m2 sec. before the experimentation. For the induction of callus, six basal media were used: Murashige and Skoog, Schenk and Hildebrand (Schenk and Hildebrandt, Can. J. Bot.50, 199 (1972)), Nilsch and Niisch, N6 (Chu et al., Scientia Sínica 18, 659 (1975)), B5 of Gamborg and means of Hoagland. Two combinations of growth regulator known to induce callus proliferation in L. gibba G3 were used: 2,4-D at 30 μM and BA at 0.02 μM, and 2,4-D at 5 μM and BA at 2 μM. For each strain, 200 ml of each basal medium was prepared with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar. The 200 ml were divided into 2 100 ml portions, and each was used to prepare the two growth regulator concentrations. The pH of sludge media was adjusted to 5.8, the media was autoclaved for 30 minutes at ? -i-- jüßí »4-! - < ft 121 ° C, they were cooled, and 4 100 mm x 15 mm petri dishes were poured from each 100 ml portion. An experimental randomized block design of 12 calibers with six media x 2 combinations of growth regulator was used., for each aquatic lentil strain tested. The design was doubled four times, with one Petri dish per replica and 6 fronds per Petri dish. For the induction of callus, 6 individual aquatic lentil fronds were placed with the abaxial side down on each medium plate for the larger fronds of the Lemma, Spirodela and Wolfiella species. For the strains within the genus Wolffia, the small fronds technically forbade plating the individual fronds; rather, small masses of fronds were used as an experimental unit. The plates were incubated at 23 ° C for 4 to 5 weeks under a foioperiod of 16 hours of light / 8 hours of darkness and with an intensity of approximately 40 μmol / m2 sec. At this time, the fronds were evaluated in terms of general health (judged by color: green to yellow, and vigor of proliferation) and the frequency of onset of callus formation of the three types: types I, II and III. The results showed a variation in the response capacity of the different species of aquilic lenses to the medium of callus induction. In general, the species and strains of the Lemna and Wolffia genera gave the best response. The five strains of Lemna gibba showed callus induction to varying degrees in MS, B5 and N6 media containing 2,4-D at 5 μM and BA at 2 μM. Strains of Lemna minor showed the same pattern, with a higher degree of induction of callus than Lemna gibba strains. The strains of Lemna miniscula showed an alpha frequency of induction of callus, with proliferation of a white callus somewhat different from Lemna minor or Lemna gibba. Lemna aequinoctialis showed curl and swelling of the frond at concentrations more alias than auxin, but the proliferation of an actual callus culture was not observed, indicating that the auxin concentrations used were not sufficiently high. Valdivian lemna showed no callus induction. In the Wolffia species, Wolffia arrhiza showed a small amount of callus proliferation in B5 medium with 2,4-D at 5 μM and BA at 2 μM. Wolffia brasiliensis and Wolffia columbinana showed induction of callus in Hoagland's medium supplemented with 2,4-D at 5 μM and BA at 2 μM. The remaining species of Wolffia, Australian Wolffia, showed no induction of callus, although the fronds showed swelling and a little abnormal growth. The species of Wolffia and Spirodela did not show callus induction. The fronds of the Spirodela species did not survive the concentrations more alias than 2,4-D, and did not grow well at the lower concentration. This response pattern is consistent with the interpretation that Spirodela is more sensitive to auxin than the Lemna and Wolffia species, and that lower auxin concentrations should be used in subsequent experiments to induce callus formation. © 8"'X ~ .¿ -"' - * • éUÁÜRO 1 Genus Species Designation of the Place of origin strain Spirodela polyrrhiza 7970 E.U.A. 4240 China 8652 China 8683 Kenya Spirodela punctata 7488 E.U.A. 7776 Ausíralia Spirodela intermedia 7178 Wolffia arrhiza 7246 South Africa 9006 Japan Australian Wolffia 7267 Tasmania 7317 Ausíralia Wolffia brasiliensis 7397 Venezuela 7581 Venezuela 8919 Venezuela Wolffia columbiana 7153 E.U.A. 7918 E.U.A. Wolffiella lingulata 8742 Argenina 9137 Brazil Wolfiella neotropica 7279 Brazil 8848 Brazil Wolfiella oblongata 8031 E.U.A. 8751 Argeníina Lemna aequinoctialis 7558 E.U.A. Lemna gibba G3 E.U.A. 6861 Italy 7784 8405 France 8678 Kashmir Lemna minor 8744 Albania 8627 Denmark Lemna miniscula 6600 California 6747 California Lemna valdiviana 8821 Argentina 8829 Argeníina EXAMPLE 10 Four auxins, naflaleneacetic acid (NAA), 2,4-D, indolbuiric acid (IBA) and dicamba, were tested for their ability to induce callus formation from L. gibba G3 fronds in three media different basics: SH, MS and N6. Two weeks of aquatic legume fronds were developed in Schenk and Hildebrandt liquid medium containing sucrose at 1% at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a light intensity of approximately 40 μmoles / m2 sec . before the experimentation. For the induction of calluses, 3 basal media were tested: Murashige and Skoog, Schenk and Hildebrandt and N6. Benzyladenine was used as a cytokinin at a concentration of 1μM. Auxin concentrations varied with the type of auxin. For the relatively strong auxins, 2,4-D and dimea, the concentrations were 0, 1, 5, 10 and 20 μM. For the weak auxins, NAA and IBA, the concentrations were 0, 5, 10, 20 and 50 μM. For each response experiment - medium dose, 2 liíros of basal medium with BA were prepared, and the pH was adjusted to 5.8. The volume was distributed in aliquots as 20 100 ml portions. To each of these portions, the appropriate amount of auxin was added, and the medium was adjusted with Gelriie at 0.15% and Difco Baccus-agar at 0.4%. The media was autoclaved for 30 minutes at 121 ° C, cooled, and 4 boxes of Pelri 100 mnfßc ^ d mm were poured from each 100 ml portion. A randomized experiment design was used at the dose of 60 days with combinations of 3 media x 4 auxins x 5 concentrations. The design was replicated twice, with a Pelri box per replica and 5 fronds per Peiri box. For the induction of callus, 5 individual aquatic leaf fronds were placed with the abaxial side down on each medium plate. The plates were incubated for 5 weeks at 23 ° C under a furoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2.sec. After 5 weeks, the fresh tissue weight of the aquatic lentil emerging from each original frond was measured, and these tissue populations were examined visually for the number of induced calli and the callus type produced. Several trends were observed in these results. First, low auxin concentrations and weak auxins promote the proliferation of fronds. This proliferation is greater than that observed without auxin present. When the fronds are proliferating, the frequency of callus induction is low. At high concentration of auxin, or with stronger auxins, curling and reduced basic proliferation of the fronds was observed. The callus formation is associated with the curling of the fronds. The auxin types were grouped (from higher curl to lower curl) as follows: 2,4-D, dicamba, NAA and IBA. N6 and MS underwent callus formation, but not SH. N6 supported a higher V proliferation than MS. Higher concentrations of auxin on N6 were required to induce callus formation than in MS medium. For the induction of compact callus type I, 2,4-D, dicamba and NAA allowed some degree of callus induction in MS medium, and in N6 medium, only 2,4-D and dicamba produced calluses. The highest callus induction was observed in MS medium conferring NAA at 10 μM.

EXAMPLE 11 Four cilocinins were tested: benzyladenine (BA), cinein, idiazuron (TDZ) and 2-PP, to determine their ability to induce callus formation from L. gibba G3 fronds in different basal media: SH, MS and N6. Two weeks of aquatic leaf fronds were developed in liquid Schenk and Hildebrandt medium containing 1% sucrose at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec . before the experiment. For the induction of calluses, 3 basal media were tested: Murashige and Skoog, Schenk and Hildebrandl and N6. 2,4-D was used as auxin at a concentration of 20 μM. The cyanocin concentrations used were 0.05, 0.1, 0.5, 1 and 5 μM. For each response experiment - medium dose, 2400 ml of basal medium was prepared with 2,4-D, and the pH was adjusted to 5.8. The volume was dissipated in aliquots as 24 100 ml portions. To each of these portions "the appropriate amount of cytokinin was added, and the medium was adjusted with 0.15% Gelrite and 0.4% Difco Bacto-agar. The media was autoclaved for 30 minutes at 121 ° C, cooled, and 4 boxes of Pelri 100 mm x 15 mm were poured from each 100 ml portion. An altered response experimental design was used at a dose of 72 treatments with combinations of 3 media x 4 types of cilocinin x 6 concentrations of cilocinin. The design was replicated twice, with a Peíri box per replica and 5 fronds per Peíri box. For the induction of callus, 5 individual aquatic leech fronds were placed with the abaxial side down on each medium plate. The plates were incubated for 5 weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2.sec. After 5 weeks, the fresh tissue weight of the aquatic lentil emerging from each original frond was measured, and these tissue populations were examined visually for the number of induced calli and the type of callus produced. Several trends were observed in these results. Proliferation of fronds did not occur at all because the 2,4-D concentration of 20 μM is too high. The rolling of the fronds was evident through all the treatments. MS and N6 showed induction of callus, with MS clearly superior. There was no callus induction in SH medium. TDZ gave the highest frequency of induction of callus in MS medium through a different scale of concentrations. There is a reciprocity between the induction of calluses of types I and II. When the induction of type I callus is alias, the induction of callus of type II is low.

EXAMPLE 12 Since strains 8744 and 8627 of Lemna minor showed higher callus induction and faster proliferation than the L. gibba strains (see example 9 and table I), a greater concentration of the conditions of culture for L. minor. The variations tested for the induction of callus included: a) selection of the basal medium composition, b) selection of the type and concentration of auxin, and c) selection of the type and concentration of cilocinin. In the selection of the basal medium, media were tested: Schenck and Hildebrandí, Murashige and Skoog, and medium F, developed by Frick (Frick, (1991) J. Plant Physiol., 137: 397-401). The supply fronds used for these experiments were developed in F medium supplemented with 2,4-D at 24 μM and 2 μP at 2 μM for two weeks before use. The callus induction means were prepared as in example 8. The fronds were separated, the roots cut, and the miiad were forced to pass through a mold (following the Frick method) before being placed in the middle of induction of callus, and the remaining milad of the fronds was deposited iníacta in plates. The fronds If "*? í88-fc 'were incubated under the conditions given in Example 8 for 6 weeks, during which time the culinos were evaluated to determine the presence or absence of callus induction, the degree to which the calluses proliferated, and the degree to which the calluses proliferated. The Murashige and Skoog medium showed the best callus induction with the strains of L. minor, the medium of Schenk and Hildebrandí could not produce calluses, and the induction of them was minimal in F. The fronds were drawn from a lamix before being deposited on plates, and there was no effect on the induction of callus In the concentration experiment and auxin type, 4 auxins were tested: 2,4-D, NAA, IBA and dicamba, each at any concentration: 2, 5, 10 and 20 μM, to determine its capacity to induce callus formation from strains 8744 and 8627 of L minor.The basal medium used was MS, and the means and the experimental protocol were basically even sa those followed in example 10. The fronds used in this experiment were developed for two weeks before being plated on callus induction medium under 3 different culture conditions: 1) SH medium without growth regulators, 2) medium F with 2,4-D at 24 μM and 2-iP at 2 μM, and 3) SH medium with 2,4-D at 24 μM and 2-iP at 2 μM. The fronds were separated, the roots cut and deposited then on plates on induction medium. The fronds were incubated under the conditions given in example 8 for 6 weeks, at which time the cultures were evaluated to determine the presence or absence of callus induction, the degree to which the callus p ^? W tatoDt and the basic morphology thereof. No induction of callus was observed in the irradiations in which the induction auxin was NAA or IBA. For strain 8744, the prolific induction of callus was observed in treatments with 2,4-D at concentrations of 5 or 10 μM, giving 2,4-D to 5 μM the best induction. The induction of callus was also observed at the concentration above dicamba, 20 μM. For Strain 8627 of L. minor, callus induction was also observed with 2,4-D and dicamba, but at lower concentrations. For 2,4-D, the induction more • 10 prolific calluses were observed at 1 and 5 μM, giving 5 μM the best induction. The useful concentrations of dicamba for callus induction were 5 and 10 μM. Regardless of the method of treatment for the induction of calluses, callus formation only came from two fronds previously developed in the middle of Schenk and Hildebrandt without growth regulators. In the experiment of the type and the concentration of cilocinin, 4 cilocinins were tested: BA; Kinetin, 2-iP and Iidiazuron, each at 5 • concentrations: 0.05, 0.1, 0.5, 1 and 5 μM, to determine their capacity to induce callus formation from strains 8744 and 8627 of L. minor. The basal medium used was MS medium, and the media and the experimental pro-isole were basically the same as those described in example 11. The fronds used in this experiment were developed during two weeks before being plated on callus induction medium under 3 different culinary conditions: 1) SH medium without growth regulators, 2) medium F with 2,4-D at 24 μM and 2-iP at 2 μM, and 3) SH medium with 2,4-D at 24 μM and 2-iP at 2 μM. The fronds were separated, the roots cut and deposited then on plates on induction medium. The fronds were incubated under the conditions given in Example 8 for 6 weeks, at which time the cultures were evaluated to determine the presence or absence of callus induction, the degree to which the calluses proliferated, and the basic morphology thereof. . For strain 8744, prolific induction of callus with 2-iP or lidiazuron was observed, each at 0.5 or 1 μM. Callus induction was only observed with fronds grown on medium F before being deposited on plates on callus induction medium. For Strain 8627 of L minor, callus induction with 2-iP or thiazuron was also observed, but at lower concentrations: 0.1 or 0.5 μM. In this strain, the induction of callus was also observed using BA at 0.5 and 1 μM.

EXAMPLE 13 The composition of basal medium was tested for its effects on callus proliferation and long-term establishment thereof using strains 8627 and 8644 of L. minor. Three compositions of basal medium were tested to determine their capacity to maintain the healthy growth of calluses: MS, medium F and SH of medium concentration. All media contained sucrose - "^ F * a 3%, and were gelified with Bacto-agar Dífco at 0.4% and Gelrile at 0.15% .The MS medium was supplemented with 2,4-D at 1 μM and BA at 2 μM, the SH medium from medium concentration was supplemented with BA at 1 μM, and medium F was supplemented with 2,4-D at 9 μM and 2-iP at 1 μM For this experiment, callus cultures of strains 8744 and 8627 proliferated at means of prior induction of callus as in example 12. The calluses were developed during a subculture period of two weeks, and evaluated for growth, color and general health.For strain 8744 of L. minor, the SH medium of medium concentration complemented with BA at 1 μM, proved to be the best to maintain the growth and health of the calluses, causing the resulting calluses areas of organizations and aberrant regeneration of the fronds. green to pale yellow The culture of the callus in MS medium or medium F gave As a result a very rapid proliferation, doubling in fresh weight every 6 days. The calluses developed on these two media showed organization and regeneration of much smaller fronds. For strain 8627, there was little effect of the basal media, and the proliferation of calluses in all three media was equally good. As in the case of strain 8744, the calluses showed greater organization when they were developed on SH medium concentration medium supplemented with BA at 1 μM.

EJE1fff > tO 14 Since Lemna minor showed higher induction of callus than Lamna gibba, an additional selection of fresh strains of L. minor was made, exceptional in leaf growth velocity and protein content, to determine if the protocol for the induction of Calluses from strains 8744 and 8627 of L. minor would be extrapolated to new strains. The strains were designated 7501, 8626 and 8745 of L. minor. The callus induction system developed in the previous examples was followed: for the induction of calluses, basal Murashige and Skoog medium supplemented with 3% sucrose, 2,4-D at 5 μM and BA at 2 μM were used, and it was gelled with Bacto-agar Dífco at 0.4% and Gelrite at 0.15%. The fronds were grown in liquid SH medium lacking growth regulators and supplemented with 1% sucrose before sowing on callus induction medium. The fronds were deposited on means of callus induction, and were evaluated 5 weeks later to determine the relative frequencies of callus induction and relative proliferation rates. For strains 8626 and 8745, callus induction did not occur during the induction period of 5 weeks; however, the subsequent culture gave a low frequency of callus proliferation. The morphology and color of the calli of strains 8626 and 8745 were quite similar to those developed from strains 8744 and 8627, and proliferated quite well when they were transferred to callus maintenance media. Strain 7501 showed a low frequency of callus induction, with corns of morphology similar to those produced from strains 8626 and 8745.

