AU3465597A - Process of producing transgenic plants in which flowering is inhibited, and dna sequences used in said process - Google Patents

Description

WO99/04003 PCT/NL97/00424 1 Process of producing transgenic plants in which flowering is inhibited, and DNA sequences used in said process. 5 FIELD OF THE INVENTION The present invention is related to recombinant DNA, particularly to recombinant DNA in relation to genetic modification of plants. The modification relates to altering 10 the flowering process of plants and specifically to inhibiting flowering in genetically engineered plants. The present invention also relates to DNA sequences which code for MADS box transcription factors, which, upon integration into a plant genome, modify flowering. 15 BACKGROUND OF THE INVENTION Plant development is characterised by a series of phase changes in which meristems are capable of generating new 20 meristems of different identity. Vegetative meristems produce roots and leaves and may give rise to generative meristems upon floral induction (evocation). In many Angiosperms, the switch from vegetative to generative phase is induced in response to many environmental conditions such as temperature, 25 light conditions, and day-length. In addition, the process of flower induction is influenced by internal factors such as the age of the plant, hormones and gene products (Bernier, 1988). Almost nothing is known, however, about the molecular and genetic controls that induce a plant to flower. 30 Following flower induction the newly formed inflorescence meristem forms flowers, whereas vegetative meristems produce leaves. The new flower meristems are formed in the axils of small leaf-like organs which are called bracts. These bracts are distinguishable from vegetative leaves with respect to 35 their shape and position in the plant. The next developmental step is the determination of the identity of the floral organs, sepals, petals, stamens and carpels, which develop as WO99/04003 PCT/NL97/00424 2 distinct primordia from the floral apex. In the stamens and carpels, the reproductive cells are produced and upon fertilisation seeds are formed giving rise to the next generation. 5 Mutants affected in the flowering process have been characterised from many species, in particular, from Arabidopsis thaliana. A number of Arabidopsis mutants are known displaying an early or late flowering phenotype (see for review, Weigel, 1995). Although the transition from vegetative 10 to reproductive phase can be delayed dramatically in some of these late flowering mutants, eventually they start to flower, indicating that the affected genes are not essential for flowering. Many of these early- and late-flowering genes are thought to modify the transduction of the external factors 15 which are involved in flower induction. The late-flowering gene CONSTANS (CO) which encodes a Zinc finger transcription factor (Putterill et al, 1995 ) is involved in light perception or transduction and, upon mutation, results in a late flowering phenotype specifically under short-day 20 conditions. Another Arabidopsis gene that affects flowering time and have been cloned is LUMINIDEPENDENS (Lee et al., 1994). A gene that is required for floral induction is the INDETERMINATE (ID) gene from maize (patent application WO 25 96/34088). This ID gene encodes a Zinc finger transcription factor and disturbance of this gene by insertion of a DS transposable element resulted in the inhibition of flowering in maize plants. To induce flowering, the so-called 'meristem identity' 30 genes that control the identity of the flower meristem can also be used. These genes are LEAFY (LFY, Weigel et al., 1992) and APETALA1 (AP1, Mandel et al., 1992) from Arabidopsis and their homologues from Antirrhinum FLORICAULA (FLO, Coen et al., 1990) and SQUAMOSA (SQUA, Huijser et al., 1992), 35 respectively. Overexpression of either LFY (Weigel and Nilsson, 1995; patent application WO 96/19105) or AP1 (Mandel and Yanofsky, 1995) resulted in early flowering in WO99/04003 PCT/NL97/00424 3 Arabidopsis. Similarly, earlier flowering Aspen were generated by expression of the Arabidopsis LFY gene under the control of the CaMV 35S promoter (Weigel and Nilsson, 1995). When either of these 'meristem identity' genes is 5 inactivated by mutations, structures resembling inflorescences are formed instead of flowers. In LFY, SQUA and FLO mutants, long indeterminate inflorescences are formed which are characterised by inflorescence shoots producing a series of bract-like leaves. The petunia mutant Aberrant leaf and flower 10 (alf) displays a similar phenotype (Gerats, 1988). In these mutants, the developmental switch from vegetative to reproductive phase (evocation) has occurred, but the subsequent transition to a floral meristem is blocked. Another strategy to inhibit flower formation has been 15 described by Landschttze et al (1995; patent application: WO 95/24487). They reported that inhibition of mitochondrial citrate synthase in transgenic potato plants resulted in a distortion of flower formation. Flower buds were formed later or were aborted at an early stage of development. 20 Nevertheless, these plants with a reduced citrate synthase level produce inflorescences. API and SQUA are both members of the MADS box gene family coding for transcription factors. These MADS box transcription factors share a highly conserved domain which is called the 25 'MADS box' and facilitates the DNA binding (Schwarz-Sommer et al., 1990). The identity of the floral organs, sepals, petals, stamens, carpels, and within the carpels, the ovules is determined by four classes of homeotic genes. These homeotic 30 genes are acting alone or in combination to determine floral organ identity. A model describing the action of these genes has been proposed first by Coen and Meyerowitz (1991) and later modified for genes controlling ovule identity by Colombo et al. (1995). Most of these organ identity genes belong to 35 the MADS box gene family. The function of these genes has been determined in a number of dicotyledon and monocotyledon species by gene inactivation and ectopic expression in WO99/04003 PCT/NL97/00424 4 transgenic plants. These studies have demonstrated that these homeotic genes are well conserved in the plant kingdom and play similar roles with respect to flower development in many species. 5 DEFINITIONS Flowering: A process in which the vegetative phase changes into the reproductive phase. This process is also known as floral induction or evocation. The reproductive phase is 10 characterised by the production of flowers which is often preceded by the formation of an inflorescence. Inflorescence: An inflorescence is characterised by a structure bearing the flowers which arises from the axils of leaf-like organs called 'bracts'. 15 Antisense construct or gene: a gene or a nucleotide sequence derived thereof, having a homology of more than 70%, preferably more than 90% to a target gene and which is linked to a promoter in the inverse 3' to 5' orientation with respect to the target gene. 20 Cosuppression construct or gene: a gene or a nucleotide sequence derived thereof, having a homology of more than 70%, preferably more than 90% to the target gene and which is linked to a promoter in the 5' to 3' orientation or which expression is not driven by an exogenous promoter. 