MXPA97004794A - Transgenic plants expressing dna constructs that contain a plurality of genes to provide resistance to the vi - Google Patents

Abstract

The present invention relates to a chimeric recombinant DNA molecule comprising: a plurality of DNA sequences, each of which comprises a promoter functional for a plant bound to a coding region, which encodes a coating protein associated with a virus, wherein said DNA sequences are preferably linked in tandem such that they are expressed in plant cells susceptible to the viruses transformed with said recombinant DNA molecule to provide resistance against said viruses, and methods for transforming plants with the constructs are provided. chimeric and to select plants that express at least one of said DNA sequences that provide resistance against vir

Description

TRANSGENIC PLANTS EXPRESSING OÜE DNA CONSTPECTORS CONTAIN A PLURALITY OF POPULAR GENES PROVIDING RESISTANCE TO VIRUSES FIELD DF 1 ATV ION This invention relates to the genetic engineering of plants and to a device and method for conferring a plurality of traits, including resistance to viruses, to a plant using a vector 'encoding a plurality of genes, such as pot'. gene-example, reverse protein imit, yen of μrotearía, or genes repilieasa. BACKGROUND OF THE INVENTION Many important crops from the agricultural point of view are successively infected by crops that can seriously damage a fruit, reduce its economic value to the grower and increase its cost or The Lonaumi do, Attempts to coni or to the infection of a crop, a plant irus have been made, yet pathogenic, and they are still a p.-obleme means to me in agriculture. ture. Scientists have repeatedly relied on devices to produce resistant plants using genetic engineering techniques. Such an approach? - > s provfc? so ^ the extent to which the genetic material that provides protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it has the ability to suppress or delay the multiplication of a virus, or the development of pathogenic symptoms. "Resistant" is the opposite of "áAa ep? Bl" ", and pued divi irl en > '1) Does it re-ai ate i *, (?) Moderate resistance, or < 3) low resistance, according to its effectiveness. Essentially, a resistant plant shows a reduced presence or absence of a symptom and the multiplication of virus within said plant is reduced or becomes increasingly severe. Several different types are known ?, host resistance to viruses. The host may be either (1) the infected person, the infection, (2) multiplies it ori of the virus, or (3) the mov ii ento vrl «The potivi us a different group of pesticides that are pathogenic to a variety of crops, and that have a strong capacity to work between my family and vegetable plants differed by L po vipjs , include the mosaic of the watermelon mosaic (WM -), strain 3 of the papaya ring stain virus, potato ring stain and mosaic I of the watermelon ípRV-p and PRV-i »), Two of the vegetables belonging to the group are vegetables that were classified at a certain time as virus viruses, but which are classified as "different" or different strains of the same virus. yellow mosaic virus of the alata Í7YMV > 5 the irus and of the potato 5 the virus left of the tobacco and other mucus, for example see table I of the published European patent publication 578,627. of particles »filamentous, fleles de d ini i? s e apt u - i a nieute 7R X 1 IHII II 'II > H. I a =, Viral particles contain urr strand APN genome which contains approximately 1.0,000 nucleotides of positive polarity < +, de difica tion, or meaning). The translation of the RNA genome of the potyviruses shows that the RNA encodes a large mica paliprotein of appropriate size 330 l-P. This polyprotein contains several proteins, one of which is a prudential of 49 specific for 13 dissociation of polypeptide at least sei =? < 6) other peptides. ta-; proteins can be found in plant cells - and they form the most important part of viral replication. One of the proteins contained »within n pol? Μl-e. { na is one. capsule \ - D or a coating that covers and p > I get the viral RNA mph degradation. Another protein ec, inclusion-protein mu.1 I'I L cmnc I also gives as a replacement, and which is the same. e - thinks that it works in the replication of the viral RNA. In the course of a poti ial infection, 1 * protein ieplicasa íó >; 0 Da, i. or also as the inclusion protein B nucl ar) and the pyrete [a protein < ? s 50 1 1 D *, also known as inclusion protein I nu »lf * a¡ > - > A protein of inclusion nu) are transported posttranslationally through the nuclear membrane in the nucleus of the cell of the plant in the later stages of the viral infection and accumulate in cough levels. Generally, the reverse protein gene is found at the 3 'end of the RNA, just before a terminal adenine nucleotide residue segment (200 to 300 bases). The location of the 49 Kd protease gene seems to be retained in these vius. Fn the furrow virus > In tobacco, the dissociation site of the pr ?: tease has been determined by = > in the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. The i.conservation: > 1e these d? Pepl? D »js like - > The dissociation in visual polymorphisms is apparent from the elements of the pottery whose value appears. l -t expression of l s »-lining protein fibers»: Jel vi rus »: iel ¡nn ^ i o of talia» o, vi rúa »of mosaic of alfalfa, i us of mo a i > _ >; > of cucumber and vi rus X of the piapía, among others, in plants -u? 'g u? > Thus, it turned out that there were still some problems in the infection due to the respec - tive condition. Some evidences have also been reported »Je pirote», jón heteró 1 •? > _ja. P »JG example, NamLia et al. , Phy t opa th »l n > jy, 82, 940 (1992) to report on. What to say of 3o =. genes of the protein of i inie o »1el v i. ru - > - 2 of the mo.iai »or > ie the watermelon well I saw you > The yellow mosaic of the courgette in transgenic tobacco plants confers protection against six other potyviruses. Bean yellow mosaic virus, potato Y virus, pea mosaic virus, clover yellow vein virus, pepper mottle virus and the virus. l furrowed ».the tobacco. Star-I et al, P i otechnol og, 1, 1257 (1989) report that the expiration ion «of potyvirus, mosaic virus > Soybeans, in transgenic plants, provided piracy against two poten- tially non-related viruses in the vineyard. : lel taba »" oy we saw the Y "of the potato, however, the fact that the ecofriendly protein does not confer in a reliable protein> hetei'ologa a pl-tnta. P »> r 'example, plants» ie labaz t rans > jén? cent dye the > .jen of pri'telna e e-.?ti The CMV-C and ue sa rsist to the strains CrlV-C, fn > are protected unha va the strains 0 highly v uentes CMV u », - luding GMV -V-27 and CARNA--. - According to the present invention, there is a need for improved methods for improving the strength of the latent COMPLEX OF THE INVENTION, the present invention, in proportion to the structure of a single molecule. DNA quiméri a re omb in nt qu > ~ omp > =? N »: le un-i pl u > a 1 i > rla» -l > -Je se »uen». The DNA each of which i.om eí »» je an operator * enl ara >:? operatively H un-i secnen:? of DNA »that encodes a protei ** as The invention relates to a coating protein (cp), a protease, or a replicase, wherein said DNA sequences are expressed in plant cells susceptible to the viruses transfused with it. > iia > 3l »-ul d DNA» e ». »J»? »I) t n < - > »» »> - e \) to prupor» unwi resistance c »&nt the infection by each of these viruses.From pr? 1 ferenc i, the DNA sequences are linked in tandem, it is to go, they exist in the head-to-tail orientation in relation to each other.Also, substantially even resistance levels are observed, which are essentially equal to the infection p > * 'r' each of said viruses in plant cells. ? "r? n "i -> with the iliit i ti id > je -, et uen" - ia ***. > 3e DNA, preferably, each sequence of the DNA is in nt also nl jól to mía is uenc ia e DNA uo asladada 3 '-that works in plant cells to cause the end of the ion and the transi tion and the addition of r ibonuc 1 éot i -ríos pol i adeni 3 a - at the 3 'end of as - => the RNA RNAs of the prefence, the virus, a virus associated with plants, such as, for example, a virus. >) r getting the DNA molecule can be used as a "construct" of the chimeric r "tomb i nanbe, or a" cassette of e-p? How to prepare the plant by means of which one can resist the increased one against the one at least at least one year. virus > ie pil antas, »orno for example» pcitivi us. The present cassettes also preferably comprise at least one > jjen selectable marker or an integrated reporter gene >Stably in the genome of plant cells transferred in association with viral genes. The marker 3IJ1I; ' I OI l b le and / »> 1 • - > - = gene-- »i > -pu > t »a? > j =. f »i 1 i tau the identification» ie trans plants and plant cells. Preferably, the set > > The human condition is flanked by two or more selectable marker genes, repoi-tero genes, or a combination of them. Ot > -c > aspec the present invention is a method to prepare a plant resistant to the viruses, as by ern i »! a »i i > ut i 1 e n, q »? e r nm í e» »». { < a) the training > of plant cells with a recombinant DNA molecule chimeric i > - • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • includes a plurality of DNA sequences, each with an r > I have a pr »- > functional engine in said plant cells, nla ao in operable manner s "" 3bre a sia of DNA, which encodes a protein associated with on vi us that can infect said plant; ib regenerates it - di ion of di has plant cells to provide a plant di ferenc? ac.3; and < • -) the i entificaci n of transformed plant that e., prea the d s s d ds s ta dias d to turn the resistant plant against infection by said-; virus, »preferentially at equal levels the resistance to infection by each of these viruses.

Another object of the present invention is to provide a method for offering resistance against virus infection in a cucurbi plant. sensitive susceptible grasp that includes: ia) 3 '* ransf' "cell membrane" of plant that »rb it r.ea» "on a DNA molecule that encodes a plurality of proteins from viruses that can infect said plant cucurbi tecea 5 (b) regenerate -these plant cells to provide a uniquely harvested plant and (c) select a selected succulent cucurbi expressing the virus proteins at levels sufficient to return the infected plant to infection. for those viruses. It is an additional object of the present invention to provide transfammed plants resistant to multiple viruses that contain stably integrated DNA sequences that encode the virus protein. It is another object of the present invention to provide transformed plant cells for viruses that contain a plurality of viral genes, i.e., 2-7 or more, in which virus proteins are made from of the same strain of virus, from different strains of virus as well as different members of the group of viruses, such as the pot ivi rus group. The present invention is primarily employed by the insertion of expression cassettes > : .le p rote í. I've been covering multiple viruses in a binari plasmid »:? and the subsequent characterization of the plasmids results before. The combination of CMV, ZYMV, WMV-2, SQMV, and cassettes of »- • x i» * »i i or I i > ie l j? ? »> - 11 '»? Ie I HJI- .I; | III [I'I | ¡? I] > ^ Pft. ' -e »» 11 o > - H I H I in the binary plasmid piPPBN. Subsequently, binary plasmids that had cassettes of multipurpose r-evestment protein cassettes were moved in Agro acter lute their use in pro d ectivities. "Plant information" L os lsi do:? binaries that had multiple expression cassettes were used to transfer two more genes *; your »ept: i 1 -a from t r-) nsf > - »rimination of viral coating protein in plants,» such as for example the family -Je la »» .u? .urb 11aceaa, junt.-i > - ?; »p lo.r. genes ma r-c aclcn es and / or rep »irteros sele ciiiuablí-.1-» associates. Accordingly, the present invention provides a methodology, "chirping" »the piyeniei'ta gen ti» -; e »ti -a» - »which range mul ti ple =» may be 'mam piul a »R y &r > treados > t a single generic insert, it is right, in the form of a '- tru tru' - > acting as a unique entity that e as a loci fi li au ».» nic. Even when the invention is carried out by means of the virus, the virus can not be used. ? mb if »gene action could be in 1 a - ar => e. Then a p>> - r ^ f ^ a block that will sign you as a group. μ example r is i = »t nc iai 1-disease, more resistance to herbicides, more extended shelf life, and similar, middle pair of the trace (of the reporter or selected selectable canvas that has if it is included in the vector e transfor-mac ion.? ^ -j.3, { e -? ». ul? ie» lo tn ?? ¡(eu ue. uand - »e H? - »? - 'rt In tandem, multiple tankers show their preference for the same degrees of effectiveness, and with a greater degree of effectiveness, where the thermal "substantial" when refers to the resi in the vir l is defined with reference to the essays desc i os in the examples that we present to inuación. Pot eje.nplo, if one x ina number ais lines »transijénicas containing» »n inje to ci pr > ? teina »Je revest i > r »? erd» ** > ZYMV and WMV-2, are »neutral or a:» line immune to infection by ZYMV is ambient immune to WMV-2 infection. From m? = =? N? r, if a 11 ne.-t presents a retard. in the related symptoms lielarmlla; with ZYMV, it also presents a retard »in the development of related symptoms • -.a WMV2. Finally, the line is in its tepid ZYMV, it will be acceptable to WMV-2. This phenomenon is unexpected. If there was not! a correlation between the effectiveness of each gene in these multiple constructions, in which approach a tool in reproducing the ion. p antas would be particularly difficult to emulate "Even with const ructs" g n ti or unique, uo <It should test "numerous lines" of transgenic plants to find a line that presents the appropriate level of effectiveness.The probability of finding a line with useful levels of expression can be between 5 and 50 * 4 (depending on the espiec ie involuda.) If the efficiency of the -ainiviv dua 1 in a state that contains multiple values was independent, the cost of finding an Transgenic line resistant to each white virus will decrease dramatically. For example, in a species in which you are seeing a probability of 10 * / * 'ie identifying a line with resistance using a single genetic insert, a single gene construct is transformed.

