AU742459B2 - Process of producing transgenic plants in which flowering is inhibited, and DNA sequences used in said process - Google Patents

Description

4, WO 99/04003 PCT/NL97/00424 1 Process of producing transqenic plants in which flowering is inhibited, and DNA sequences used in said process.

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 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.

BACKGROUND OF THE INVENTION Plant development is characterised by a series of phase changes in which meristems are capable of generating new 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, 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.

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 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 WO 99/04003 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.

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 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 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 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 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' genes that control the identity of the flower meristem can also be used. These genes are LEAFY (LFY, Weigel et al., 1992) and APETALA1 (API, Mandel et al., 1992) from Arabidopsis and their homologues from Antirrhinum FLORICAULA (FLO, Coen et al., 1990) and SQUAMOSA (SQUA, Huijser et al., 1992), respectively. Overexpression of either LFY (Weigel and Nilsson, 1995; patent application WO 96/19105) or API (Mandel and Yanofsky, 1995) resulted in early flowering in WO 99/04003 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 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 (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 described by Landschtze 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.

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 '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 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 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 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.

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 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 Consupression construct or gene: a gene or nucleotide sequence derived thereof, having a homology of more than 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 identical to the amino acid composition in the MADS boxes of AGAMOUS and DEFICIENS.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

\\melbfiles\home$\amyo\Keep\34655-97 speci amendments.doc 1/11/01 4a It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION 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 *ooo \\melb files\homeS\amyo\Keep\34655-97 speci amendments.doc 1/11/01 WO 99/04003 PCT/NL97/00424 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 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 method for reducing flowering or completely abolishing the formation of inflorescences and flowers.

It was found that inhibition of the petunia MADS box gene resulted in transgenic petunia plants in which flowering was inhibited. Moreover, it has been found that the said 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 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 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.

The DNA sequence of the invention can also be characterized in that it comprises the FBPIO gene or corresponding cDNA having the nucleotide sequence given in SEQ ID NO:l or a functionally homologous gene or an essentially identical nucleotide sequence or part thereof or derivatives 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

II

WO 99/04003 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.

Further the invention provides processes of producing transgenic plants in which flowering is inhibited, comprising inhibiting the expression of endogenous FBP10 gene or a homologous gene.

Still further, the invention provides recombinant doublestranded 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 transgenic plants.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the cDNA sequence of the petunia FBP10 gene and 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 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 tissues as indicated: leaf, stem, bract, sepal, petal, stamen, style/stigma, ovary and leaf, stem, shoot and flower from the cosuppression plant.The SmaI/HindIII DNA fragment (nucleotide 325-885) of FBP10 cDNA was used as probe.

Figure 4 shows the expression of FBP10 by in situ hybridisations using a digoxygenin labeled antisense RNA probe (Figures 4B, 4D, and 4F) derived from the SmaI/HindIII fragment (nucleotide 325-885) of FBP10 cDNA. Sense control WO 99/04003 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, 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 sense construct. This binary vector, designated pFBP113, was used to generate transgenic petunia plants in which expression was inhibited.

Figure 6 shows the phenotype of a wild type petunia plant (a left) and representative plants in which the expression of was inhibited (a right and b).

DETAILED DESCRIPTION OF THE INVENTION The DNA sequence of the invention encodes a MADS box protein indicated FBP10 or a fucntionally homologous protein 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 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 FBP10 and a homologous gene may vary. The functionally homologous gene from a species related to petunia may have more than sequence identity, whereas the functional homologue from a non-related species may have only 65% or less sequence identity.

The invention also relates to derivatives of the gene or a homologous gene, which can be derived from the parent sequence by insertion, deletion or substitution of one WO 99/04003 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 flowering is inhibited, the expression of endogenous FBP1O 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 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 FBPIO 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 tissuespecific, or being induced by promoter elements controlling the expression of the introduced DNA, c) the expression of endogenous FBPIO gene or homologous 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 FBPIO 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 FBPIO gene or homologous gene sequence. The minimum similarity should be 70%, preferably more than In a second embodiment the expression of endogenous FBPIO or a homologous gene is inhibited through the use of sense/cosuppression. The process comprises the following steps: a) a DNA which is the FBPIO gene or homologous gene or a sequence having at least 70% sequence similarity to said FBPIO or homologous gene, or a fragment thereof having at WO 99/04003 PCT/NL97/00424 9 least 15 base pairs, is introduced in a plant cell, b) said DNA being expressed constitutively or tissuespecific or being induced by promoter elements controlling the expression of the introduced DNA in such a way that transcription produces sense RNA, or being introduced without the use of a promoter, c) the expression of endogenous FBPIO gene or homologous gene and the introduced gene are inhibited by the cosuppression effect, 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 base pairs, and sequences having a similarity of at least 70%, preferably at least 90%, can be used.