25 MADS box gene: a gene coding for a transcription factor having a region of 56 amino acids which is homologous to a similar region in the Arabidopsis AGAMOUS protein and Antirrhinum DEFICIENS protein. This region is called the 'MADS box'. At least 50% of the amino acids in this region should be 30 identical to the amino acid composition in the MADS boxes of AGAMOUS and DEFICIENS. SUMMARY OF THE INVENTION 35 Inhibiting flower formation can be advantageous in plants which multiply in predominantly vegetative manner. Flowers are strong sink tissues and therefore, prevention of flowering may WO99/04003 PCT/NL97/00424 5 lead to an increased deposition of stored substances in storage organs and increased growth of vegetative organs such as leaves, stems and roots. It therefore appears desirable to provide a method to inhibit flowering independent from 5 external factors and without exogenous application of substances. The present invention provides a method to produce genetically engineered plants in which the flowering process is altered. More specifically, the inventors have found a 10 method for reducing flowering or completely abolishing the formation of inflorescences and flowers. It was found that inhibition of the petunia MADS box gene FBPO10 resulted in transgenic petunia plants in which flowering was inhibited. Moreover, it has been found that the said 15 genetically modified plants produce long indeterminate vegetative shoots bearing leaves and eventually becoming much taller than non-transformed plants. Within the scope of the present invention, inhibiting flowering means that the transformed plants do not produce 20 inflorescences, neither inflorescences bearing flowers nor inflorescences without flowers, develop fewer inflorescences or short inflorescences with a single or a few flowers. Accordingly, this invention provides an isolated DNA sequence which encodes a MADS box protein indicated FBP10 and 25 having the amino acid sequence given in SEQ ID NO:2 of the sequence listing hereinafter or a functionally homologous protein or part thereof, wherein inactivation of gene function of said DNA sequence, if present as an endogenous gene in a plant, results in inhibition of flowering. 30 The DNA sequence of the invention can also be characterized in that it comprises the FBP10 gene or corresponding cDNA having the nucleotide sequence given in SEQ ID NO:1 or a functionally homologous gene or an essentially identical nucleotide sequence or part thereof or derivatives 35 thereof which are derived from said sequence by insertion, deletion or substitution of one or more nucleotides. In another aspect, the invention provides an RNA sequence WO99/04003 PCT/NL97/00424 6 encoded by any of the above defined DNA sequences. In a further aspect, the invention provides a protein encoded by any of the above defined DNA sequences. 5 Further the invention provides processes of producing transgenic plants in which flowering is inhibited, comprising inhibiting the expression of endogenous FBPO10 gene or a homologous gene. Still further, the invention provides recombinant double 10 stranded DNA molecules comprising an expression cassette to be used in the above process. Finally, the invention provides transgenic plants showing inhibited flowering, and also plant cells, seeds, tissue culture, plant parts or progeny plants derived from said 15 transgenic plants. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the cDNA sequence of the petunia FBP10 gene and 20 the deduced amino acid sequence. The MADS box region is underlined. Figure 2 is a comparison of the petunia FBP10 protein sequence and known homologous protein sequences from potato (POTM1), tomato (TDR4), and Arabidopsis (AGL8). The overall amino acid 25 sequence, the MADS box and the K-box sequences have been compared. Figure 3 shows the expression of FBP10 in wild type petunia plants (line W115) and in FBP10 plants as determined by Northern blot analysis. The blot contains total RNA from the 30 tissues as indicated: leaf, stem, bract, sepal, petal, stamen, style/stigma, ovary and leaf, stem, shoot and flower from the cosuppression plant.The Smal/HindIII DNA fragment (nucleotide 325-885) of FBPO0 cDNA was used as probe. Figure 4 shows the expression of FBP10 by in situ 35 hybridisations using a digoxygenin labeled antisense RNA probe (Figures 4B, 4D, and 4F) derived from the Smal/HindIII fragment (nucleotide 325-885) of FBP10 cDNA. Sense control WO99/04003 PCT/NL97/00424 7 probe has been used for figures 4A, 4C, and 4E. Figures A and B show longitudinal sections through an apical meristem, C and D show an axillary vegetative meristem and in E and F floral buds are depicted. a=apical mersitem, am=axillary meristem, 5 l=leaf, b=bract, f=floral meristem, l=sepal, 2=petal, 3=stamen. Figure 5 depicts a schematic presentation of the T-DNA region between the borders of the binary vector containing the FBP10 sense construct. This binary vector, designated pFBP113, was 10 used to generate transgenic petunia plants in which FBP10 expression was inhibited. Figure 6 shows the phenotype of a wild type petunia plant (a left) and representative plants in which the expression of FBP10 was inhibited (a right and b). 15 DETAILED DESCRIPTION OF THE INVENTION The DNA sequence of the invention encodes a MADS box protein indicated FBP10 or a fucntionally homologous protein 20 or part thereof. The cDNA sequence of the FBP10 gene and the deduced amino acid sequence are given in Fig. 1 and in SEQ ID No:l and 2. The term "functionally homologous" in the present description and claims should be understood to mean proteins and genes belonging to the MADS box family and being 25 functionally equivalent to FBP10 protein and FBP10 gene, respectively. Inhibition of the endogenous FBP10 gene in a plant results in inhibition of flowering. Homologous sequences are, for example, sequences derived from other organisms than petunia. The percentage of sequence similarity of FBPO10 and a 30 homologous gene may vary. The functionally homologous gene from a species related to petunia may have more than 90% sequence identity, whereas the functional homologue from a non-related species may have only 65% or less sequence identity. 35 The invention also relates to derivatives of the FBP10 gene or a homologous gene, which can be derived from the parent sequence by insertion, deletion or substitution of one WO99/04003 PCT/NL97/00424 8 or more nucleotides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or recombination. In the process of producing a transgenic plant in which 5 flowering is inhibited, the expression of endogenous FBPO10 gene or homologous gene is inhibited. Said inhibition can be effected in several ways. In one embodiment the expression of endogenous FBPIO gene or a homologous gene is inhibited by the use of anti-sense RNA. The 10 process comprises the following steps: a) a DNA, or a DNA fragment having at least 15 base pairs, which is complementary to a degree of at least 70% to the FBP10 gene or homologous gene is introduced in a plant cell, 15 b) said introduced DNA is transcribed into anti-sense RNA, said DNA being expressed constitutively or tissue specific, or being induced by promoter elements controlling the expression of the introduced DNA, c) the expression of endogenous FBP10 gene or homologous 20 gene is inhibited because