AU740653B2 - Plants with controlled side-shoot formation and/or controlled abscission area formation - Google Patents

Abstract

Disclosed are nucleotide sequences coding polypeptides which are responsible for controlling side-shoot formation and/or petal formation and/or abscission area formation, in addition to polypeptide and amino acid sequences coded by nucleotide sequences. Disclosed are also plants with controlled side-shoot formation and/or petal formation and/or controlled formation of abscission areas, wherein the expressible DNA sequence, fragment or derivative thereof responsible for side-shoot formation and/or petal formation and/or abscission area formation is integrated in a stable manner into the genome of the plant cell or the plant tissue. Further disclosed are methods for the production of plants with controlled side-shoot formation and/or petal formation and/or controlled formation of abscission areas, wherein the expressible DNA sequence or fragment or derivative thereof responsible for side-shoot formation and/or petal formation and/or controlled formation of abscission areas is integrated in a stable manner into the genome of plant cells or plant tissue and the plant cells or plant tissue thus obtained is regenerated to form plants. The invention also relates to plants and the seeds of plants which can be obtained according to the inventive method.

Description

PLANTS WITH CONTROLLED SIDE-SHOOT FORMATION AND/OR ABSCISSION ZONE FORMATION The present invention relates to nucleotide sequences encoding polypeptides which are responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation as well as to the polypeptides and amino acid sequences encoded by said nucleotide sequences. Furthermore, the present invention relates to plants having controlled side-shoot formation and/or petal formation and/or controlled formation of abscission zones, wherein the expressible DNA sequence or fragment or derivative thereof responsible for side-shoot formation and/or petal formation and/or abscission zone formation is integrated in a stable manner into the genome of the plant cell or the plant tissue. Further, the invention relates to methods for the production of plants having controlled side-shoot formation and/or petal formation and/or controlled formation of abscission zones, wherein the expressible DNA sequence or fragment or derivative thereof responsible for side-shoot formation and/or petal formation and/or abscission zone formation is integrated in a stable manner into the genome of plant cells or plant tissues and the resulting plant cells or plant tissues are regenerated to form plants. Moreover, the invention relates to plants and seed stocks of plants, which are obtainable according to the method of the invention.

Technical Background The performance characteristics of economic and ornamental plants are considerably determined by their architecture. While the basic structure of a plant manifests in the embryonic development, the post-embryonic phase is characterized by the activity of apical meristems. Of fundamental importance is the ability of the shoot apical meristem (SAM) of higher plants to initiate shoot branches and to control their development. As a result, the habit of a plant and thus an essential performance feature is characterized by the number, arrangement and developmental intensity of its sideshoots. The branching of the shoot may occur terminally as well as laterally. The terminal branching in which the SAM is separated into two portions mainly occurs in lower cormophytes and has been described for only a few flowering plants (Steeves and 77 Sussex, 1989, Patterns in Plant Development, 2 nd Edition, Cambridge University Press, Cambridge). The lateral branching typical for flowering plants is based on the formation of new shoot apical meristems in the leaf axils, which are derived from SAM cells, the meristemic character of which remains preserved in contrast to surrounding cells which are involved in the development of leaf primordia. In the further course of development, a side bud is formed from said residual meristems, which besides some leaf primordia contains an apical meristem, the activity of which is subject to the control by the main shoot SAM.

The analysis of plant mutants revealed that branching of the shoot system is controlled by genetic factors. Thus, in tomato (Lycopersicon esculentum) for example, there have been described a number of mutants, the side-shoot formation of which is inhibited in different stages blind, blind-2, torosa, lateral suppressor). A morphological characterization showed that the production of axil buds is disturbed in the tomato mutants blind, blind-2 and torosa (Tucker, 1979, Ann. Bot. 43: 571-577; Mapelli and Lombardi, 1982, Plant Cell Physiol. 23: 751-757). In contrast, in plants which are homozygous for recessive lateral suppressor (ls) mutation, the initiation of most of the side buds does not occur (Brown, 1955, Rep. Tomato Genetics Cooperative A histological analysis (Malayer and Guard, 1964, Amer. Jour. Bot. 51: 140- 143) shows that cells directly derived from SAM in the axils of the leaf primordia, on the meristemic activity of which the formation of side shoots is based, are missing in the lateral suppressor mutant. If a lack of side shoots in all leaf axils results in a termination of the shoot axis in the first inflorescence, the transition to floral development shows that the ability to establish axil meristems is not completely lost in the mutant. In the axil of the leaf primordium established directly before the inflorescence a meristem often is established in homozygous Is mutants as well. The establishment of this meristem which is necessary for the sympodial structure of the shoot axis is often associated by the formation of a side bud in the axil of the next older leaf. Following the transition to the floral phase, the development of the Is mutant is characterized by a smaller number of flowers per inflorescence (Williams, 1960, Heredity, 14: 285-296), the missing establishment of petal primordia (Szymkowiak and Sussex, 1993, Plant J., 4: 1-7) and an aberrant number of stamens and carpels (Groot et al., 1994, Sci. Hort., 59: 157-162). Furthermore, a reduced fertility in the mutant is observed, which also results in the reduction of yield and which is the reason that the Is mutant did not reach any significance for yield-oriented cultivation.

A further phenotypic change of the Is mutant relates to the formation of abscission zones in the flower and fruit stems. While wild type plants have a region of 5-10 layers of smaller cells, at the distal ends of which the non-pollinated flower or the ripe fruit comes off the plant (Roberts et al., 1984, Planta, 160: 159-163), this abscission zone is not formed in the ls mutant and during harvest the fruit comes off the plant without residues of the fruit stem and sepals.

The observed phenotypic changes are correlated with disorders in the equilibria of particular plant hormones on a physiological level. In comparison with the wild type, lower cytokinin concentrations were measured in the shoot tips of ls mutants (Maldiney et al., 1986, Physiol. Plant, 68: 426-430; Sossountzov et al., 1988, Planta, 175: 291- 304), while the amounts of P-indolylacetic acid (IAA)-like compounds as well as gibberellic and abscisic acids are markedly increased (Tucker, 1976, New Phytol., 77: 561-568). Attempts to remedy the deficiencies of the Is mutant by introducing an isopentenyl transferase gene from Agrobacterium tumefaciens resulted in an increase of endogenous cytokinine concentrations, but not in a normalization of the side-shoot development (Groot et al., 1995, Plant Growth Regulation, 16, 27-36).

Due to the great interest of breeders in single stem tomato varieties there have been early efforts to render the Is mutant usable for commercial cultivation. Since the DNA sequence of the gene (Ls gene) responsible for side-shoot formation and/or petal formation and/or abscission zone formation has so far not been known, it was repeatedly attempted by genetic methods to separate the desired effects on the side-shoot formation from the non-desired effects on fertility and yield. However, up to now none of these efforts have been successful.

For the isolation of genes which are only characterized by a mutant phenotype and their position on the genetic map, the strategies of insertional mutagenesis and positional cloning have been preferably used during the past years. The insertional mutagenesis uses mutant alleles formed by the insertion of a known sequence for the isolation of genes which in this manner are labeled on a molecular level. In plants, the T-DNA from Agrobacterium tumefaciens (Koncz et al., 1992, Plant Mol. Biol., 20: 963-

T-,

9 7 6 as well as transposable elements (Gierl and Saedler, 1992, Plant Mol. Biol., 19; 39- 49) were used for insertional mutagenesis (Jones et al., 1994, Science 266: 789-793).

Since the transposable elements Ac and Ds from maize preferentially transpose to coupled positions on the same chromosome (Knapp et al., 1994, Mol. Gen. Genet., 243: 666-673) a transposon mutagenesis is particularly promising when a starting line is available in which the transposable element is present in close coupling with the gene of interest. Since such a tomato line is not available, a transposon mutagenesis for the isolation of the Ls gene is not very promising.

The strategy for positional cloning was developed for the analysis of the molecular principles of hereditary diseases in mammals and inter alia used for the isolation of human genes for Duchenne's muscular dystrophy (Koenig et al., 1987, Cell, 509-517), Cystic Fibrosis (Rommens et al., 1989, Science, 245: 1059-1065) and Huntington's Disease (Huntington's Disease Research Group, 1993, Cell 72: 971-983).