CZW and each > jen presents an indefinite level of efficiency, the probability of finding a line with resi tence -i CMV, ZYMV and WMV-2 would be 0.1 X O.t X. I - 0.0 1, 3 is 0.1 * 4. However, the fact that equivalent genes are not independent of each other- ", the probat > I can find a line with a resistance to CMV, TYMV, and WMV - 2 -if it is still 10 * 4 in ve: d .1 * 4. Obviously, this advantage is more noticeable when they are used »OIHÍ u > : t »" js that contain -3 or more genes BRFVF DESCRIPTION OF THE FIGURES l fi-jura 1 rsn the binary erp tab figu la 2 r eta the est u ura > Je ve »tm bin-jr m pPPPfJ, figure Z preaent, the structure of pPPCPW Figure 4 presents the structure of the binary pletsmido pEPG321 Figure 5 presents the structure of the plasm? »: the binary pEPG106. The structure 6 presents the structure dlp >] sítud > buiapp pEPGlU Fig. 7 shows the structure of the binary plasmid pEPG109. Figure 8 shows the structure of the binary lasmida pEPGl15, Figure c shows the structure of the pl binary signal pEPG21 Figure 10 shows the structure of the binary p > -> binary pEPG113, f igure 11 presents the binary code pGA482GG b Figure 12 presents the structure of the binary populate pEPG 28. FSPCRTPCIO DETAILED OF THE INVENTION I The viral resistance conferred to 3 < *? plants of the present invention is provided by the. e: pressure on 1-3 plant of an isolated DNA Qje c-omprende nu »; read i > -jc »s that c.fn 11 fican a pluralid-ad, is dc ii, from 2 to 7 pr t í na s» le vi ru. », co or for example protrúa?» de re eiino, protea = »Ay / or rep» 1 a sa. Representative viruses for the settings these DNA sequences can be isolated include, but are not limited to, potato virus X (PVX), piotivirus, such as, for example, virus Y from the pap > a < PVY), cucomo irus ÍCMV), vein mottle virus »from 'aba» i », i us d» - osa? > < ~ > e 1 - • »- n > »> The yellow mosaic of the zucchini TYMV), cold mosaic ciphon virus, yellow mosaic virus of the fri jo , soy mosaic irus, virus »1the peanut mottle, virus > of the beetle moss, irus of the wheat mosaic mosaic, i us of the sicicole an ..leí ma i z, irus ciel mole of the sorghum, virus > Jel mosaic of 1: t c-tña d * - a .-? Car, v i ru > > of the mos i o »iel ... irg» * »» ie Al'-p'i, virus of rottenness »d > -? 1 plum, rum from the tobacco furrow, from the feathery moth of sweetpotato, virus: the mosaic "J sweet potato, and VH" from the ring spot of the papaya ÍPPV *, or omovi us, introducing CMA and comovirus . Gene »* -» 1 mind, a pot i i rus is a single-strand APN pus surrounded by monomer p r-oteiuasea repe i i o, which are known as reverse rot. PJ ", the encapsulated virus has a flexible rod michrophology, most of the pot i vi rus are transmitted non-p rsist n by Af-rs. As can be seen from the beginning of]: '»M? 1 ic? N ngo»: ie c ech -? Ceptü. Ias by pol> vir us », the range of hosts includes diverse families of plans such as So3: •» na > - eae, ^ r ipodiaceae, Gra ineae, "Compo itae, Legumi nu ae, Diose oe ^ ceae, Cucur i aceae, and Cari caceae. As used here, with respect to a DNA sequence or b in "gene", the term "isolated" is defined to mean that the "s". uef? c i - »> * »Bieu -» e e; -t? E e »u co j e¡ t > - in the genoii- viral p »:» r chemical means and it is purified to modify insofar as it can be introduced in the vectors present in the appropriate orientation, that is to say *, -entid > D antisense. As it is written here, the term "chimeric" refers to the linkage of DNA to DNA sequences that are derived from different sources, strains or species, it is to go from bacteria and plants, to Ltien of two or more > s DNA sequences > : ie the same spei is linked so that nc »occurs in the born genome vi. Also, the DNA sequences useful in the present invention may occur naturally, "in a semi-formal or tuteral manner." The sequel ». the one of DNA can be linear or circular, that is to say, it can be located in a linearly raised or intact plastic, as for example l »;? s p l binary m ations described to i i > nt i nuac on. As used here, the term "het er'ól ogo" * = > and defines to dignify an independent party either in the same or identical way, for example different in sequence of "." > or anunoScidci. As it is written, the term "e, pressure" refers to the string: > > ~ r i? > Ion or 11 ansc »-? pc i ón followed by translation» of a r particle molecule of AD. 1. 5 Most of the recombinant DNA methods employed in the practice of the present invention are standard procedures, well known to those skilled in the art, and to those skilled in the art. - > > "." N alle e, r. »» Ree »ila, 1 European patent application published under number 223,452, published on November 2, 1986, which is incorporated herein by reference. Obi have commercial sources and are used in accordance with the recommendations of sell- or else:> Go through familiar variations in the tea.National references that contain such standard techniques include the following: P. Wu, e., J. 979) Methods in Er.ay ol, vol. &C; JH Mil ler (1 72) E 'eriments ni Molecular Geneties; J "Sambmol- et al. (1989) Molecula Clonin -J: A Labor Manual, 2nd ed., DM G3, >? er, ed. (1985) DNA Cloning v. TT; HG Polites &i-, Marat i (1987) "To this-my prntocol f nr>; DNA s nthes i s," Ijn progressive protocol for the DNA of cDNA), Biptheciuques 4; 514-520; S.B. Gelvin and R.A, Schi lfneroort, eds. Jntrciduit ion, E,? Pr is i an, 3 d An l and yes? of Gen Products in Plants, which will be used here by reference. In order to practice the present invention, a "isolated" gene can be isolated. Enoma i l inserted in a vect »? r» u contains the necessary genetic regulatory sequences to express the inserted gene. Therefore, a vector can be constructed to provide the regulatory sequences in such a way that they become functional upon insertion of a desired gene. When the constructor vwc tor is assembled from e: pre 31 ón / in? I - "-? To, = e in» p 1 w to tra f »jrni3 r cells> of plants that are then used to regenerate-plants. These transgenic plants carry the viral gene in the pressure vector construct, and the gene is run in the plant and thus provides increased resistance against viral infection, several different courses isolate a gene. For this, a person can be certain about my subject matter in informing him about the genetic organization of the poti irus, the virus or the c om t GU-? »ia ubi r- and isolate the yen from the reverse protein or the genes of the nuclear inclusion body.The gene» df the prole hm> Je reverse in l »_rs potí virus * They refer to the erythromycin of AP, UST: "before"; • • • • • • • • • • • • • • • • • • • • • cnpi or F "(Nlb)» of? ncU? s? 4fl- nuclear is located just 5 'of the revol.protein gene, and the gene of the • inclusion body A (Nia) nm- lear e- = > 5 'the NTb gene "Ad i > "Finally, 1 * informs": ion related to the elements and dissociation nroteol í 11 ca s "? employs μara det rm nr the interiio N»: iel »3t-n >" Protein of coating of pn I , ivi rus and the e :, tremo N and C of the genes of pr »Dteína of non-coating. 1 ns pratease recognition sites are conserved in the viruses and have been determined y3 either as the dipeptide Gln-Ser, Gln-Gly or Gin-Ala. The connections between the leotides that code these signals can be determined. Using methods well known in the art, a quantity of virus is cultured and harvested. The viral APN is then separated and the viral gene is isolated using numbered known procedures. A cDNA library was used using the viral APN, by methods known in the art. The viral APN is i? T ubad »> -an initiators that are made with the viral APN and reverse transpnptasa, and a DNA molecule is observed as i. Ur »'-'i l nto e ADN > Te] 3 molecules of complementary DNA s produced and this sequence represents a copy of the DNA (cDNA) of the molecule »APN vira *} original. Fl > The DNA component can be produced in a way that in a double-stranded cDNA '' meo > . *) Well can you em > -pneumerase chain reactions in order to amplify the DNA > that codifies DNA > - > : > n the use of ini iadores ol 1 gomero especfi o; »p-? r < It's the viral gene. These can be explained in detail in the specifics, objectives, and improvements in the improvement of the system. By cons 1 gu 1 entity. a molecule of DNA is generated ».ie double strand that with 1 has 1 sequence information of the APN vi.

These RNA molecules can be cloned in plasmid vectors > E. coli after additions of restriction enzyme enzyme molecule by DNA ligase. The various fragments are displayed in vectors "-» »* > - «s» - l »? I? A? For example, they can be used, for example, for well-spiked pylides, which are then used to transfuse E. coli to create a Li-Ii and DNA tag. Since po and virus genes are generally conserved, they are based on an analogue gene from a previous isolate or a> fragment is analogous to a gene. a previous isolate that can be used as a screening wave to screen the cDNA library for deletion if any of the trans formed bacteria contain DNA strands with the viral sequences appropriate. The cDNA inserts in any bac and i nna that hybridize with these conces can be removed. The viral gene is present in its entirety in colonies »? U» have s uen what is understood from 5 'to sequences that hate the uncle and di »» ocia »in 0 proteol í t 3, -rí» I say "N" and ":" I would like to know if I have a sil x of its »? Protective ion of i? "* Mn C for the gene: ie you are nt ernatively, the DNAs of the DNA can be found in the orientation of the senses in the same. Antibodies against a viral protein can be used to screen the DNA expression library, and the gene can be isolated from colonies expressing the protein.The nucleotide sequences are encoded by the reverse-protein genes. Inclusion genes for numerous viruses have been determined and genes have been inserted into vectors of expression.Expression vectors contain the genetic regulatory sequences necessary for the expression of an inserted gene.The coating pratein gene is inserted in such a way that these regulatory sequences are functional and the genes can be expressed when they are incorporated into a plant genome.