EXAMPLE 15 Since Lemna miniscula showed significant induction of callus in the first selection (see example 9), callus induction was repeated with strains 6600 and 6747 of Lemna miniscula. Callus induction medium was prepared, and the fronds were culíivaron as described in example 14. Strains 6600 and 6747 of Lemna miniscula showed very common callus induction frequencies, proliferating callus from virtually any frond. The onset of callus formation occurred rapidly in these strains, and calluses were observed for the first time 2 to 3 weeks after sowing. The calli were pale in color, and proliferated more slowly than those produced from strains 8744 or 8627 of Lemna minor (see example 14). 8 # EXAMPLE 16 Based on the investigations described in the previous examples, the preferred methods for the induction and growth of callus in Lemna are the following: The induction and growth of callus and the regeneration of the fronds of aquatic lentil plants are achieved through incubation in the appropriate environment and manipulation of growth regulator types and concentrations at specific stages of development to promote formation, growth and reorganization of the callus until totally differentiated plañías. Typically, for species within the Lemna genus, the preferred means for induction of callus are N6 and MS, with MS being more preferred. The fronds are incubated in the presence of an auxin and a cytokinin, the auxins being preferred, NAA and 2,4-D, and the preferred cytokines BA and TDZ. The concentrations of these growth regulators vary on a wide scale. For auxins, the preferred concentrations are 5-20 μM, the most preferred are 5-10 μM, and for the kinocinins, the preferred concentrations are 0.5-5 μM, with 0.5-1 μM being more preferred. The fronds are incubated during an induction period of 3 to 5 weeks in medium containing growth regulators with means of callus proliferation during this time. For the growth of calluses, the preferred means are the same as those used for the induction of callus, but the concentration of auxin is reduced. For the years, the preferred concentrations are 1-5 μM, and for cytokinins, the preferred concentrations are 0.5-1 μM. The subculculum period was also reduced from 4 to 5 weeks for the induction of callus, and 2 weeks for the growth of callus in the long term. Callus growth can be maintained in solid gelled medium with agar, Gelrile, or a combination of both, with the preferred combination of Difco Baclo-agar at 0.4% and Gelrite at 0.15%, or on liquid medium. By using this method, callus cultures can be kept healthy for indefinite periods. 10 EXAMPLE 17 Strains of the genus Wolffia respond to the growth regulator concentrations for induction of callus, in a similar way to the observed for the denlro strains of the genus Lemna. Therefore, additional strains of Wolffia were investigated to determine their ability to grow calluses. Four strains of Wolffia arrhiza were tested: 7246, 8853, 9000 and 9006, and four strains of Wolffia brasiliensis: 7393, 7581, 7591 and 20 8319 to determine their ability to proliferate calluses in response to growth regulators. The basal medium used was MS medium supplemented with 3% sucrose, 5 μM 2,4-D, 5 μM BA and cinnamin, and 65 μM phenylboronic acid. The cultures were plated and incubated in callus induction medium for 5 weeks, and then evaluated for callus proliferation. No callus proliferation was obtained during this 5-week incubation period from any of the strains tested. However, the morphology of induction prior to callus was easily evident in several strains, including 8853, 9000 and 9006 of Wolffia arrhiza and 7581 of Wolffia brasiliensis. With these strains, the thickening of the frond was evident, a response that is frequently observed in fronds before the formation of the callus becomes evident, and indicates that the concentration of auxin used was insufficient to sustain the proliferation of calluses.

Transformation This section covers experiments that belong to the methods used for the real transfer of genes. There are several sections: (1) leaf transformation using the gene gun, (2) Agrobacterium-mediated Iransformation using aquatic lentil fronds, and (3) Agrobacterium-mediated transformation using aquatic lentil callus. The frond transformation experiments were used to optimize the parameters that affect the real transfer of genes: (a) bacterial growth, (b) inclusion of an acetosyringone, (c) bacterial concentration, (d) solution to resuspend bacteria and the effect of osmotic shock, (e) co-culture medium for fronds and calluses, (f) duration of inoculation, (g) co-cultivation time for fronds and c and (h) lighting conditions during co-cultivation. The proolocol developed with fronds was applied to transform the callus cultures obtained using the optimized tissue culture procedure. It is this transformation transformed what is taken to carry out the selection, and then to the regeneration to obtain transformed fronds.

EXAMPLE 18 Transformation mediated by means of a gene gun G3 leaflets of Lemna gibba were subjected to microcarrier bombardment to test their ability to express constructs of injected genes. For the proliferation of fronds, 60 ml of medium of high concentration of salts were prepared (De Fossard, TISSUE CULTURE FOR PLANT PROPAGATORS 132-52 (1976)) supplemented with 3% sucrose and 0.8% agar; the pH was adjusted to 5.8, and the medium was autoclaved for 20 minutes at 121 ° C, cooled, and used to pour 6 Pelri boxes of 60 mm x 15 mm. A frond was inoculated for each box of Pefri. The fronds developed during two weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec.

M4 For the bombardment, 1.6 μm gold microcarriers were repaired, and the plasmid pRT99 DNA was precipitated on the microcarriers following the protocols of the manufacturer's gene gun (Bio-Rad). Plasmid pRT99 (Topfer et al., Nucleic Acid Res. 16, 8725 (1988)) codes for the gene for neomycin phosphotransferase and the gene for β-glucuronidase (GUS; Jefferson et al., EMBO J. 6, 3901 (1987 )), both under the control of the 35S promoters of the CaMV. The fronds of the aquatic lentil were turned with the abaxial side upwards, and were bombarded with the microcarriers coated with DNA at four levels of helium pressure: 56.24, 42.18 and 28.12 kg / cm2. It was carried out 24 hours after bombardment hislochemical staining for GUS activity using 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) as substrate, following the Stomp method (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (SR Gallagher ed.1991)). The frequency of GUS-positive staining bands was directly proportional to the pressure used for the bombardment, with the highest number of cells expressing GUS in the treatment at 56.24 kg / cm2, the frequency varying from 4 to 20 cells stained. /frond. In all the raids, the bombardment resulted in the removal of more than half of the fronds. .8ß EJ LO 19 The G3 fronds of Lemna gibba were subjected to microprojectile bombardment to test the effect of microcarrier size on the frequency of expression of injected genes. For the proliferation of fronds, 200 ml of medium were prepared with ally salt concentration, supplemented with 3% sucrose and 0.8% agar; the pH was adjusted to 5.8, and the medium was autoclaved for 20 minutes at 121 ° C, cooled, and used to pour 20 Peiri boxes of 60 mm x 15 mm. A frond was inoculated for each Petri dish. All the fronds were developed during two weeks at 23 ° C under a photoperiod of 16 hours of light / 8 hours of darkness and with a luminous intensity of approximately 40 μmoles / m2 sec. Two microcarrier handles, 1.0 and 1.6 μm, were tested at 3 helium pressure levels: 28.12, 56.24 and 84.36 kg / cm2, using a PDS-1000 / He gene gun manufactured by DuPont. Gold microcarriers were prepared, and the pRT99 DNA was precipitated on the microcarriers following the methods provided by the manufacturer (Bio-Rad). The aquatic legume fronds bombarded for GUS expression were tested 24 hours after bombardment using Slomp histochemical staining method (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (SR Gallagher ed.1991) ). The highest frequency of GUS expression was found in fronds bombarded with microcarriers of 1.6 μm and helium pressure of 56.24 kg / cm2. The number of positive events to GUS varied from 1 to 21 per frond. Transgenic aquatic lentil plants are regenerated from aquatic leech calli transformed by ballistic bombardment. The callipers of callus I are developed as described in example 42 below. Typically, 20-30 pieces of aquatic lentil callus, approximately 2-4 mm in diameter, are disseminated uniformly through the bombardment area on MS medium (MS medium described in example 42). Gold particles (1.6 μM diametre) and bombardment (helium pressure of 56.24 kg / cm2) are used and as described in example 18 and in example 19. The bombardment DNA consists of an expression plasmid containing the gene of interest (eg, GUS, another marker gene, a gene that codes for a mammalian protein, or a gene that codes for a bacterial, fungal, plant or mammalian enzyme) and a gene that codes for a marker gene selectable, for example, nptll (resistance to kanamycin), hptll (resistance to hygromycin), sh ble (resistance to zoecin), and bar (resistance to phosphinocyrrhine), as well as ofras necessary sequences for gene expression (for example, sequences of promoter, and termination sequences). After bombardment at 56.24 kg / cm2, the callus is incubated in the dark for 2 days (or longer if necessary), followed by incubation under a luminous intensity of 3-5 μmol / m2 for 4-6 weeks. The corns are transferred to new medium every 2 weeks with the selectable agent added to the medium 2-4 weeks after the bombardment. The selection of 8? Resistant corns are continued for 8-16 weeks, until completely resistant calli are produced. The regeneration of fronds and transgenic plants is carried out as described in example 42.

EXAMPLE 21 Transformation with Agrobacterium using aquatic lentil fronds.

Lemna gibba aquatic lentil fronds were used to test the susceptibility of aquatic lentil to Agrobacterium tumefaciens using two different media for co-cultivation, Schenk and Hildebrandí and Murashige and Skoog. Agrobacterium tumefaciens strain AT656 and A. tumefaciens strain A136 were used to inoculate the leaflets of the aquatic lentil. Strain AT656 is constructed from strain EHA105 (Hood et al., Transgenic Res. 2, 208 (1993)) which contains the pTiBo542 region on a disarmed pTiBo542 plasmid. The T-DNA is carried on a binary plasmid, pCNL56 (Li et al., Pl.Mol. Biol 20, 1037 (1992)). This binary plasmid is obtained from pBIN19, and modified as such carries a gene for neomycin phosphotransferase under the control of the nopaline synthetase promoter and a nopaline synthetase terminator, and a β-glucuronidase (GUS) gene (Janssen and Gardner, Plant Mol. Biol. 14, 61 (1989)) under the control of the mas2'-CaMV35S promoter and an octopine synthetase terminator. The coding region for GUS contains a dense incle of the coding sequence of the gene to prevent the expression of GUS (Vancanneyi et al., Mol.Genet, 220, 245 (1990)). Strain A136 is obtained from the host broad spectrum strain, C58. When C58 develops at temperatures above 30 ° C it loses its Ti plasmid becoming A136 avirulenia. Two strains AT656 and A136, were grown overnight on minimal AB medium (Chilton et al., Proc.A Nat. Acad. Sci. USA 71, 3672 (1974)) solidified with 1.6% agar and supplemented with 100 μM of Aceyosyringone at 28 ° C. The aquatic lentil fronds were grown in Hoagland liquid medium containing 3% sucrose for two weeks at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 «sec. before the experiment. For co-cultivation, 500 ml of Schenk and Hildebrandí medium containing 1% sucrose and 0.6% agar were prepared, the pH adjusted to 5.6, autoclaved at 121 ° C for 30 minutes, and cooled. 500 ml of Murashige and Skoog medium containing 3% sucrose and 0.6% agar were also prepared, pH adjusted to 5.8, autoclaved at 121 ° C for 30 minutes and cooled. To both media, a sterilized acetosyringone solution was added by filtration to a final concentration in the medium of 20 mg / L. The medium was poured into 20 Pelri boxes of 100 mm x 15 mm from each cold medium. For each bacterial strain, the bacteria from a 100 mm x 15 mm Petri dish were resuspended for at least one hour before being used in 100 ml of the following solution (Hiei et al., The wt Plant J 6 , 271 (1994)): B5 ^ Bamborg salts, Murashige and Skoog vitamins, glycine (8 mg / l), aspartic acid (266 mg / l), arginine (174 mg / l), glulamine (876 mg / l) , casamino acids (500 mg / l), sucrose (6.85%), glucose (3.6%) and acetyringone (20 mg / l). The solution was prepared, adjusted to pH 5.8 and sterilized by filtration before the addition of the bacteria. A completely factorial experimental design of 2 bacterial strains x 2 co-cultivation media (4 tramadolins in lolal) with 5 repetitions was used, with 2 Petri dishes per repetition and 20 fronds for each Petri dish. For the inoculation, the aquatic leaf fronds were floated in the bacterial solution for several minutes. For co-cultivation, the fronds were transferred either to Schenk and Hildebrandl or Murashige and Skoogal medium and as described above. The fronds were incubated at 23 ° C under a flashlight period of 16 hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec for four days. The fronds were then transferred to fresh medium of the same composition except that aceylosingone was absent and 500 mg / l of timentin and 50 mg / l of kanamycin sulfate had been added to the medium. Histochemical staining was used to determine GUS activity following the method of Stomp et al., Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (S.R: Gallagher ed.1991)) to confirm gene transfer in fronds. The staining of the fronds inoculated with A136 was done as a control to test the fronds inoculated with bacteria in terms of the endogenous GUS activity. Staining done 10 days after inoculation did not show staining for GUS in the controls of A136 inoculated and showed aliases of staining frequencies in fronds inoculated with AT656, without taking into account the basal medium MS or SH used for co-culture. Iransformation frequencies greater than 70% were observed in the original inoculated fronds, showing GUS cells somewhere in the dense fronds.

EXAMPLE 22 Lemna gibba G3 fronds were used to determine the effect of injury on the frequency of GUS expression after co-cultivation. Aquatic legume fronds were developed in the middle of Schenk and Hildebrandl liquid containing 1% sucrose for two weeks at 23 ° C under a furoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 after the experiment. For cocultivation, a liter of Murashige and Skoog medium containing 3% sucrose, 0.6% agar, 20 μM 2,4-D, 2 μM BA, and 20 mg / l acetosyringone was prepared. adjusted the pH to 5.8, autoclaved at 121 ° C 30 minutes and cooled down. A sterilized acetosyringone solution was added by filtration to a final concentration in the medium of 20 mg / l. The cold medium was poured into 40 Peiri boxes of 100 mm x 15 mm. r For the inoculation, Agrobacterium tumefaciens strain was harvested AT656 and developed overnight at 28 ° C over minimum medium AB (Chilíon et al., Proc. Nat. Acad. Sci E.U.A. 71, 3672 (1974)) containing 50 mg / l of kanamycin sulfate and 20 mg / l of acetyringone. For the inoculation, the basins from a 100 mm x 15 mm Petri dish were resuspended as described in example 21. A completely factorial experimental design of 2 treatments was used for injury x 2 bacterial inoculations with 5 repeats, with 2 Pelri boxes per repetition and 20 fronds per Petri dish. For the treatments for injury, masses of aquatic lentil fronds of the SH medium were re-irrigated on wet sterile filter paper. The masses were separated into individual fronds, the fronds were turned with the abaxial side upwards and were injured by one of two ways: 1) cross-sectional cutting from the crown of the frond, cutting in this way from the adjacent merisiemálicas regions from left to right, or 2) cut on each side of the hill, cutting from east to west long way from each meristemic region. For bacterial rats, both classes of injured fronds were floated on: 1) resuspended AT656 or 2) in resuspension fluid without bacteria. For the inoculation, the fronds were floated for 10-30 minutes. For co-culture, the fronds were transferred to Murashige and Skoog medium as described above with 3% sucrose, 20 μM 2,4-D, 2 μM BA, 100 μM aceyosyringone and 0.6% agar. The fronds were incubated at 23 ° C under a photoprobe of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 sec for four days. A subsample of frond was used to determine GUS following the procedure of Synp (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (SR Gallegher ed.1991) .The staining of co-cultivated fronds four days after inoculation showed that the direction of injury did not affect the frequency of the fronds with GUS staining, which had an average of approximately 70% Confrol, damaged fronds inoculated with bacterial resuspension solution without bacteria did not stain for GUS The number of fronds with denier staining of the meristemic regions gave an average of approximately 40%.