Figure 1 schematically illustrates the course of a positional cloning. For this strategy the integration of the classical genetic locus into a map of molecular markers is of fundamental importance. The use of restriction fragment length polymorphisms (RFLPs) as genetic markers (Botstein et al., 1980, Am. J. Hum. Genet., 32: 314-331) enables the identification of closely coupled DNA fragments from the environment of the gene to be isolated. These fragments subsequently serve as hybridizing probes in Southern analysis by means of pulsed field gel electrophoresis (Chu et al., 1986, Science, 234, 1582-1585) of separated high molecular weight DNA to transform the relative genetic distance into an absolute value for the physical distance which has to be bridged by the so-called "chromosome walk". Starting with flanking markers as starting points the environment of the desired gene is isolated in the form of overlapping DNA fragments. Depending on the distance of the flanking markers in the genetic map the DNA fragments are YAC or cosmid clones (Burke et al., 1987, Science, 236: 806-812). RFLP maps with high marker density have been developed by Nam et al., 1989, Plant Cell, 1, 699-705, and Tanksley et al., 1992, Genetics, 132: 1141-1160. Grill and Somerville, 1991, Mol. Gen.

Genet., 226: 484-490, and Martin et al., 1992, Mol. Gen. Genet, 233: 25-32, describe the preparation of YAC-libraries.

In the classical genetic map of tomato the Ls locus is mapped on the long arm of chromosome 7 (Taylor and Rossall, 1982, Planta, 154: Schumacher et al., 1995, Mol. Gen. Genet, 246: 761-766, describe an integration of the Ls locus into the RFLP map, wherein the Ls locus was mapped within a 0.8 cM interval near the distal end of chromosome 7. Furthermore, Schumacher et al. describe that the Ls locus is bounded by the RFLP markers CD61 and CD65. The physical mapping by means of pulsed field gel electrophoresis showed that CD61 and CD65 are not more than 375 kb apart from each other.

With respect to agricultural cultivation the formation of side shoots is not desired in many economic plants due to various reasons: 1. Firstly, the young side shoots are "sink" organs (organs of consumption) and thus reduce the yield of the main shoot.

2. Highly branched shoot systems often represent a hardly surmountable obstacle for mechanical treatment harvest with machines).

For these reasons there have been early attempts to cultivate varieties without side shoots in a conventional manner. This has been successful in individual economic plants sun flower). However, in many other dicotyledonous economic plants (e.g.

tomato, cucumber, apple-tree, pear-tree) the single stem would be desirable, but this has so far not been realized in efficient culture varieties. Also in monocotyledonous economic plants, such as maize and sugar cane, suppression of side shoot formation is advantageous and highly desired for commercial use. At present, the single stem e.g. of tomato is achieved in green house cultivation common in Central and Northern Europe by manually removing the side shoots. Since the removal of the side shoots cannot be done with machines this is associated with enormous costs. Furthermore, at the wound site the plants are very susceptible of infections by pathogens, such as pathogenic bacteria, viruses and fungi. Thus, the removal of side shoots contributes to the spreading of diseases in green house.

In many ornamental plants, however, the additional formation of side shoots and thus an enhanced formation of flowers is desired. Enhanced formation of side shoots is also highly beneficial in many economic plants, such as potato, coffee or tea plant. Thus, there is a need for cost-effective, efficient economic plants and ornamental plants, in which the formation of side shoots is increased or suppressed.

Inhibition of the formation of abscission zones is of interest in a number of plants. Thus, the premature abscission of fruits in citrus plants resulted in losses of yield which could be prevented if no abscission zones were formed. Similar results may be found in other fruit species, such as cherry, peach or black currant. Further, an inhibition of the formation of abscission zones, e.g. in tomato, is advantageous. If the abscission zones are not formed, the fruit comes off the plant during harvest without residues of the fruit stem and sepals. This feature is desired when tomatoes are harvested with machines and are subsequently processed to products such as tomato puree, since sepals and fruit stems deteriorate the quality of the tomato products.

In ornamental plants, an increased formation of abscission zones may be useful, since flowers would fall off by themselves after fading and there would be no need to remove them manually, such as with many balcony and garden plants. If this does not occur, the formation of new flowers is suppressed.

Short Description of the Invention Isolation and cloning of the Ls gene would offer the possibility to change the activity of said gene in a targeted manner and thus to suppress or increase the formation of side shoots in transgenic plants. Further, one may suppress or increase the formation of abscission zones and/or petals by changing the activity of the Ls gene in a targeted manner. Accordingly, the object underlying the present invention is to isolate the Ls gene or a DNA fragment containing said gene, determine its sequence and provide a method for the preparation of transgenic plants in which the activity of the Ls gene was varied in a targeted manner to suppress or increase the formation of side shoots and/or the formation of abscission zones and/or petals.

The object of the present invention is solved by providing the nucleotide sequences according to SEQ ID NO: 1, 9 or 13 and the nucleotide sequences hybridizing to the nucleotide sequence according to SEQ ID NO: 1, 9 or 13, wherein said nucleotide sequences according to SEQ ID NO: 1, 9 or 13 and said nucleotide sequences hybridizing to the nucleotide sequence according to SEQ ID NO: 1, 9 or 13 encode polypeptides which are responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation. According to the present invention, the term "hybridization" is directed to conventional hybridization conditions, preferably "hybridization" is directed to such hybridization conditions in which the TM value is in the range from TM 45 0 C to TM 68 0 C. The term "hybridization" is particularly preferably directed to stringent hybridization conditions. The invention further relates to polypeptide and arnino acid sequences encoded by said nucleotide sequences.

A further aspect of the invention is solved by a method for preparing plants having controlled side-shoot formation and/or petal formation and/or abscission zone formation, wherein the expressible DNA sequence or fragment or derivative thereof responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation is integrated in a stable manner into the genome of plant cells or plant tissues and the resulting plant cells or plant tissues are regenerated to form plants.

The invention further provides a nucleotide sequence according to SEQ ID NO: 1, 9 or 13 which is responsible for controlling side shoot formation and/or petal formation and/or abscission zone formation, the fragment or derivative thereof or a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence according to SEQ ID NO: 1, 9 or 13 and which is responsible for controlling side-shoot fornmation and/or petal formation and/or abscission zone formation.

In the present invention a method is preferred in which the integrated DNA suppresses the side-shoot formation and/or petal formation and/or abscission zone formation. Particularly preferred is a method in which the integrated DNA is expressed in an antisense orientation with respect to the complementary endogenous sequence 20 controlling side-shoot formation and/or petal formation and/or abscission zone oo* formation. Also particularly preferred is a method in which the integrated DNA is expressed in a sense orientation with respect to the complementary endogenous i: sequence controlling side-shoot formation and/or petal formation and/or abscission zone formation. Furthermore, particularly preferred is a method in which side-shoot formation and/or petal formation and/or abscission zone formation is suppressed by a ribozyme comprising the DNA sequences or fragment or derivative thereof according to the present invention. Particularly preferred is also a method in which the DNA sequences or fragment or derivative thereof according to the invention are used to switch off ("knock-out") the endogenous gene in plants by way of homologous recombination.

In the present invention a method is further preferred wherein the DNA _R integrated into the genome of the plants enhances side-shoot formation and/or petal preferred is also a method according to the present invention for the preparation of transgenic plants, wherein the DNA integrated into the genome of the plants comprises the sequence according to SEQ ID NO: 1, 9 or 13 or fragment or derivative thereof or which is complementary to said sequence or fragment or derivative thereof, or which hybridizes with the sequence according to SEQ ID NO: 1, 9 or 13 or fragment or derivative thereof and encodes a polypeptide having the biological activity of side-shoot formation and/or petal formation and/or abscission zone formation.

The invention further relates to transformed plant cells or transformed plant tissue, wherein an expressible DNA sequence or fragment or derivative thereof responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation is integrated in a stable manner into the genome of the plant cell or plant tissue. Further, the invention relates to plants as well as to seed stocks of plants obtainable according to the method of the present invention.

The invention is further illustrated by the following figures, wherein: Figure 1 schematically shows the course of a positional cloning.

Figure 2 illustrates in a portion of the RFLP map published by Tanksley et al., 1992, Genetics, 132: 1141-1160. In the Ls region according to Schumacher et al., 1995, Mol. Gen. Genet., 246: 761-766, is integrated into this map.

Figure 3 shows the mapping of cDNA and cosmid clones from the Ls region.