Selected references in the. Literature to methods for isolating, cloning and expressing viral genes appear in the list in Table I, below. TABLE I. Genes cloned from irus of APN. Viral gene. Reference Protein of fl.M. Eitch et al., Bio / Technology, coating of 10, 146 > (1992) ring spot of papaya Protein cié. L i ng et al., Bio / Technolgy,, cover of 752 (1991); X virus from Papia A. Hoel-rema et al., Bio / Technology, 7, 273 (1989) Protein by H. Quemada et al., J. Gen. Virol., Coating of virus 71, 1451 (1990)? II of the mosaic of S. Namba et al., Phytopathology, "* N.1- 82, 940 í 1-? < -? '- * •) Protein of S. Namba et al., Phytopathology, coating of the virus 82, 940 (1992) of the yellow blood of the cyanaceum Protein of P. 5. Nelson et al., Coating of the virus Big T;; »/ Tec hnology, *», 40? »(1988) 5 of the tobacco rail mosaic P. P» 3 »? Fel l Abel et al., Science, 232, 738 < 1 «Í») Pro ica of h-Fríes et al., EHB0 T ", £ > , coat of virus 1845 f 1987 ^ 5 of the mosaic of N.E. Turner et al., EMBO xT. , or, alfalfa 1181 M987) Protein of D.Í1. Start et al., Biotechnology ogy, coat of vi ur »7, 1257 (1909)» l soy mosaic Prot J a > of H.Q. Quemada et al., Mol c. Plant coating of the Pathol., 81, 794 ("1991) strain C of the virus> ridge of the cucumber Protein of UpJohn C". "PCT W090 / 21 C5- ~ coating of the strain WL 'irus mosaic cucumber Protein There are et al., Virology, 147, 30c? Reveal 1 mi of the -irus C1 85) of the tobacco furrow inclusion pratein J.C. rarrington et al., J. Virol., nuclear virus 61, 25 AO ("1 87) furrowed tobacco Protein from WG ougherty et al., Virology, virus coating 146, 282 i1 85 mottle of the protein of DD ShuM = »et al., Virology, lr" »2, virus coating 118 (l-'Qifi) Y >; "the potato Inclusion protein Nuclear European Patent Application for the virus Y of 578,627 1 a pa» Protein of r * "Lawson et al., Putt e> - hn» >] ogy, 8, reverse of the virus 127 ("1990) Fr" -teine CM, Dun mt 31., Virology, 164, coating "" I saw 383 C1988) from the vein of] tabai or »'TSV * < To express the viral gene , the necessary genetic regulatory sequences must be provided, since the p »r» .te.} ns r »c» d? fi »ada-1 in n ijni ti-ima» p p »» livii us produced by the post-translational process 1 of a polyprotein, a viral gene isolated from viral APN does not contain the necessary transcription and translation signals to its expression once it has been transferred and integrated into a plant genome. Therefore, it must be manipulated to contain a promoter that can be used in plants, a start codon of ra? to? a (? TG) and an "inlet". poli ) functional plant AAT AA) Z 'of its stop codon of t rasl a »: i ón. In the present invention, a viral gene is inserted into a vector which does not include cloning par = * i serc i n 3 'of the > "odón": Je initiation and 5 'of the _ > eñ3l of poli ("A). The promoter is 5 'from the initial »-o» ion in such a way that when the structural genes are inserted into the cloning site, a uni »1a» i fu? T is formed? > .n? al where 1 c »s gene = > inserted are under the cont rol > of the various sec uen ».. i a-- regulates to a yenéticas. The DNA segment • onoct * - »co o el pro oto» -., Find re .-? P > »» »Ns-» bl> e the legul- ization of DNA transection in APNm Numerous pro - nodes that function cells of p 1 xf n 1 - • > are >They can be added to the technique and can be used in the practice of the present invention, which can be obtained from various sources such as, for example, plant viruses or plants, and can include, without limitation, , isolated promoters of the caol imov i rus group, such as the 35S pomotor of the vi rus l mo-sa i • _ »-» of the II flu 5 «CaMV35S), the promoter 3 ^ 8 »: ie! I rus of the mosai or of the increased colloriform ípaM), the promoter of the transcript of total length of the mosaic virus of the scrofulapa { FMV35S), and the pr '' n »? T» ot to island »in of the nl ace / b protein of the chlorine» -) fi 1a. P ro-j pm uers of '' di 1 es iin luyen promotores' J that can r ^ ar 1 ^ s proteins poti vius inducible or Ipen in the form e, ptfi »-a for tissue in» "i 'a ? tipi »lular ü where - ^ e ri b acurre 1 H infection. For example, lso »t? P tor * is indi-able from the feml alanina amoni al ti, lialcona si rdasa, &rt;> Tension, proton-n related to the pathogenesis (for example, PP? -ta * - a. = í as? nh?? »Jc» r of the? r? gt? the P.-> pa may be the preferred promoters for their u => or <> self-expression cassettes of the present visual genes include the p > r. > motore-> constitutivos »ie CaMV, the genes i» t ñopa li n- ^ i nta- ^ a CBevau et al., Nuc le, »- A» "ids Res, II, 369-3R5 (1983 )) I to insintasa íDepi »ler et al. , J. Mol. Appl. Genet , 1, 561-56-3 í 1982) 5, and the > T i de de alma alma alma - - - - - - fr fr fr fr fr señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales señales "señales" the present cassettes. The particular promoters selected are capable of preferably causing an e p? he??? 'd 1 f í u in i-- 1 -3 s - > "Ue | n la-i de» »» »I ii» .-) &on the DNA on which they are linked in an operable manner, to result in the production of effective amounts of APN proteins for provide a viral resistance, but not excessive as to negatively accept the cells in which they are expressed.The selected promoters could be able to function in tissues that include, not limited to, hepatic, vascular, and mesophilic peptides. Does not the promoter itself is not a critical feature insofar as it has sufficient transferability to achieve the expression of the preselected proteins? »? well antiseptide APN, and subsequently confers viral resistance to plants. The non-translated leader sequence can be derived from any appropriate source and can be modified specifically to increase the rate. ", I n >of 1 mRNA. The untranslated region 5 'can be obtained from the promoter selected ptara e; to hold the gene, a promoter not re-acted, the gene sequence sequence or the region of od i f i > .to". i ó »_? to the ivsar, viral APNs, genes eucaí '?? t i eos hin do, > - > bi n a synthetic genetic sequence. The present invention is not limited to the constructs presented in the following examples. The end region or non-translated region 3 'that is used is a region > that causes the termination of the transcription and the addition of ol lyonucleótidot »pol iadem 1 to the 3 'trems of the sequence of transcribed APN, the termination region can be native to the promoter region, native to the gene It is possible to derive the other source, and preferably includes an "operation" and a terminator for pollination, such as "n" and "n". »Transposed 3 'dde ua -» 3 of the plant chimeric gene include, without limiting 3 it-s, (1) the regions not transla tted as 3' that. " They have the signal, in addition to the genes of the ulcers, which are used in the T? or? T?) e Agr -'- ibactep u, umm por-e. CG synthase "', and (2) ge * nes plant» :: o? t »?" »pr example protein genes of soybean 79. Gene?» selectable markers can be incorporated into cass of the present invention, and the use of p-ar-i spien * mnai the cellulose plants that have been formed. To press the resis in. »; -! -» to an anonymous,? »J > 7» »for example c * na ic ma, genta mi ma, T? IB, i gi-omi cin, it is re omicin, spectacle and not, tetracycline, > "loriferatur, &suri i 1 a > e • > . 0! The abovementioned methods could be used to »-temAs > e this? »or in a way l erna i a, co o for example a gene coding n * a la 2 ¿> Tolerance to herbicides such as tolerance to glyphine, its use? Reason, phosph i not r ic ina, or bromine: - my 1. Additional means »of selection could include the i i -te» - i a al etot r ¡? t > », At 1 cc» m ls comp i em ac i n providing frototropia to uxotrófico host, and -similar. For example, see Table 1 of PCT WO / 91 1072, mentioned above. The present invention also includes the replacement of all genes as viruses with a set of maternal genes are readable. The particular sea 'pl a or be-; »a ma rr-dor that will allow the selection > "Transformed cells as opposed to 1: it" cells that were not transfered according to the different hue-apied spectr e nr? Different selections would be useful in selecting the different hue-set, and would be well known to the experts in the field, a marker that could be improved, or bi-gen. reporter »-)" co ") for example the gene beta-gl».?> urorudasa »; > well the gene luer-asa pmede emplerse * - instead of a marker it will be» -_ » - One-to-one, together in the - Transformed cells: This one can be identifi ed by the production of a product. »Nn 5 -b» »? Mo-4- c 1 ar a- - ~ in» ioi 1 --beta-D-glncuroni or ¡X - 81 »» »-.). Fu development of the const of 1-present ei-pressure, the various components of the construct expressed by, for example, DNA sequences, eula ad Or, fragments of it will normally be inserted in a conventional cloning vector, such as by e plu p 1 -ic-im »t. or phage, which can be replicated in a host! bacterial, as an example E. Cul i. There are numerous vectors that have been described in the literature. After each cloning, the cloning vector can isolate -, e and undergo l = t additional manipulation, > -omr > po * example c ^ »ic. ion, insertion of new fractions: j ents, ligation, motion, reception, insertion, mu ne ne m n o r 11, addition of fragments of pol l ector, and sim iers for ptr > ?? p »- > »-c to a veer that meets a specific need». i f? > =? . In the case of the t r -insí or? »» A »- i ón to me by A» -ji - »-.» Bac teri um, the d-is d e; -. P. resónón will be a luido in a ctor, and flan u do by f ragmept »-» = • ciel p] the gone Ti u Pi de A roba ter ium, which represents the right edges and, »? Pc? > juamente i, .qu? e »* d'-? of DNA tr 'ufepdo by the p »l ^ s?» i »? Ti or Ri ÍT-DNA). Fst »: > the illegality of the DNA of the DNA that is present in the genomes of the host cell will also be included in the database. they facilitate the replication of the plasma in the cell of Agricola, in this way, in the cell of F, and in all of them. ions, DNAs are typically carried out in the cells of E. cli, and the final DNA that carries the expression cassette of the virus is displaced in the cells of Agrobacter i? m by ct transformation of DNA, conjugation and the like, these Agrobaterium cells contained a second plasmid, also derived from pldsmido-a Ti or Pi.This second plAs will remove all the vir genes required to transfer into DNA in the cell. Plant vectors Suitable plant transformation cloning vectors include those derived from a plated Ti of Agrobacterium tumefaciens, presented in general terms and n Gl ssmao et al., North American No. 5,258,300). Besides lo = > presented, for example, in Herrera-Estrella, Na tu re, 03.209 í 1 c? 83), (PCT Solved PCT WD / 91 l5, and US Pat. No. 4,940,838, requested a Se hi 1 eroort et al. Vain techniques are available for the introduction of genetic material into the plant cellular host or the t ran- »fc >; r < -f > . i ón d »to said cellular * vegetal host. However, the particular form of introduction -Je! Seeing the plant in the host is not a critical factor for the practice of the present invention, and any method that can provide an efficient way can be employed. A »JemAs of the information using .ec. tures de t cansí arma »: i óp de plants--« derivatives of pl as > rn »; i» > s that i u in tumors ti) or that induce roots < R? ) > At Agrobacterium, alternative methods could be used to insert the DNA constructs of the invention into plant cells. Such methods may include, for example, the use of a chemical, which increases direct DNA uptake (Pasrfc.w I. et al., EMBO J., 3.2717 (1984)), initiation injection Cr-osswa et al., M »D1. Gen. Bepet. , 202, 179 (1985)), elec troor-ac t ion (Fromm et al, P ** oc Na i, Acad S i USA, 82, 824 ("85)), well m? C» * oproye, ile--, of at speed (Mein et al., Nature, 32": *, 70 (1987)). I. = »choice» ie source »ie teiid '» of plant or branch of the rhine-to-thirteenth-century culi ivadas par-, t r an- = > fr »rm-n i n p > in »d A of 1 - > naturale a »ie the plant host of the ro t o 1 o of transformation i n. Fuent ?? te s' i 'or lime il include the cells, ivo cul in -uspen i n, pratopl ast years, segm nto-, - |? E leaf, stem segments, -j i'till' -'j,? »jleu, emhr?» - »ne-, hipor óti los, segment» »-» de lubricarulo, reuiuiie-, i'i yes e - ^ i »as, and si mi 1 ares. i 'rú ce nt re Dyeing μ.i "jener to r'se in and go in retaining apa c r i ad' er '' 3r pl SWTR NTE, ils de-spiué.- faith, ls ra n --fo mar i ón. l t ra ns f > go sc ón _ ** »e leads to» p ba o »-» _? nc¡? r? one? »l? r? »jid3-» ha », ta l eji or plant of .-. je». ». ion. L - elula-, »-» bi n »-.»] Pl rrr mruneri tissue to the DNA that arrives, '3 the ca: -set te rl »-» t-pressure of last yen present d * - n aide a perto »1» cash of time. This can be located within a pulse of electricity of less than one second in the case of electroporation, up to a counting of two to three days in the cells of Agr. = "•" - »i do-3. Regulators and staffs will vary according to. source of plant tissue and protorrolo »ie transformation. A lot. »Proto > - Transfers use an > ~ T ap-feed culture cells in suspension (eg taba "oo bi nl * Air Meixicano Black) - = '') br-and the floor area .., of me solid ios, separated by a d ? = ¡> . or filter paper s é il »ie l s cells» Je plant or h? »- n tissues that - =» e are t > - n-í • go to ndc », J3e :, ué-; "Tr t -jel my noc '?" "DNA, the plant cells or bi ji n t' J ''! And plant can be cultured for varying periods of time before l selection, to bierr can e.xti.jn Immediately refer to a selection agent, for example, as described above. Protocols that involve the e-xposic 1 ón a Agí oba > Also, a substance that inhibits the secretion of Agrobacterium cells. Compounds commonly m l eadc > = > they are antibiotics > . > . »M or» - inj) i .j cefot "i.-x-iua and" 1 heni c 111 ua I or "and" iio- > e '* r' pi ea "l"?. > b in the sele »1. jón pue» Jeu formulate = »for mautenei >" --ll »_) transformed» * »ijien > ~ > "- 1u 1 ^ 'n 1e snspeinión culture in a non diferenr 1' Jo or your n pai to allow produ» »:: 1 ng > of suckers par'tir 'the lime,. Leaf or stem components, tuber discs, and the like: Cells or cells that grow in the presence of normally inhibitory agents of the agents, the e> g-1 g = >They are transformed and can sub-several times more in the same medium to remove the non-resistant ions.The cells can be assayed either to determine the presence of a viral gene or they can be tested. to regeneration protocols and plant nodes. In principle, they involve the direct production of suckers, the suckers that appear in medium selective lime are construed and can shorten and anayze, and at the same time a sele »Ie >;) - for the production of rices, or bi n 'sim lm nt suniei-g i in the shoot in a compound that induces the root form and planting it di rec rm nt in vr ul ia In order to produce trapsgenic plants that show multiple viral resistance, the viral genes must be absorbed in the cells of the plant and integrated in an effective way within the plant. Cell tissues from plant selected by > A representative of an agent, and a group of people who have acquired the ma r > : a »_i» * > What is the reasonableness of the treatment? »This resistance to the treatment >;! and i. r-ansfcu mac i? n. Since the marker is commonly linked to viral genes, it can be considered that viral genes have been acquired in a similar way. The analysis of Southern tinsion hybridization using a specific probe for the viral genes can be used to confirm that the "- > They have been absorbed and integrated into the genome of the plant cell. This technique can also provide certain indications regarding the number > : ie copies of the gene that have been incorporated. A successful transcription of the gene involved in APN can be assayed in a similar manner using the analysis of Northern Tinsion hybridization of the total cellular APN and / or cellular RNA which has been inoculated in a poly region. ? 1 ada. The nMCA molecules encompassed within the scope of this invention are molecules that contain specific viral derivatives. of the viral genes p esen is in the vector * transformed »* they have the same p larity as the genes of the viral genomic APN» ie such that they can be coupled with bases specific viral APN of the opposite polarity polarity > iel RNA gen i i vi rail ba e »» oí id i > : cinemas described in Chapter 1, what 7 of Ritmb ot-et = »1. (198c?). Molecules of mRNAs also encompassed within the scope of the invention are molecules that contain specific viral derivatives derived from the genes present in the vector that has been determined to be stable. : He polar? Cl- < »J opposite the polarity of the APN genó» * n? c -'i ral in such a way that they can form bases of bases with viral genomic APN ba or conditions in Chapter 7 of S mbr > : * > > : *) et al. (1989). The presence of a viral gene can also be detected by assays. " .- "? , "For example," j? Jlu low eu-? Ayo- =? - »» i "n" h ci double antibody descripts by- Namba et al., Gene, 107, 181 (1991) as modified by Clart et al., T. Gen.