EXAMPLE 23 Lemna gibba G3 fronds were used to determine the effect of inoculation time for injured fronds in bacterial resuspension medium on the frequency of GUS expression after co-culling. Aquatic leaf fronds were grown in liquid Hoagland medium containing 1% sucrose to a density of approximately 120 fronds per 25 ml of medium in a 125 ml flask at 23 ° C under a 16 hour light / photoperiod. 8 hours of darkness with luminous intensity of approximately 40 μmol / m2-sec before the experiment. For co-cultivation, 1500 ml of Schenk and Hildebrandt medium were prepared with 1% sucrose and 0.6% agar, the pH was adjusted to 5.6, autoclaved at 121 ° C for 30 minutes and cooled. A sterilized acetyringone solution was added by filtration to a final concentration in the medium of 20 mg / l. The medium was poured into 60 Petri dishes of 100 mm x 15 mm. A randomized block experimental design was used with 4 treatments of inoculation time, with 3 repetitions, with 5 Peiri boxes per repetition, and 25 fronds per Petri dish. For inoculation, Agrobacterium tumefaciens strain AT656 was used and developed overnight at 28 ° C on minimal AB medium containing 50 mg / l of kanamycin sulfate and 20 mg / l of acetosyringone. For inoculation, the bacteria from a 100 mm x 15 mm Peiri box was resuspended as described in example 21. For inoculation, individual fronds were separated from the masses, each was turned with the abaxial side up and was injured with a sterile scalpel in the merisiemal regions, then they were transferred to bacterial suspensions and incubated for 15, 30, 45 or 60 minutes. For co-cultivation, the fronds were transferred to Schenk and Hildebrandt co-culture medium as described above. All 60 Petri dishes were incubated at 23 ° C under a fofoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 sec for six days.

The repeated repetitions were made over a period of four days. The submuesíras of fronds co-cullivadas provenieníes of each time of incubation (15, 30, 45 or 60 minutes) were taken after 2, 3 and 6 days of co-culíivo. The subsampled fronds were blended to deduce GUS expression following the procedure of Synp (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (S.R.

Gallagher ed. 1991)). The results are presented in table II.

TABLE II Repetition Time Time Total number Number of co-cultivation incubation of fronds dyed samples days 15 1 27 27 30 1 27 27 45 1 28 26 60 1 28 26 days 15 2 30 29 30 2 28 28 45 2 25 24 60 2 27 26 days 15 3 27 24 30 3 26 22 45 3 23 21 60 3 30 28 Although the staining to determine GUS on stem injury was evident after 2 days, staining to denoseline GUS in the meristematic regions was not evident after 2 days of cocultivation. The meristematic staining was greater at 3 days of co-culture and decreased around 6 days of co-culture. The incubation time of the aquatic lentil fronds in the bacterial suspension solution did not have a significant effect on the frequency of GUS total expression after co-cultivation.

EXAMPLE 24 Lemma gibba G3 fronds were used to determine the effect of the Agrobacterium strain and the gene construct introduced on the frequency of GUS expression after co-culling. • 10 Two strains of Agrobacterium tumefaciens were used: AT656 and C58sZ707pBI121. C58sZ707pBI121 is a disarmed C58 broad-range host strain (Hepburn et al., J. Gen. Microbiol. 131, 2961 (1985)) in which pBI121 has been transferred. The binary plasmid pBI121 is obtained from pBIN19 and its T-DNA encodes a neomycin-15-phospholransferase gene under the control of the nopaline-synliase promoter and a nopaline-sinielase enerminator, and for a β-glucuronidase gene (GUS) ) low conírol of a CaMV35S promoter and an oxyopin-synlelase enerminator.

• AT656 was seeded in rows in a minimum AB medium containing kanamycin sulphate at 50 mg / l and C58sZ707pBI121 was streaked in AB medium minimum containing sclerephomyomycin at 500 mg / l, speclinomycin at 50 mg / l and kanamycin sulfate at 50 mg / l. Both bacterial strains developed overnight at 28 ° C.

The aquatic lentil fronds were grown in Hoagland liquid medium containing 1% sucrose for 1 week at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with light intensity of approximately 40 μmol / m2 sec before the experiment . For co-cultivation, 500 ml of Schenk and Hildebrandt medium were prepared with 1% sucrose and 0.6% agar, pH adjusted to 5.6, autoclaved at 121 ° C for 30 minutes and cooled. A sterilized acetosyringone solution was added by filtration to a final concentration in the medium of 20 mg / l. 20 Pelri boxes of 100 mm x 15 mm were poured from the cold medium. A randomized block experimental design with 2 bacterial strain was used, with 2 replications, with 5 Peiri boxes per repeat and 25 fronds per Petri dish. For the inoculation, the bacteria coming from an AB plate of each strain were resuspended and as described in example 21. For the inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side towards above and were injured with a sterile scalpel in meristematic regions, then transferred to bacterial suspensions and incubated for 15-30 minutes. For co-cultivation, the fronds were transferred to the Schenk and Hildebrandt co-culture medium as described above. All 20 Peiri boxes were incubated at 23 ° C under a flashlight 16 hours / 8 hours darkness with luminous intensity of approximately 40 μmol / m2 sec for 6 days. A submuesíra of fronds was lowered at 6 days of co-cullívo and fry to determine the expression of GUS. With AT656, 12 of the 13 masses of aquatic leaf fronds sampled showed staining for GUS, however none was observed in the merislemálica region. With C58sZ707pBI121, all masses of aquatic legume fronds showed extensive staining. Incubation was continued for all remaining fronds for one week after transfer to new medium containing kanamycin sulfate. For transfer after co-culture, 1500 ml of Schenk and Hildebrandt medium containing 1% sucrose and 0.6% agar was prepared, the pH adjusted to 5.6, subjected to the auíoclave at 121 ° C for 30 minutes and cooled. Two titanium and kanamycin sulfate antibiotics were added as solutions sterilized by filtration to the cold medium to a final concentration in the medium of 500 mg / l and 2 mg / l, respectively. The cold medium was used to pour 60 Peiri boxes of 100 mm x 15 mm. After one week, the fronds were evaluated for growth on kanamycin and GUS expression. The proliferating fronds showed 3 categories of response to kanamycin: (1) about 20% of the fronds arising from those originally co-cultured with the bacterial strain AT656 showed vigorous growth in the presence of kanamycin and about 30% of the fronds from those originally co-cultured with the bacterial strain C58sZ707pBI121 showed vigorous growth in the presence of kanamycin, (2) other group of fronds did not clearly proliferate and were lacking chlorophyll and were dying, (3) an intermediate group of fronds showed some proliferation in presence of kanamycin but the fronds were half bleached, indicating sensitivity to kanamycin. The results of the determination to determine GUS indicated that the active enzyme was still present at a high frequency in the originally co-cultivated fronds.

EXAMPLE 25 Lemna gibba G3 fronds were used to determine the effect of Agrobacterium strain, introduced gene construct and frond pre-irradiation on the frequency of GUS after co-culture. Two strains of Agrobacterium tumefaciens were used: AT656 and EHA101 pJR1. EHA101 pJR1 is a strain of Agrobacterium tumefaciens that conies an unsettled pTiBo542 plasmid that hosts the hypervirulence region of the wild type strain, Bo542, and a small binary plasmid that hosts a hygromycin-phospholransferase gene under the control of an alcohol dehydrogenase. increased, promoted CaMV35S and a β-glucuronidase gene constructed as in AT656. These two strains were seeded on potato dexory agar with 50 mg / l of kanamycin and developed overnight at 28 ° C. Aquatic legume fronds were grown in Schenk and Hildebrandf liquid medium containing 1% sucrose with and without 10 μM indoleacetic acid (IAA), a sufficient concentration to increase the rate of proliferation. The fronds were grown in aliquots of 25 ml of medium in 125 ml flasks at 23 ° C under a photoperiod 16 hours light / 8 hours dark with luminous intensity of approximately 40 μmol / m2'sec. For the co-cultivation 500 ml of Schenk and Hildebrandl medium containing 1% sucrose, 0.8% agar, 20 mg / l aceyosyringone and with and without 10 μM indoleacetic acid, pH adjusted to 5.6, were prepared. Autoclave at 121 ° C for 30 minutes and cooled. Acid-syringone and acety-syringone and indoleacetic acid solutions were sterilized by filtration into the cold medium, to the appropriate final concentrations. 20 Pelri boxes and 100mm x 15 mm were poured from the cold medium. Experimental design by random blocks was used with 2 trays with bacterial strain x 2 frond growth media, with 5 repetitions, with one Pelri box per repetition and 20 fronds per Peiri box. For the inoculation, the bacteria of each strain were resuspended separately and as described in example 21. For the inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side upwards and they were injured with a sterile scalpel in the merislemáic regions, then they were transferred to bacterial suspensions of either AT656 or EHA101pJR1, and incubated for 10-15 min. For the co-cultivation, the fronds were transferred to Schenk medium and Hildebrandt solid with 1% sucrose, 0.8% agar and 100 μM of no.

Aceyosyringone without and with 10 μM of rholalacetic acid as described previously, with the abaxial side down. The fronds were co-cultivated for 4 days at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 »sec. The fronds from 2 boxes of each of the 4 ripenings were stained to determine the expression of GUS. Table III shows the results of the GUS stain.

TABLE III • 10 Medium Strain Numerical number Number of dyed fronds SH AT656 61 12 SH EHA101pJR1 62 4 SH + IAA AT656 66 32 15 SH + IAA EHA101pJR1 68 2 • Without taking into account the presence or absence of IAA, the fronds co-cultured with EHA101pJR1 had much lower frond frequencies that showed GUS expression. An effect of IAA in the middle of incubation was detected with medium containing IAA giving 48% of co-cultivated fronds that showed GUS expression compared to 20% of co-cultivated fronds in medium without IAA.

TO EXAMPLE 26 Lemna gibba G3 fronds were co-cured during five different times: 12.5, 18.5, 40.5, 82 and 112 hours, with the bacterial strain AT656 to test the effect of co-culture time on GUS expression after co-culture . The aquatic lentil fronds were grown for two weeks in liquid Schenk and Hildebrandf liquid containing 1% sucrose and 10 μM indoleacetic acid at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 * sec before the experiment. For the co-culio, 750 ml of Schenk and Hildebrandt medium were prepared with 1% sucrose, 0.8% agar, 10 μM indoleacetic acid and 20 mg / l aceyosyringone, pH adjusted to 5.6, the medium autoclaved at 121 ° C during 30 minutes and cooled. A solution of acetosyringone and indole acetic acid was sterilized by filtration at the final concentration of the medium. 30 100 mm x 15 mm Peiri boxes were poured from the cold medium. Bacterial strain AT656 was seeded on potato-dextrose agar with 50 mg / l of kanamycin sulfate and developed overnight at 28 ° C. An experimental design was used by random blocks with 5 incubation times, with 6 repetitions, with one Pelri box per repetition and 60 fronds per Petri dish. For inoculation, the bacteria were resuspended as described in example 21. For the inoculation, the individual fronds were separated from the masses and each was turned with the abaxial side up and injured with a sterile scalpel at the ends. meristematic regions. The fronds were then transferred to the bacterial resuspension solution and incubated for approximately 10-15 minutes. For the co-cultivation, the fronds were transferred to Schenk medium and solid Hildebrandt as described above, with the abaxial side down. The fronds were co-cultivated under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 »sec. At appropriate times, 10 fronds of each Peiri box (6 samples) were filtered and their chemistry was determined to determine GUS expression. Table IV gives the results of the GUS stain: TABLE IV Time (hr) Total number of Illegal number of fronds dyeing 12.5 61 0 18.5 61 0 40 61 0 82 75 24 112 67 25 S, The co-culture time had a significant effect on the frequency of fronds with GUS expression. Before 40 hours, no GUS expression was detected. Around 3.5 days (82 hours) the GUS expression could be easily deleted. Prolonged co-culture did not significantly increase the frequency, intensity, or tissue association patterns with GUS expression in aquatic lentil fronds. It was concluded that 3.5-4 days is the shortest co-culture time that will give the maximum frequency of gene transfer in aquatic lentil fronds.

EXAMPLE 27 Bacteria of strain AT656, grown in different bacterial media were used: minimal AB, potato-dexfrosa and Luria broth with mannitol glutamine, to co-culminate fronds of Lemna gibba G3 that had been developed with and without indolelic acid before co-cultivation. cultivation, in light and darkness to test the effects of those traversals on the expression of GUS after co-cultivation. The fronds of Lemna gibba G3 were developed for two weeks in Schenk and Hildebrandí liquid medium containing 1% sucrose and with and without 10 μM of indoleacetic acid in 25 ml aliquots in 125 ml mats, at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 * sec before the experiment. For co-cultivation, 900 ml of I & 4-Schenk and Hildebrandt medium containing 1% sucrose, 0.8% agar, with and without 10 μM indoleacetic acid and 20 mg / l aceyosyringone, adjusted pH were prepared to 5.6, the medium autoclaved at 121 ° C for 30 minutes and cooled. Sterilized acetosyringone and indoleacetic acid solutions were added by filtration to the appropriate final concentrations in the medium. 36 Petri dishes of 100mm x 15 mm were poured from the cold medium. Three bacterial media were prepared: 1) minimal AB that contained 1.6% agar (AB), Difco potato agarose medium with 1.6% agar (PDA), and Luria broth medium with mannitol-glulamine (Roberts and Kerr, Physiol. Plant 4.81 (1974) with 1.6% agar (MGL, Miller, EXPERIMENTS IN MOLECULAR GENETICS 433 (1972)), subjected to auíoclave at 121 ° C for 20 minutes and cooled, sterilized solutions were added by filtration of sulfate. kanamycin and acetosyringone to the cold medium to final concentrations in the medium of 50 mg / l and 20 mg / l, respectively The AT656 strain was streaked in these three media and incubated overnight at 28 ° C. A design was used. completely factorial experimental with 3 bacterial media x 2 plant media x 2 light condition traiamientos (12 cases in toal), with 3 repetitions, with a Pelri box for repeiición and 20-25 fronds per box Pelri. For inoculation, the bacíerias people from each medium came back They were suspended separately and as described in example 21. For inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side upwards and was injured with a 1S. . sterile scalpel in the sphygmist regions. The fronds were then transferred to the Baciferian resuspension solution and incubated for approximately 10-15 minutes. After inoculation, the fronds were transferred to solid Schenk and Hildebrandt co-culture medium as described above. The fronds were co-cured for 4 days under a 16 hour dark light / 8 hours dark period with a luminous intensity of approximately 40 μmol / m2 'sec for light tracing or placed in dark darkness for dark trafaction. After co-culling, all the fronds were knotted to determine the expression of GUS following the • 10 procedure of Slomp et al. (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (S. R. Gallagher ed.1991)). Table V gives the results of the GUS stain: • CUADRp V Medium Medium Light or Number Number Number baclerian vegelal Oral darkness of toíal de o o frondas frondas meristem dyed d stained PDA SH O 63 60 5 PDA SH L 67 66 8 MGL SH O 66 65 7 MGL SH L 70 65 9 AB SH O 58 58 7 AB SH L 62 60 6 PDA SH + IAA O 61 61 14 PDA SH + IAA L 68 63 14 MGL SH + IAA O 62 61 11 MGL SH + IAA L 46 39 2 AB SH + IAA O 62 61 6 AB SH + IAA L 58 53 3 The bacterial medium had a significant effect on the frequency of GUS expression after 4 days of co-culture. The medium AB gave the lowest frequency of GUS expression and the PDA medium the highest. Developing fronds on indoleacetic acid before inoculation increased the frequency of GUS expression after co-culture. The presence of light during co-cultivation did not significantly affect the frequency of GUS expression after co-cultivation in plants using fronds developed without indoleacetic acid, however, the co-cultivation in the dark increased the frequency of expression in GUS in areas that used fronds developed in the presence of indoleacetic acid. When averaging frequencies from PDA and MGL in all the aquatic lentil fronds developed in the middle of Schenk and Hildebrendí with indolaceic acid, a frequency of GUS expression in meristematic tissue of approximately 17% is obtained.