of the anti-sense effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected. As appears from step a) instead of the complete FBP10 25 gene or homologus gene sequence, partial sequences thereof can be used to obtain antisense inhibition. Sequences up to a minimum length of 15 base pairs can be used. It is also possible to use DNA sequences which have a high degree of similarity to the FBP10 gene or homologous gene sequence. The 30 minimum similarity should be 70%, preferably more than 90%. In a second embodiment the expression of endogenous FBP10 or a homologous gene is inhibited through the use of sense/cosuppression. The process comprises the following steps: 35 a) a DNA which is the FBPO10 gene or homologous gene or a sequence having at least 70% sequence similarity to said FBP10 or homologous gene, or a fragment thereof having at WO99/04003 PCT/NL97/00424 9 least 15 base pairs, is introduced in a plant cell, b) said DNA being expressed constitutively or tissue specific or being induced by promoter elements controlling the expression of the introduced DNA in such 5 a way that transcription produces sense RNA, or being introduced without the use of a promoter, c) the expression of endogenous FBP10 gene or homologous gene and the introduced gene are inhibited by the co suppression effect, 10 d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected. As in the antisense method, partial sequences of at least 15 base pairs, and sequences having a similarity of at least 15 70%, preferably at least 90%, can be used. In a third embodiment the expression of endogenous FBP10 or a homologous gene is inhibited by the use of dominant negative mutations. The process comprises the following steps: 20 a) a DNA which encodes a modified FBP10 protein or homologous protein or an essential part thereof, whereby said modified protein or part thereof is suitable for inhibiting the function of the endogenous FBP10 protein or homologous protein, is introduced in a plant cell, 25 b) said introduced DNA is transcribed into RNA and translated into a polypeptide, said DNA being expressed constitutively or tissue specific, or being induced by promoter elements controlling the expression of the introduced DNA, 30 c) the function of the endogenous FBP10 protein or homologous protein is inhibited by the dominant-negative mutation effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected. 35 The transgenic plants produced by this process express an altered FBP10 protein or homologue thereof. As a result said WO99/04003 PCT/NL97/00424 10 plants are changed in their flowering behaviour. This strategy has been successful for the inhibition of the Arabidopsis MADS box protein AGAMOUS by expressing a c-terminal truncated AGAMOUS protein (Ma et al, 1996). 5 The invention also relates to recombinant double-stranded DNA molecules for use in the above processes for producing transgenic plants. Accordingly, in a first embodiment the invention provides a recombinant double-stranded DNA molecule for use in the 10 anti-sense method, comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is FBP10 gene or a homologous gene as defined above, which is fused to the 15 promoter in anti-sense orientation so that the non coding strand is transcribed, and if necessary iii) a signal fucntional in plants for the transcription termination and polyadenylation of an RNA molecule. 20 In a second embodiment the invention provides a recombinant double-stranded DNA molecule for use in the sense/cosuppression method, comprising an expression cassette comprising the following constituents: i) a DNA sequence which is FBP10 gene or a homologous gene 25 as defined above, ii) optionally a promoter functional in plants, which is fused to the DNA sequence in sense orientation, and if necessary iii) a signal functional in plants for the transcription 30 termination and polyadenylation of an RNA molecule. In a third embodiment the invention provides a recombinant double-stranded DNA-molecule for use in the dominant-negative mutation method, comprising an 35 expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is a modified FBP10 gene or WO99/04003 PCT/NL97/00424 11 homologous gene as defined above, which is fused to the promoter in sense orientation, and if necessary iii) a signal functional in plants for the transcription termination and polyadenylation of an RNA molecule. 5 A preferred promoter to be used in any of the above processes or recombinant double-stranded DNA molecules for expressing the said recombinant polynucleotide that is active in the shoot apical meristems comprises the cauliflower mosaic 10 virus (CaMV) 35S promoter. In another preferred embodiment, the promoter is an inducible promoter active in the shoot meristem or a tissue-specific promoter active in the shoot meristem. In another preferred embodiment, an inducible promoter is 15 used which can be activated or suppressed in response to external stimuli. Activation or suppression of the promoter results in plants displaying characteristics according to the present invention. The present invention provides plants without 20 inflorescences or flowers. Because flowers and seeds and fruits produced thereof are high energy demanding tissues ('sink-tissue'), prevention of flowering saves energy that can be used for the formation of vegetative organs or deposition of stored substances in storage organs. Therefore, the present 25 invention provides a method to increase the total biomass of vegetative tissues, such as roots, tubers, stems and leaves. The present invention can be used in Gymnosperms and Angiosperms. The present invention is especially useful for plant species for which vegetative propagation is possible. 30 In another preferred embodiment, parent plants carrying transgenes related to the DNA sequence shown in figure 1 (SEQ ID NO:1) are crossed resulting in progeny plants which display characteristics of the present invention. The parent plants have a wild-type phenotype. 35 The present invention is especially useful for plant species for which the vegetative part of the plant is used as the economical product such as vegetables (e.g. lettuce, WO99/04003 PCT/NL97/00424 12 spinach, chicory, etc.), sugar beet, potato, trees for wood production (e.g. Eucalyptus. oak, willow, etc), tobacco, grasses, plants for nitrogen fixation, ornamental plants for production of cuttings. 