In a third embodiment the expression of endogenous FBPIO or a homologous gene is inhibited by the use of dominantnegative mutations.

The process comprises the following steps: 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, b) said introduced DNA is transcribed into RNA and translated into a polypeptide, said DNA being expressed constitutively or tissuespecific, or being induced by promoter elements controlling the expression of the introduced DNA, 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.

The transgenic plants produced by this process express an altered FBP10 protein or homologue thereof. As a result said WO 99/04003 PCT/NL97/00424 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).

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 anti-sense method, comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is FBPIO gene or a homologous gene as defined above, which is fused to the promoter in anti-sense orientation so that the noncoding strand is transcribed, and if necessary iii) a signal fucntional in plants for the transcription termination and polyadenylation of an RNA molecule.

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 FBPIO gene or a homologous gene 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 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 expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is a modified FBPIO gene or WO 99/04003 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.

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 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 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 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 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.

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.

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 lettuce, WO 99/04003 PCT/NL97/00424 12 spinach, chicory, etc.), sugar beet, potato, trees for wood production Eucalyptus. oak, willow, etc), tobacco, grasses, plants for nitrogen fixation, ornamental plants for production of cuttings.

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 (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 cattle. Inflorescences of grasses contain relatively low quantities of carbohydrates and large quantitives of lignin, which cannot be digested by cattle.

EXAMPLES

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., 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 l(FBP1) and FBP2 (Angenent et al., 1993). The hybridizing 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 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 WO 99/04003 PCT/NL97/00424 13 determined by the dideoxynucleotide-mediated chain termination method and is depicted in Figure 1 (SEQ ID NO:1). The cDNA clone has a length of 1131 nucleotides and encodes for a polypeptide of 246 amino acid residues. All characteristics of 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 and homologues from Arabidopsis, potato and tobacco is 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 AG18 have not been determined yet.

Example 2: Expression of 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.

Using 10 jig of total RNA from various petunia tissues, expression of FBP10 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 situ hybridization using a DIG labeled antisense RNA probe corresponding to the SmaI/HindIII fragment of the FBP10 cDNA.

(Figure 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.

Strong hybridizing signals were observed in the vegetative apical meristem, floral meristem and weaker signals in the developing leaves.

Example 3: 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 WO 99/04003 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 SKvector (Strategene lambda ZAP excision vector) was cut with BamH1 and Xhol and reinserted in the binary vector yielding plasmid pFBP113 (Figure Example 4 Generation of transgenic petunia plants: 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.

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 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 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).

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 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, WO 99/04003 PCT/NL97/00424 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 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 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 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).

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 FBPIO. Twenty offspring plants from the self-pollinated T30.009 transgenic plant were used for Northern blot analysis.

Total RNA was extracted from leaf tissue and hybridized to an FBPIO 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 FBPIO or at a very low level. No expression of FBP10 was detectable in the non-flowering plants (see Figure 3).

Example 6: Flowering behaviour of FBP10 mutant after cold treatment Vernalization (cold treatment) is a well known inducer of 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 WO 99/04003 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 The cold treatments did 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 mutants remained vegetative just like the untreated mutant plants.

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 described specifically herein. Such equivalents are intended to be encompassed in the scope of the attached claims.

WO 99/04003 PCT/NL97/00424 17

REFERENCES:

Angenent, Busscher, Franken, Mol, J.N.M. and van Tunen,.A.J. (1992). Differential expression of two MADS box genes in wild-type and mutant petunia flowers. Plant Cell 4, 983-993.

Angenent, Franken, Busscher, Colombo, L., and Van Tunen, A.J. (1993) Formation of petals and stamen in 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, 175-219.

Canas, Busscher, Angenent, Beltran, J-P and van Tunen, A.J. (1994). Nuclear localization of the petunia MADS box genes protein FBP1. Plant J. 6, 597-604.

Coen, Romero, J.M. Doyle, Elliott, Murphy, and Carpenter, R. (1990) Floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell, 63, 1311-1322.