EXAMPLE 28 Six times of co-culture and the presence or absence of light duranie and co-culture were examined for their effect on GUS expression after co-culture. Lemna gibba G3 fronds were grown for 17 days in Schenk and Hildebrandt medium containing 1% sucrose and 10 μM indoleacetic acid at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec. For co-culture, 150 ml of Schenk and Hildebrandt medium containing 1% sucrose, 1% agar, 10 μM indoleacetic acid and 20 mg / l acetosyringone, pH adjusted to 5.6, autoclaved for 30 min. Were prepared. and cooled. Sterilized acezosyringone and indoleacetic acid solutions were added to the cold medium by filtration to the final final concentrations in the medium. The medium was used to pour 6 Pelri boxes of 100 mm x 15 mm. The strain of Agrobacterium tumefaciens AT656 was seeded by esírías in minimal AB medium that con ganized kanamycin sulphate at 50 mg / l and 5 20 mg / l aceyosyringone and developed overnight at 23 ° C. A randomized experimental block design was used with 6 days of co-culling time, with 6 repetitions, with one Petri dish per repetition and 30 fronds per Pelri box. For the inoculation, the trays from a Petri box were resuspended and as described in example 21. »For the inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side upwards and they were injured with a sterile scalpel in the merisiematic regions. The fronds were transferred to bacterial suspensions and incubated for 10 minutes.

For co-cultivation, the fronds were transferred to Schenk co-culture and Hildebrandl as described above. Fresh boxes were wrapped with aluminum foil to achieve complete darkness and all boxes were incubated at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 sec for 6 points. time: 13, 23, 36, 49, 73.5 and 93 hours. After co-cultivation for the appropriate time, 5 fronds were removed from each of the 6 plates (3 dark samples and 3 light samples) and stained to determine GUS expression following the procedure of Slomp et al. (Histochemical * fQT localization of beta-glucuronidase, in GUS PROTOCOLS 103 114 (S.R. Gallagher ed.1991)). The results showed that GUS expression became evident at 23 hours after co-culture, with the expression deíecíada only in the broken end of the failures. At 36 hours, staining was detected in cells surrounding the lesions and in the broken ends of the failures. The staining was globally more intense, however the level of staining intensity was higher in the fronds incubated in the dark. At 49 hours, the difference in staining intensity and pairing of staining was evident in the dark conditions with light. Staining was more extensive in fronds incubated in the dark, however the frequency of fronds showing GUS expression and the frequency of meristematic regions expressing GUS was not significantly different between light and dark conditions. At 73.5 hours the staining pattern and the staining frequency did not differ significantly between light and dark treatments except that the staining of the injured tissue was greater in the dark treatment. At 93 hours (approximately 4 days), the largest number of merisiemal regions expressing GUS was detected, being the traffic in darkness definitely superior to the treatment with light. An intense staining was still present in the injured cells.

EXAMPLE 29 Lemna gibba G3 fronds were used to determine the effect of bacterial resuspension solutions, the osmotic potential of these solutions and the leaf lesion on the frequency of GUS expression after co-cultivation. Fronds were developed in the middle of Schenk and Hildebrandí liquid that contained 1% sucrose at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2 sec. For the co-cultivation, 1800 ml of Schenk and Hildebrandl (SH) medium were prepared with 1% sucrose, 0.8% unwashed agar, and 20 mg / l of acetosyringone, the pH adjusted to 5.6, subjected to auoclave during 30 minutes at 121 ° C, and cooled. Thermolabile acetosyringone was added to the cold medium subjected to autoclaving as a sterile solution by filtration. The cold medium was used to pour 72 Pelri boxes of 100 mm x 15 mm. Agrobacterium strain AT656 was seeded in minimal AB medium containing 20 mg / l of acetosyringone and 50 mg / l of kanamycin sulfate and developed overnight at 28 ° C. A fully factorial experimental design was used with 12 solutions of bacterial resuspension x 2 lesion treatments (24 procedures in total), with 3 repetitions, with a Peiri box per repetition and fronds per Petri dish. Ten combinations of 2 different bacterial resuspension solutions were tested: (1) Gamborg 5B salts, Murashige and Skoog vinyls, glycine (8 mg / l), aspalic acid (266 mg / l), arginine (174 mg / l) , glutamine (876 mg / l), casamino acids (500 mg / l), sucrose (6.85%), glucose (3.6%), and acetosyringone (20 mg / l), and (2) Schenk's and Hildebrandt's medium with 1% of sucrose, each at 5 different concentrations of mannitol: 0, 0.2, 0.4, 0.6 and 0.8 M, in terms of its efficiency in gene transfer. In addition, two other solutions were tested: (3) Gamborg B5 salts, Murashige and Skoog vitamins, glycine (8 mg / l), aspartic acid (266 mg / l), arginine (174 mg / l) glutamine (876 mg / l), casaminoacids (500 mg / l) and acetosyringone (20 mg / l), and (4) Schenk and Hildebrandt's medium with sucrose (6.85%), glucose (3.6%), and acetyringone (20 mg / l) . All bacterial resuspension solutions were sterilized by filtration before use. For inoculation, the bacteria from a box of AB medium were resuspended in 100 ml of each of the 12 resuspension solutions at least 1 hour before using them. The importance of injuring fronds before inoculation was also tested. For each lesioned or undamaged frond, the individual fronds were first separated from the masses. For the injury, the fronds were turned towards the abaxial side upwards and cut with a sterile scalpel in the meristematic regions. The undamaged fronds did not receive additional tracing after separation in individual fronds. For inoculation, 120 fronds, 60 injured and 60 unharmed, were floated on each of the 12 bacterial resuspension media for 10 minutes, inoculating the injured fronds separately from the undamaged fronds. For co-cultivation, the fronds were transferred to Schenk medium and solid Hildebrandt as described above. All the trays were co-cultivated for 4 days in the dark. After 4 days of co-culture, two boxes of each treatment were randomly selected and stained to determine GUS expression. The results indicated that without taking into account the medium, 0.6 M of mannitol gave the frequencies plus alias of GUS expression after co-cultivation. The simplest formulation of Schenk and Hildebrandí also worked out the same way as the more complex medium using the 5B salts of Gamborg. The lesion gave a measurable, but not statistically significant, increase in the frequency of fronds showing GUS expression and did not increase the frequency of staining in the meristematic region.

EXAMPLE 30 Lemna gibba G3 fronds were used to test the effect of bacterial concentrations during inoculation on the frequency of GUS expression after co-cultivation. Aquatic lentil fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose and 10 μM indoleacetic acid in 25 ml aliquots in 125 ml flasks at 23 ° C under a «. *? -s? The photoperiod of 16 light hours / 8 hours of darkness with luminous intensity of approximately 40 μmol / m2 sec lasts two weeks before use. For co-cultivation, 750 ml of Schenk and Hildebrandl medium were prepared with 1% sucrose, 1% agar, 20 mg / l of aceyosyringone and 10 μM of 5-idoladelic acid, the pH adjusted to 5.6, subject to autoclave at 121 ° C for 30 minutes and cooled. Solutions of sterilized indolesyringone and indoleacetic acid were added by filtration to the cold medium to obtain the final concentrations in the medium. The cold medium was used to pour 30 Pelri boxes of 100 mm x 15 mm. Agrobacterium strain AT656 was seeded by esírías in agar papa- dexírosa- culture broth Luria manitol- glutamine of medium concentration with 1.6% of Difco Bacto-agar, 20 mg / l of acetosyringone and 50 mg / l of kanamycin sulphate and were developed during the night at 28 ° C. An experimental randomized block design with 10 levels of bacterial concentration, with 3 repetitions, with one Petri dish per repetition and 20 individual fronds or masses of frond for each Petri dish. For the inoculation, bacteria from a Peiri box were resuspended as described in example 21. This bacterial solution constituted the sample "undiluted" and was the beginning of a series of serial dilutions for the following dilutions: 1/3, 10"1, 1/33, 10" 2, 1/333, 10 ~ 3, 1/3333, 10"4, 10" 5. The 1/3 dilution had an OD540nm of 1,006, which corresponds to approximately 1.6 x 109 bacteria / ml.

! ", S & s *? '? *' S?, - #? R '? Lí ^ * 5 For the inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side upwards and they were injured With a sterile scalpel in the meristematic regions, the fronds were then transferred to each of the 10 different concentrations of bacterial resuspension solution and incubated for approximately 10-15 minutes.For co-culling, the fronds were transferred to medium. Schenk and Hildebrandi cocultivation described above, with the abaxial side down, the fronds were co-cultured for 4 days at 23 ° C in the dark.After co-cultivation, all the fronds were stained to determine GUS expression following the procedure of Sípp et al. (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (SR Gallagher ed.1991).) The results showed that the GUS expression frequencies varied through the concentration bac The highest frequency of GUS expression was observed in the highest bacterial concentration tested. At dilutions greater than 10"3, no GUS expression was detected.

EXAMPLE 31 Lemma gibba G3 fronds were used to test the effect of quartering of co-culture media on GUS expression using a primozyme transformation technique.

The fronds were grown in Schenk and Hildebrandí liquid medium that contained 1% sucrose and 10 μM indoleacetic acid at 23 ° C under photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec. For co-culture, four media were used: 1) Murashige and Skoog medium (MS) with 20 μM of 2,4-D and 0.1 μM of BA (MS1), 2) MS medium with 20 μM of 2,4-D and 1 μM of BA (MS2), 3) MS medium with 1 μM of 2,4-D and 2 μM of BA (MS3), and 4) Schenk and Hildebrandí medium (SH). For each medium, 100 ml were prepared with the appropriate plant growth regulators containing 3% sucrose, 0.15% gelrite and 0.4% Difco Bacid-agar., the pH was adjusted to 5.6, subjected to auíoclave at 121 ° C during 20 minutes. A sterilized acetosyringone solution was added by filtration to each cold medium to a final concentration of 20 mg / l. Each medium was used to pour 4 Peiri boxes of 100 mm x 15 mm. Bacterial strain AT656 was seeded with striae on papa-dextrose agar with 20 mg / l of aceyosyringone and 50 mg / l of kanamycin sulphate and developed overnight at 28 ° C. An experimental randomized block design was used with 4 media treatments with four repeiitions, with one Petri dish per repetition and 20 fronds per Petri dish. For the inoculation, the bacteria from a Petri dish were resuspended for one hour before using them in 100 ml of sterilized SH medium by filtration with 0.6M mannitol and 20 mg / l aceyosyringone at pH 5.6. For inoculation, the individual fronds were separated from the masses and floated on the resuspended bacteria for 8-1 min. For co-culture, the fronds were transferred to the co-culture medium described above (MS1, MS2, MS3, SH). The fronds were co-cultivated at 23 ° C in the dark for four days. After four days of co-cultivation all the fronds were stained to determine the expression of GUS. The frequency of fronds that show GUS expression ranged from 80-90% across all regions. The co-cullive medium did not have a significant effect on this frequency. The intensity of staining for GUS varied from light to intense. The stain was associated with root tips, stems, • 10 stems, stems and lesions, merismatic regions and leaf margins.

EXAMPLE 32 The transformation of fronds using Agrobacterium was achieved through the manipulation of the speed of cell division of the fronds? F before the inoculation, the medium in which the Agrobacteria were developed, the optimization of the co-cullive parameters including secondary melaboliths such as aceyosyringone, the concentration of Agrobacteria, the osmolarity of the inoculation fluid, the duration of the co-culture period and the intensity of the co-culture period. Based on the studies described in the previous examples, a preferred method of frond transformation and selection is as follows. Typically, the fronds are grown in medium containing an auxin that increases the proliferation rate of the fronds, with NAA, IBA and IAA being the preferred auxins and the preferred concentrations in the range of 0.2-1 μM. The Agrobacteria are grown in a medium that does not contain enriched nulrilive supplements and include such secondary metabolites as acetosyringone, with potato-dextrose agar and Luria broth with mannol-glutamine as the preferred media. The frequency of transformation is determined by the composition of the inoculating fluid, with the preferred fluid being the basal salts of • 10 MS or SH supplemented with 0.6M mannitol and 100 μM aceyosyringone. The concentration of resuspended Agrobacteria in this inoculation fluid also affects the frequency of transformation, with the preferred concentration being of the order of 1 x 109 bacteria per ml. The inoculation time can vary, with the preferred time being 2-20 minutes.

The co-culture time also affects the frequency of transformation, with a time of 3-4 days being preferred. The co-culture can be performed under light or in dark conditions with dark (for example, dim light) being preferred. The development of transformed fronds also depends on the preferred conditions. The means MS and SH are the preferred means. The fronds are decontaminated from the Agrobacteria that infected them using the appropriate antibiotics at high concentrations, typically 100-500 mg / l, frequently transferring the infected tissue, being the transfer to new medium with antibiotic every 2-4 days the preferred method of transfer . Incubation is preferred under low light intensity, with the preferred range being 1-5 μmol / m2. sec, for an initial rest / recuperation period of 3-6 weeks. Selection by development in the presence of the selection agent can be initiated at varying times, with the preferred time being 1-3 weeks after inoculation. Also preferred is the initial selection under reduced light levels and low concentration of selection agent with ranges of light levels of 1-5 μmol / m2.sec and appropriate low concentrations for the selection agent as determined from the toxicity studies for the specific agent. For kanamycin sulfate, the typical range is 2-10 mg / l.

EXAMPLE 33 The provenin fronds of 10 varieties of aquatic lentils are: Lemna trisulca 7315, Lemna minor 7101, Lemna japonica 7427, Lemna turionifera 6601, Lemna gibba G3, Lemna valdiviana 7002, Lemna aequinocitalis 7001, Lemna miniscula 6711, Lemna obscura 7325, and Spirodela punctata 7273, were tested for their ability to give GUS expression after co-culture using the transformation protocol developed with Lemna gibba G3. «-fif All lentil strains except L. gibba G3 were grown in liquid Schenk and Hildebrandt with 1% sucrose in 25 ml aliquulas in 125 ml flasks. The Lemna gibba G3 strain was grown in Schenk and Hildebrandt medium with 1% sucrose and 10 μM indoleacetic acid. All aquatic legume cultures were incubated at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec. For co-culture, 400 ml of Schenk and Hildebrandt medium containing 1% sacorose, 0.9% agar and 20 mg / l acetosyringone, pH adjusted to 5.6, were prepared. • 10 subjected to auíoclave at 121 ° C for 30 minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cold medium to obtain the medium with the final concentration. The cold medium was used to pour 10 Petri dishes of 100 mm x 15 mm. The AT656 strain of Agrobacterium tumefaciens was streaked with potato-dextrose concentration agar media mixed with Luria broth medium with medium concentration mannitol-glulamine containing 20 mg / l of aceyosyringone and 50 mg / l of • kanamycin sulfate and developed overnight at 28 ° C. A randomized block experimental design was used with 10 treatments of aquatic lentil strain with one repetition, with a box Pelri per repetition and 20-25 fronds per Petri dish. For inoculation, the feces coming from a Pelri box were resuspended as described in example 21.

For inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side up and was injured with a sterile scalpel in the meristematic regions. The fronds were transferred to bacterial suspensions and incubated for approximately 10 minutes. 5 For the co-cultures, the fronds were transferred to the Schenk and Hildebrandí co-cultivation medium described above and incubated at 23 ° C in the dark for four days. After co-cultivation, the fronds were stained to determine GUS expression. Of the 10 strains tested, 8 showed GUS expression • 10 in a pattern identical to L. gibba G3 and the frequencies varied from 14% to 80%.

EXAMPLE 34 Twenty strains of aquatic lentils from the 4 genera of Lemnaceae in terms of their ability to give GUS expression after co-cultivation with Agrobacterium strain AT656 using the protocol Transformation developed with L. gibba G3. The twenty strains were: Wolffiella lingulata strains 8742 and 9137, Wl. Neotropic strains 7279 and 8848, Wl. oblongata strains 8031 and 8751, Wolffia arrhiza strains 7246 and 9006, Wa.

Australian strain 7317, Wa. brasiliensis strains 7397, 7581 and 8919, Wa. columbiana strains 7121 and 7918, Spirodela intermediate strain 7178, S. polyrrhiza strains 7960 and 8652, S. punctata strains 7488 and 7776, and L. gibba G3. yy. «. ",) -!«% .- á * i «* - * a? * -t 'ff All the strains were developed in the middle of Schenk and Hildebrandí liquid with 1% of sucrose, pH of 5.6 during two weeks before the experiment . For the co-culfivo, 1500 ml of Schenk and Hildebrandl medium containing 1% sucrose, 0.8% agar and 20 mg / l aceyosyringone, the pH adjusted to 5.6, autoclaved at 121 ° C for 30 days were prepared. minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cold medium to obtain the final concentration of the medium. The cold medium was used to pour 60 Petri dishes of 100 mm x 15 mm. The bacterial strain, AT656, was seeded on potato-dextrose agar with 20 mg / l of aceyosyringone and 50 mg / l of kanamycin sulphate and developed overnight at 28 ° C. For inoculation, the trays from a Petri dish were resuspended for at least one hour before using it in Schenk and Hildebrandl medium with 0.6M mannitol, 20 mg / l acetosyringone, pH 5.6, which was sterilized by filtration before use it An experimental randomized block design was used with 20 trays of aquatic lenses, with 3 replications, with a Petri dish for replication and 20 individual fronds or masses of fronds for each Petri dish. For the inoculation, they were separated from the individual fronds masses of Spirodela and Wolfiella strains and L. gibba G3. For the Wolffia strains, the fronds were inoculated as masses because of their small size which makes individual separation difficult. For inoculation, the fronds of each aquatic leaf strain were floated in the bacterial suspension solution for 2-5 minutes. For co-culture, the f22 fronds were transferred from the bacterial solution to Schenk co-culture medium and solid Hildebrandt described above. For the strains of Spirodela and Wolfiella and for L. Gibba G3, 20 individual fronds were transferred to each of the 3 repetition boxes; for the Wolffia strains, 20 small masses of fronds were transferred to each of the 3 repetition boxes. All strains were co-cultured in the dark at 23 ° C for 4 days. After co-cultivation, 2 boxes from each strain were stained to determine GUS expression. The results of the staining showed that all but one of the tested species and most of the strains of aquatic lenses within species gave some expression of GUS 4 days after co-cultivation. Of the 4 Wolffia species tested, iodine showed variable frequencies of GUS expression. The three strains of Wolffia brasiliensis showed the highest frequencies of GUS expression, varying from 50-75%. Through the six strains within the genus Wolfiella the frequency of GUS expression was lower, varying from 5-12%. Two of the fres species of Spirodela gave GUS expression of 10 and 35%; the third did not indicate expression of GUS. Lemna gibba G3, serving as the positive conlrol had a GUS expression frequency of approximately 50%.

«Y¿? Tv f- EXAMPLE 35 Transformation by Agrobacteria using callus cultures Type I calli produced from Lemna gibba G3 fronds were used to test their ability to express GUS using the optimized transformation process developed with L. Gibba G3 fronds and to test the effect of vacuum infiltration. Type I calluses were produced by developing fronds in the middle of Murashige and Skoog solid with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) , and 2 μM of benzyladenine (BA). Induction of callus and all subsequent cultures were done at 23 ° C and under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec. After 4 weeks of callus induction, callus masses type I were grown separately on the same medium with the concentration of 2,4-D reduced to 1 μM. The calluses were subcultured in fresh medium every two weeks until sufficient callus proliferated for experimentation. For co-culling, 400 ml of Murashige and Skoog solid (MS) medium were prepared with 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacto-agar, 1μM 2,4-D, and 2μM BA, the pH adjusted to 5.6, autoclaved at 121 ° C for 20 minutes and cooled. A solution of sterilized acetosyringone was added by filtration to a final concentration in the medium of 20 mg / l. The cold medium was used to pour 16 Pelri boxes of 100 mm x 15 mm. Agrobacterium strain AT656 was seeded with dexative potato agar with 20 mg / l of aceyrosyrone and 50 mg / l of kanamycin sulfate and developed overnight at 28 ° C. An experimental randomized block design was used with two treatments of vacuum infiltration with 4 repetitions, with two Peiri boxes per repetition and 10 pieces of callus per box Peiri. For inoculation, the bacteria were resuspended in Schenk and Hildebrandt medium sterilized by filtration containing 0.6 M mannitol and 20 mg / L acetosyringone at pH 5.6 for at least 1 hour before use. The inoculation with the bacteria was done with and without vacuum infiltration. Without vacuum infiltration, small pieces of type I calli were placed in the bacterial solution for 10 minutes, then dried by blotting and transferred to MS co-culture medium as described above. With vacuum infiltration, the calluses were placed in bacterial solution, a vacuum of 25.4 cm of mercury was applied for 10 minutes, then the calluses were dried by means of bloiíing and transferred to MS co-culture medium. All boxes were co-cultivated in the dark at 23 ° C. After 4, 6 and 9 days of co-culture, approximately 40, 20 and 20 pieces of callus, respectively, were set to determine GUS expression. The results showed that the frequencies of the callus pieces that present expression of GUS did not vary with respect to the vacuum infiltration system, nor did the frequencies vary with the co-culture time. Without vacuum infiltration at all time points, GUS staining ranged from 25 to 78% and with vacuum infiltration the frequencies varied from 25 to 74%. The inity of staining for GUS ranged from dark blue to light blue and had no correlation with irradiation.

EXAMPLE 36 Different means of co-culinary differences were tested in terms of their effect on the frequency of GUS expression following the callus co-cultivation of type I with Agrobacteruim strain AT656. I-I callus were produced by developing Lemna gibba G3 fronds in solid Murashige and Skoog medium containing 3% sucrose 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 μM 2,4-D, and 2 μM of BA. The induction of callus and iodine the subsequent cultures were done at 23 ° C and under a photoperiod of 16 light hours / 8 hours of darkness with light intensity of approximately 40 μmol / m2.sec. After 4 weeks of callus formation, callus masses type I were cultured separately in the same medium with the concentration of 2,4-D reduced to 1 μM. Calluses were subcullated in fresh medium every two weeks until sufficient callus proliferated for experimentation. For co-culture, four media were tested: Murashige and Skoog medium (MS) with 20 μM of 2,4-D and 0.1 μM of BA (MS1), MS medium with 20 μM of 2,4-D and 1 μM of BA (MS2), MS medium with 1 μM of 2,4-D and 2 μM of BA (MS3), and Schenk and Hildebrandí medium (SH) without plant growth regulators. 50 milliliters of each medium was prepared with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar, the pH was adjusted to 5.6, autoclaved at 121 ° C for 20 minutes, cooled and added a sterilized acetosyringone solution by filtration to the cold medium to a final concentration of 20 mg / l. The medium was used to pour 24 Pelri boxes of 100 mm x 15 mm. Agrobacterium strain AT656 was seeded by slices on potato dextrose agar containing 20 mg / l of aceyosyringone and 50 mg / l of kanamycin sulfate and developed overnight at 28 ° C. An experimental randomized block design was used with 4 isolations of co-culture media with two repeats, with one Petri dish per repetition and 20 pieces of callus per each Petri dish. For inoculation, bacteria from a Peiri box were resuspended in sterilized SH media containing filtration containing 0.6M mannitol and 20 mg / l aceyosyringone at pH 5.6 at least 1 hour before use. For the inoculation, callus type I pieces were placed in bac terial solution for 8 minutes, dried by blotting and then transferred to 4 different co-culture media. All boxes were co-cultivated at 23 ° C in the dark for 4 days. After co-cultivation, all calluses were stained to eliminate GUS expression following the procedure of Slomp e to al. (Histochemical localization of beta-glucuronidase, in GUS PROTOCOLS 103-114 (S.R. Gallagher ed.1991)). The co-culture medium did not have a significant effect on the frequency of the callus pieces that show GUS expression. In all cases, the frequency of GUS expression ranged from 70 to 85%. The intensity of GUS expression varied with staining in a range of dark blue to light blue.

EXAMPLE 37 Two different co-culture times, two and four days, were tested in terms of their effect on the frequency of GUS expression after co-cultivation of callus I I with Agrobacterium strain AT656. Type I calluses were produced by developing fronds of Lemna gibba G3, in the middle of Murashige and Skoog solid containing 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacid-agar, 5 μM 2,4-D and 2 μM BA. The induction of callus and all subsequent cultures were done at 23 ° C and under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2. sec. After 4 weeks of callus induction, the callus masses type I were cultured separately in the same medium with the concentration of 2,4-D reduced to 1 μM. The calluses were subcultured in fresh medium every two weeks until sufficient callus proliferated for experimentation. For co-cultivation, 400 ml of Murashige and Skoog solid (MS) medium were prepared with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 μM 2,4-D and 2 μM of BA, pH adjusted to 5.6, autoclaved at 121 ° C for 20 minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cooled medium to a final concentration of 20 mg / l. The cold medium was used to pour 16 Peiri boxes of 100 mm x 15 mm. Agrobacterium strain AT656 was streaked with potato-dextrose agar with 20 mg / l of acetosyringone and 50 mg / l of kanamycin sulphate and developed overnight at 28 ° C. 5 Experimental design by randomized blocks was used with two treatments of co-culture time, with two repetitions, with four Peiri boxes per repetition and 10 pieces of callus per each Petri dish. For the inoculation, the basins were resuspended in the middle of Schenk and Hildebrandl sterilized by filtration that coniined 0.6M of manilol and 20 mg / l of acetosyringone at pH 5.6 at least one hour before use. For inoculation, callus type I pieces were placed in bacterial solution. For co-cultivation, the pieces were dried by bloíting, then transferred to MS co-culture medium described above. All boxes were co-cultured in the dark at 23 ° C for either 2 or 4 days. After 2 or 4 days of co-culling, all calluses were stained to eliminate the expression of GUS. m The results showed that the GUS expression frequencies did not vary with respect to the co-culture time. In all cases, the GUS expression frequencies ranged from 50 to 70%. The intensity of the GUS stain ranged from dark blue to light blue. However, excessive bacterial overgrowth was present after 4 days of co-cultivation and it was discovered that this bacterial coating inhibits GUS staining.

** - EXAMPLE 38 A different gene construct was used to test the efficacy of the I-I callus co-culture protocol with an additional strain of Agrobacterium, C58sZ707pBH21. I-I calli were produced by developing Lemna gibba G3 fronds in Murashige and Skoog solid media containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 μM 2,4-D and 2 μM of BA. Induction of callus and all subsequent cultures were done at 23 ° C and under a photoperiod of 16 light hours / 8 hours of darkness with luminous intensity of approximately 40 μmol / m2 'sec. After 4 weeks of callus induction, the callus masses type I were cultured separately in the same medium with the concentration of 2., 4-D reduced to 1 μM. The calluses were subcul- livated in new medium every two weeks until sufficient callus proliferated for experimentation. For co-culture, 400 ml of Murashige and Skoog solid (MS) medium were prepared with 3% sucrose, 0.15% Gelrite, 0.4% Difco Baccus-agar, 1 μM 2,4-D and 2 μM of BA, pH adjusted to 5.6, subjected to auíoclave at 121 ° C during 20 minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cold medium to a final concentration of 20 mg / l. The cold medium was used to pour 16 Pelri boxes of 100 mm x 15 mm. Agrobacterium strain C58sZ707pBI121 was seeded on strips on potato-dextrose agar with 20 mg / l aceyosyringone, 500 mg / l of «& ü Sclerephomyomycin sulfate, 50 mg / l of espectinomycin and 50 mg / l of kanamycin sulfate and developed overnight at 28 ° C. A randomized block experimental design was used with a bacterial strain treatment with four repetitions, with four Pelri 5 boxes per repeat and 10 pieces of callus per each Peiri box. For inoculation, dishes from a Petri dish were resuspended in Schenk and Hildebrandt medium sterilized by filtering 0.6M of mannitol and 20 mg / L of acetosyringone at pH 5.6 at least one hour before use. For the inoculation, the callus pieces 1 were placed in • 10 bacterial solution lasts 8-10 minutes. For co-culture, the pieces were blotted and then transferred to co-culture medium MS described above. All corns were co-cultured in the dark at 23 ° C for two days. After the co-culíivo, two pieces of callus were selected from one of the boxes by repetition (8 pieces in ioíal) and were fixed for to eliminate the expression of GUS. All callus pieces showed variable GUS expression from dark blue to pale blue. Residual calli were transferred from the MS co-cull medium to MS medium containing 500 mg / l of cefotaxime during the first two weeks and 500 mg / l of each of cefotaxime and carbenecillin, after that, to clean the tissue of the bacterial conies. All callus tissues were transferred to new MS medium containing cefotaxime and carbenecillin at two week intervals. After each transfer, a callus sub-sample was selected to determine the expression of GUS. The frequencies of GUS expression decreased slightly but remained elevated with 70-95% of the pieces showing some expression of GUS.The visual inspection of the calluses in • medium with antibiotics showed no indication of bacterial contamination after 4 weeks of culture.

EXAMPLE 39 Callus II and II were tested for their ability to • 10 give expression of GUS after co-cultivation in the presence of Agrobacterium strain AT656. Both types of callus were induced by cultivating fronds of Lemna gibba G3 in solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacid-agar, 30 μM 2,4-D and 0.02 15 μM of BA at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with a luminous intensity of approximately 40 μmol / m2 'sec. After • for one to four weeks, type II and III callus were separated from the original fronds and transferred to solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelriffe, 0.4% Difco agar-agar, 10μM of 20 2 , 4-D and 0.01 μM of BA to maintain the calluses under the same conditions of lemperairy and light. The calluses were subcultured in new medium every two weeks until sufficient callus proliferated for experimentation.

The Agrobacterium strain AT656 was seeded by smears on potato-dextrose agar containing 20 mg / l of acetosyringone and 50 mg / l of kanamycin sulphate and developed overnight at 28 ° C. For co-cultivation, 200 ml of Murashige and Skoog (MS) medium were prepared with 3% sucrose, 0.15% Gelriie and 0.4% Difco Bacto-agar, 10 μM 2,4-D and 0.02 μM BA, the pH was adjusted to 5.6, autoclaved at 121 ° C for 20 minutes, and cooled. A solution of acetyringone sterilized was added by filtration to the cold medium to a final concentration of 20 mg / l. The cold medium was used to pour 8 Peiri boxes of 100 mm x 15 mm. An experimental randomized block design was used with two callous type isolates. 40 masses of green callus were inoculated, transferred, uniformly to 4 Peip boxes and 9 masses of white callus, uniformly transferred to 4 Peiri boxes. For inoculation, the bacteria were resuspended in Schenk and Hildebrandí medium sterilized by filtration containing 0.6M mannilol and 20 mg / l aceyosyringone at pH 5.6 at least one hour before inoculation. For inoculation, pieces of green calluses, and white calluses were immersed in the bacterial solution for 2-5 minutes. For co-culling, the callus pieces were blotted and then transferred as masses to MS co-culture medium described above. All calluses were incubated at 23 ° C in the dark for 2 days. After co-cultivation, all white calluses and 3 green callus pieces per box were randomly selected and stained for &2 f s determine the expression of GUS. Of 19 white callus masses, 7 masses showed GUS expression with variable intensity. Of 12 green callus pieces, 6 showed GUS expression with variable intensity.