5 The use of the present invention is of particular interest to sugar beet, since "bolting" can be prevented by inhibition of inflorescence formation. This flower induction process is induced by vernalization (cold treatment) and can be circumvented by planting relatively late in the year 10 (April/May). Inhibition of inflorescence formation by applying the present invention will allow an earlier planting of the sugar beet resulting in an increase in yield. The use of the present invention is of particular interest for grasses to improve its quality as feed for 15 cattle. Inflorescences of grasses contain relatively low quantities of carbohydrates and large quantitives of lignin, which cannot be digested by cattle. EXAMPLES 20 Example 1: Isolation of FBP10 cDNA Petunia MADS box cDNA clones were isolated from a cDNA library made from young petunia pistils (Angenent et al., 25 1993) The cDNA library was constructed using the lambda ZAP cloning vector (Stratagene) The library was screened under low stringency hybridization conditions with a mixed probe comprising the MADS box regions of Floral binding protein gene I(FBP1) and FBP2 (Angenent et al., 1993). The hybridizing 30 phage plaques were further purified using standard techniques. Using the in vivo excision method, E.coli clones which contain a double-stranded Bluescript SK-plasmid with the cDNA insertion between the EcoRl and Xhol cleavage site of the polylinker were generated. Cross-hybridization of the purified 35 clones revealed 10 independent clones designated FBP2, FBP6 14. One of the clones obtained was designated FBP10. The nucleotide sequence of a full length clone (clone C10) was WO99/04003 PCT/NL97/00424 13 determined by the dideoxynucleotide-mediated chain termination method and is depicted in Figure 1 (SEQ ID NO:l). The FBP1O cDNA clone has a length of 1131 nucleotides and encodes for a polypeptide of 246 amino acid residues. All characteristics of 5 a MADS box protein are present in FBP10: a N-terminal located MADS box region which shows a high degree of similarity with other MADS box proteins, and a K-box in the middle of the protein with an alpha helical structure. The alignment of FBP10 and homologues from Arabidopsis, potato and tobacco is 10 shown in Figure 2. FBP10 is most similar to the potato MADS box protein POTM1 (Kang and Hannapel, 1995). The functions of POTM1, TDR4, and AGl8 have not been determined yet. Example 2: 15 Expression of FBP10 The expression of FBP10 was determined by standard Northern blot hybridization experiments according to Angenent et al. (1992). A SmaI/HindII DNA fragment containing nucleotides 325 to 885 of FBP10 cDNA was used as a probe. 20 Using 10 pg of total RNA from various petunia tissues, expression of FBPO10 was detectable in young leaves, stems, bracts, and all floral organs except anthers. No expression was detectable in roots and old leaves (Figure 3). The expression in the apical meristem was determined by in 25 situ hybridization using a DIG labeled antisense RNA probe corresponding to the Smal/HindIII fragment of the FBP10 cDNA. (Figure 4). In vitro antisense RNA transcripts were made using T7 RNA polymerase. A standard protocol for in situ hybridization was used as described by Cands et al., 1994. 30 Strong hybridizing signals were observed in the vegetative apical meristem, floral meristem and weaker signals in the developing leaves. Example 3: 35 Construction of Chimeric FBP10 construct for co-suppression The full length FBP10 cDNA was subcloned into the binary vector pFBP20 (Angenent et al., 1993). This binary vector WO99/04003 PCT/NL97/00424 14 contains the CaMV 35S promoter, the adh intron, a multiple cloning site for insertion of the cDNA and the nos terminator. The full length cDNA clone (C10) present in the bluescript SK vector (Strategene lambda ZAP excision vector) was cut with 5 BamHl and Xhol and reinserted in the binary vector pFBP20 yielding plasmid pFBP113 (Figure 5). Example 4 Generation of transgenic petunia plants: 10 The binary vector containing the FBP10 cDNA in the sense orientation behind the CaMV 35S promoter (construct pFBP113) was transferred to Agrobacterium tumefaciens strain LBA4404 by triparental mating. The plasmid was transferred from E.coli HB101 to LBA4404 using a strain containing plasmid pRK2013. 15 Plasmid DNA from the Agrobacterium conjugates were isolated and the structure of the binary vector was verified by restriction analysis. Agrobacterium conjugants were used to transform Petunia hybrida leaf disks as described by Horsch et al. (1985). Leaf disks were prepared from top leaves of young 20 Petunia hybrida variety W115 plants. After shoot and root induction on Kanamycin selection media, plants were planted in soil and transferred to the greenhouse. Example 5: 25 Analysis of transgenic plants Twelve independent transgenic petunia plants were generated and examined for inflorescence structure and any other morphological alterations. Wild-type petunia plants have indetermined inflorescences of the raceme type (Figure 6a). 30 In the axils of two bracts, which are positioned opposite to each other, a flower is formed and the inflorescence continues. Under normal greenhouse conditions, this inflorescence maintains an inflorescence identity and never reverses to the vegetative phase. Vegetative leaves are not 35 positioned opposite to each other like bracts, but are arranged in a spiral phylotaxy. Two plants were selected which showed abberations in inflorescence structure. Occasionally, WO99/04003 PCT/NL97/00424 15 part of these inflorescences reverted to short vegetative shoots. No abberations were observed in the flowers. These two selected plants (T30.009 and T30.012) were self pollinated and the offspring were analyzed. A few plants of the offspring 5 population of T30.012 exhibited the same mild alterations as were observed in the primary transformant. The offspring of T30.009 could be divided into three classes: 8 plants with a wild-type phenotype, 12 plants with a phenotype resembling the primary transformant, and 5 plants with severe alterations in 10 inflorescence development. Plants from the latter class are affected in the switch from vegetative to inflorescence phase, in that vegetative shoots are produced instead of indetermined inflorescences (Figures 6a and 6b). Occasionally, a single flower is produced which might be due to the leaky nature of 15 sense cosuppression. The vegetative shoots produce small leaves which are arranged in a spiral phylotaxy like vegetative leaves. These mutant plants continue to grow and reach two to three times the size of a normal wild-type petunia plant (line W115). 20 Northern blot hybridization experiments were performed to examine the expression of FBP10 in the transgenic plants and to confirm the linkage of the phenotype with the suppression of FBP10. Twenty offspring plants from the self-pollinated T30.009 transgenic plant were used for Northern blot analysis. 25 Total RNA was extracted from leaf tissue and hybridized to an FBP10 specific cDNA fragment. Normal expression levels were observed in 6 plants. These plants were indistinguishable from wild type plants. The remaining 14 plants do not express FBP10 or at a very low level. No expression of FBPO10 was detectable 30 in the non-flowering plants (see Figure 3). Example 6: Flowering behaviour of FBPO10 mutant after cold treatment Vernalization (cold treatment) is a well known inducer of 35 flowering for many species. Therefore, we analysed the stability of the non-flowering phenotype of the FBP10 mutant by exposure of the plants to cold conditions (4 oC) for 4 and WO99/04003 PCT/NL97/00424 16 8 weeks. Wild type plants (line W115) were treated simultaneously. The plants (4 plants of each) were transferred to the cold about two weeks before they start to flower under normal greenhouse conditions (20 oC). The cold treatments did 5 not affect the timing of flowering, neither for the wild type plants nor for the FBP10 mutants. About two weeks after the treatment wild type plants started to flower, while the FBP10 mutants remained vegetative just like the untreated FBP10 mutant plants. 10 Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention 15 described specifically herein. Such equivalents are intended to be encompassed in the scope of the attached claims.