The cosmid clones A, B, C, D, E, F, G and Las well as YAC clone CD61-5 are symbolized by bars. The positions of the cDNA clones cl0, c21, y25 and ET are illustrated by open rectangles. The dashed lines represent recombination sites in F2 plants 23, 24, 865 and 945.

Figure 4 shows the autoradiograph of a Southern blot analysis for the detection of Ls-related genes in different plant species. Genomic DNA from tomato (Lycopersicon esculentum), potato (Solanum tuberosum) and snapdragon (Antirrhinum majus) was treated with the restriction enzyme EcoRI and hybridized with the cDNA clone ET.

Figure 5 shows the nucleotide sequence and the amino acid sequence derived therefrom (one letter code) of the Ls wild type gene from tomato (Lycopersicon esculentum).

Figure 6 shows the nucleotide sequence and amino acid sequence derived therefrom (one letter code) of the Ls homologous gene from potato (Solanum tuberosum).

Figure 7 shows the nucleotide sequence and the amino acid sequence derived therefrom (one letter code) of a 687 bp DNA fragment of the Ls homologous gene from Arabidopsis thaliana.

Figure 8 shows an alignment of amino acid sequences of the Ls polypeptide from Arabidopsis thaliana (LsAt), Lycopersicon esculentum (LsLe) and Solanum tuberosum (LsSt). The one letter code was used for amino acids. Identical amino acids are shaded in black, similar amino acids are shaded in gray. The dash represents missing sequence information, a dot represents an additional amino acid in a polypeptide. An asterisk represents a stop codon on nucleic acid level.

Detailed Description of the Invention The method of cloning DNA fragments being several hundreds of kilobases in length as artificial yeast chromosomes (Yeast Artificial Chromosome: YAC) in Saccharomyces cerevisiae (Burke et al., 1987, Science, 236: 806-812) enables the transformation of the physical map into a number of overlapping YAC clones spanning the gene to be isolated. From a YAC library of tomato (Martin et al., 1992, Mol. Gen.

Genet., 233: 25-32) clones containing the RFLP marker CD61 were isolated. By mapping the YAC terminal fragments with respect to the RFLP markers flanking the Ls gene as well as to the recombination break points and to the Ls gene itself the position of the isolated DNA fragments in the Ls region was determined. Thus, YAC clone CD61-5 was found to hybridize both with CD61 and with CD65 and therefore contains the entire genomic region including the Ls gene. Figure 3 schematically illustrates the position of the marker and of the YAC clone.

For identification of coding regions localized within the YAC clone this clone was used as a radiolabeled probe to screen a cDNA library (Simon, 1990, doctoral thesis, University of Cologne, Cologne, Germany). The cDNA library used is made from RNA of both vegetative and floral shoot tips and thus represents expressed genes of the tissues in which the phenotype of the Ls mutation manifests itself. A characterization of cDNA clones by cross hybridization revealed that the purified clones represented a total of 29 different transcripts. The subsequent fine mapping of the cDNA clones relative to the recombination break points in interval CD61-CD65 revealed that only cDNA clone y25 cosegregated with the Ls gene and is a possible candidate for said gene. After the establishment of a cosmid contig also cosmid clones were used as probes to isolate further cDNA clones from the CD61-CD65 interval, which in screening with YAC clone CD61-5 as a probe were not detectable due to the high complexity of the probe. In these experiments three additional cDNA clones (cI0, c21 and ET) were isolated which also cosegregated with the Ls gene and were possible other candidates for the Ls gene. Thus, a total of four cDNA clones were identified from the Ls region, which were candidates for the Ls gene. In Figure 3 said clones are represented by open rectangles.

In order to clone the Ls gene together with the promoter sequences necessary for the regulation of expression, the cDNA clone y25 was used as a starting point for the isolation of shorter genomic DNA fragments of the Ls region. For this purpose a genomic cosmid library from tomato was established in vector pCLD04541 (Bent et al., 1994, Science, 265: 1856-1860). Said vector contains the T-DNA border sequences necessary for plant transformation and thus allows for an introduction of isolated DNA fragments into plant cells without further cloning steps. From this library a number of overlapping cosmid clones was isolated in several typical cloning steps. Mapping of said cosmid clones relative to the recombination break points in the tested interval showed that the isolated genomic DNA fragments spanned a genomic region of about 60 kb. The position of the cosmid clones is schematically illustrated in Figure 3.

To investigate the question whether a gene from the genomic DNA region isolated as cosmid contig is able to compensate for the biological function for formation of side shoots, petals and abscission zones which is missing in the Is mutant (complementation experiment), said Is mutant was transformed with the cosmid clones A, B, C, D, E, F, G and L. In all transgenes made by introduction of the cosmids A, B, C, D, E and F, no alteration of the phenotype could be observed. In contrast, in eight independent transgenic plants containing either cosmid G or L a partial or complete recovery of the wild type phenotype could be observed. The results of the complementation experiments are illustrated in Table I.

Cosmid number of transformed plants number of complemented plants pCLDO4541 8 0 A 5 0 B 15 0 C 5 0 D 7 0 E 2 0 F 8 0 G 5 3 L 11 Table I: Complementation experiments of ls mutant via cosmid transformation These transgenic plants form side shoots during vegetative development and again petals and abscission zones in the floral development. A Southern blot analysis of transgenic plants containing cosmid G or cosmid L revealed that in plants showing no complementation the T-DNA was only incompletely transferred. Thus, it has been shown that introduced DNA fragments are able to complement the genetic information for formation of side shoots, petals and abscission zones, which is absent from the mutant.

By using complementation experiments with subfragments of cosmid G the DNA region in which the Ls gene is localized could be determined in more detail. While following transformation with DNA fragments containing the previously identified gene c21 no complementation of the ls phenotype could be observed, the wild type phenotype could be recovered in eight independent transgenic plants by the introduction of an approx. 6 kb fragment bearing the ET gene. A DNA sequence analysis revealed that the ET gene of the Is' mutant harbours a 1550 bp deletion which removes the first 185 amino acids of the protein and 865 bp of the sequence which is localized upstream. A second independent mutant allele Is 2 contains a 3 bp insertion and several point mutations in a short DNA portion, one of which results in a termination of the protein after 24 amino acids. The complementation experiments and isolation and mapping of the cDNAs as well as the sequence analyses of the ET gene from the wild type and two independent Is alleles revealed that the cDNA clone ET represents the entire coding sequence of the mRNA of the Is gene.

To address the question whether similar or homologous genes are present also in other plant species the cDNA clone ET was employed as hybridization probe in Southern experiments under reduced stringency. The term "plant", as used herein, comprises monocotyledonous and dicotyledonous economic and ornamental plants. The term "reduced stringency", as used herein, is directed to typical hybridization conditions with the modification that hybridization temperature was between 50 0 C and 55 0 C. In potato (Solanum tuberosum) and snapdragon (Antirrhinum majus) several DNA fragments could be detected. From snapdragon several genomic clones were isolated by hybridization at 55 0 C. A DNA sequence analysis revealed that the isolated snapdragon clone has significant sequence homologies to the Ls gene. Thus, genes homologous to the tomato Ls gene may be isolated according to conventional methods by using the cDNA clone ET as a probe. Using gene specific primers the Ls homologous gene was isolated from genomic DNA of potato (Solanum tuberosum) via PCR. The Ls homologous gene from potato shows a sequence identity of approx. 98% to the Ls gene of tomato on the DNA level as well as on the protein level. From genomic DNA of Arabidopsis (Arabidopsis thaliana) a 687 bp DNA fragment of the Ls homologous gene was isolated via PCR using degenerate primers. On DNA level the Arabidopsis thaliana DNA fragment exhibits a sequence identity of about 63% to the tomato Ls gene. On protein level about 55% of the amino acids are identical.

The present invention is further directed to DNA sequences which are derived from a plant genome and code for a protein necessary for controlling side-shoot formation and/or petal formation and/or formation of abscission zones. Upon introduction and expression in plant cells the information contained in the nucleotide sequence results in the formation of a ribonucleic acid. By means of said ribonucleic acid a protein activity may be introduced into the cells or an endogenous protein activity may be suppressed. Particularly preferred is a DNA sequence according to SEQ ID NO: 1 from Lycopersicon esculentum shown in Figure 5, a DNA sequence according to SEQ ID NO: 9 from Solanum tuberosum shown in Figure 6 and a DNA sequence according to SEQ ID NO: 13 from Arabidopsis thaliana shown in Figure 7.