Vi rol., 34, 475 (1979). See also, Namba et al., Phytophat.il ogy, 82, 940 (1992). The resistance to the vi s can be tested by means of a study * of i n fec 1 v i > Jad is »as generally reported by Namba et al,, ibid. , d ». * > The plants are classified as mp toméctica s cu- > nd > . »Any ho a in ula presents clarification of, siut» more than m »» t «? > or else uecr ótn os.

S ent] e > - | e that the invention that GH 1 ii rse is voiced or transcribed as a specific viral APN of either antisense from the a.st ter »ari expression was described, F_ > to say - _ there is no specific molecular "ipi mc». ") -t 11 to the genotype ui-s t lcí and / or ai > je? ¡-? t, i po most rado s. Therefore, the protec '. I n- - against the viral retina, it may be a pair of lesqu i er of v po-s meca ni-s. It is possible that the virus can be purified by the use of any gene encoded visibly, therefore, by transgenic plants. <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> <tb> a transgenic plant of PPV Nia protease was found to be resistant to challenge with PPV (r Table 7, example 171), a transgenic plant that has a strain WMV-2 FL of protetna gene resistant to challenge coating with a heterologous strain of virus , WMV-2 NY (Tables 1-8, examples l-TV), and a tigers plant that has a re-stimulation protein gene CMV-C was a bit more resistant to challenge with ZYMV ( "piara informs": on uin lal, see the Application »of Patent? ropend? e lo--, assignees of loa Sol ic i t nt e N. ds -, er 1 e entitled "Transgeni Plants F-? H ib 11 ug Heter »» 3 ogous I saw the Prn ecti r »" (Pl ntxas Tr nsgétp ca presenting a Heterologous Vital Protection * • presented on December 12, 1 9-, incorporated to ».ju i por re f ren»;?) Seeds »He f» 3 plants regenerated from titul crops grown in the > - ampo and - au topiol inia? T pi ra Generate verd-nJeras plants »» He reproduction, I < * progeny of these plants became true reproductive lines that evaluate for resistance to what is happening in the csnipu within a r ngo e »oud ict ons environmentally - the value of the plant - 1 of the plant -» re ~ > i = 'ten tees 3 the viruses ea s-yor _s i much is L wibuiai ions > J if erents > -Je hibri-Jos with ivsctence are available for sale. typically grow more than one type > of hybrids based on such differences in maturity; resistance to disease and insects, heat and other agronomic traits.

Are 101, 1, and 1, and are not available to another party due to differences in such characteristics as they mature? , tolerance to disease and insects, or public demand for specific varieties in given geographical locations. Due to this, it is necessary to reproduce the i-nsi-alem. The virus in a large number of lines is so large that many combinations of hybrids can be produced. L - > The digestion of viral resistance to agronomically elite lines is achieved in a positive way. j n e when the genetic control of resillen- is understood. ? s Csto r wants to cross the plant- »resistant __. • sensitive and of the study i »? from the pattern of hpr ni t to flee to > ie segregate generates »-, i in s p ra df'ternunar if the theistic face s exp sa as' dominant or re 'is? va ?, the t» *' »e» > ) of the genes involved, as well as any possible in ect ion of the genes, require the use of one for the pressure. As for the typical plants of the type presented here, do they generate a c: o r t = m? nt >; »The nd of the genetic, dominant, genetic, can part, -e the e *? Perto: - > Initially, I will convert the lines to ronomically the other but still sensitive in the resten tive lines of the backward conversion process, it is brought to > ab > . *) by crossing the original resistant line with a sensitive line and the crossing of the origin to the relative - > on = »ble. The pu »> »Jen? s- a piai t i de eat »- > »- t u > - e - »e will segregate in such a way» - * that some plants carry the resistance genes or genes that others do not. The plants that carry the s-genes of 1-i resistance will cross another p.- with the sensitive relative, which results in progeny that shows the sensitivity and sensitivity. This is to be refuted until the original sensitive relative is involved in a second residence, and yet still possesses all of the important items at the end of the day. ad sn the sensitive lens pair. U th ogr ama sep a raa dos de r * .- t roe r-u: .am? In this case, it is important that the line is sensitive and that it becomes a virus resistant virus. After the retrocruiramiepto, the new resistant lines and 1 a-- > > -Beautifully appropriate lines that form good hybrids, and they are evaluated to determine their resistance. > viruses, as well as a set of important agronomic traits. 3 pro »ju in hybrios and l inn ,, t --.- 1 steptes that are fjel > to the type of the original lines and hybrids = > en- > tbl e- ,. The evolution of the evolution of a range of environmental conditions under which the line or the hybrids will be traded will be monitored. I -t = s l ír > For the production of hybrids that perform in a sati-factory manner, they are increased and used for the production of hybrids using standard production practices. The invention will be further described with reference to 3 following detailed examples. Axis pilpos I. Varieties of cal abara with resistance to virus ipiles. A. Viewers of Pl A m i two. binary DNA that was transferred in the genomes. The plant is contained in the bina fl ies. Bevan, Nucleu ris Pe,, 11, 7, 9, 9, 1 ^ B- ".).). The second parallel was PGA4R2, constructed by G. An, Plant Physiol. , 81, 8 /> (1986). This vector 'contains the T-DNA border sequences from pTiT37, the marker gene sle > - ci onab le Nos-NPT-7? (containing the? r »» engine of the nopal ina gene which can be fed into the plant fused with the PT 17 ba terian obtained from "Tp5 * -, a region of clpnapon m" '? l tiple, and The train of the phage lambda.f pl -> sm.do pPRBoriC? iM (figure 1) = - • = • den vó clel? l - '' = > ? »Io P8A482 of the following inane i: a bad eater b-t t.-rn.uo selecc ensble, ta re -.- 1 - =. t er > c a a a g nt - * m > »I u ^ ÍR, All insberger, e al. , Mole-- - "- * n" Rertet., 1 8, 514 .'19P5 * - * 'was inserted to »lyac nte s 1 hoi right (BR), fiero to the region of T-ADf-i . Fl -3 in Nos- NPT IT was dismissed: - »oi ado v the multiple cloning site (MCS) was regenerated adjacent to BR, just inside d» s > the T-DNA region. Next, a ge * r cassette? of beta-glucuronidase that can be e? p? »ibar plant (BUS) (R -A-- Tef fer on, et al., RIBO T., ¿, 3901 (1987)) was inserted into the T-DNA region adjacent to the pBP322 origin of rep lication. Finally, an NPT 71 igen that can be expressed in plant within the T-DNA region adjacent to the left bar (BL) was inserted. This NPT 17 gene was produced by inserting the NPT 77 coding region into the caste of the expression of the pl.-jx-.rn i or of F.. cal i pD1-1 1 (R. Lay t al., Nucí.