WO99/04003 PCT/NL97/00424 17 REFERENCES: Angenent, G.C., Busscher, M., Franken, J., Mol, J.N.M. and van Tunen,.A.J. (1992). Differential expression of two MADS 5 box genes in wild-type and mutant petunia flowers. Plant Cell 4, 983-993. Angenent, G.C., Franken, J., Busscher, M., Colombo, L., and Van Tunen, A.J. (1993) Formation of petals and stamen in 10 Petunia is regulated by the homeotic gene fbpl. Plant Journal, 4 101-112. Bernier, G.(1988). The control of floral evocation and morphogenesis. Annu.Rev.Plant Physiol.Plant Mol. Biol, 39, 15 175-219. Cans, L.A., Busscher, M., Angenent, G.C., Beltran, J-P and van Tunen, A.J. (1994). Nuclear localization of the petunia MADS box genes protein FBP1. Plant J. 6, 597-604. 20 Coen, E.S., Romero, J.M. Doyle, S., Elliott, R., Murphy, G., and Carpenter, R. (1990) Floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell, 63, 1311-1322. 25 Coen, E.S., and Meyerowitz, E.M.(1991). The war of the whorls: genetic interactions controlling flower development. Nature, 353, 31-37. 30 Colombo, L., Franken, J., Koetje, E., van Went, J., Dons, J.J.M., Angenent, G.C. and van Tunen, A.J. (1995). The petunia MADS box gene FBP11 determines ovule identity. Plant Cell,7, 1859-1868. 35 Gerats, A.G.M., Kaye, C., Collins, C., and Malmberg, R.L. (1988) Polyamine levels in petunia geneotype with normal and abnormal floral morphogenesis. Plant Physiology 86, 390-393.