EXAMPLE 40 Type I calluses established from two different fast growing strains of Lemna gibba (strain 6861 and 7784) and a strain of Lemna gibba with AT656 were co-cultured to determine the frequency of transformation with the established protocol using Lemna gibba G3. The Agrobacterium strain AT656 was streaked with potato dextrose agar containing 20 mg / l aceyosyringone and 50 mg / l kanamycin sulfate and developed overnight at 28 ° C. For co-cultivation, 200 ml of Murashige and Skoog (MS) medium were prepared with 3% sucrose, 0.15% Gelriie and 0.4% Difco Bacto-agar, 10 μM 2,4-D and 0.02 μM BA, the pH was adjusted to 5.6, autoclaved at 121 ° C for 20 minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cold medium to a final concentration of 20 mg / l. The cold medium was used to pour 8 Petri dishes of 100 mm x 15 mm. For the inoculation, the bacteria were resuspended in Schenk and Hildebrandt medium sterilized by filtration that contained 0.6M of manilol and 20 mg / l of aceyosyringone at pH 5.6 at least one hour before inoculation. For inoculation, approximately 10-15 pieces of type I calli from the aquatic lenses and L. Gibba G3 (positive control) were immersed in the bacterial solution for 2-5 minutes. For the co-cultivation, the callus pieces were dried by blotting, then transferred as masses to two boxes (for each strain of aquilic lentil) of co-culture medium, as described above. All calluses were incubated at 23 ° C in the dark for 2 days. After co-cultivation, all callus pieces from the two strains of Lemna gibba and fresh pieces of tripe from the Lemna minor strain were randomly selected and stained to determine GUS expression. All the callus pieces showed small multiple spots of cells with GUS staining two weeks after co-cultivation, consistent with the successful transformation.

EXAMPLE 41 Selection using fronds Lemna gibba G3 fronds were used to test the effect of fresh co-culinary media to rescue fronds that express GUS and grow them in the middle of selection with kanamycin. The fronds were grown for 3 days in Schenk and Hildebrandt liquid medium containing 1% sucrose and 10 μM indoleacetic acid before use. The bacterial strain, AT656, was grown overnight on potato-dextrose agar containing 20 mg / l acetosyringone and 50 mg / l sulfate e ^ kanamycin at 28 ° C. 3 solid media were used for co-cultivation: 1) Schenk and Hildebrandt medium (SH) containing 1% sucrose, 1% agar, 20 mg / l acetosyringone and 10 μM indoleacetic acid, 2) Murashige medium and Skoog (MS) containing 3% sucrose, 1% agar, 20 mg / l aceyosyringone and 50 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 3) Murashige and Skoog medium that it contained 3% sucrose, 1% agar, 20 mg / l of aceylosyrose, 5 μM of 2,4-D, 10 μM of naphthalene-clenic acid, 10 μM of gibberellic acid G3, and 2 μM of benzyladenine. The media was prepared, the pH was adjusted to 5.6 (SH) or 5.8 (both types of MS), subjected to auyoclave, cooled, the heat-labile components acetyringone, indolelic acid and gibberellic were added as sterile solutions by filtration, and the medium was poured into 100 mm x 15 mm Petri dishes. For each medium, 20 Petri dishes (500 ml) were prepared. A randomized block experimental design was used with three treatments of co-culture medium with 4 repetitions, with 5 Petri dishes per repetition and 20 fronds for each Petri dish. For inoculation, bacteria from a Peiri box were resuspended as described in example 21. For inoculation, the individual fronds were separated from the masses, each one turned with the abaxial side upwards and were injured with a sterile scalpel in the meritematic regions. The fronds were transferred to bacterial suspensions and incubated for 10 minutes. For the crop, the fronds were transferred to the three co-cultivation media described above. All boxes were incubated for 5.5 days in the dark at 23 ° C. After 5.5 days of co-cultivation, the fronds from two Peiri boxes were interlaced to determine GUS expression. The results showed that the expression of GUS was presented in a greater degree in co-cultivated fronds in Schenk and Hildebrandt medium, less staining was observed with fronds in the middle of Murashige and Skoog. The remaining fronds of the other 18 boxes of each medium (18 boxes x 3 media = 54 boxes) were transferred to solid media as liquids of the same composition without acetosyringone, and with 500 mg / l of timenine. The three-case, foliar fronds were transferred in 3 flasks with 25 ml of liquid medium and developed at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μmol / m2 sec. The fronds in 15 boxes of solid media were divided into 2 groups: 1) the fronds of 10 original boxes were transferred to 12 new boxes and incubated in the dark (12 boxes), 2) the fronds of 5 original boxes were transferred to 6 new boxes and incubated under low light conditions of less than 5 μmol / m2 • sec. After 11 days of development, subsamples of fronds were taken to dye them to determine GUS expression. The results showed that without taking into account the irradiation of light or the medium heat, the expression of GUS was still present. All the fronds, without a ^ f3 take into account the means, in © afeadfeéß under low light showed the intensity beyond GUS expression. The fronds incubated in the dark showed an intermediate level of GUS expression and the fronds incubated in the light showed very low levels. The fronds incubated in the middle of 5 Schenk and Hildebrandt showed the frequencies more alias of GUS positive tissue, however no GUS expression was associated with freshly expanded fronds. In the middle of Murasíghe and Skoog formulated to induce callus, the staining pattern was restricted to individual cells and very • 10 small ones The fronds in MS medium containing 2,4-D, NAA, GA3 and BA showed more intense staining than those incubated in MS medium containing only 2,4-D. Callus formation in both mediums with MS base, with plant growth regulators adjusted to induce calluses, did not occur in the dark, but was initiated in MS medium with 2.4-15 D, NAA, GA3 and BA under light. faint. Based on these results, the fronds in the middle of Schenk and Hildebrandt were excluded from the experiment. f All the remaining fronds in the middle of MS in the dark were transferred to dim light conditions to continue the incubation. All tissues were kept in the same training medium but transferred to medium fresh with timentin and the incubation was continued under dim light conditions for approximately 5 weeks. Seven weeks after co-culture, the remaining tissues were transferred back to fresh medium and kanamycin sulfate was included at 10 mg / l (approximately 25% tissue) or 2 mg / l (approximately 75% of the remaining tissue). A week later, a submuesira of tissue from kanamyric traiamientos was deinered to eliminate the expression of GUS. The following types of staining were observed: 1) staining associated with the original, co-cultivated fronds, 2) staining associated with type I calluses, and 3) staining associated with callus of the lll. The frequency of callus staining was not high, estimated at approximately 5-8 fronds, giving rise to cultures resistant to kanamycin per one hundred co-cultivated fronds. Incubation and subcutaneous tissue was continued for another 5 weeks under normal light. At twelve weeks, all the Residen tissues were from culinae in MS medium containing 2,4-D, NAA, GA3 and BA. The tissue was transferred to Murashige and Skoog medium with 1 μM of 2,4-D, 2 μM of BA, 0.15 g / l of Gelrile, 0.4 g / l of Difco baculo-agar, 500 mg / l of timentin and 10 mg / l of kanamycin sulfate. The heat-labile components were sterilized by filtration and added to the medium subjected to cold auctoroclave. The healthy fruit that had proliferated from each of the co-cultivated fronds was originally transferred to an individual Peiri box. All tissues were incubated at 23 ° C and changed from light to full light of approximately 40 μmol / m2 sec and a photoperiod of 16 light hours / 8 dark hours. At this time, a small submuesira of tissue was stained to determine GUS expression and the results showed a low frequency of GUS staining associated with lll-type callus. After two weeks it became evident from the visual observation that the transfer to full light had increased the resistance to kanamycin from of kanamycin sensitive material. Callus growth in kanamycin was continued for another 4 weeks of 16 weeks) by transfer of sludge from living tissues to fresh medium. 5 Between weeks sixteen and twenty after co-culture, the callus lines resistant to kanamycin were established. These compact lipo I and lipo lll characterized by growing in 10 mg / l of kanamycin in the light. Eight cultures of callus resistant to kanamycin were proliferated from 360 co-cultivated original fronds. As these eight lines are • 10 developed, submueslras of callus were transferred to medium Schenk and Hildebrandí medium concentration that contained 0.5% sucrose to regenerate fronds. Of these eight, three fronds regenerated in the absence of kanamycin, the regeneration of fronds would not occur in the presence of kanamycin. None of these fronds showed GUS expression when stained.

EXAMPLE 42 • Selection of callus cultures and regeneration of transformed fronds Type I calluses were tested for their ability to give expression of GUS and cultures resistant to kanamycin sulfate after co-culture in the presence of Agrobacterium strain C58sZ707pBI121.

Type I callus were produced by developing Lemna gibba G3 fronds in Murashige and Skoog solid media containing 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacfo-agar, 5 μM 2,4-D, and 2 μM BA. The induction of callus and all subsequent cultures was done at 23 ° C and under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2.sec. After 4 weeks of callus induction, the callus masses of type I were separately cultured in the same medium with the concentration of 2,4-D reduced to 1 μM. The calluses were subcultured in new medium every two weeks until sufficient callus proliferated for experimentation. For co-culling, 400 ml of Murashige and Skoog solid (MS) medium were prepared with 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacillus agar, 1 μM 2,4-D and 2 μM BA, the pH was adjusted to 5.6, subjected to auíoclave at 121 ° C for 20 minutes and cooled. A sterilized acetosyringone solution was added by filtration to the cold medium to a final concentration of 20 mg / l. The cold medium was used to pour 20 Pelrí boxes of 100 mm x 15 mm. The Agrobacterium strain C58sZ707pBI121 was seeded on potato-dextrose agar with 20 mg / l of acetosyringone, 500 mg / l of schympicomycin, 50 mg / l of spectinomycin and 50 mg / l of kanamycin sulfate and was developed overnight at 28 ° C. Experimental design was used for random blocks with a strain of baciferian strain with one repetition, with 20 Pelri boxes per repetition and approximately 10 pieces of callus per each Petri dish. For the inoculation, the bacteria become infected. to be suspended in medium of Schenk and Hildebrandt sterilized by means of filtration containing 0.6 M of mannitol and 20 mg / l of acetosyringone at pH 5.6 for at least one hour before use. For the inoculation, the pieces of callus I I were placed in bacterial solution. For co-culture, the pieces were blotted and then transferred to MS co-cull medium described above. All callus pieces were co-cultured for two days at 23 ° C in the dark. After co-culling, a submueslra of callus pieces was stained histochemically to determine GUS expression. The results showed a high frequency of GUS expression of varied ininess. The approximately 200 remaining callus pieces were transferred to deacylamylation medium. For the decontamination, 500 ml of solid MS medium containing 3% sucrose, 0.15% Gelriie, 0.4% Difco Bacfo-agar, 1 μM de2.4-D, and 2 μM BA were prepared, the pH was adjusted to 5.6, it was subjected to auíoclave for 20 minutes at 121 ° C and cooled. A solution containing sterilized cefotaxime was added by filtration to the cold medium to a final concentration in the medium of 500 mg / l. The cold medium was used to pour 20 Pelri boxes. Approximately 10 pieces of callus, each one, were transferred to the 20 Peiri boxes of disinfection medium. All Pelri boxes were incubated at 23 ° C in the dark. Weekly subcultures of the callus pieces were done in identical new medium and the calluses were incubated under the same conditions. At week 5, a small sub-sample of cat was taken to reduce the expression of GUS. The expression is present in a high frequency and with variable inlensity. In week 5, the resideníes pieces of callus were transferred to the selection medium. For the selection, 500 ml of MS medium with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar, supplemented with 1 μM of 2,4-D and 2 μM of BA, the pH adjusted to 5.6, were prepared. , subjected to auíoclave during 20 minutes at 121 ° C and cooled. A solution containing cefoximax was added, carbenicillin and kanamycin sulphate sterilized by filtration in the cold medium had a final concentration in the medium of 500, 500 and 2 mg / l, respectively. The cold medium was used to pour 20 Pelri boxes. Approximately 9-10 pieces of callus were transferred to the 20 Petri dishes of the selection medium. All calluses were incubated at 23 ° C under a 16 hour light / 8 hour darkness with a dim luminous intensity of approximately 3-5 μmol / m2 • sec. After a week of incubation, the calli were transferred to the same medium except that the concentration of kanamycin was increased to 10 mg / l. The callus cultures were continued under the same incubation conditions for the last week, once they were subculivated to a new medium of identical composition. At the end of these two weeks, the selection period with kanamycin, approximately 25% of the original callus pieces showed healthy callus growth with the rest in decline.

At week 7, 64 d of the healthiest callus were transferred to solid MS medium with 35 sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 μM de2.4-D, 2 μM BA, 500 mg / l, each, of carbenicillin and cefotaxime, and four different concentrations of kanamycin sulfate: 10, 20, 40, and 80 mg / l. 160 ml of the medium was prepared, the pH was adjusted to 5.6, autoclaved and cooled. Solutions sterilized by filtering the ferrous-metal antibiotics were added at the appropriate concentrations. The cold medium was then used to pour 16 Pelri boxes of 60 mm x 15 mm. Approximately 4 pieces of callus were transferred to each box and 4 boxes were prepared for each kanamycin concentration (16 pieces of callus per concentration of kanamycin). Incubation of calluses continued at 23 ° C under dim light. At weekly intervals, 4 boxes were transferred, one from each of the kanamycin concentrations to a light intensity greater than 40 μmol / m2. sec. In week 9, without considering the light conditions, all the calluses were transferred to a new medium of identical composition as the previous subculture. By week 12, all calluses were under a luminous intensity greater than 40 μmol / m2. sec. Callus culture was continued for 4 weeks (until week 16), with subcultures in new medium at two week intervals. At week 16, a small submueslra of the healthy callus were stained to determine the expression of GUS. All pieces of healthy callus showed GUS expression with complete pieces of callus showing uniform staining and segregation of callus that express GUS of callus that do not express it. By this time, most calluses had died, but more than 10% showed variable degrees of healthy callus proliferation. Three callus lines, A, B and C were identified and transferred to medium to promote regeneration of fronds. After further subculturing the growing callus pieces in selection medium, 6 more callus lines, D-1, were idenified and transferred to the regeneration medium. Eight of the 9 lines were discovered in medium containing 10 mg / l of kanamycin. The exception was line D which showed good development in kanamycin at 40 mg / l. After the subsequent subculture, the 6 callus lines continued to develop: A, B, D, F, H and i- Two of the nine lines identified regenerated fronds that were positive for GUS expression when they were stained and that easily proliferated in the presence or absence of kanamycin. For regeneration, agar was prepared in water from 100 ml of distilled water with 0.4% Difco Bacfo-agar, 0.15% Gelriie, the pH was adjusted to 5.6, and the medium was subjected to auíoclave for 18 minutes at 121 minutes. ° C. This medium was used to pour 10 Peiri boxes of 60 mm x 15 mm. Small pieces of provenanal calli from lines A, D, F, H, and I were transferred to two Pelri boxes, each one, of the media. The calli were incubated at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 40 μmol / m2. sec. The culture of callus in agar in water was continued for six weeks, - ^ • fm subculturing on agar in water every two weeks. By week six, the calli from all the lines had turned yellow and brown. The callus was transferred at the end of week 6 to medium Schenk and Hildebrandt medium concentration either solid or liquid containing 0.5% sucrose and 0.8% Difco Bacto-agar (only solid medium). After 4-6 weeks the callus had organized green nodules that differed in thick leaf-like structures. Since the fronds could be detached from the callus masses, these were transferred to Schenk and Hildebrandí medium of full concentration that 0 contained 1% sucrose, with incubation at 23 ° C under a furoperiod of 16 light hours / 8 hours darkness with luminous intensity of approximately 40 μmol / m2.sec. These fronds proliferated in liquid SH medium indefinitely. The fronds proliferated in the same way in the middle of SH with or without kanamycin. Blanching of the fronds was not observed in the presence of 5 kanamycin. Frond submuesirs were taken periodically and stained to determine GUS expression. All fronds showed GUS expression. To confirm the transformation, Southern hybridization analysis was performed on DNA isolated from lines A and D. The 0 preparations of aquatic lentil DNA were prepared from untransformed L. gibba G3 and transformed lines A and B using Doyle's CTAB procedure and Doyle (Amer. J. of Botany 75, 1238 (1998)). The isolated DNA was digested with the restriction enzymes EcoR1 and Hind III, and with both Jtlzz. A-_ -? E zfriL. enzymes, and the fragments were subjected to separation by electíü oresís in 0.7% agarose gel. The gel was applied by means of bloling to a nylon membrane following the method of Sambrook, SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (1989). For the probe, the plasmid DNA from pBI121 was isolated using an alkaline SDS procedure from Sambrook. Id. The 12.8 kb plasmid DNA was digested with the restriction enzymes EcoR1 and Hind III to produce a fragment consisting of 3.2 kb of the gene for β-glucuronidase and a fragment of approximately 9 kb which conended the neomycin phosphoransferase gene.