J ». This provided a signal d pol i =? Den? 1 ation of the 3 * 3g promoter of the.-Ol i fl mosaic virus (CaMV). Fl pl? »Sm? o pPRBN íf? ura ura 2 * s derived:: pPPBoriG as follows; The piPPBopGN region of the mico, the coding sequence of GUS up to Bl was removed, and by OII-J, the one or one of the RUS gen and the ass were removed. tte 35S NPT TT, F- »t the region was then replaced by a» f »ration» :-) consisting of the asset, t, e 358 / NPT TT only the net result of .. »-. ),., ^ -. s was the removal of l?.] «n R! 18 j a-i short region of ho-i-log? a.> p? BR322, d jan» J » "the ge»> of UFT II that can x resa sn rime adjacent to Bl .. F. Gen »don nte-, 1. Virus 2 of the mosaic of watermelon Uu gene WMV2 that can be expressed in plant was built by the use of specific oligonucleotide primers to generate a fragment consisting of the re in ci cod if?> ac ion ep Read the rev stimi »-» n I Wf1V2 t from strain WMV-2 FL and restriction sites of AatlI (5 ') and Bg 111 (3') flank This fragment was linked to pl) C19B2 digested by Aa 11 T E »» -> 1 T T, which is Pl & smi or pUC19 modified to contain the restriction enzyme Bg 17 T in its multiple cloning region. The resulting result, called p > UCWM2P25, it was mod > f i rado ad i > : i nally by the addition of the CaflV 358 promoter and the polynuclear signal obtained by par p > ! C1813 / CP19, (T.I. Slightom, Gene, 1O0, 2 1 Í19 <) t)) to produce a cassette »: Je protein > Je estient that can be emitted in the plant. the prole- Jna produced by the expression of this gene should be a fusion between the reverse side of WMV2 and 1 to extreme part of the NH3 of the HV rsvirus protein, Fs e ca s! Faith (PPW) was then cut off before ingestion with £ a > * r > HI and ligad »-) to the si of E * g 111 of pPRBor-iGN to produce the binary assignated pl pPRCPW (figure 3). 2. I saw you > Jel mp? Aicn yellow zucchini La ». Innation and characterization of the gene.-Je 1 a pi rot.-_ •! na »rle reve i ient» :) »le 7YMV from the >; -; ep? a 7YMV FL used here are:: ies > -pbe in H. Oue ada et al3 T "Gen. Vi rol. , 71, 1451 (1990). The strategy employed in the construction of a plant rotational gene for ZYMV that can be expressed in plants is described by -7. L. Slighton, (1991) above, and S. Namba et al., Phy topa tology, 82, 945 (1 9). 3. Cucumber Moss Virus The cloning, characterization and engineering of the CMV reversal protein gene used in our experiments are described in H. Quemada et al., J. Gen. Virol., 70, 1065 (1989) in the 1991 document cited above. 4. Pumpkin mosquito virus G0MV is a seed-borne virus that is targeted by beetles, Jel cucumber, or mottled (Acalym a spp. And Diabrotica spp.). The insect acquires the virus within 5 minutes and the virus is conserved for up to 20 days. The range of guests is limited to cucurbi spp. This virus consists of isometric particles of 3 n diameter which contain RNA > The single strand is divided into two functional parts called M-RNA and B-APN (Provv i cieut i, in Plant Viruses of the Crops or the Tropics and Bubtropics, Taiman, Rep. E China (198o)). pages 20-36). Fl isolation, sequence determination < of DNA, mod i f ica »: - i ón y- expression of these genes in plant cells is described in Hu et al., Arch. Vi rol., 130, 17 (1993). In summary, after isolation and sequencing, the genes were manipulated genetically in the cassette of the plant pressure, piJClG ».- pe: < .p} ress, according to 8i? ghtom, Gene, 100, 251 (1991). The use of this methodology and the expression cassette produced SQMV coating protein clones attached in frames to the untranslated leader 5 'of the cucumber mosaic virus. The mergers are driven by the promoter 358 and the use of the terminator 358 (Figure 4). The modified genes were then isolated after digestion by HindIII in a single step, introduced in the f11 asmid ». *? nap john river pGA482GG (Figure 11. This - plasmid is a derivative of pGA482 (An, Methods m Fnzymol., 153, 292 (1987)) .The two coating protein cassettes are oriented in the same "direction". that the gene of NPTII. ls genes are present as single copies. "Spot virus" "the papaya ring A PPV gene that can e., resarse in pilanta was isolated by polymerase chain reaction. For more information, please refer to the Application for Co-pending Claims of the Applicants before Serial No. ......... entitled "Papayí P? ng- »pot Vi rus Coat Protein Gene" (Virus Coating Fringe Protein Generator »from the Ring Spot> of the Papaya) filed on December 30, 1994, which is incorporated herein by reference. of the sequence, the gene was genetically manipulated in the, - .ia .- > tt >? of e-pi ' €? .ion f plant pLJC18..pe, ie i; se »jún Slightom, ibid. H991). 6. Multiple coat protein constructions Expression cassettes ».ie multiple coat proteins were placed together in various combinations to obtain binary plasmids, in several combined piara ions obtain pl if» 3S binary capiaces to transfer more than one • expression gene »of plant in geoma of plant. ía) 7YMV72 WMBN-22 ZYMV72 WMBN22 is derived from the binary pásmidc pFBBN, in which cassette »: ie expression fiara the piroteíns» of coating ZYMMV and WMV2 have been inserted. The cassettes of «.pressure were inserted sequen nc i =? 1 mind on the site > restriction »: le B»; »1? T single»: ie pPRBN. In order to achieve this, it was int edujo n - \ -3? T? »:? Ba HI 5 'to the promoter 358, a site ».» Bgll? It was introduced 3 'to the oenc i a »of a» diction poli A of the a t ci ei-pressure > of WMV-2 and 7YMV. The sites Ba HT and BglTT were introduced by the >I do not know how to use it - appropriate for ol i gonu > ~ lécot i > d ':? s during a pil i f i cation in the chain to join the l »-» as ett ^, i s »product of the reaction in scienas» of polymer * were »i i > jep »: i»:? s with Ba Hl and B »3lII to produce the appropriate ends. The WMV-2 cassette carrying the BamHI BglII ends was inserted into the unique RamHI BglII termination site to provide 7YMV72 / UMBN22 igfm to 5). Fl. i ?? tc -, e ll-cr.a? W. 5 ib) CMV73 ZYMV72 WMBN22 The coating protein expression cassette CMV-c was inserted into the unique HindT? I site of 7YMV72 WMBN22 to provide CMV73 / 7YMV72 / WMBN22 (FIG. 5). This cassette ter Lar i o - ^ > e call C7W. 10 (c) CMV-WL 41 / ZYMV72 WMBN22 (C-WLZW) L cassettes »-; The solution for CMV leaflet strain (WL), ZYMV, and protein genes "" ie the WMV2 strain was inserted into the l * - »s?» 1st biuapu pPRB par-j to obtain CMV-- Wt 41 / ZYMV72 / WMV2 (C-W17W) .For msfald e, ta c »-» mb i na »- i ón ele electronic cassettes ». > t of r-evest i my ent o .je virus in pPPBN,, and inserted a • 5 set of ^, - presyón of prototype gene coating of white leaf ceps- CMV in 7YMV72 WMVN22 (Verdar was going to the »cu> g> truc> ton ci ZYMV72 / WMBN2). For »Rinsf i-ui r the dr» = »et ie d ¡, n jón lie protein rie "- reverse CMV-Wl, Nsmba et al., Grane, 17, 181 (1991) inserted the CMV-Wl. nc? e -press reverse protein coding ion. A Hiudlll fragment > ^? contains »al t a sset»? dea x ression > of CMV-Wl. was inserted into the HmdlTT site »1e 7YMV72 WMBN22 to get 5 CMV-Wl 41 / 7YMV72 WMB 22 íf igura - * > , This pl ^ s i.to tunan »:» was ll mad »or CWLZW. (d) WM310 / ZYMV47 482G A fragment HindlII q ie has the expression cassette of the ii 'iteina ele? evest? o n »-)» le 7YL1V si ib * »ler. ,, ',, -. was installed on the single site H nd? IT of pGA482G trust to obtain ZYMV47 / 482G. Then, a BamHT fragment having the WMV2 cassette described above was ipser-side in the only Bgl II of 7YMV47 / 482G to obtain WMV310-'482G. Fste C'ius' riicti ") -» e »»? Nc "» - ». or W7. íe * > PP *. | .wm1? G / W7WI 41 / 7Y "? 2 WMBN22 Cassettes of e? pre -»? on for the PPV-FL strain (for information about -irrigation and referral to the R l "? D of P ten" -. >? »- nd ine of 1 os Ce-, tona ri ei-s > of the Gol? Ci ta rd ^ N» n. De Ser? R .. ....., t »spin" Fi si R t Virus C ».» a!, Proteiu Ge »? e" (Gen > .ie Protegí na na Rev -. »l imi to d V¡» -us de la Mancha »de An? ll» D de la P p ^ ii e- = > en > the »dfa 30» ie r?? e ».- I have 1Q94, which is incorporated here?» p_ refer-en »ia), strain and white sheet of CMV, genes of p * rol» - *! na of reve = »t?» ni ent of 7YMV and WMV-2 were inserted in the pl < sm? > ! »: Binary pPPBN p» - ^ get PPVcpu? M4óS CWl 41 / 7Y7 WNBN22 (f igur'a 9) »oí» - ** t ru »_ 1 or was de i nado PC7W.) PNIa22 / CWl 1 7Y72 WMBN22 Itib ca r »-:> et tes de e¡, resi to the gene PíJTa de] J, pa PRV -P (leg? Nfor» -rta »additional information refer i 1? Sol ici ud of Patent copen »! i enl e of the Transferors of the Implementing Agencies No.