WO99/04003 PCT/NL97/00424 18 Horsch, R.B., Fry, J.E., Hoffman, N.L. Eichholz, D. Rogers, S.G. and Fraley, R.T. (1985). A simple and general method for transferring gene into plants. Science, 227, 1229 1231. 5 Huijser, P., Klein, J., Lonnig, W-E., Meijer, H., Saedler, H., and Sommer, H. (1992). Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS box gene squamosa in Antirrhinum majus. The EMBO 10 J.11, 1239-1249. Kang, S-G, and Hannaple, D.J. (1995) Nucleotide sequence of novel potato MADS box cDNAs and their expression in vegetative organs. Gene 166, 329-330. 15 Landschttze, V., Willmitzer, L., and Muller-Rober, B (1995) Inhibition of flower formation by antisense repression of mitochondrial citrate synthese in transgenic potato plants leads to a specific disintegration of the ovary tissue of 20 flowers. EMBO J. 14, 660-666. Lee I., Aukerman, M.J., Gore, S.L., Lohman, K.N., Michaels L.M., Manorama, C.J., Feldmann, K.A. and Amasino, R.M. (1994) Isolation of LUMINIDEPENDENCE: A gene involved in 25 the control of flowering time in Arabidopsis. The Plant Cell, 6, 75-83. Mizukami, Y., Huang, H., Tudor, M., Hu, Y and Ma, H. (1996). Functional domains of the floral regulator AGAMOUS: 30 characterization of the DNA binding domain and analysis of dominant negative mutations. The Plant Cell 8, 831-845. Mandel, M.A., Gustafson-Brown, C., Savidge, B., and yanofsky, M.F. (1992), Molecular characterization of the 35 Arabidopsis floral homeotic gene APETALA1. Nature, 360, 273 277.