Moreover, the present invention relates to the use of the DNA sequences or fragments or derivatives according to the present invention which are derived from said DNA sequences by insertion, deletion or substitution in the transformation of plant cells.

The DNA sequences according to the present invention may be employed using different methods to suppress the formation of side-shoots and thus of branches of the shoot system and/or petals and/or abscission zones: 1. To suppress the formation of side-shoots and/or petals and/or abscission zones the DNA sequence according to the present invention may be cloned in an antisense or a sense orientation into conventional vectors plasmids) and thus combined with control elements for expression in plant cells, such as promoters and terminators. By using the prepared vectors, plant cells may be transformed with the aim to prevent the synthesis of the endogenous protein. For this purpose, shorter parts of the DNA sequence according to the invention, i.e. fragments, or DNA sequences having a sequence similarity of from 50% to 100%, i.e. derivatives, may also be used. Thus, the Ls homologous gene isolated from Arabidopsis may be employed for example to suppress the formation of side-shoots and thus of branches of the shoot system and/or petals and/or abscission zones in the related species Brassica napus (rape). The targeted suppression of a genetic activity in plant cells by the introduction of antisense or sense constructs is a common method which has been successfully employed in many cases (Gray et al., 1992, Plant. Mol. Biol., 19: 69-87).

2. Furthermore, the formation of side shoots and/or petals and/or abscission zones may be inhibited by expressing a ribozyme constructed for this purpose using the DNA sequences according to the present invention. Preparation and use of ribozymes are disclosed in de Feyter et al., 1996, Mol. Gen. Genet., 250: 329-338 for tobacco mosaic virus resistant tomato and tobacco plants.

3. Furthermore, the DNA sequence according to the present invention may be used to inactivate the endogenous gene. By using the DNA sequences of the present invention oligonucleotides may be synthesized to test plants in the context of mutagenesis experiments by means of PCR technique for the presence of insertions (e.g.

transposable elements or T-DNA from Agrobacterium tumefaciens) in the Ls gene.

Generally, the genetic activity will be blocked by such insertions (Koes et al., 1995, Natl. Acad. Sci. USA, 92: 8149-8153).

4. The DNA sequence according to the invention may be also employed to switch off ("knock-out") the endogenous Ls gene by means of homologous recombination. This method was successfully employed in mice and is also described for use in plants by Miao and Lam, 1995, Plant. 7, 359-365.

In contrast to tomato and other economic plants, in ornamental plants (e.g.

geraniums, fuchsias and chrysanthemums) phenotypes are often preferred which exhibit a bushy growth due to a strong development of the side shoots. In order to generate said growth forms today, the plants are either decapitated, which promotes the initiation of side axes, or are treated with particular chemicals. However, said practice is also associated with considerable costs. In these cases, the preparation of transgenic plants having bushy growth forms according to the present invention represents a more costeffective alternative.

In ornamental plants an enhanced formation of abscission zones my be used such that after fading the flowers fall off by themselves and must not be manually removed as with many balcony and garden plants. If this does not occur, the formation of new flowers often is suppressed.

For the preparation of transgenic plants with strong side-shoot formation and/or abscission zone formation the DNA sequence or fragment or derivative thereof according to the invention which is derived from said sequence by insertion, deletion or substitution, is introduced into plasmids in a sense orientation and combined with control elements for expression in plant cells. Using said plasmids plant cells may be transformed such that a translatable messenger ribonucleic acid (mRNA) is expressed which enables the synthesis of a protein stimulating the formation and development of side shoots and/or petals and/or abscission zones.

The DNA sequence or fragments or derivatives thereof according to the present invention which are derived from said sequence by insertion, deletion or substitution may be used to isolate homologous or similar DNA sequences from the genome of tomato or other plants, which DNA sequences influence the formation of side shoots and/or petals and/or abscission zones as well. For this purpose the DNA sequence or fragments, e.g. oligonucleotides, or derivatives according to the present invention may be employed as probe molecules to screen cDNA libraries or genomic DNA libraries of the plants to be screened according to conventional methods. Alternatively, degenerated or non-degenerated oligonucleotides (primers) may be derived from the sequence according to the present invention, which may be used to screen said cDNA libraries or genomic DNA libraries on a PCR basis. Similar to the DNA sequences according to the present invention, the thus isolated related DNA sequences may be employed for inhibition or stimulation of side-shoot formation and/or petal formation and/or abscission zone formation in plants.

For expression of the DNA sequences according to the present invention in sense or antisense orientation in plant cells on the one hand transcription promoters and on the other hand transcription terminators are necessary. A great number of promoters and terminators have been described in the literature K6ster-T6pfer et al., 1989, Mol.

Gen. Genet., 219: 390-6; Rocha-Sosa et al., 1989, EMBO 8: 23-29). The transcriptional initiation and termination regions may be derived either from the host plant or from a heterologous organism. The DNA sequences of the transcription initiation and transcription termination regions may be prepared synthetically or obtained naturally or may contain a mixture of synthetic and natural DNA components.

Methods for genetic modification have been described for dicotyledonous and monocotyledonous plants (Gasser and Fraley, 1989, Science 244: 1293-1299; Potrykus, 1991, Ann. Rev. Plant. Mol. Biol. Plant. Physiol., 42: 205-226). In addition to the transformation by means of Agrobacterium tumefaciens (Hoekema, 1983, Nature, 303: 179-180; Filatti et al., 1987, Biotech, 5:726-730), DNA may be introduced by transformation of protoplasts, microinjection, electroporation or ballistic methods into plant cells. For selection of transformed plant cells the DNA to be introduced is coupled with a selection marker which imparts resistance against antibiotics kanamycin, hygromycin, bleomycin) to the cells. From the transformed plant cells whole plants may then be regenerated in a typical selection medium. Regeneration of plant cells is described for example in EP-B-0 242 236, which is incorporated herein by special reference. The plants thus obtained are tested for the presence and intactness of the introduced DNA by means of conventional molecular biological methods. Once the introduced DNA is integrated into the genome, it is generally stable and is transmitted to the offspring. By using conventional methods seed stocks may be obtained from the resulting plants.

The following examples are meant to illustrate the present invention and are not construed to be limiting. If not mentioned otherwise, molecular biological standard procedures were used, as described by Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Southern hybridizations were carried out in 6 x SSPE (0.9 M NaC1, mM NaH 2

PO

4 x H 2 0, 5 mM EDTA, 0.1% BSA, 0.1% Ficoll, 0.1% PVP, 0.5% SDS, 100 jlg/ml of calf thymus DNA) with a Hybond N+ membrane (Amersham). Plaque hybridizations were performed in 6 x SSPE (1.08 M NaCI, 60 mM NaH 2

PO

4 x H 2 0, 6 mM EDTA, 0.1% BSA, 0.1% Ficoll, 0.1% PVP, 0.1% SDS, 200 g/ml of calf thymus DNA) with a Hybond N+ membrane (Amersham).

Example 1 Isolation of YAC clones from the Ls region of tomato From a tomato YAC library (Martin et al., 1992, Mol. Gen. Genet., 233: 25-32) clones were isolated containing CD61 marker (Schumacher et al., 1995, Mol. Gen.

Genet., 246: 761-766). For this, DNA mixtures which were derived from a microtiter plate with 96 YAC clones were first tested by using the conventional PCR method.

Thus, from 144 of such DNA mixtures nine could be identified which yielded a PCR product with the CD61-F and CD61-R primers (Schumacher et al., 1995, Mol. Gen.

Genet., 246: 761-766). The isolation of single clones was carried out by means of colony hybridization or PCR, wherein the DNA of clones of a row or column of a microtiter plate was used as a mixture. Thus, from 96 clones of a plate single clones were identified using 20 PCR reactions. In total, five YAC clones were identified, the insert size of which was determined to be 280 320 kb by pulsed field gel electrophoresis (Chu et al., 1986, Science, 234: 1582-1585). It was shown in PCR and Southern experiments that YAC CD61-5, in addition to CD61, also carried the second flanking marker CD65 and thus spanned the Ls locus.

Example 2 Isolation of cDNA clones of the Ls region from tomato For preparation of a hybridization probe DNA from the YAC clone CD61-5 was isolated following separation by means of pulsed field gel electrophoresis. However, separation on said pulsed field gel only allowed for a relatively rough preparation, such that the probe used, in addition to the YAC clone CD61-5, also contained portions of the DNA from yeast chromosome 1n (360 kb) and VI (280 kb). Following radio-labeling said DNA was used as a probe to screen 5 x 10 5 pfu (plaque forming units) in a conventional plaque hybridization. Hybridization with the YAC probe provided a plurality of signals of different intensity. For rescreening 50 plaques of different signal intensities were selected and 44 purified clones could then be grouped by means of cross hybridization. 23 of 44 clones which resulted from rescreening were present only once.