Coen, and Meyerowitz, E.M.(1991). The war of the whorls: genetic interactions controlling flower development.

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Colombo, Franken, Koetje, van Went, Dons, Angenent, G.C. and van Tunen, A.J. (1995). The petunia MADS box gene FBP11 determines ovule identity. Plant Cell,7, 1859-1868.

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(1988) Polyamine levels in petunia geneotype with normal and abnormal floral morphogenesis. Plant Physiology 86, 390-393.

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Rogers, S.G. and Fraley, R.T. (1985). A simple and general method for transferring gene into plants. Science, 227, 1229- 1231.

Huijser, Klein, Lonnig, Meijer, H., Saedler, 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 J.ll, 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.

Landschutze, Willmitzer, 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 flowers. EMBO J. 14, 660-666.

Lee Aukerman, Gore, Lohman, K.N., Michaels Manorama, Feldmann, K.A. and Amasino, R.M. (1994) Isolation of LUMINIDEPENDENCE: A gene involved in the control of flowering time in Arabidopsis. The Plant Cell, 6, 75-83.

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(1996). Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations. The Plant Cell 8, 831-845.

Mandel, Gustafson-Brown, Savidge, and yanofsky, M.F. (1992), Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature, 360, 273- 277.

WO 99/04003 PCT/NL97/00424 19 Putterill, Robson, Lee, Simon, 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.

Mandel, M.A. and Yanofsky, M.F. (1995) A gene triggering flower formation in Arabidopsis. Nature 377, 522-524.

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WO 99/04003 PCT/NL97/00424 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Centre for Plant Breeding and Reproduction research (CPRO-DLO) STREET: Droevendaalsesteeg 1 CITY: Wageningen COUNTRY: The Netherlands POSTAL CODE (ZIP): 6708 pB TELEPHONE: 31-317 477000 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: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) WO 99/04003 PCT/NL97/00424 21 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1131 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: FBP10 cDNA STRAIN: petunia W115 TISSUE TYPE: flower CELL TYPE: pistil (vii) IMMEDIATE SOURCE: LIBRARY: CLONE: (ix) FEATURE: NAME/KEY: CDS LOCATION:65..802 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCACAGCTCT CTCTCTATAG TATAGTTTAA TTTATTCTGC ACTATACTT TTTGTTAGAC AAAA ATG GGA AGA GGA AGA GTG CAG ATG AAG AGA ATT GAG AAT AAA ATT Met Gly Arg Gly Arg Val Gin Met Lys Arg Ile Giu Asn Lys Ile AAT AGA CAA GTT Asn Arg Gin Val

ACT

Thr TTT TCA AAA CGT Phe Ser Lys Arg TCT GGA TTA TTG Ser Gly Leu Leu AAG AAA Lys Lys 109 157 205 253 GCT CAT GAA Ala His Glu TTT TCT ACT Phe Ser Thr

ATC

Ile TCT GTG CTT TGT Ser Val Leu Cys

GAT

Asp 40 GCT GAA GTT Ala Glu Val AAA GGC AAA CTC Lys Gly Lys Leu GAG TAT GCT ACT Glu Tyr Ala Thr GGT TTA ATT GTT Gly Leu Ile Val GAT TCT TGC ATG Asp Ser Cys Met GCT GAG AGG CAG Ala Glu Arg Gin GAG AGG Glu Arg ATT CTT GAA AGA Ile Leu Glu Arg

TAT

Tyr GAA AGA TAC TCA Glu Arg Tyr Ser

CTT

Leu GTT TCT ACT GAT Val Ser Thr Asp AGC TCC CCG GGA Ser Ser Pro Gly TGG AAT CTG GAA Trp Asn Leu Glu

CAT

His GCA AAA CTT AAG Ala Lys Leu Lys

GCC

Ala 100 AGA ATT GAG GTT Arg Ile Glu Val CAG AGA AAC CAA Gin Arg Asn Gin AGG CAT Arg His 110 397 445 TAT ATG GGA Tyr Met Gly

GAA

Glu 115 GAT TTG GAC TCG Asp Leu Asp Ser

TTA

Leu 120 AGT ATG AAA GAC Ser Met Lys Asp AAA CAC ATT CGA Lys His Ile Arg 140 CTT CAG AAT Leu Gin Asn 125 TCA AGA AAG Ser Arg Lys TTA GAA CAA CAG Leu Glu Gin Gin 130 CTG GAT TCT Leu Asp Ser TCT CTT Ser Leu 135 WO 99/04003 WO 9904003PCT/NL97/00424 AAC C.AA Asn Gin 145 TTG ATG CAT GAG Leu Met His Giu