Series titled "Papaya Rmgspot Virus Co t P ro te in Gene "(Gene of the Pr»; Virus Theory of the Ring Spot of 1 =? Papaya) set on December 30, 1994, and which - = »and incorpor q > < í by rferen », ia ** and lo '» cassettes ».1e ge &je the coating protein for the cep > CMV-Wl. ZYMV and WMV-2 were grafted onto the binary plPidoBPPBN to obtain PNTa22 / CWL 41 / ZY72 / WMBN22 (Figure 10). This l uustrur.to is named PNIa C7W. íg) S021.'S042 / WMBN22 7y72 / PRVcp? ifmló, s CWL 41 e-.Prescription cassettes fi xed the piar-to-y proteins y rev ii nto of WMV-2, 7YMV, PRV-Fl and strain CHV-W1. and the protein species for the SqMV coating and inserts for the binary 10 pGA4R2GG (FIG. 12). Fste ronsi? U »I > . »» E »I have the name SWZPC. C. Tr ansf or'iiia »? Au de? After the removal of the seeds, the seeds, the seeds were sicily removed for 20-25 minutes in one solution. ? of h? p »-» - l »?? i) - »j of so» * i? > . "> (Cl» > r »"),) which contained tt-ie in 20 (200 ul l 0 mis). The disinfection was uyunli by three rinses in 10 ml of water and rinse. 1 as semi l la - »ger marorr in culture tubes! R-.0 25 mm qu» -. contained 2 l- = »e a me» u or »jan?» - or me nor im (MS) > Je Mura without ge and Si oog -i una fuer, - a 1/4 -jcil idif i »Sito i on 0.8 / i de Agar Di ugly Bact o. Dismissal of the trees and the tips of the shoots were cut and transferred in containers GA7 (Magenta C). > rp.) containing 75 mis of MS medium sun with 1.5 of Di fo Bac to Agar. Unless indicated to be 'cintrated', all 1 '' = crops were incubated in a growth stage at a temperature of "C with a photoperiod of 1 hour of lu: The light was supplied with cold-light fluorescent lamps (Phil lp» F40CW) and lamps. General Flecf ri »: F4 - PF). Pe'la, 's leaf (0.5 cm) were the second to part of plant 3 in vit.royma ado in broth culture A 'i rubri read in tum fae? N = »(OD 6 0 .1 - <; ", *" ** > and!, ».in fer i 'is =, 100' 0 IIH? I ready to go? - C '¡t» JU í - Ad tt ~? l - »of a measure» MS '' imfil n a »i > > ? n 1.2 > j''l i i n i ü > - i or 2,4,5-t i'i '-.1 oi-of in > »? J o. the, i > »» (2,4,5-T) and O,.? »? _» L? tr »? ».ie * > . t ben »** i 1 a?» ti no (RAP) (MS-T)? H 200 ¡JM AS. The plates are attached to "^" ^. Desfiues of two s \ r:, »J í = f =. ,! »> -, p > .-. ia..v > "? = -» J -.- hoj-a were t.annfei two in a year MS-I that has nf. To 500 mg -'l 11? *? I of c = rben i &- 11 i na, 2Í "* O mg / li I r"? »Je. efota-i ua and 15 mg / l i tro = ,. > 3 to e c an-urp ». ina (MS-IA). After dld (- * • = >, I-, fled- were f, ransf er &das - ni-] me io io MS-IA fresh, D? - 'S i> e, el t jí. ! »» Was transferred to? »N ^ lio MTS-TA ft-f-ac aa trea". S m-; .- > -? S. D »? ~.? >»? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .- = > the »:: auam? > _ _ i cia rju" was l.rarnfei 'to redi lio-a tube »that» ontetrían me »». > minimum organic MS omplemented liquid with 500 g / liters of wood and 150 mg / liter of sulphate of canami and 1.03 mg / l of CAC12.2H20.

C -ai-r ».h? Td» J x L i '- * 1 f1- > f > - 1 l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l »1 lll» I »1 - 'MS that had 20 mg of AgN0. The embryos in germinae ion were submerged 111 in milling until they were obtained or are rooted. L - = »s plantita-» were transferred to the soil piara the firoduc ».- ón dej seed Rl. D. Au iste plant The most resistant to the canami were analogous to the determination of the MPT II gene using FL TSA. :! »-» a cou.iun tei of elements p ra llt-var out l anal i-iis Fl ISA rne rial tríente di sponb le (5 '-, 3'-, B uldej-- rn; , Rea > laugh-, n "~ a" -ther? A? Il? Ol? Imer-isa?? Piple? 1 * »if neither appropriate takers were ealiz os to amplify the npt NPT TT (adjacent to on the right edge, and the protein of the vestigatio n over the year to the point of departure. There is no doubt that the analysis of the Southern Timer, iae: pire-si ón> iel> jn > le pyr-oteina »ie viral coating on plant-, putatively I r-j ?? - »f? > rm adas was detected by FI.TSA u-j-ando antf. uerpo-j > played with phosphatab alcali r »in accordance The pr? _? T? C.? L »J of M.F.Cl-ll'k -i l. , J. Red »Vtr.jl,, ZA, 47"" M977). Anti-sera for CMV-C, WMV-2-MY, and 7YMV-R, are available by D. oncal ves (Cimel 1 llniversi ty, Geneva, Nem Yarl-). The appearance or absence of the T-DNA in Pl and subsequent generations was determined by the tests > -Je The TSA iri i a > -l ge ,, mat 'a »l» »? I-PT II -nr-l. > . ? ou = »bie. - .. used chain reaction »Je pol i mer-asa or analál i Southern Pia followed the hei gum on line ZW20 whose advanced generations did not have the NPT gene TI, D. P roced i rn i t i ng c o nc y The seg e ation > from l to p »'». > geu? a Rl ».? well P2 together with the lines = »of contr-cjl a rop i -» rl - = * were ger- ned in the vernadero, before the inoculation, the users of coti ledons were I H-ciy i da .-, for tests. Fl 1 SA d »- NPT II. Cpt i 1 edones t-spol JLH eadu-, > ou carborundo were? noc: ulados me »atrit amente c-n? J =? f > i i t.as de seí a tía5 with a sejlm. ion 1/10 weight / volume CMV strain C, 7YMV strain FL, or WMV - 2 »-epa NY ('available in D. UIHÍ I V-Ü, C me 11 Un i ver--, ify 5, which were pr oduced err Co.-umi a sati us, Cc >. < nb it, to pepo and Pha seo 1 vulgar? s re-ape »-ti men» e. ls? laul a 3 fuer n inoc ula os with V¡ \ gt; .-- Í »in the inverse api'o: i Alarically 7-tO days ago, the elane »: n" nla »ion, the plants were 1 ran-jpl - »litadas in the field. Fn to I »**} a »: > ** -. essays plants of i .iiit i 'il uo i U'ic: -cla s f? »er» »r» intúluid?:. Do you have to fight to fight against the PI? by ^ f i > -j »> -s. Data on the "a to r- I ~ J 13 o s > nt >") ma t ¡c o were • ¡. Umulated before reviewing '»- > - > resellers of the ISA of NPT II, in such a way that the qualification was made without knowledge of the statistic.; > transgenic of the individual segregant that was being evaluated. The plants were able to reduce the disease from 0 to 9 on the basis of the symptoms of the foliage (0 - non-infectious, 3 = symptom-: »ho ho inoculated and / or very mild symptoms in shoots , 5 = disseminate moderate systemic ion 7 - systemic di ina ination, 9 - sεtem dissemination: to atrophy). Fruits were also rated according to the severity of l > -_? s symptom: - »(0 - n» 3 symptomatic, Z = man »has green li of the fruit, 5 - of» moderate coloring 7 - fade »" go to »ri ón -evera , ^ - discoloration and di splay of the fruit.) The line receives a one-to-one episode of disease for fruit and fruit that was an average Pileas i ividual s F. Di s rie) cié the plot of ensa o de po Field essays:: »e real i carón with permission:» granted by Apimil and Plant Heal h I -.pect icid Ser ir-e (APHIS) of the Departament Neirteamwr i »- year» Je Agriculture (IJSDA). It was used for every year that it consisted of a tra nsm line. was coupled with one of your lines or contained your i out rapa r I in »-», arr-sgém »-» »- >; - > mo »ontiol. Each row 'insisted on 1 storeys, a se- en enta and an entímetr s and ejisxt ne la, with 152 entimet ros' .ie distsru. i e eutie la-i rows. Be a. Orpo aron two to three rep 1 i cations of each transgenic line in cacta pr-test. The plots were surrounded by an edge zone of a minimum of nine meters of non-transgenic calábalas plants to reduce the flow of pollen 1 an g > "; n? c» i - "» .''.- * ra del? i t o. ' > I was going to monitor viral dispersion in the field. The transgenic material incorporated in the test included progenin Rl and P2 from endochrananic lines of cal aba *? the twisted yellow of: .utopol i nac i ón or of ret rocruzami ento. In some cases, an endogenous tragic line was eroded with the endovenous line. 11 a n-i-jéni ca apiropí ada a »-produced ir l s' ion s t i 3 a n i c y of the hybrid comers. i to 1 is the pumpkin, Pa or D I; x le. F. Pesul ta ».i» DS The line 'ZW20 was de facto to start off' -je a plant Po transformed with 7YMV72 / W N22. It observes real ions,: ada'cku before the tests of 1 mpe * »1 and 2 in the?» -) b! a * i ón R1 revealed that all tires containing the inse or MPT II remained in the 7YMV and WMV-2 during the course of the event. lliwj 7W19 was also from - »ar» ol lada starting from a plant Ru q >? e was I r ~ us fot mad-a in :: YM .'72 / Wp 2"In Clin! with 1 i l ine 7W-20, 7W-19 prcipnrcionado a reduction d 3 »desa rtol 1 o de 1 > > s symptoms cu < .ndp f?.? and inoculated with either ZYMV or MV-2 (Tabbla 1). TABLE 1 Develop 1 or symptoms in squash lines of t-etor neck »gone anta i. lia eudogs-mi'-? > i -iii éni 's i'íC,) 4 < After the inoculation with a 1/10 dilution weight / volume of strain ZYMV-FL or strain WMV-2 NY carried out during our 1991 and 1992 trials. (* /.) Plant symptomatic) NPT II line 1991 trial 19 < *? 2 trial 1 92 trial WMV - 2 im? > -? ZYMV ZW-19 + - - 1 '14 (1O0) 11/11 »Í10O) 1¿. / 1¿, (100) 19/19 (100» 7 -. 7 -20 + 0/1 () 0 '' (0) 18 (28) 1 -'25 (40) mild Cort str »_ cough CW Pumpkin plants that had the vei-torev» e; pressure. rt yy of coating protein - - e e CMV-C and WMV-2 f uet ou inoculated already s to f ur. MMV strains V27, V 3, or bi n V7 »'? For additional infnrnination, as the Sun i itud efe Patente Conend i erd.e» _Je the Cesi ps ri ».» S de l'h Solí »I tantes -to. of Se íe "..... itulada" P 1 a n t =. Pe s i a n t! »_ > V ' \ > ZZ or V Btraius c > f Cucumber Mosaic Virus "(plants resistant to strains V27, V33 or V34 of the Cucumber Mosaic Virus) filed on December 30, 1994, which is incorporated here by reference), which can infect plants 11 -an gene-s that e, p »p-? 11 the coatings of CMV-C, a majority of the transgenic plants but resistant to challenges with these strains of heterologous CMV, unlike the transgenic lines that have a CMV reverse protein. C. Only the remaining plants that were infected showed very reduced symptoms in comparison with trapsgenic plants with a CMV-C s-wing coating. the line C? U ~ ~ 3 3 from-> a plant Po transformed.-on CMV73 / 7YMMV72 / WMBN22.Prmaneci ttoma en esayos e ampio »-.of» infection of CMV-C, 7YMMV- F! Or WMV-2, which applies each mo »ul» in in individual or bi n in a set containing the following: ig nt s viral (Table 2) "Line CZW-40 was .jpsa r-ro 1 ada from a Po plant transformed with HWl 41. / 7l 1V-72 / WMBUr422. He did not provide any anti-infective pt s when inoculated with CMV-C, 7 MV-FL or lne-n k'MV- ~ * - NY (T- <bl 2). TABLE 2 Development > ie symptom in plants of praise * a after inoculation with urta »i? 1/10 weight / volume er e CMV-C, ZYMV-FL or WMV- - Y ca lining of disease i n ntx'í 1 c a f »il 1 a je i" i'i 1 a Line P to V ratio, CZW-3 CMV-C 1/13 0.7 0.4 0.0 6/6 1 0 8.5 - CZW- CMV-C 4/4 100 5.0 - 4¡ "<" 11/11 1 0 9.0 - 0 C7W- ZYMV- 0/1 0 .0. FL 4/4 1 0 8, 7.0 C7W- Z ZYYMMVV-- QQ ,, '' JJ 1 1 (10 ° .o 40 F FLL 5 5 // 55 1 1 00 7.0 C7W- + WW? M1VV ~ -22-- 0 0 '' 4400 0 0. O (i, í) NY 1 r / 15 1 3,0 C7 .L-1- W WMMVV - 2; 1 111 '' 1i1l 1 10000"/.or 40 N NYY 3/3 1 1000 7.0 * Screening in ínvei'nadei or ** < ) The fruit and the folie- e p »'» set up ratings of illness and O, En »r > »Nt r aste, the 1 14» ie l »_- > s segregants protetna de r ves i i n or roll out symptoms sever »> -, viral infest both in its foll e »-like in the fmt-i, la-, - • > classifying ions of enf r age piara 1 > : > The segregating protein of reversal protein was between 7.0 and 9.0 (Table 3). This trial showed that by placing the reverse protein genes in combination, one can get r sistent id cuntía in e »heardm. s'mulf '«nea =. p? »? v i? -u -_, different, both in hybrid lines and endcigaßncas. TABLE 3 Results of field trials Development of symptoms in pumpkin plants 55-days later of inoculation if late with a mixture of 1/10 weight / volume of CMV-C, ZYMV-Fl and WMV-2 NY.