In total, 29 different transcripts were identified in this screening. Following establishment of a cosmid contig the cDNA library was again screened with the cosmid clones to isolate additional cDNA clones which were not detectable in screening with YAC61-5 as a probe due to the high complexity of the probe. In these experiments, three additional cDNA clones were isolated. In total 32 different transcripts were detected.

Example 3 RFLP mapping of isolated cDNA clones from tomato Of 30 identified transcripts 22 showed typical hybridization patterns for single or low-copy sequences which enabled RFLP mapping. In a first RFLP analysis the isolated cDNA clones were hybridized against filters which carried DNA from L. esculentum, L.

penellii as well as from the back crossing line IL83 digested with the restriction endonuclease enzymes EcoRI, EcoRV and XbaI (Eshed et al., 1992, Theor. Appl.

Genet., 83: 1027-1034). This line, in which the distal terminus of chromosome 7 is derived from L. pennellii while the rest of the genome is composed of L. esculentum chromosomes, enables a first rough mapping in the presence of a polymorphism between L. esculentum and L. pennellii. If a polymorphous DNA fragment was derived from the Ls region, the line 1L83 exhibited the L. pennellii allele, whereas the L.

esculentum allele was present for fragments from the remaining genome. In this manner four cDNA clones were identified which were not derived from chromosome 7. Fine mapping of the 18 remaining cDNA clones derived from chromosome 7 was carried out via RFLP analysis of the plants W23 and W24 which contained recombination events in the interval CD61-Ls and Ls-CD65, respectively. Since in this analysis candidates for the Ls gene in plant W23 exhibited the L. esculentum as well as the L. pennellii specific fragment, while in plant W24 only the L. esculentum specific fragment was present, the cDNA clones were hybridized against filters carrying genomic DNA digested with EcoRI, EcoRV or XbaI of both parental species as well as of both recombinants W23 and W24. In this manner a total of four cDNA clones was identified which cosegregated with the Ls gene and thus, were possible candidates for the Ls gene.

Example 4 Preparation and screening of a genomic cosmid library of tomato DNA of the T-DNA/cosmid vector pCLD04541 (Bent et al., 1994, Science, 265: 1856-1860) was isolated according to the protocol of Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2 n d Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, purified via two CsCI gradients and dialyzed against TE for 3 days. The DNA was completely digested with BamHI and subsequently dephosphorylated with alkaline phosphatase to prevent self ligation of the vector. 200 ng of genomic tomato DNA partially digested with MboI and 2 mg of vector DNA were ligated with T4 DNA ligase in 10 ml at 16 0 C over night. 3 ml of said ligation assay were employed for packaging and transfected into E. coli SURE (Stratagene). This assay resulted in 6 x 106 independent recombinant bacteria. Each of 100 plates were plated with 2500 cfu (colony forming units) and rinsed off with 10 ml each of LB medium. In each case a glycerol culture was made from this material and a DNA preparation was carried out. These 100 DNA pools were screened by means of PCR analysis. Positive pools were then subjected to colony filter hybridization to identify positive single clones.

Example Cloning and sequencing of the Ls gene from tomato The insert of the cDNA clone ET which was isolated as a probe in screening of the cDNA library with cosmid G was cut out with EcoRI and cloned into vector pGEM- 11Zf(+). The missing 5' terminus of the gene was isolated by means of the RACE technique (Frohman et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002). Here, starting from an oligonucleotide specifically binding to known regions of the gene, a DNA complementary to RNA (cDNA) was prepared. Subsequently deoxycytosin nucleotides were attached to the cDNA using terminal transferase. With a second gene specific primer and a primer binding to the polydeoxycytosin tail the 5' end of the cDNA was amplified via PCR and cloned into the plasmid vector pGEM-T. Subsequently the longest of the RACE clones were sequenced. Simultaneously with the analysis of cDNA clone ET subfragments of the respective genomic region of cosmid G were isolated and recloned into the plasmid vectors pGEM-4Z and pSPORTI. Overlapping subfragments were then sequenced. The genomic sequence did not show any difference from the sequence of the cDNA clone, which means that the Ls gene does not contain any intron.

Moreover, the respective genome regions of both mutants Is' and Is 2 were amplified from the genomic DNAs via PCR using suitable primers and cloned into the pGEM-T vector. Sequence analysis of said products exhibited a deletion of 1.5 kb in the Is' allele compared to the wild type sequence. Besides the loss of nucleotides 1-685 of the open reading frame the Is' mutant also lacks 865 base pairs of the region located 5' of the open reading frame, which is thought to have a regulatory function (promoter) for expression. Therefore, it may be assumed that the ls' mutant is no longer able to form a functional protein from the Ls gene. In the Is 2 allele an insertion of 3 base pairs as well as 3 base exchanges were found in the 5' region of the open reading frame. One of these base exchanges leads to a stop codon resulting in a termination of the amino acid chain after 24 amino acids. Again a protein without any function is to be assumed. The vectors pGEM-1 pGEM-4z, pGEM-T were purchased from the company Promega Corp., Madison, vector pSPORTI was purchased from the company Life Technologies, Eggenstein, and used according to the manufacturer's instructions.

Example 6 Transformation of plants with Ls cDNA constructs of tomato Ls cDNA was isolated with gene specific primers CD61-13 TTAGGGTTTTCACTCCACGC-3'; SEQ ID NO: 3) and CD61-28 TCCCCTTTTTTTCCTTTCTCTC-3'; SEQ ID NO: 4) by means of the conventional PCR method and cloned into plasmid vector pGEM-4z (GSET8). For preparation of the transformation constructs the Ls cDNA was cut off from plasmid GSET8 with SalI/SstI (for sense construct) and XbaI/SstI (for antisense construct) and ligated into the plant transformation vector pBIR digested with SalI/SstI (sense construct) and XbaI/SstI (antisense construct), respectively (Meissner, 1990, doctoral thesis, University of Cologne, Cologne). In the resulting clones the cDNA is present either in sense or in antisense orientation between promoter and polyadenylation site of the 35S gene of cauliflower mosaic virus. The resulting sense and antisense plasmids were introduced into the Agrobacterium tumefaciens strain GV3101 (Koncz and Shell et al., 1986, Mol.

Gen. Genet., 204: 383-396) by direct transformation. Subsequently the T-DNAs of the two different constructs were transformed into leaf pieces of tomato and tobacco according to Fillatti et al., 1987, Biotech, 5: 726-730. Different transgenic 'plants containing the Ls antisense construct show a reduction of side-shoot formation Example 7 Isolation of a Ls related gene from snapdragon (Antirrhinum majus) With cDNA clone ET as a probe a genomic phage library from Antirrhinum majus was screened. Hybridization was carried out at 55 0 C, i.e. under reduced stringency. In this experiment 14 clones were isolated, clone HH13 of which showing the strongest hybridization signals was further characterized. The sequence analysis carried out following recloning the phage insert into the plasmid vector pGEM-11 zf(+) showed that the isolated Antirrhinum majus gene has high sequence homology to the Ls gene from tomato. Within both sequences regions could be identified, in which the derived amino acid sequence is totally conserved.

Example 8 Isolation of an Ls related gene from potato (Solanum tuberosum) In a Southern blot experiment under reduced stringency at 55 0 C using cDNA of the Ls gene as a hybridization probe, a DNA fragment could be detected in genomic DNA from Solanum tuberosum (Fig. Using gene specific primers CD61-24 TTTCCCACTCAAGCCAACTC-3'; SEQ ID NO: CD61-6 GGTGGCAATGTAGCTTCCAG-3'; SEQ ID NO: P01 TCGAGGCGTTGGATTATTATAC-3'; SEQ ID NO: 7) and PO5 GGCCCCCATATCTTTTTCC-3'; SEQ ID NO: 8) from Ls gene overlapping genomic DNA fragments were isolated from conventionally isolated DNA from Solanum tuberosum by using the PCR method. The PCR reactions were carried out as follows: Denaturation at 95 0 C for 30 seconds, annealing at 60 0 C for 1 minute, elongation at 72 0

C

for 2 minutes. This cycle was repeated 30 times. The resulting PCR products were cloned into the plasmid vector pGEM-T. A sequence analysis revealed that the isolated DNA fragments from Solanum tuberosum bear the sequence information for an open reading frame having a coding capacity of 431 amino acids (Fig. The DNA sequence is shown in SEQ ID NO: 9 and the amino acid sequence encoded by the DNA sequence is illustrated in SEQ ID NO: 10. On DNA level as well as on protein level the Ls homologous gene of potato exhibits a sequence identity of about 98% to the Ls gene of tomato.