TCC

Ser 150 ATT TCT GAG Ile Ser Giu CTT C.AA Leu Gin 155 AAG AAG Lys Lys 170 AAA AAG GAC AAA LyS Lys Asp Lys 541 589

TCA

Ser 160 TTG CAA GAG Leu Gin Giu CAA AAC Gin Asn 165 AAC CTr CTT TCA Asn Leu Leu Ser GTG AAG GAG Val Lys Giu

AGG

Arg 175 GAG AAA GAG TTG Giu Lys Giu Leu CAA CAA ACT CAA Gin Gin Thr Gin GAG CAG CAG AAT Giu Gin Gin Asn AAT CAT Asn His 190 CAT GAG ATI' His Giu Ile TCT CCT CAC Ser Pro His 210

AAC

Asn 195 TCA TCA TCT TCA Ser Ser Ser Ser Phe 200 GTT 'ITG CCA GAG Vai Leu Pro Gin CCA TTG GAC Pro Leu Asp 205 AAT GGA GAA Asn Gly Giu CTA GGG GAA GCA Leu Giy Giu Ala CAG AGC ACA GTA Gin Ser Thr Val

GAC

Asp 220 GTA GAA Val Giu 225 GSA GCT TCA GAG Gly Ala Ser Gin

CAG

Gin 230 CAA CCT GCT Gin Pro Ala AAT ACA Asn Thr 235 ATG CCA CCA TGG Met Pro Pro Trp

ATG

Met 240 CTI' CGC CAT CTT Leu Arg His Leu AAT GGC Asn Gly 245 TAAGTTTTTG GTGGTCTAAG AATTAGGTAA 832 AGCACCTTCA AACTCAACTA GTAATGTGTA AGITAGGTCC ATATCACGGG 'ITCGAAGCTr GCTACAGATT AAAAACTACA GGTATITTAG TA7=TAGTG GAGAAGGATA GTTATATCAA.

CCAGAATI'TG CTGGCCCTAG AAGATTTCTC! GATTATAAAA ATAAATGATA GAITTATATC TAAT'rrATAT TIATATAAAT ATATAGATGG GCTAGCTGTr TGTAAA6ACAA TATGTAACAT GATCTTATIT ACTGTATCAG CAGCCTTGCC TTGAATAACT TAAATATTCT GAATGATCT 892 952 1012 1072 1131 WO 99/04003 .23 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 246 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: PCT/NL97/00424 Met Gly Arg Gly Arg Vai Gin Met Lys Ile Giu Asn Lys Ile Asn Arg His Ser Arg Val Lys Met Giu Gin i4 5 Leu Lys Giu Pro Giu 225 Gin Val Giu Ile Thr Lys Ile Leu Ser Thr Leu Lys Gly Giu 115 Gin Gin i3 0 Leu Met Gin Giu Glu Leu Ile Asn 195 His Leu 210 Giy Ala Thr Ser Giy Giu Asp Ala i00 Asp Leu His Gin Aila iso Ser Gly Ser Phe Val Lys Arg His Arg Leu Asp Glu Asn 165 Gin Ser Giu Gin Ser Leu Leu Tyr 70 Ser Ile Asp Ser Ser 150 Asn Gin Ser Ala Gin 230 Lys Cys Phe 55 Glu Ser Giu Ser Ser 135 Ile Leu Thr Ser Tyr 21i5 Arg Arg 25 Asp Aia 40 Glu Tyr Arg Tyr Pro Gly Val Vai 105 Leu Ser 120 Leu Lys Ser Giu Leu Ser Gin Trp 185 Phe Val 200 Gin Ser Ser Giu Aia Ser Ser 90 Gin Met His Leu Lys 170 Giu Leu Thr Giy Val Thr Tyr 75 Trp Arg Lys Ile Gin 155 Lys Gin Pro Val Leu Gly Asp Al a Asn Asn Asp Axg 140 Lys Vai Gin Gin Asp 220 Leu Leu Ser Giu Leu Gin Leu 125 Ser Lys Lys Asn Pro 205 Asn Lys Lys Ile Val Cys Met Arg Gin Giu His Arg His 110 Gin Asn Arg Lys Asp Lys Giu Arg 175 Asn His 190 Leu Asp Gly Giu Aia Phe Giu Leu Ala Tyr Leu Asn Ser 160 Giu His Ser Val Gin Pro Ala Asn Thr Met Pro Pro Trp Met 235 240 Leu Arg His Leu Asn Gly 245

Claims (14)

1. An isolated DNA sequence which encodes a MADS box protein indicated Floral Binding Protein 10 (FBP10) and having the amino acid sequence given in SEQ ID NO:2 or a functionally homologous MADS box protein or functional 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.