CF line r. i ni'om * 1 c o alification of illness 11 a c i or 11 foll J1 * -1 f u a Pi ¡x l CZW- »» '> . / 0. 0. 0 1 8.4 7. Turkey CZW 0/27 0 .0 0 »t ~ -r /" '100 7.0 JOCZW- Í "Í / 4Í") 0 { "• .0 0.0 1 ^ 15 100 7. ? 3.0 P i ¡i e 1/14 100 8. ' '0 P o '-o 4í /.' • 2- > 4 100 8.9 3.0 For the third test, a P2 generation of C7W-3 was produced by the replication of a CZW-3 segregant of Rl positive for coat protein. Two hybrid transgenic lines, equivalent to the uniifiable hybrids. Pa or D '> > - * f i > > -. i ..i .111, i. »»] »I. ota-. using this line in »iogami»: a transgenics or one of the parents. The progeny of the inbred line showed the rela- tion of spatial segregation (in t &g and NPT II ELISA) of 3: 1 for the inserted gene, while both hybrid lines present a relationship expected 1: 1. The inbred progeny and hybrid tr ans. ru c a fuer inoculated with a me ... 'cl de i no', u 1 »-» ud a dilution l / l p »- -, o / volumenlele 1».) -. is u? u «, CMV-C, WMV - 2-NY and 7YMV-F1. l Table 3 shows that they were completely resistant to infection by three viruses. The results presented here confirm that the "jenes" of the ?? > »Reversing theory propitiates a viral resistance when they are grafted on» -or »» b i nac t ion. Far eiempJn, the line i < an-g n? or to CZW- remained ¿tomatico en tod »_). ** > the tests with the CMV res virus, 7YMV, WMV-2l < in drinde they were moated with each virus in an indefinite manner. They remained intact when they were inoculated > .-on l r »three vi r LIS simul anly, the e ap e aciency and get-lines rjne? est, have a peep infection »- JI? I, multiple-,» J ~; esenc ral piara the »Je-.at c» 'il the ultivo »de cal aba.- a c tome rc. The first is that, under commercial conditions, it is common to find infection with more than one virus during a growing season. In these assays, it was also observed that when multiple peptide genes are inserted in a single construct, tod-DS the > jenes in the cortstructo proporcan ourtan simile levels are-a »of effectiveness. The CZW-3 line that provides a high degree of resistance to CMV-C provides: it also has a high level of resistance to 7YMV-FL and WMV-2-MY. Fn contrast, transgenic lines for example ZW-l1 ?, > that (they showed only moderate resistance (that is, a development> fears the "slight" symptoms) for WMV-pt eserd-or also a resistance only moderate to ZYMV. Greenhouse gases in the ttansgérin line to C7W-40 'demo that "this line, which did not offer re - = -»? - > te? v_ iaa CMV-C, no pr-opoi »? nor does it refer to ZYMV-FL and WMV-2-NY. The level of action between genes in a multiple genetic code may reflect the effect of the action. »^ The location within the yeuom of the plant in which the lines are inserted. In any way, the phenomenon provides a method for greatly increasing the probability of encountering lines of transmission. a = »md ii dnß 1 &» r »> ou alt s mueles > resisting resistance against multiple times - E TEMPLE II: INTRODUCTION OF CASSETTES DE GEN DF PROTEINA DF MULTIPLF COATING IN MFl ON 1. Transform i melon ions Melon endo lines were transformed with the multiple gene harvests listed above using a miiiJif n a > . i ón del p > • r ed ino in your Fan-j and ~ ~ j- i.mt-d Mole,. Plant 5 Microbe Interact íons, 6, 358 (1 93). Ingrained transformed plants were transferred to the greenhouse and Pl was produced. Plant analysis / inoculation procedure. Transgenic plants were analyzed and inoculated in accordance with what was described in the above mentioned. 2. Resul ta ».3» js Constructs 7W. line CA7Ó-- 7W- 102-2"pu-opor-». i ort resi tence to the fee: »- 1 ort both 7th and YNV-FI as per WMV-2-NY.

In cu all the other .- > 1 Line-a > , u pt on .. »» - »- loriaron 5 resistance contri infection either by 7YMN or WMV- (Table 4). TAP l A - »xi r role 1 or symptoms ert p > lar »tat? of melon t r-tnsgém cas after »ie the inoculation with a 1/10 weight / volume dilution? of ZYMV-Fl or WMV-2 - NY Si ntomS t í'-o ca 11 f icac i o n t i n t NPT TI Peto relac io n "of illness CA-7W-102-29 + 7YMV-FL 0/30 0 0.0 r? + WMV-2-NY 0/9 or 0.0 - 2/2 100 5.0 CA-ZW-11.5-38 + ZYMV-FL 0/30 0 0.0 to V - ^ - 9/17 > "O - 6 / P, 75 4.0 PCZW Constructure. The CA95 PX7W-1 line provided resistance to CMV-C, ZYMV-FI infection. and WMV-2. The traditional resistance. PPV in such a way that- »13 ef i ca > "" ia > of the insert »Je PRV no pu» lo > I mean m. Fu contrast, triastic lines e- P07W unm > e »-c? sa -» no propeire ionaron res i - »t encí a. CMV- or 7YMV -Fl. I to inoculation e -.-. t - * - l íue-t, - , ... ... n WMV- "- Y .-. Igue in progress (Table 5), Df-'SAPPOLi p DF SYMPTOMS FN t TNF AS DF Fi 0N TPANS0ENTCAS AFTER DF 1 TN0CM1 ATION WITH I INA DILUTION I / I PFS0 / V MJMFN OF 7YMV-FI, WMV-2- Y, PTF CMV-C LINE NPT II Challenge relay% C A95 - PC 7 W- 93351- 1 + CMV-C 1/12 (OR) 3/3 (1 0) + WMV-2- 1/9 f 1 * < NY 2/2 í0OO) - 6/6 (1 0) C795 ~ PCZW-9335¿, ~ 1 CMV-C 9/9 (10) 4/4 (100) YMV 10/1 O f 100) 5/5 ( 1 oo) CA95-PC7W-93356-6 CMV-C 9/9 (1 O) 1/1 (100) +: YMV 10/1 (i oo> 1 / l (100) L f i? Ea ca 1 i f i c ac i ón > ie eufermí? d3 CAC -FC: w- 0, 6 6.Z .1 .0 4.2 9.0 C795-PCZW -93356-1 7.0 7.0 7.0 CA9 * - "» "FC7 l-9335 .'-, -, 7. 9.0 7.0, or Constructed SWZPC. Even if these lines have not been validated to end their residence, the analysis > of the pul imei-asa chain reaction showed that 27/36 (75%) of the melon lines produced with this construct contained IC-JS sei gencas »Je protein 'coating plus the selectable marker gene 1 e NPT II. This showed that 1 pior mediated transfiguration: > > j steal ten um can i-mip 3 ea? -s > -e to transfer to inenn-j seven (fiero prob-ibl men many more) genes enl a.; - acts in urt p > l? s? tt? >; l > > binary IO to cells of plants with recuperation, ion. "plant sequence" intact that ciiitt leu rt tos "seven inserted".) - > * you in r »canned. FTFMPI O Til. INTRODUCTION DF PROTEIN GENE CASSETTES DF PEVFSTTMTFNTO MUL TPIRS FN CUCUMBER 1. TRANSFORMATION OF CUCUMBER Cucumber - »were tr •) n -, f orinados» c »" go the cori-itinctus e neu "1e prnteiuc» de revesti my multi et al) stacks • above, using an in difh ation of the p? ct'di? n? enl.o de Sarmentado et al., Plsnt Cell Ti sue and Organ Culture, 31, 105 (1992). were transferred to the greenhouse = produce nls - ^ - miles Rl, Plants t ran:> jén 11 as was »rou ßu-tl iisilan ej nocu! xa s of onforinidad on the description u? -l example I above 2. RFC! ÜTADOS CONSTPUCTOS C7W GA715 C7W lines 7,95,33,99 are resistant to both ZYMV-FL and WMV-2-NY (these lines have been traditionally reproduced for resistance against CMV-O, in such a way that it was not possible to determine the effectiveness of the tiiaT-rto of p? i »jt» e í na ».». »-» -. 'e: - 11 mieut »j CMV (Tahli o *. TAPIA 6 DFSARROTLO DF SYMPTOMS EH LINEAS DE PEPINO TRANSGE NICAS AFTER INOCULATION WITH A DILUTION 1/10 WEIGHT / VOLUME OF 7YMV-FL, ZYMV-CA, 0 WMV-2-NV Without t inático line NPT TI Rete »ratio V, GA715 C7W - 7 -i 7YMV-FI / E) "? T i (-.O + WMV-2-NY 0 0 / / 5 or R, 'Q CA71' -0ZW-33 to 7YMV-FI,, ' A 9/9 1 < "or WMV - ^ - NY 0 • '6 0. tnl 00 R? 71 -CZW-9C > a- 7YMV-FI. { < "'.. • \ i X i" - 2 / -'22 1 0- • + WMV-7- AND O / I -' 1 0 RA71 -C7W-99 + 7YMV-F / S O 6/6? r + WMV - ^ - NY 0/7 .0 100 l -, Hf (f »»? tiie »1 GA71F» CZW-7 0.0 6.4 0.0 4.4 CA715-C7W- 0.0 6.9 0"0 GA71, r.-CZW-95 or "or 5. O. O 5.0 GA715-CZW-99. or 6.7 O. O, or CONSTPUCTO PNl to CZW. The line 0A71 PNTa C? W-21 was resistant to rr-IV-C, ZYMV-FL PRV-P-HA while the line GA715 PNI CZU- ~ 1r-. e s »> scep t i ble a • "" YMV-Fl and WMV - ^ - NY (Table 7).