Example 9 Isolation of an Ls related gene from Arabidopsis thaliana For the isolation of the Ls homologous gene from Arabidopsis thaliana the degenerated primers CD61-38 (5'-CARTGGCCNCCNYTNATGCA-3'; SEQ ID NO: 11)* and CD61-41 (5'-TGRTTYTGCCANCCNARRAA-3'; SEQ ID NO: 12)* were made and used for PCR reactions with genomic DNA from Arabidopsis thaliana isolated in a usual manner. The PCR reactions were carried out as follows: Denaturation at 95°C for 30 seconds, annealing at 50 0 C for 1 minute, elongation at 72 0 C for 1 minute.

This cycle was repeated 35 times. In this manner a DNA fragment of about 700 bp could be amplified which was subsequently cloned into the plasmid vector pGEM-T. A sequence analysis showed that the isolated DNA fragment from Arabidopsis thaliana (SEQ ID NO: 13) was 687 bp in length and has a high sequence similarity to the Ls gene from Lycopersicon esculentum. On the DNA level the Arabidopsis thaliana DNA fragment shows a sequence identity of about 63% to the Ls gene of tomato. On the protein level about 55% of the amino acids are identical The amino acid sequence encoded by the isolated DNA fragment (SEQ ID NO: 13) is illustrated in SEQ ID NO: 14. By using the isolated DNA fragment the Ls homologous gene from Arabidopsis thaliana may be isolated using conventional molecular biological standard methods.

In the description of the degenerated primers the WIPO standard St. 23 was used: 22

R=A+G

N=A+G+C+T

Y=C+T

23 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Nikolaus (Klaus) Theres STREET: Schiffgesweg CITY: Pulheim STATE: NRW COUNTRY: Germany POSTAL CODE: 50259 TELEPHONE: 49 2234 89386 (ii) TITLE OF INVENTION: PLANTS WITH CONTROLLED SIDE-SHOOT FORMATION AND/OR ABSCISSION ZONE FORMATION (iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPA) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1729 Base pairs TYPE: Nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETIC: NO (vi) ORIGINAL SOURCE: ORGANISM: Lycopersicon esculentum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCTCTGTCCT TCCCCCCAGG TCCCCTTTTT TTCCTTTCTC TCTCTCCTTT ATTTCTCTTT