WO99/04003 PCTINL97/00424 19 Putterill, J., Robson, F., Lee, K., Simon, R., Coupland, G. (1995). The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to Zinc finger transcription factors. Cell 80, 847-857. 5 Mandel, M.A. and Yanofsky, M.F. (1995) A gene triggering flower formation in Arabidopsis. Nature 377, 522-524. Schwarz-Sommer, Z., Huijser, P., Nacken, W., saedler, H. 10 and Sommer, H., (1990) Genetic control of flower development: Homeotic genes in Antirrhinum majus. Science 250, 931-936. Weigel, D. (1995) The genetics of flower development: From floral induction to ovule Morphogenesis. Ann. Rev. 15 Gentics 29, 19-39. Weigel, D., Alvarez, J., Smith, D.R., Yanofsky, M.F. and meyerowitz, E.M. (1992) Leafy controls floral meristem identity in Arabidopsis. Cell, 69, 843-859. 20 Weigel, D. and Nilsson, O. (1995) A developmental switch sufficient for flower initiation in diverse plants. Nature 377, 495-500.

WO 99/04003 PCT/NL97/00424 20 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Centre for Plant Breeding and Reproduction research (CPRO-DLO) (B) STREET: Droevendaalsesteeg 1 (C) CITY: Wageningen (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 6708 pB (G) TELEPHONE: 31-317 477000 (H) TELEFAX: 31 317 418094 (ii) TITLE OF INVENTION: Process of producing transgenic plants in which flowering is inhibited, and DNA sequences used in said process. (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) WO 99/04003 PCT/NL97/00424 21 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1131 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: FBP10 cDNA (B) STRAIN: petunia W115 (F) TISSUE TYPE: flower (G) CELL TYPE: pistil (vii) IMMEDIATE SOURCE: (A) LIBRARY: c10 (B) CLONE: c10 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:65..802 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCACAGCTCT CTCTCTATAG TATAGTTTAA TTTATTCTGC ACTATACTTT TTTGTTAGAC 60 AAAA ATG GGA AGA GGA AGA GTG CAG ATG AAG AGA ATT GAG AAT AAA ATT 109 Met Gly Arg Gly Arg Val Gln Met Lys Arg Ile Glu Asn Lys Ile 1 5 10 15 AAT AGA CAA GTT ACT TTT TCA AAA CGT CGA TCT GGA TTA TTG AAG AAA 157 Asn Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys 20 25 30 GCT CAT GAA ATC TCT GTG CTT TGT GAT GCT GAA GTT GGT TTA ATT GTT 205 Ala His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Val 35 40 45 TTT TCT ACT AAA GGC AAA CTC TTT GAG TAT GCT ACT GAT TCT TGC ATG 253 Phe Ser Thr Lys Gly Lys Leu Phe Glu Tyr Ala Thr Asp Ser Cys Met 50 55 60 GAG AGG ATT CTT GAA AGA TAT GAA AGA TAC TCA TAT GCT GAG AGG CAG 301 Glu Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gln 65 70 75 CTT GTT TCT ACT GAT CAT AGC TCC CCG GGA AGC TGG AAT CTG GAA CAT 349 Leu Val Ser Thr Asp His Ser Ser Pro Gly Ser Trp Asn Leu Glu His 80 85 90 95 GCA AAA CTT AAG GCC AGA ATT GAG GTT GTG CAG AGA AAC CAA AGG CAT 397 Ala Lys Leu Lys Ala Arg Ile Glu Val Val Gln Arg Asn Gln Arg His 100 105 110 TAT ATG GGA GAA GAT TTG GAC TCG TTA AGT ATG AAA GAC CTT CAG AAT 445 Tyr Met Gly Glu Asp Leu Asp Ser Leu Ser Met Lys Asp Leu Gln Asn 115 120 125 TTA GAA CAA CAG CTG GAT TCT TCT CTT AAA CAC ATT CGA TCA AGA AAG 493 Leu Glu Gln Gln Leu Asp Ser Ser Leu Lys His Ile Arg Ser Arg Lys 130 135 140 WO 99/04003 PCT/NL97/00424 22 AAC CAA TTG ATG CAT GAG TCC ATT TCT GAG CTT CAA AAA AAG GAC AAA 541 Asn Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Asp Lys 145 150 155 TCA TTG CAA GAG CAA AAC AAC CTT CTT TCA AAG AAG GTG AAG GAG AGG 589 Ser Leu Gln Glu Gln Asn Asn Leu Leu Ser Lys Lys Val Lys Glu Arg 160 165 170 175 GAG AAA GAG TTG GCT CAA CAA ACT CAA TGG GAG CAG CAG AAT AAT CAT 637 Glu Lys Glu Leu Ala Gln Gln Thr Gln Trp Glu Gln Gln Asn Asn His 180 185 190 CAT GAG ATT AAC TCA TCA TCT TCA TTT GTT TTG CCA CAG CCA TTG GAC 685 His Glu Ile Asn Ser Ser Ser Ser Phe Val Leu Pro Gln Pro Leu Asp 195 200 205 TCT CCT CAC CTA GGG GAA GCA TAC CAG AGC ACA GTA GAC AAT GGA GAA 733 Ser Pro His Leu Gly Glu Ala Tyr Gln Ser Thr Val Asp Asn Gly Glu 210 215 220 GTA GAA GGA GCT TCA CAG CAG CAA CCT GCT AAT ACA ATG CCA CCA TGG 781 Val Glu Gly Ala Ser Gln Gln Gln Pro Ala Asn Thr Met Pro Pro Trp 225 230 235 ATG CTT CGC CAT CTT AAT GGC TAAGTTTTTG GTGGTCTAAG AATTAGGTAA 832 Met Leu Arg His Leu Asn Gly 240 245 AGCACCTTCA AACTCAACTA GTAATGTGTA AGTTAGGTCC ATATCACGGG TTCGAAGCTT 892 GCTACAGATT AAAAACTACA GGTATTTTAG TATTTTAGTG GAGAAGGATA GTTATATCAA 952 CCAGAATTTG CTGGCCCTAG AAGATTTCTC GATTATAAAA ATAAATGATA GATTTATATC 1012 TAATTTATAT TTATATAAAT ATATAGATGG GCTAGCTGTT TGTAAAACAA TATGTAACAT 1072 GATCTTATTT ACTGTATCAG CAGCCTTGCC TTGAATAACT TAAATATTCT GAATGATCT 1131 WO 99/04003 PCT/NL97/00424 23 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 246 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Gly Arg Gly Arg Val Gln Met Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20 25 30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Val Phe 35 40 45 Ser Thr Lys Gly Lys Leu Phe Glu Tyr Ala Thr Asp Ser Cys Met Glu 50 55 60 Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gln Leu 65 70 75 80 Val Ser Thr Asp His Ser Ser Pro Gly Ser Trp Asn Leu Glu His Ala 85 90 95 Lys Leu Lys Ala Arg Ile Glu Val Val Gln Arg Asn Gln Arg His Tyr 100 105 110 Met Gly Glu Asp Leu Asp Ser Leu Ser Met Lys Asp Leu Gln Asn Leu 115 120 125 Glu Gln Gln Leu Asp Ser Ser Leu Lys His Ile Arg Ser Arg Lys Asn 130 135 140 Gin Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Asp Lys Ser 145 150 155 160 Leu Gln Glu Gin Asn Asn Leu Leu Ser Lys Lys Val Lys Glu Arg Glu 165 170 175 Lys Glu Leu Ala Gln Gin Thr Gln Trp Glu Gin Gin Asn Asn His His 180 185 190 Glu Ile Asn Ser Ser Ser Ser Phe Val Leu Pro Gin Pro Leu Asp Ser 195 200 205 Pro His Leu Gly Glu Ala Tyr Gln Ser Thr Val Asp Asn Gly Glu Val 210 215 220 Glu Gly Ala Ser Gln Gln Gln Pro Ala Asn Thr Met Pro Pro Trp Met 225 230 235 240 Leu Arg His Leu Asn Gly 245