Both fragments were isolated from the agarose gel and radioactively labeled by random priming using the Príme-a-Gene (Promega) kit. Using these probes, hybridization was carried out with blotts carrying DNA from aquatic lentil without transforming and DNA from any of the transformed A lines or transformed D line. The hybridization reaction was carried out at 65 ° C overnight in a hybridization oven. The membrane was washed under stringent conditions of 0.1X SSC, 0.1% SDS. The blott was then placed in convention with BIOMAX MS film (Kodak) and the radiograph was exposed for 2 days at -70 ° C. The results of the hybridization experiments demonstrated that the DNA that hybridizes to GUS and NPTII was present in the lines A and D of aquatic lentil, but not in the DNA from the untransformed aquatic lentil (results for line D shown in figure 1). Double digestion of transformed aquatic lentil DNA provided hybridization • aas.-f at the expected molecular weight. Individual digestion demonstrated that the hybridization was associated with DNA fragments of unexpected molecular weight, indicating that the hybridizing DNA was not of bacterial origin and was integrated into the vegetative DNA. When sounding the same blotts with the labeled virulence region probe, the absence of hybridization was demonstrated, indicating that the positive signals for GUS and NPTII came from the plant, not from the bacteria.

EXAMPLE 43 • 10 Type I calli were tested for their ability to give GUS expression and cultures resistant to kanamycin sulfate after co-culture in the presence of Agrobacterium strain C58sZ707pBII21. Type I calluses were produced by developing fronds of Lemma gibba G3 in Murashige and Skoog medium containing 3% sucrose 0.15% Gelrite, 0.4% Difco Bacchus agar 5 μM de2.4-d, and 2 μM • BA. The induction of callus and all the subsequent culfivos was done at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with light intensity of approximately 40 μmol / m2.sec. After 4 weeks of In the case of callus induction, the callus masses of type I were grown separately in the same medium with the concentration of 2,4-D reduced to 1 μM. The calluses were subcultured in new medium every two weeks until enough callus proliferated for experimentation.

For co-curing, you will prepare 750 ml of Murashige and Skoog solid (MS) medium with 3% of flavor, 0.15% of Gelriie, 0.4% of Difco Baclo-agar, 1 μM of 2,4-D. and 2 μM of BArf the pH adjusted to 5.6, autoclaved at 121 ° C for 20 minutes and cooled. A solution of sterilized acetosyringone was added by filtration to the cold medium to a final concentration of 20 mg / l. The cold medium was used to pour 30 Peiri boxes of 100 mm x 15 mm. The Agrobacterium strain C58sZ707pBII21 was seeded on potato-dextrose agar with 20 mg / l of acetosyringone, 50 mg / l of streptomycin, 50 mg / l of speclinomycin and 50 mg / l of kanamycin sulfate and developed overnight 28 ° C. An experimental randomized block design was used with a repeat strain of bacterial strain, with 30 Pelri boxes per repetition and approximately 5 pieces of callus per each Pefri box. For inoculation, the baths were resuspended in sterilized MS medium by filtration containing 0.6 M mannitol and 20 mg / l aceyosyringone at pH 5.8 at least one hour before use. For the inoculation, the callus pieces of lipo I were placed in bacterial solution.

For co-cultivation, the pieces were dried by blotting and then transferred to MS co-culture medium described above. All the callus pieces were cocultivated during 2 days at 23 ° C in the dark. After co-culture, a sub-sample of callus pieces was stained histochemically to de-expression of GUS. The results showed a high frequency of GUS expression.

The approximately 50 pieces of rerifiarrant calli were transferred to decontamination media. For the decontamination, 750 ml of solid MS medium was prepared -pe coneníaía 3% of sucrose, 0.15% of Gelrite, 0.4% of Bacto-agar Difco 1 μM of2,4-D, and 2 μM of BA, the pH 5 adjusted at 5.8, autoclaved for 20 minutes at 121 ° C and cooled. A sterilized solution was added by filtration containing cefotaxime and carbenicillin to the cold medium to a final concentration in the medium of 50 mg / l, each. The cold medium was used to pour 30 Pelri boxes. Approximately 5 pieces of callus were transferred to each of the 30 • 10 Petri dishes of decontamination medium. Then the calluses were incubated at 23 ° C in the dark. Weekly subcultures of the callus pieces were made in identical new medium and the calluses were incubated under the same conditions. The selection for callus lines resistant to kanamycin was started at week 5. For the selection, 750 ml of MS medium containing 3% sucrose, 0.15% Gelrile, 0.4% Difco Baclo-agar, 1 μM 2,4-D, and 2 μM were prepared. of BA, the pH adjusted to 5.8, subjected to • Autoclave for 20 minutes at 121 ° C and cooled. A sterile solution was added by filtration containing cetofaxime, carbenicillin and kanamycin to a final concentration in the medium of 500 mg / l, 500 mg / l and 2 mg / l, respectively. The cold medium was used to pour 30 Petri dishes. Approximately 5 pieces of callus were transferred to each of the 30 Petri dishes of the selection medium. they were then incubated at 23 ° C in the dark. For weeks 6 and 7, the calli were transferred to identical new medium with the increased kanamycin concentration at 10 mg / l. Incubation was continued in the dark at 23 ° C. At the beginning of week 8, the kanamycin concentration increased. Murashige and Skoog medium of identical composition to that of previous subcultures was prepared with the medium medium containing kanamycin at 10 mg / l and the half with kanamycin at 40 mg / l. Approximately half of the remaining calli were transferred to each kanamycin concentration. The incubation lighting conditions were changed in the same way. All calluses were incubated at 23 ° C under a photoperiod of 16 light hours / 8 dark hours with luminous intensity of approximately 3-5 μmol / m2 sec. The calluses were manuvered under these medium conditions and incubation for two weeks. After two weeks at the illumination level, the callus was transferred to a new medium of identical composition containing kanamycin at either 10 or 40 mg / l and the light intensity was increased to 40 μmol / m2 sec. The calluses were run on a two-week subculture regimen in identical medium and identical incubation conditions. Around week 12, approximately 10% of the calluses remained healthy and developing. Of the 15 remaining proliferative calli lines, half developed in kanamycin at 10 mg / l and the rest in 40 mg / l. The hischiochemical staining of small submuesíras of calluses coming from 6 lines showed expression of GUS in sectors of the pieces of the calluses. Fronds were regenerated following the procedure of example 42. Leaf growth was normal when developed in the presence or absence of kanamycin sulfate, and blanching of the fronds was not observed. The fronds also showed intense hislochemical staining of GUS. The Souíhern hybridization analysis showed the presence of DNA sequences of the fragment size expected for both the GUS and neomycin phosphotransferase genes, and the absence of DNA sequences from the vir region. The appropriate restriction enzyme analysis was performed as in Example 42, and the results were consistent with a discovery of gene integration introduced in the vegetative genome.

EXAMPLE 44 Based on the above examples, the following method is preferred for transforming aquatic leaf tripe with Agrobacterium, followed by selection and regeneration of the transformed plants. In general, Lemma minor has a particularly vigorous callus system, which makes it easier to regenerate fransformed plañís from these species. Typically, Callus Transformation, Leaf Selection and Regeneration is dependent on a well-maintained callus system as described in Example 16. The .Agrobacteria develop (in Papa-Dexlrosa Agar with appropriate antibiotics and 100 μM acetosyringone ) and are resuspended as in Example 32, except that the preferred resuspension means is MS instead of SH. The callus pieces are inoculated by immersing them in the solution of resuspended bacteria for a minimum of 2-5 min., Drying by blotting to remove excess fluid and sowing in a co-culture medium consisting of MS supplemented with auxin and cytokine. optimized to promote the development of callus and 100 μM aceyosyringone. The inoculated calli are incubated in the dark for 2 days. After co-cultivation, the callus is transferred to a new medium containing antibiotics to decontaminate the cultures of the Agrobacteria that infect them. The preferred medium is MS with 3% sucrose, 1 μM 2,4-D, 2 μM BA, gelled with 0.15% Gelrite and 0.4% Bacto-agar Dífco and antibiotic (antibiotics). The callus is incubated under low light of 3-5 μmol / m2-sec.

The callus is transferred every 2-5 days, 3 days are preferred, to new medium of the same composition. The total period of recovery lasts 2-3 weeks, 3-6 subcultures. The callus selection comes after the recovery period. The callus is transferred to MS medium supplemented with 1 μM of 2,4-D, 2 μM of BA, 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, and 10 mg / L kanamycin sulfate. . The callus is incubated under low light of 3-5 μmol / m2 sec, with transfer to new medium of the same composition every 2 weeks. The callus stays in this way for 4-6 weeks. Then the callus is incubated under complete light of 40 μmol / m2 sec, 5 in the same medium. The selection is considered complete when the callus shows vigorous development in the selection agent. The calluses that show vigorous growth in callus maintenance in the presence of the selection agent are transferred to the regeneration medium to organize and produce plants. In general, the aquatic lentil is regenerated in poor medium. For L. Minor, it is medium SH with an average concentration of 1% sucrose; for L. gibba it is agar in water. Typically, the selection agent is not present in the regeneration medium. The calluses are incubated, under complete light, in regeneration medium for 2-4 weeks until the fronds appear. The compielamenie organized fronds are transferred to liquid SH medium with 1-3% sucrose and without plant growth regulators and incubated with full light for an additional clonal proliferation. • EXAMPLE 45 The effect of light intensity and concentration of kanamycin sulfate was tested with respect to its effect on the frequency of transformation of Lemma minor callus cultures.

The fronds of Lemma «lli were developed in the middle of Schenk and Hildebrandí liquid that contained 1% sucrose during two weeks at 23 ° C under a photoperiod of 16 light hours / 8 hours dark with luminous intensity of approximately 40 μml / m2 sec before of the induction of calluses. The induction of callus was achieved as in example 14 using provenin fronds of Lemna minor strain 8744. The calluses were grown in MS medium which contained 3% sucrose, 1 μM 2,4-D, 2 μM BA , 0.4% Bacto-agar and 0.15% Gelrife last 13 weeks after the co-culinary. Calluses were subcultured in fresh medium every 2 weeks during this 13-week period. Agrobacterium strain C58sz707 that houses the T-DNA that conies to the binary plasmid from strain AT656, as described in example 21, was developed in PDA containing 50 mg / l of kanamycin sulfate, 50 mg / l of specinomycin and 500 mg / l of scopicomycin for 2 days at 28 ° C. For co-cultivation, solid MS medium was prepared with 3% sucrose, 1 μM 2,4-D, 2 μM BA, 0.4% Bacto-agar, and 0.15% Gelrite, the pH was adjusted to 5.6, the medium was autoclaved at 121 ° C for 20 minutes and cooled. Sterilized acetosyringone solution was added by filtration to the cold medium to a final concentration of 100 μM. The cold medium was used to pour 8 Petri dishes of 100 mm x 15 mm. For inoculation, the Agrobacteria were resuspended in sterilized MS medium by filtration containing 0.6 M of mannitol and 100 μM of acefosyringone at pH 5.6 at least one hour before inoculation.

For inoculation, type I in the callus solution. For the co-culture, the deca-pieces were dried by blotting, then masses were transferred as masses to the co-cull medium, 20 masses of callus per each Peiri box of 100 mm x 15 mm. All inoculated calli were incubated at 23 ° C in the dark for 2 days. For the selection, 200 ml of MS medium containing 1 μM of 2,4-D, 1 μM of BA, 3% of sucrose, 500 mg / l of carbencycline, 500 mg / l of cefotaxime, 10 mg / l of kanamycin sulfate, 0.4% Bacto-agar and 0.15% Gelrife, the pH was adjusted to 5.6, the medium was autoclaved at 121 ° C for 20 minutes and 8 Petri dishes of 100 mm x 15 mm were poured. Antibiotics were added to the cold medium subjected to auíoclave as a solution sterilized by filtration just before pouring. The co-cultivated callus masses were transferred to fresh selection medium, 20 masses of callus per each Pelri box. 80 callus masses (4 boxes) were incubated under low light of less than 5 μmol / m2.sec and the other 80 callus masses (4 boxes) were transferred to a light intensity greater than 40 μmol / m2.sec.

For 3 weeks, the calluses were subcultured each week in new medium which was anthelmintic. At week 4, the calli from the calli (40 callus masses) from each light treatment were transferred to fresh medium in which the concentration of kanamycin was increased from 10 mg / l to 40 mg / l. The remaining 40 masses of callus were transferred to fresh medium maintaining the original kanamycin concentration of 10 mg / l. Incubation under luminous conditions or kanamycin alias was continued for 2 more weeks, with weekly subcultures. At 6 weeks, after inoculation, all the samples were transferred to fresh medium and incubated under full luminous intensity. From this moment on, the subculculum was at 2-week intervals. After 12 weeks of kanamycin culinae, the callus with vigorous development was transferred to fresh regeneration medium. The regeneration medium of fronds consisted of Schenk medium and Hildebrandl medium concentration medium that contained 1% sucrose, 0.4% Bacio-agar and 0.15% Gelriie. The callus masses were transferred to new medium of the same composition every 2 weeks. The fronds regenerated from callus masses 3-6 weeks after transfer to the regeneration medium. Two lines of transformed clonal fronds were regenerated from this experiment. Both lines showed histiochemical staining of GUS, had different levels of enzymatic activity for GUS (0.31% and 0.14% of excretable protein) and as measured in a soluble test using methylumbelliferone-glucuronic acid (MUG) as the substrate, and had levels detectable of the enzyme neomycin-phosphotransferase as measured using an ELISA test. Soulhem hybridization analysis confirmed the presence of DNA sequences introduced into high molecular weight DNA, which when digested with the appropriate resizing enzymes gave the expected fragment sizes. Upon new application of the separate blot probes with DNA sequences representing the virulence region of the original Agrobacterium, no hybridization could be detected.