TCATAAGCAT

ATTGAATGAT

TGAAGAATCT

CACCACCACC

GGAGTTGATT

TAACTCATCT

TTCCCTTCGT

AACAACTCCA

ATTCTTTCTC

ATGTTAGGAT

TCTGATCATC

ATCACTACCT

TCGCAGTCCG

CCTTTTGGTG

CTCAACCGCT

ACTGATTCTT

TCTCTAGGGT

CCTTTGGTTC

ATCAACAGCG

CACCAGCTAT

ATTTCTCGGC

ATTCAACTGA

ATATATCGTC

CTTCTTCGTC

TTTCACTTTC

TTCATCATCT

TAGATTCACC

TCAAATCCGC

CGCGAAAAGA

ACGGTTAGTC

AACCACCAAT

ATCATTAGCT

ACCTGAAATA

CAATCTCACC

GCTACTGCTA

CAGCTACTCA

CTCCTTACTA

CATCAATTTA

CATTTCATGA

CTAATTCAAT

GTGTTGTTAA

CTCATCATGA

CAACTATCAC

TTAGCTGTGC

TATTATCAAC

CTCGCGCACT

CACCTGTTGA

CATCATATCT

ATCTCTAAAC CAAGTTACCC CTTTCATAAG GTTTACTCAA TTAACCGCTA ATCAAGCGAT 600

TTTAGAAGCG

CGGGGTTCAA

TCGAATCACC

TAAATTTGCT

TAACCACGAT

AACCCTAGCT

AAGGATTTTT

GGAAGCAAAT

TACAGCTGTG

AGTTGAACAA

AAGGAAAGAA

TAGTAATGTT

TTATCCTTCT

AAATCAACCC

TCAGAGGGTA

AAAACCCTAA

GAACAATATT

TGTTTTAAAA

GTCTTTGTAT

ATTAACGGTA

TGGCCACCGT

GGTACTGGAA

CACTCATTAG

CACGATGAAG

ATCAACTGTG

TTGCATAGGG

CATAACCATC

TTTGATTCAC

GTGTGGTTTG

AGACATGAAA

GCTTTAAGCC

GAAGGCTATC

CTTTTCTCCA

ATTAAGACTA

ATAACCAGAT

GAAGAGGTAT

TTTTTAACAT

AACGCAAGAT

ATCATCAAGC

TAATGCAAGC

ATGACCTTGA

GGTTGAGATT

ATCCTTCTAT

TTTTCTACCT

TTAAGTCAAT

CTCTTTTTTT

TGGAAGCTAC

GGAGAGAGAT

GGTTTAGATC

CTTTTGCATT

AACTCGGAGT

TCTCGTCTTG

CTGATAGTTT

TTTCTAATGA

TGAAATTTCA

AGAGGACTAG

CTTGATCAAC

24

AATCCACATC

ACTAGCTGAT

TACCCTTCGT

TCAATTCCAT

TATTTCCTCC

CCACCGCCTT

GAACCCTAAA

ACAAAGATTC

ATTGCCACCG

TGTTGATATC

ATGGGAAGTT

ATCACAAGCT

TTCGAGTAAT

GCGT TGAGAA

AGGAGGGATC

AGTTGTAGTA

TGTTTTTTTT

GTTGATGATA

TTATTTTTAT

GTTGATTTCG

CGTTACCCTG

AGAACAGGTG

CCTCTTTATA

ATTGTACTAC

TTAAAAGACC

ATTGTTACAA

ATCGAGGCGT

GGTAGTCGAG

GTTGCGATGG

ATGTTGAGGA

AAGCTTCTTT

TCTTTCTTCT

AAACTATCAA

TGAAGAAAAC

GTAGAAATTT

GTTTTACTTA

TATAGTATTT

TTTTAATTA

ACATTAATCA

CTCCCACTCT

ATCGTTTAGC

TAGCCAATAA

TCCCTGATGA

GCGAAAACTT

TCGCGGAGAA

TGGATTATTA

AGAGGATGAC

AAGGAGATAA

GTTGTGGATT

TGAGACTTCA

TAGGTTGGCA

ATAGCCAACT

GCGTGGAGTG

GCATGGTGAA

TTGATATGAA

AAGTTAACTA

660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1729 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 428 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Protein vi) ORIGINAL SOURCE: ORGANISM: Lycopersicon escuientum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Leu Giy Ser Phe Gly Ser Ser Ser Ser Gin 1 5 10 Asp Glu Glu Ser Ser Asp His His Gin Gin Arg Ser His Pro His His Ala Thr Thr Ile Thr Thr Thr Thr Ile Arg Gin Leu Leu Ile Ser Cys 55 25 Ile Arg Phe Thr Ala Thr Ser Pro Ala Ile Gin Thr Thr Ala Glu Leu Ile Gin Ser Asp Leu Leu Thr Ile Leu Ser Thr Asn Ser Ser Phe Ser Ala Ala Lys Arg 70 Pro Leu Met Leu Phe 145 Ile His Pro Leu Leu 225 His Glu Asp Pro Leu 305 Phe Thr Met Glu Phe Ser I Thr Ala 130 Ile Asn Gly Ala Arg 210 Arg Asp Thr Arg Lys 290 Phe Asp Val Glu Val Gly Leu Pro 115 Leu Arg Gly Val Pro 195 Arg Phe Glu Leu Glu 275 Ile Leu Ser Glu Gly 355 Met Asp Arg 100 Val Ile Phe Asn Gin 180 Thr Thr Gin Asp Ala 260 Lys Val Gin Leu Gin 340 SAsp SLeu Ser Leu Glu Gin Thr His 165 Trp Leu Gly Phe Pro 245 Ile Leu Thr Arg Glu 325 Val Lys Arc Thr Asn Thr Ser Gin 150 Gin Pro Arg Asp His 230 Ser Asn Arg Ile Phe 310 Ala Trp SArc Ser Glu Arg Thr Ser 135 Leu Ala Pro Ile Arg 215 Pro Ile Cys Ile Ala 295 Ile Thr Phe Lys SCys 375 Arg Tyr Pro 120 Tyr Thr Ile Leu Thr 200 Leu Leu Ile Val Phe 280 Glu Glu Leu Gly Glu 360 GlI Leu Ile 105 Thr Leu Ala His Met 185 Gly Ala Tyr Ser Phe 265 Leu Lys Ala Pro SArg 345 Arg Phe Val 90 Ser Asp Ser Asn Ile 170 Gin Thr Lys Ile Ser 250 Tyr His Glu Leu Pro 330 Glu His Ser His C Ser I Ser Leu Gin 1 155 Val Ala Gly Phe Ala 235 Ile Leu Arg Ala Asp 315 Gly Ile Glu Asn ;ln 'hr Ser Asn Ala Asp Leu Asn Ala 220 Asn Val His Val Asn 300 Tyr Ser Val Arg Val 380 Phe Thr Ser 125 Gin Ile Phe Ala Asp 205 His Asn Leu Arg Lys 285 His Tyr Arg Asp Phe 365 Al Thr I Asn 110 Ser Val Leu Asp Asp 190 Leu Ser Asn Leu Leu 270 Ser Asn Thr Glu Ile 350 SArg i Leu Arg His Ser Thr Glu Ile 175 Arg Asp Leu His Pro 255 Leu Met His Ala Arg 335 Val Ser Ser Ala Phe Ser Pro Ala 160 Asn Tyr Thr Gly Asp 240 Asp Lys Asn Pro Val 320 Met Ala STrp SPro v 370 Phe 385 Ala Leu Ser Gin Ala 390 Lys Leu Leu Leu Leu His Tyr Pro Ser 400 26 Glu Gly Tyr Gin Leu Gly Val Ser Ser Asn Ser Phe Phe Leu Gly Trp 405 410 415 Gin Asn Gin Pro Leu Phe Ser Ile Ser Ser Trp Arg 420 425 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 20 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:-3: TTAGGGTTTT CACTCCACGC INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 22 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID b TCCCCTTTTT TTCCTTTCTC TC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TTTCCCACTC AAGCCAACTC INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 20 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear O: 4: NO: (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGTGGCAATG TAGCTTCCAG INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 22 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:-7: TCGAGGCGTT GGATTATTAT AC 22 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 19 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGCCCCCATA TCTTTTTCC 19 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 1296 Base pairs TYPE: Nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETIC: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGTTAGGAT CCTTTGGTTC TTCATCATCT CAATCTCACC CTCATCATGA TGAAGAATCT TCTGATCATC ATCAACGGCG TAGATTCACC GCTACTACTA CAACTATCAC CACCACCACC 120

ACAACGACCT

TCGCGGTCCG

CCTTTTGGTG

CTCAACCGCT

ACTGATTCTT

TCTCTAAATC

TTAGAAGCGA

GGGGTTCAAT

CGAATCACCG

AAATTTGCTC

AACCGCGATC

ACCCTAGCTA

AGGATTTTTT

GAAGCAAATC

ACAGCTGTGT

GTTGAACAAG

AGGAAAGAAA

AGTAATGTTG

TATCCTTCTG

AATCAACCTC

CACCAGCTAT

ATTTCTCGGC

ATTCAACTGA

ATATATCGTC

CATCTTCGTT

AAGTTACCCC

TTAACGGTAA

GGCCACCGTT

GTACTGGAAA

ACTCATTAGG

ACGGTGAAGA

TCAACTGTGT

TGCATAGGGT

ATAACCATCC

TTGATTCATT

TGTGGTTTGG

GACATGAAAG

CTTTAAGCCC

AAGGCTATCA

TTTTCTCCAT

TCAAATCCGC

CGCGAAAAGA

ACGGTTAGTC

AACCACCAAT

GCCATCGTCA

TTTTATAAGG

TCATCAAGCA

AATGCAAGCA

TGACCTTGAT

GTTGAGATTT

TCCTTCTATT

TTTC TAT CT C

TAAGTCAATG

TCTTTTTTTA

GGAAGCTACA

GAGAGAAATT

GTTTAGATCA

TTTTGCATTA

ACTCGGAGTT

CTCGTCTTGG

28

CAGCTACTCA

CTCCTTACCA

CATCAGTTTA

CAT TTCATGA

TCATTAGCTC

TTTACTCAAT

ATCCACATCG

CTAGCTGATC

ACCCTTCGTA

CAATTCCATC

ATTTCCTCCA

CACCGCCTTT

AACCCTAAAA

CAAAGATTTA

TTGCCACCGG

GTTGATATCG

TGGGAAGTTA

TCACAAGCTA

TCGAGTAATT

CGTTGA

TTAGCTGTGC

TATTATCAAC

CTCGCGCACT

CACCTGTTGA

TAATTCAATC

TAACCGCTAA

TTGATTTCGA

GTTACCCTGC

GAACAGGTGA

CTCTTTATAT

TTGTACTTCT

TAAAAGACCG

TTGTTACAAT

TCGAGGCGTT

GTAGTCGTGA

TGGCGATGGA

TGTTGAGGAG

AGCTTCTTTT

CTTTCTTCTT

GGAGTTGATT

TAACTCTTCT

TTCCCTTCGT

AACAACTCCA

ATCATATCAT

TCAAGCGATT

CATTAATCAC

TCCTACTCTT

TCGTTTAGCT

CGCCAATAAT

CCCTGATGAA

CGAAAAATTA

CGCGGAGAAG

GGATTATTAT

GAGGATGACA

AGGAGATAAA

TTGTGGATTT

GAGACTACAT

AGGTTGGCAA

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1296 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 431 amino acids TYPE: amino acid- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: ORGANISM: Solanun tuberosun (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Leu Gly Ser Phe Gly Ser Ser Ser Ser Gin Ser His Pro His His 1 5 10 Asp Glu Glu Ser Ser Asp His His Gln Arg Arg Arq Phe Thr Ala Thr 25 Thr Thr Thr Ile Thr Thr Thr Thr Thr Thr Thr Ser Pro Ala Ile Gin 40 Ile Arg C Phe Ser Pro Phe Leu Ser Met Thr Ser Ser 130 Val Thr 145 Leu Glu Asp Ile Asp Arg Leu Asp 210 Ser Leu 225 Asn Arg Leu Pro Leu Leu Ser Met 290 Asn His 305 Thr Ala Glu Arg Ile Val Arg Ser 370 ;ln Ala Gly Leu Pro 115 Ser Pro Ala Asn Tyr 195 Thr Gly Asp Asp Lys 275 Asn Pro Val Met Ala 355 Trp Leu Ala Asp Arg 100 Val Leu Phe Ile His 180 Pro Leu Leu His Glu 260 Asp Pro Leu Phe Thr 340 Met Glu Leu Lys Ser Leu Glu Ala Ile Asn 165 Gly Ala Arg Arg Gly 245 Thr Arg Lys SPhe Asp 325 Val Glu SVal Ile S Arg I 70 Thr C Asn Thr Leu Arg 150 Gly Val Pro Arg Phe 230 Glu Leu Glu Ile Leu 310 Ser Glu SGly SMet Leu 390 ier C i5 Leu I ;lu Arg Thr Ile 135 Phe Asn Gln Thr Thr 215 Gin Asp Ala Lys Val 295 Gin Leu Gin Asp Leu 375 ;ys Leu Arg Tyr Pro 12C Glr Thr His Tr Lei 20( Gl1 Phi Pr Il Le 28 Th Ar Gl Va Ly 36 Ar 29 Ala C Thr Leu Ile 105 Thr 1 Ser SGn 3 Gn p Pro 185 u Arg 0 y Asp e His 3 Ser e Asn 265 u Arg 0 r Ile g Phe u Ala 1 Trp 345 s Arg ;0 g Ser ;lu lie Val 90 Ser Asp Ser Leu Ala 170 Pro Ile Arg Pro Ile 250 Cys Ile Ala Ile Thr 33C Phe Lys Cy; Leu Leu 75 His Ser Ser Tyr Thr 155 Ile Leu Thr Leu Leu 235 Ile Val SPhe Glu SGlu 315 SLeu e Gly 3 Glu s Gly s Leu 395 :le Ser G1n Thr Ser His 140 Ala His Met Gly Ala 220 Tyr Ser Phe Leu Lys 300 Ala Pro Arg Arg Phe 380 Ser Thr Phe Thr Ser 125 Ser Asn Ile Gin Thr 205 Lys Ile Ser Tyr His 285 Glu Leu Pro Glu His 365 Ser Arg Asn Thr Asn 110 Ser Leu Gin Val Ala 190 Gly Phe Ala Ile Leu 270 Arg Ala Asp Gly Ile 350 Glu Asn Ser Ser Arg His Leu Asn Ala Asp 175 Leu Asn Ala Asn Val 255 His Val Asn Tyr Ser 335 Val Arg Val Asp Ser Ala Phe Pro Gin Ile 160 Phe Ala Asp His Asn 240 Leu Arg Lys His Tyr 320 Arg Asp Phe Ala His 400 Ser Pro Phe Ala Ser Gin Ala Ly Leu Leu Arg Leu Tyr Pro Ser Glu Gly Tyr Gin Leu Gly Val Ser Ser Asn Ser Phe Phe 405 410 415 Leu Gly Trp Gin Asn Gin Pro Leu Phe Ser Ile Ser Ser Trp Arg 420 425 430 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 20 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CARTGGCCNC CNYTNATGCA INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 20 Base pairs TYPE: Nucleotide STRANDEDNESS:single TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TGRTTYTGCC ANCCNARRAA INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 687 Base pairs TYPE: Nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETIC: NO (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAGAGGTCAT CAAACCCTAG CAGTCCACCT CCATCTCTCC GCATAACCGG ATGCGGTCGA GATGTAACCG GATTAAACCG AACTGGAGAC CGGTTAACCC GGTTCGCTGA CTCTTTAGGT 120 CTCCAATTCC AGTTTCACAC GCTAGTGATC GTAGAAGAAG ATCTCGCCGG ACTTTTGCTA 180 ^1\ 'T I-Li 'Ar n^