Claims (16)

1. An isolated DNA sequence which encodes a MADS box protein indicated FBP10 and having the amino acid sequence given in 5 SEQ ID NO:2 or a functionally homologous protein or part thereof, wherein inactivation of gene function of said DNA sequence, if present as an endogenous gene in a plant, results in inhibition of flowering. 10

2. DNA sequence according to claim 1, characterized in that it comprises the FBP10 gene or corresponding cDNA having the nucleotide sequence given in SEQ ID NO:1 or a functionally homologous gene or an essentially identical nucleotide sequence or part thereof or derivatives thereof which are 15 derived from said sequence by insertion, deletion or substitution of one or more nucleotides.

3. RNA sequence encoded by the DNA sequence of any of claims 1 and 2. 20

4. A protein encoded by the DNA sequence of any of claims 1 and 2.

5. A process of producing a transgenic plant in which 25 flowering is inhibited, characterized in that the expression of endogenous FBP10 gene or a homologous gene as defined in claims 1 or 2 is inhibited.

6. A process according to claim 5, characterized in that the 30 expression of said endogenous gene is inhibited by the use of anti-sense RNA.

7. A process according to claim 6, characterized in that a) a DNA, or a DNA fragment having at least 15 base pairs, 35 which is complementary to a degree of at least 70% to the WO99/04003 PCT/NL97/00424 25 FBP10 gene or homologous gene is introduced in a plant cell, b) said introduced DNA is transcribed into anti-sense RNA, said DNA being expressed constitutively or tissue 5 specific, or being induced by promoter elements controlling the expression of the introduced DNA, c) the expression of endogenous FBP10 gene or homologous gene is inhibited because of the anti-sense effect, d) plants are regenerated from the transgenic cells, and 10 e) plants exhibiting inhibited flowering are selected.

8. A process according to claim 5, characterized in that the expression of said endogenous gene is inhibited by the use of sense/co-suppression. 15

9. A proces according to claim 8, characterized in that a) a DNA which is the FBPO10 gene or homologous gene or a sequence having at least 70% sequence similarity to said FBP10 or homologous gene, or a fragment thereof having at 20 least 15 base pairs, is introduced in a plant cell, b) said DNA being expressed constitutively or tissue specific or being induced by promoter elements controlling the expression of the introduced DNA in such a way that transcription produces sense RNA, or being 25 introduced without the use of a promoter, c) the expression of endogenous FBP10 gene or homologous gene and the introduced gene are inhibited by the co suppression effect, d) plants are regenerated from the transgenic cells, and 30 e) plants exhibiting inhibited flowering are selected.

10. A process according to claim 5, characterized in that the function of said endogenous gene is inhibited by the use of dominant-negative mutations. 35

11. A process according to claim 10, characterized in that a) a DNA which encodes a modified FBP10 protein or WO99/04003 PCTINL97/00424 26 homologous protein or an essential part thereof, whereby said modified protein or part thereof is suitable for inhibiting the function of the endogenous FBP10 protein or homologous protein, is introduced in a plant cell, 5 b) said introduced DNA is transcribed into RNA and translated into a polypeptide, said DNA being expressed constitutively or tissue specific, or being induced by promoter elements controlling the expression of the introduced DNA, 10 c) the function of the endogenous FBP10 protein or homologous protein is inhibited by the dominant-negative mutation effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected. 15

12. Recombinant double-stranded DNA molecule for use in the process of claim 7 comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, 20 ii) a DNA sequence which is FBP10 gene or a homologous gene as defined in claims 1 or 2, which is fused to the promoter in anti-sense orientation so that the non-coding strand is transcribed, and if necessary iii) a signal fucntional in plants for the transcription 25 termination and polyadenylation of an RNA molecule.

13. Recombinant double-stranded DNA molecule for use in the process of claim 9 comprising an expression cassette comprising the following constituents: 30 i) a DNA sequence which is FBP10 gene or a homologous gene as defined in claims 1 or 2, ii) optionally a promoter functional in plants, which is fused to the DNA sequence in sense orientation, and if necessary 35 iii) a signal functional in plants for the transcription termination and polyadenylation of an RNA molecule. WO99/04003 PCT/NL97/00424 27

14. Recombinant double-stranded DNA molecule for use in the process of claim 11 comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, 5 ii) a DNA sequence which is a modified FBPO10 gene or homologous gene as defined in claims 1 or 2, which is fused to the promoter in sense orientation, and if necessary iii) a signal functional in plants for the transcription 10 termination and polyadenylation of an RNA molecule.

15. Transgenic plants containing recombinant DNA molecules according to any of claims 12 to 14 and showing inhibited flowering. 15

16. Plant cells, seeds, tissue culture, plant parts or progeny plants derived from a transgenic plant according to claim 15.