EXAMPLE 46 The effect of the Lemma minor genotype on the frequency of rescue of transformed fronds was tested using callus cultures from strain 8627 of Lemma minor. The maintenance of calluses before inoculation, the bacterial strain, the bacterial growth for inoculation, the bacterial resuspension, the callus inoculation procedure and the co-culine for two days in the dark were performed as in example 45. For the selection with kanamycin, after the co-cultivation, 180 callus masses were transferred to MS medium containing 1 μM of 2,4-D, 2 μM of BA, 500 mg / l of carbenicillin, 500 mg / l of cefotaxime and 10 mg / l of kanamycin sulfate. All calluses were incubated under light levels of less than 5 μmol / m2.sec. In the second week after inoculation, the callus pieces were transferred to a new selection medium in which the concentration of kanamycin sulphate was increased from 10 mg / l to 40 mg / l, and the rest was transferred to selection medium. new one containing 10 mg / l of kanamycin sulfate. The weekly subculture was continued until the week 5 after the inoculation, the operation in which the subcultures were done every two weeks. To regenerate transformed fronds, callus lines with vigorous development in kanamycin and showing GUS expression using histochemical staining after 12 weeks were transferred to frond regeneration medium containing Schenk and Hildebrandt medium concentration medium with 1% sucrose, 0.4% Bacio-agar and 0.15% Gelrite. Fronds regenerated after 3-4 weeks in regeneration medium. The regenerated fronds were grown in SH medium with 10% sucrose. In this experiment, three lines of transformed clonal fronds were generated. The 3 lines showed histochemical staining for GUS, variable levels of GUS activity as measured by the MUG test (0.2-0.3% extractable protein), and detectable levels of neomycin phosphotransferase protein as measured in an ELISA. Southern hybridization was used to confirm the Jfc transformation and integration of DNA sequences introduced into the DNA of the aquatic lentil.

EXAMPLE 47 The effect of the composition of the medium on the regeneration of Provençal fronds of L. Minor callus culinae was also tested. Seven media formulations were tested: (1) agar in water, (2) agar in water with 100 μM of adenine sulfate, (3) agar in water with 10 μM of BA, (4) agar in water with 10 μM of BA and 1 μM of IBA, (5) medium SH medium concentration, (6) medium SH with medium concentration with 10 μM BA, and (7) SH medium concentration medium with 10 μM BA and 1 μM IBA. Callus cultures from both strains 8744 and 8627 were used, which prolifed in a previous callus induction medium as in example 12 for that experiment. The calluses were incubated in the seven different media for 8 weeks, with continued observation for the development of fronds. The regeneration of fronds was only achieved with treatments in SH medium concentration medium. When the SH medium concentration medium was supplemented with 10 μM of BA only, the development of callus was faster than that which was planted in SH medium concentration medium without plant growth regulators, however, the regeneration was lighter than in the middle SH medium concentration. The addition of IBA to the medium had no effect on the time or availability of the callus to regenerate fronds.

EXAMPLE 48 The efficiency of the aquatic lentil system for mammalian gene expression was tested using a gene construct for β-hemoglobin and a P450 oxidase construct.

Two strains of Agrobacterium were used to inoculate the type I callus of Lemna minor strain 8627. For the transformations of β-hemoglobin, strain C58 C1, which hosts 3 plasmids: pGV3850, pTVK291, pSLD34 was used. pTV291 contains the supervirulence gene G from 5 of pTiBo542. pSLD34 is a binary plasmid of Agrobacterium obtained from pBIN19, consists of a neomycin-phospholransferase gene under the control of the CaMV35S promoter, and a gene for human β-hemoglobin driven by the super-mac promoter. For the transformations of P450-oxidase, the strain was used • 10 C58 C1, which houses 3 plasmids: pGV3850, pTVK291, and pSLD35. The T-DNA is carried in the binary plasmid pSLD35, which has clearances similar to pSLD34, with the exception that pSLD35 does not contain the gene for β-hemoglobin and instead contains a DNA sequence encoding 3 proteins: an oxidized P450 of human, an oxidoreductase and a cyclochrome 15 B5. Each gene is driven by a super-mac promoter. Plasmid pSLD35 contains both selectable marker genes for hygromycin and f kanamycin. Several experiments were used with the basic experimental design of 2 bacterial strains x 2 light intensities during the initial selection x 2 concentrations of kanamycin during the design of experimental selection (8 isolates in íofal) with 3 repetitions, with 2 Petri dishes per repetition and 10 pieces of callus per each Peiri box. Callus cultures produced from Lemma minor strains 8627 and 8744 and Lemma * - »# gibba strain G3 were used in these experiments. The maintenance of calluses before inoculation, the bacterial strain, the bacterial growth for inoculation, the bacterial resuspension, the procedure of inoculation of callus, and the co-cullive during 2 days in the dark were performed and as in example 45, with the exception that the bacteria were grown in PBA containing 50 mg / l of kanamycin, 50 mg / l of gentamicin, 100 mg / l of carbenilicillin and 100 μM of acetosyringone before inoculation. For the kanamycin selection, after co-culture, the callus masses were transferred to MS medium containing 1 μM of 2,4-D, 2 μM of BA, 500 mg / L of carbenicillin, 500 mg / L of cefotaxime. and two concentrations of kanamycin: 10 mg / l and 40 mg / l. The calí culíivos were later divided during the incubation with the milad of the callus pieces in each kanamycin concentration in dim light and the oíra miíad incubated under complete light. The calluses were subcultured in new medium of the same composition at weekly intervals during the first four weeks after co-cultivation. In week 5, the culinos were incubated under full luminous intensity during 6 weeks, with the subculture in new medium every two weeks. The regeneration of fronds was achieved using the appropriate medium for regeneration of fronds from strains of L. gibba G3 or L.minor lal and as described in example 42 and in example 47. The fronds were regenerated after 3- 4 weeks in the middle of regeneration. *. * * • && amp; The regenerated fronds were grown in SH medium with 1% sucrose. In all the experiments, more than 20 lines of transformed clonal fronds were rescued. More lines were found using 5 kanamycin at 10 mg / l as the selection concentration, opposite to 40 mg / l. The dim luminous intensity during the selection proved to be advantageous. All the lines showed vigorous callus growth in kanamycin, had variable and variable levels of neomycin phosphotransferase pro as measured by an ELISA test. The presence of DNA, RNA and / or The prolein of P450-oxidase and β-hemoglobin was detected in aquatic leaf plants stably transformed by any method known in the art, for example, Souihern, Northern and Western hybridizations, respectively. All publications and patent applications mentioned in The specifications are indicative of the level of the experts in the technique to which the invention belongs. All publications and patent applications are incorporated in the présenle for reference to the same degree as if each individual publication or patent application was specifically and individually indicated to be incorporated as a reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (55)

1 $ 3 NOVEDAD DÉ L ^ LLWENCION CLAIMS

1. - A method for transforming lentil with a nucleotide sequence of interest, characterized in that the nucleolide sequence comprises at least one expression cassette comprising a gene which confers resistance to a selection agent, the method comprising the steps of: a) provides a target tissue of aquatic lentil, including tissue cells from the aquatic lens cell walls, and b) boosting the nucleotide sequence towards the target tissue of lentil in a sufficient velocity to perforate the cell walls and deposit the sequence of nucleotides within a tissue cell, whereby a transformed tissue is produced, wherein the nucleotide sequence is carried by a microprojectile; and where the sequence of nucleóíidos is impelled in íejido impelling the microproyecíil towards íejido.

2. The method according to claim 1, further characterized in that it comprises the step of culturing the ransform with the selection agent.

3. The method according to claim 2, further characterized in that it comprises the step of regenerating transformed aquilic lentil plants. t &

4 A.- The conflux method with claim 1, further characterized by the fact that the material is callus tissue.

5. The method according to claim 1, further characterized in that the material is merisiemal tissue.

6. The method according to claim 1, further characterized in that the material is woven frond.

7. The method according to claim 1, further characterized in that the tissue of the aquatic lentil is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella and genus Lemna.

8. The method according to claim 1, further characterized in that the tissue of the aquatic lens is selected from the group consisting of a species of Lemna minor, Lemna miniscula and a species of Lemna gibba.

9. The method according to claim 1, further characterized in that the microprojectile comprises a melicic particle.

10. The method according to claim 1, further characterized in that the microprojectile comprises a metal particle having a diameter of about half a micrometer to about 3 micrometers.

11. The method according to claim 1, further characterized in that a plurality of microprojectiles are provided, each of the microprints having the nucleotide sequence immobilized therein, and each of the microprojectiles being fired at the objective tissue. of the plant.

12. The method according to claim 1, further characterized in that the nucleotide sequence codes for a protein or pee that is selected from the group consisting of insulin, growth hormone, α-inérferferon, β-glucocerebrosidase, prolein of relinoblasíoma , p53 protein, angioslatine, le and serum albumin.

13. The method according to claim 1, further characterized in that the nucleoid sequence encodes at least one proinin or pee subunit of a multimeric proiein.

14. A method for transforming aquatic leia with a sequence of nucleoids of interest, characterized in that it comprises the steps of: a) inoculating an aquatic plant tissue with Agrobacterium comprising a vector which comprises the sequence of nucleolides, wherein the nucleotide sequence comprises at least one expression cassette comprising a gene which confers resistance to a selection agent; and b) co-culturing the tissue with Agrobacterium to produce transformed tissue.

15. The method according to claim 14, further characterized in that it comprises the step of culturing the ransform in a medium comprising an antibiotic in an amount sufficient to inhibit the development of Agrobacterium.

16. - The method according to claim 14, further characterized in that it comprises the step of culturing the tissue transformed in a medium comprising the selection agent.

17. The method according to claim 16, further characterized in that it comprises the step of regenerating aquatic leaf plants transformed from the transformed form.

18. The method according to claim 14, further characterized in that the tissue is callous tissue.

19. The method according to claim 14, further characterized in that the tissue is merisfematic tissue.

20. The method according to claim 14, further characterized in that the fabric is drawn frond.

21. The method according to claim 14, further characterized in that the fabric of the aquatic lens is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella and genus Lemna.

22. The method according to claim 14, • characterized further by the fact that the acid of the aquatic lens is selected from the genus Lemna.

23. The method according to claim 14, further characterized in that the leaflet of the aquatic leaf is selected from the group consisting of a species of Lemna minor, Lemna miniscula and a species of Lemna gibba.

1. The method according to claim 14, characterized in that the Agrobacterium is Agrobacterium tumefaciens.

25. The method according to claim 14, characterized in that the Agrobacterium is Agrobacterium rhizogenes.

26. The method according to claim 14, further characterized in that the vector is a superbinary vector.

27. The method according to claim 14, further characterized in that the vector is a vector derived from C58.

28. The method according to claim 14, further characterized in that the gene that confers resistance to a selection agent is selected from the group consisting of neo, bar, pat, ALS, HPH, HYG, EPSP Hm1.

29. The method according to claim 14, further characterized in that the nucleotide sequence comprises two genes of interest.

30. The method according to claim 14, further characterized in that the nucleolide sequence encodes a protein or peptide selected from the group consisting of insulin, growth hormone, α-interferon, β-glucocerebrosidase, retinoblastoma protein , p53 protein, angiostaine, leptin and serum albumin.

31. The method according to claim 14, further characterized in that the nucleoid sequence encodes at least one proinin or peptide subunit of a multimeric protein selected from the group consisting of hemoglobin, collagen, P450 oxydise and a monoclonal antibody. .

32.- A method for transforming an aquatic lentil cell with a nucleophilic sequence of interest, characterized in that the nucleoid sequence comprises at least one expression cassette comprising a gene which confers resistance to a selection agent, comprising method is the step of introducing the nucleolide sequence into an aquatic lentil cell by electroporation.

33. The culture of transformed aquatic lentil tissue produced in accordance with the method of claim 3.

34.- The cultivation of transformed aquatic lentil tissue produced in accordance with the method of claim 17.

35.- The plant of Transformed aquatic lentil produced in accordance with the method of claim 3.

36.- The transformed aquatic legume plant produced in accordance with the method of claim 17.

37.- The Iransformated lentil plant produced in accordance with the method of Claim 32.

38.- A transformed aquatic lentil plant comprising a heterologous nucleic acid of interest incorporated in its genome.

39.- The aquatic lentil plant transformed according to claim 38, further characterized in that said nucleic acid of His interest is flanked by T-DNA border sequences incorporated into his genome.

40.- The aquatic lentil plant transformed in accordance with claim 39, further characterized in that said aquatic lentil plant comprises less than 5 copies of said heirologous nucleic acid of interest.

41. The aquatic lentil plant transformed in accordance with claims 38 or 39, further characterized in that said aquatic lentil plant is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella and genus Lemna.

42. The aquatic lentil plant transformed in accordance with claim 39, further characterized in that said aquatic leia plant is selected from the genus Lemna.

43.- The aquatic lentil plant transformed according to claim 39, further characterized in that said aquatic lentil plant is selected from the group consisting of a species of Lemna minor, Lemna miniscula and a species of Lemna gibba.

44. The transformed aquatic lentil plant according to claim 39, further characterized in that said nucleic acid comprises at least one expression cassette comprising a gene which confers resistance to a selection agent.

45.- The aquatic lentil plant Iransformed according to claim 44, further characterized in that said gene which confers resistance to a selection agent is neo, bar, pat, ALS, HPH, HYG, EPSP and Hm1.

46.- The aquic leia form transformed in accordance with claim 39, further characterized in that said nucleic acid 5 comprises two genes of interest.

47. The aquatic lentil plant transformed in accordance with claim 39, further characterized in that said nucleic acid encodes a protein or peptide that is selected from the group consisting of insulin, growth hormone, -interferon, β-glucocerebrosidase, proinin of reíinoblasíoma, p53 protein, angiostafina, lepíina and serum albumin.

48. The aquatic lentil plant transformed in accordance with claim 39, further characterized in that said nucleic acid encodes at least one protein or peptide subunit of a 15 mullimeric prolein that is selected from the group consisting of hemoglobin, collagen, P450 oxidase and a monoclonal antibody.

49.- A method for producing recombinant proteins or peptides, characterized in that it comprises the steps of: a) cultivating a transformed aquatic plant that expresses at least one 20 protein or peptide helerologist; and b) collecting at least one protein or peptide from the aquatic lentil cultures. ^^^^^^^^^^^^^^^^^^^^^

50. - The method according to claim 49, further characterized in that the aquatic lentil plant is transformed into waste water.

51. The method according to claim 49, further characterized in that the Iransformated aquatic lentil plant expresses and assembles all the subunits of a multimeric protein.

52. The method according to claim 51, further characterized in that the mulimeric protein is selected from the group consisting of collagen, hemoglobin, P450 oxidase and a monoclonal antibody.

53. The method according to claim 49, further characterized in that the transformed aquatic lentil plant is grown in a bioreactor vessel.

54. The method according to claim 49, further characterized in that a recombinant proiein or peptide is produced.

55. The method according to claim 49, further characterized in that at least one prolein or heterologous peptide is a proiein or therapeutic peptide. 56.- The method according to claim 49, further characterized in that at least one protein or peptide is selected from the group consisting of insulin, growth hormone, - interferon, β-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin , lepina and serum albumin. 57 - The method according to claim 49, further characterized in that at least one heterologous protein or peptide 5 is an enzyme. 58 - The aquatic lentil plant transformed according to claim 49, further characterized in that said aquatic lentil plant is selected from the group consisting of the genus Spírodela, genus Wolffia, genus Wolfiella and genus Lemna. 59 - The transformed aquatic lentil plant according to claim 49, further characterized in that said aquatic lentil plant is selected from the group consisting of a species of Lemna minor, Lemna miniscula and a species of Lemna gibba. 60.- The method according to claim 49, Further characterized in that at least one heterologous protein or peptide is secreted by the transformed aquatic lentil plant. 7, .. - * ~ -? ** »**" V * »- * ------- 17 $ Methods and compositions for the efficient transformation of aquatic lentils are provided; preferably, the methods involve transformation by either ballistic bombardment or Agrobacterium; in this way, any gene or nucleic acid of interest can be introduced and expressed in aquatic lentil plants; plants, cells and tissues of transformed aquatic lentil are also provided; Also described is tissue culture of Iransformed aqueous sheet plant and methods for producing 10 proteins and recombinant peptides from transformed aquatic lentil plants. ^ MG / JT / fpm * jtc * pbg * mmr * ald * xa abg * ltf * sff * aom * yac * eos * mvh. P99 / 1417F