CAGATCCGAT

CACTTCCTCC

ATCAAGAGCT

CACTCGTTCT

TTGGAAGCGA

GGTAAGGAGA

AGGTTTGAGA

AGCTTTGCTT

AATCTTCAGT

TGTTAGCTCT

ACAAAATATT

TAAACTCTAG

TGAATAGATT

CGTTGCCGCC

TTTTGGATGT

TTTGGGAAGA

TGTCTCAAGC

TCCTTAACA.A

CTCAGCCGTA

TAACGACGAT

AATCGTTACA

CTCTGAGGCA

AAATAGCCGA

TGTGGCGGCG

GATGATGAAG

TAAGCTTCTT

TTCTTTG

CAAGGAGAGA

GGAGATATGA

ATGGCAGAGA

GTGGATCATT

GAGAGACTAA

GAAGAGACGG

AGGTTTGGTT

CTTAGACTTC

CCATTGCCGT

TCGGTCACTT

GAGAAGCTAA

ACATGGCGAT

CCCTAGAGCA

AGAGAAAGCA

TCGTTAACGT

ATTATCCTTC

CAATTGTGTT

CTTGTCAGCG

TCATGGAGAT

CTTTGATTCG

ACGGTGGTTC

AAGACATCGG

TCCTATTGGA

AGAAGGT TAT 240 300 360 420 480 540 600 660 687 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 229 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Giu 1 Arg Ser Ser Asn Pro Ser Ser Pro Pro Ser Leu Arg Ile Thr Giy Cys Gly Arg Thr Arg Phe Ala Val Ile Val Glu Asp Val Thr Gly Leu 25 Leu Arg Thr Gly His Thr Leu Asp Ser Leu Gly 40 Gin Phe Gin Phe Giu Asp Ala Leu 55 Gin Ala Gly Leu Leu Leu Gin Ile Arg Leu Leu His Leu Ser Ala Gly Glu Thr I le 75 Gly Ala Vai Asn Cys Val1 Phe Leu His Ile Phe Asn Lys Ser Leu Asp Asp Asp Met Ile Gly His Phe Leu Ser Glu Arg Giu 115 Glu Ala Val 130 Leu Pro Pro 145 Asn His Gly Asp 120 Ala Asn Ser Arg Ile Val Thr Met Ala 105 110 His Ser Phe Leu Asn Arg Phe Ser 125 Ile Phe Asp Ser Leu Giu Ala Thr 140 Leu Thr Leu Glu Gin Arg Trp Phe 155 160 Asp His Tyr Met 135 Asn Ser Arg Glu Arg 150 32 Giy Lys Glu Ile Leu Asp Vai Vai Ala Ala Giu Giu Thr Giu Arg Lys 165 170 175 Gin Arg His Arg Arg Phe Giu Ile Trp Glu Giu Met Met Lys Arg Phe 180 185 190 Gly Phe Val Asn Val Pro Ile Gly Ser Phe Ala Leu Ser Gin Ala Lys 195 200 205 Leu Leu Leu Arg Leu His Tyr Pro Ser Glu Gly Tyr Asn Leu Gin Phe 210 215 220 Leu Asn Asn Ser Leu 225

Claims (13)

1. A nucleotide sequence according to SEQ ID NO: 1, 9 or 13 which is responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation, the fragment or derivative thereof or a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence according to SEQ ID NO: 1, 9 or 13 and which is responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation.

2. A nucleotide sequence as illustrated in SEQ ID NO: 1,9 or 13.

3. A polypeptide having an amino acid sequence as illustrated in SEQ ID NO: 2, 10 or 14. 15 4. A vector comprising a nucleotide sequence according to any one of claims 1 to 2. A transformed plant cell or transformed plant tissue, wherein an expressible DNA sequence responsible for controlling side-shoot formation .:ooo e S 20 and/or petal formation and/or abscission zone formation, or fragment or derivative thereof according to claim 1 is integrated in a stable manner into the genome of the plant cell or the plant tissue.

6. A plant cell or plant tissue according to claim 5, which may be regenerated into a seed producing plant.

7. A method for the preparation of plants having controlled side- shoot formation and/or petal formation and/or abscission zone formation comprising stable integration of a least one expressible DNA sequence responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation or fragment or derivative thereof according to claim 1 into the genome of plant cells or plant tissues and regeneration of the resulting plant cells or plant tissues into plants. X:\Elisabeth\PJC\NODELETE\82060-98.doc 34

8. A method according to claim 7, wherein for integration a DNA sequence or fragment or derivative thereof is used which suppresses side-shoot formation and/or petal formation and/or abscission zone formation.

9. A method according to claim 8, wherein the integrated DNA sequence or fragment or derivative thereof is expressed in an antisense orientation relative to the endogenous sequence responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation.

10. A method according to claim 8, wherein the integrated DNA sequence or fragment or derivative thereof is expressed in a sense orientation relative to the endogenous sequence responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation. 15 11. A method according to claim 8, wherein the side-shoot formation and/or petal formation and/or abscission zone formation is suppressed by a ribozyme comprising the integrated DNA sequence or fragment or derivative thereof. S 20 12. A method according to claim 8, wherein the DNA sequence or fragment or derivative thereof is integrated into the genomic region of the homologous endogenous gene by homologous recombination.

13. A method according to claim 7, wherein for integration a DNA sequence or fragment or derivative thereof is used which enhances side-shoot formation and/or petal formation and/or abscission zone formation.

14. A method according to claim 13, wherein the integrated DNA sequence or fragment or derivative thereof is expressed in a sense orientation relative to the endogenous sequence responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation. A method according to any one of claims 7 to 14, wherein as a plant a tomato plant, a rape plant, a potato plant or a snapdragon plant or the cell or tissue thereof is used.

16. A plant obtained according to any one of claims 7

17. Seed stocks obtained from plants according to claim 16 wherein the seed incorporates a transgene responsible for controlling side-shoot formation and/or petal formation and/or abscission zone formation.

18. A nucleotide sequence according to claim 1 substantially as hereinbefore described with reference to any one of examples 1-3, 5, 7-9. 1 19. A method for the preparation of plants according to claim 7 15 substantially as hereinbefore described with reference to example 6. DATED: 19 September, 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: NIKOLAUS THERES :\Elisabeth\PJC\NODELETE\82060-98.doc