TABLE 7 DEVELOPMENT OF SYMPTOMS IN LINES OF TANSGENIC CUCUMBER DFSPUES OF INOCULATION WITH A DILUTION I / IO WEIGHT / VOLUME DF 7YriV-Fl, WMV-2-NY, OR PPV-P-HA Si ntomático 1. TNA NPT II P o? * »- 'the»: i on GA715 PNIaC7.W-21 4- 7 YMV- 0/7 0 - Fl 100 + CMV.' A o - G rrir- - 5 4/11 A A- H- WMV - 2 NT NT - NY NT NT + PRV - p 0/3 - HA 6/6 i o or i CYMV-. 100 - p 12/1 1 or + WMV --- * - 4/4 1 oo - NY 10/1 1 and PR '-F- NT NT _ NT NT -? - CMV-C NT * NT - Ca ii. -go. NT NT. -_. . l í n.-s-1 ifn * i on l -i in f r a lle to GA715 PNIaCZW-21 O. O 7.0 .0 NT NT 0.0 3.0 6A715 PNIa C7W-15 3.0 10. 3. 3. O NT NT 1 NT NT EIEMPLO IV. INTRODUCTION OF CASSETTFS DE PROTFTNA GENE ? 0 REVESTI TION MUÍ TTPl E IN SANDIA 1. TRANSFORMATION OF SANDIA Ranilla -.- »:: > n c sset tes, ele gen e protein de r v < = »S t uu i -nto úl ti le-", W7 l a ss aba va, using an iiodi f? "" Ac t ion of the proceeding to this - * 5 by Cbeii t al., Plant Cell Peports, 344 Í i Q * < c 2. ANALYSIS OF PL ANTAIN / PROCEDURE FOR INOCULATION Transgenic plants were ani lized and inoculated according to that described in the example above, 3. PESl.it TADO.S CONSTPUCTO WZ. The WA3W7-20-14 lines were reviewed by ZYMV-FL and WMV-2-NY (table 8). TAPI R DFSARROUO OF SYMPTOMS FN I IAS OF SANDIA TRANSGFNTCAS AFTER INOCUTATION WITH A DTUJCTON I -'I PESO / VOLUME DG z YMV-FL or PIGN UMV-2-NY or I n Oitt ?. ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡! ! . { , "- e 11 i f i i a i i 11 i l ent entied d WM» / '-. or, , > (I * I? Pn li > - 3c ions, pt enf I '? &Tt.? S. Pa. Terite .-. And here incorporated p > »: &rt; r reference, > If they were to be incorporated by reference, the invention has been described with reference to modalities and techniques -. > - > i í f I '-, p 1 &I »'| ti -t - •» SI, - »ltb a > J > > ei! -I JO e. I! II .ij? - ?, iii, o it can lead to many variations and modi fi cations within the "spirit vl" - m >of the utmost intention.

Claims (1)

CLAIMS 1. A recombinant DNA molecule that comprises: a plur ality of DNA sequences, each one of 13 s of which 1 is a pyro »» l '> r -anla 'Hi'i de m t re i »jpe? able. on im t DNA sequence that t ocl 1 f n to a viral role, »: J» .- > f f of bliss- ifrijiant those of DNA ~ se n • elnlas de plant *, su .-. »ceμ! ibs to the irus r nsforma »the can tiicba-. ole »ui ^ s of? DN ree: o? To start with, you can use the 1 rife »» i ón > ie c! 1 »lias c elula-, par -í \ '? > ~? ie or hur »i rus. 2. I -. < m ».» l écul a »of quintér DNA 1 -1 > him the > and i v 1 u i 1 »a» 1 o 1, liiind la :. _,"-•" ".?in the . D - • - > nl -i, ~ \ n in tAnd '? lt, 3. I -1 »r» ») DNA label uu onéi J. -,» | e? -f) U f' "&rt; 'rii 1 da »J e? I | i 1 a? Ei? I ud t ca > i ón 1,» .l ») u» i> - »the ep» -e-jt ón d- 'd icha ae »»? ru ».? a do DN i opit» Jtina m, '»sl»' -? - -t dit »• to the inut i gua 1 e-, of tr • -ii -xte» ». ia >: »" »Nf» a > 111 f e- > > .. i or n, 4, 1 -i! i »'il. * a > ula »faith DNA > r 'i ^ ci' "of l n- i nrli,» in 1, tltvit'd »> 1 < J e¡¡¡¡¡¡¡¡ ha pi n »-1> at DNA p» »» »» »» »» »» »» »" "" "" "" "" "" "" "" • » L a it.olé. ¡1 a > of DNA '' Je l a »i '- i r» > ! i c C i i on 1, doml »- h - s »» *? iiou I? -? e? DN COIL l »-? * t» len deitiA- .. i ... »je?» mal "1 '-iit'H - • i-dui i í'ii'abl »-», i »p > j n r ep »» r l t .. »-». ? . a »> thousand > i »> i 'i »!» - • -, 111 = • »MI i -?» to a »? ne pei mi I .. .. - n' ifoaiiuo» 111 a - »d» - fil xOif -,! r 't ex., formed - »> »U d o lía» - > . > ! é u 3 - »of DNA. 6. i "ñu < ule DNA of the» »-ovi nd i e-3c r on 5, cleip of the sect uenc ia- * of? DN est af fi ar? the per cent, markers sel e> g onab 1 e 5, two reporter genes or one of the same ones 7. I j ni ».i 1 '' ' 1 • < 'I - 1 AO i J?' 3 ii • i, 'ii "i' • a» i, •, 1,. I »I. id - • I viral proteins buy at least two of the coatings of the v i rua II coating» Jel sa ic o d * - the a rdla, vir-n- »Jel > ??? o -.- »3 i» •) of the pep i no, or L »ien v? u < -, of > n > - »?, -. ? > c »amari l lo of O-I I I ta. The DNA code of the re vindicates the value of the cell. The cells of a cell are a cell of a molecule. * »> ie 3 - f a ¡it i l i rt »ie l -» »e u. i-i j i! c »- a s > . ! U me! > .? .. p- .-, pu'o r »?» -, n = »r r i st n» »a cini r,. ? , n-, • fli'i 1! 1 j) 1 e - -i > »> "! '" > ?the ! a., n-? .'id ilil »? -i lii, i r > i-i, i iue . . > .n i »~» r > »" "5 > - > ,: - '3 -s t r - f'-jf or »? T > a »> or rt of 1 -. -he, > .1 -_. -, .j »-. . i ». h-, u "= ni ¡sn-.i ntibie c on ni to the ula of DNA rei-nml.» »-» -t? d "e, ¡ni». ,, - ',, t, - jc? i? »'». itijt reí »» Je u ?? - < »? L ut, < l i e - > e > »In» i a - d DNA,. -? > j "?» r »a mp l'epd 1 e? Uf »l O I I n! ni or pirin pl ant a and nlaj de man * -? -t Du 'le a ur »to be. I t > - • DNA »] i i» - i. id 1 f t C a iifi i p t »» t < from my cu -, e 3 p?, j; •. E. r. f, - t, < . > 11 > - it 1 pl nt a ,, i * t. * the pui er - "> I f, ¡." i o. I ..! i a -,, _vi,] i, 3 a;,. E »» l a f »f, i i-a • a p > »» > »» »» I »11 ca t nn-t pl añí a > i i f e. - > ..-. «..I ^ < ? • 1 -i d '-' d1 '> f l > a »i n tle ,, f., < nl -xU '-i f,! =? n --- 'Ji- | l1 a. | -t a »., e, p > > ~ - -i a-, -, pt i.μiit i -? -, ».!» • »DNA '- .. > »11 ^ > '> ^ > v? ust a ac-e l a pl »> ? r,? _. becomes resistant to infection ». ion pior > d? irus rites. 1 n The whole of the circle »J icac onion 9, d» - »nof the sp» - * io n of the DNA sequence cod i f i cator p opiorc i ona ni eles -} < j - »t l le i a 1 tlu-'i 11 j IJI» - t > '- 11' - > " " -.1 . I -. 'I i > 011 I I-t I n 'o. i i i, i i, t i C3 ».ia ur» »-) > of cl i > . bc »s UI? I-J, 11. The method of re l a n d d ica tion 9, where -1 l to go to the menu of the one > : le dt »-h" i- »s uen»:; d DNA eudí fua ci an provide i '^ si ^ t' - 'm. tá::::? g? t? -a a plura I nity ed? cin »s 1 * "* -. Fl method dla re.», Jf ».J i < a.?? N 9 where the pl an e e, n n d icut i 3 educing 13. E3 > (? É?? D ?) i '»' 1 go í» i ndi »ai i? n 9, &! -?») .- »the» and? o3é »ni i.1». DNA ea pi rejeje »ji? Ti a -,. »» »» Ti ti tii »-»? »? L -.» 1 u 3 as ij, .. ,? 1 a r »I,: 'M', i e» r iit-i i »- t f .-, f or > t »a. (i r i me »i I l. The uléi Otlt i de l a t. or - > > »1 > > "a > 1 r, i. <" **, rioitcl-- '3 a -i - »e» pam. -j _ "DNA> -»? m |) r »'i" ?? | rJ? go. N otan sea »-i» t > ~ > »- r. l e »_ t o?» - < ¡3 e or, -n tjofi r i r t? - r ')? | N > '? e.a- »o te 3 a i ent i f i a» i o > i? ch- > l =? u! r n-i f ornt > ia "15, The method '!» -' l -? re i,. ' i fi »: l esc i < • '. n ", onde di > b. ¡s" ^, ne i * ia • > e? DN »rf > c in addition to -3 l» - * nu- »_ |» »_. »!» - l -, r »» .. ne-,? | Ei óte ». {Na> g» 1 »- • t» -, f, 1 m 1 in t ».» Net Vi rus 31 '"i» .-- 3 m »-? - i» > ? > f- the "-.- a.1 id i 1, v ire» »i '- l» »to-.? &.»? »iel ucpi 1 no»? bin' 1 > u- > ».i» - 1 , '"j -» -i I' t) 11 tita 1 I 1 t »> > Je 3 -? 1 a 1 a I 1 > 1. 1 í, Í, 16. E3 étt» »!» »Rf» - '3.1 I ei? 1 ridl' -i> l 7, where 3 l nta cta nta b e sible b ille. -. - »» e-? => 17, A plant fc an 3 form 1a re a ra r r f f a n d the method of 1. < re ivi < td ic at ton ^. 1? A, t JIM ». 1 > < 1 '" -le u 1 in > a > "• n- = foim -» ¡,,, ... >., »I a. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -. ? r »r» - »of the plant t ri n-» f orm of re i 'i nd ita > i o »t 17. 2 - l.ln pia uta hf bi ¡tre ¡trepaía» "! - to nao t * > r »le 3 a pi 'anta]? v i i nd 11 ac i on 1" • ". une »- -, r > -'- 3 I a; > - »n t. ,? j f- »»? ? > ? Nobody »3?»!? »? -. Id RF9HMEN OF THE INVENHION La p? Re: -ente? n > 'eit »ton pir oppo» *, i orta an iitciléoila of DNA i e > oii't) i na nte qn iitiéi i »cornpirende; a lm -? ; > > • - '. lien »» a- e DNA ,, »-" ií »> tn <1 - • .-» t '' - 3 > • ': - t C'1'1' l itiii-1? ltt I would like to say that I would like to say that I would like to say that I have not been able to do so for a while. i »! e í na i eve-> 11 I am a -» »» -!> j i nn - 'iii - ->, where ui ».t? aa se» * u »- - n > i as le-1 AD'N e eila 'llt e' rei en »1 - > en t ^ *»? »_ i» - • "!" e tsl m'tne »a -» ee,? »e-.eu n 1» '»3113 -,,? -' n? -, ta 3e- > ? - »? -? ppl l le ».1 3o-, j» -u-, ! i ':? n-, fti?' ?? i,? |, -j -, 1. oft »| l > '< ? i ") 3 t ul a > I» - DN re > htr > -ai > faith pa 1 au ro í 111 i 011 a 1 r »'-» 1 - >' ii > - > '>? > • »- - < > d 1» iv > > >,' 11 n., - • "s ~, 010.-Nti

1.» »P» 1 » »1 >» '> 11? I »é I o» lo., P -, laf »af» -xf »i |,» ix, j > 3 -. N I a -, »». tf. 1 ».-, 1 1 H, -, 't'i 11 111 j ijp I .IIH | 11? I-, p -i. a »e 1 e» > (olí 3 t 1) 1 to 111"a -x Uno »'| I [H | f '| p 1 op »11» 1 > > C? -11 f > -x t - »f e? i. \ - 1 or I 1!, R t- 1 rn-,