2. A DNA sequence according to claim 1, wherein said DNA sequence comprises the FBP10 gene or corresponding cDNA having the nucleotide sequence given in SEQ ID NO:1 or a nucleotide having more than 70% of the nucleotide sequence 15 indentical to the nucleotide sequence given in SEQ ID NO:1 or a functionally homologous gene or a functional part *0 thereof or a functional derivative thereof which is derived from said sequence by insertion, deletion or substitution of one more nucleotides.

3. An RNA sequence encoded by a DNA sequence according to claim 1 or claim 2.

4. A protein encoded by the DNA sequence according 25 to claim 1 or claim 2. A process of producing a transgenic plant in which flowering is inhibited, wherein the expression of endogenous FBP10 gene or homologous gene according to claim 1 or claim 2 is inhibited.

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

7. A process according to claim 5 or claim 6, wherein eeSc .\\melbfiles\home$\amyo\Keep\34655-97 speci amendments.doc 1/11/01 OFF&- 25 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, 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 gene is inbited because of the anti- sense effect, d) plants are regenerated from the transgenic cells, and 15 e) plants exhibiting inhibited flowering are selected. 4

8. A process according to claim 5, wherein the expression of said endogenous gene is inhibited by the use of 20 sense/co-suppression. 4

9. A process according to claim 5 or claim 8, wherein a) a DNA which is the FBP10 gene or homologous gene or a sequence having at least 70% sequence similarity to 25 said FBP10 or homologous gene, or fragment thereof having at 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 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 e) plants exhibiting inhibited flowering and selected. \\melbfi es\home$\axyo\Keep\34655-97 speci asendments.doc 1/11/01 26 A process according to claim 5, wherein the function of a said endogenous gene is inhibited by the use of dominant-negative mutations.

11. A process according to claim 5 or claim 10, wherein a) a DNA which encodes a modified FBP10 protein or homologous protein or a functional part thereof is suitable for inhibiting the function of the endogenous FBP10 protein or homologous protein, is introduced in a plant cell, b) said introduced DNA is transcribed into RNA and translated into a polypeptide, said DNA being expressed constitutively or tissue- ee 15 specific, or being induced by promoter elements controlling the expression of the introduced DNA, 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.

12. A recombinant double-stranded DNA molecule for use in the process according to claim 7 comprising an expression 25 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 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 functional in plants for the transcription termination and polyadenylation of an RNA molecule.

13. Recobinant double-stranded DNA molecule for use in the process according to claim 9 comprising an expression cassette comprising the following constituents: i) a DNA sequence which is FBP10 gene or a homologous \\melbfies\home$\amyo\Keep\34655-97 speci amendments.doc 1/11/01 27 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 iii) a signal functional in plants for the transcription termination and polyadenylation of an RNA molecule.

14. Recombinant double-stranded DNA molecule for use in the process according to claim 11 comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which encodes a modified FBP10 protein or homologous protein or a functional part thereof and is suitable for inhibiting the function of the 15 endogenous FBP10 protein or homologous protein, 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.

15. A transgenic plant containing recombinant DNA molecules according to any one of claims 12 to 14 and showing inhibited flowering. 3. 25 16. Plant cells, seeds, tissue culture, plant parts or progeny plants derived from a transgenic plant according to claim \\melb_files\homeS\amyo\Keep\34655-97 speci amendments.doc 1/11/01 28

17. An isolated DNA sequence according to claim 1, substantially as herein described with reference to the examples and figues. Dated this 1st day of November 2001 CENTRUM VOOR PLANTENVEREDELINGS- EN REPRODUKTIEONDERzOEK (CPRO-DLO) By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia Goes &so- so&*~ \melbfies \home$\aMYO\ Keep\ 3 4655- 97 speci amendments.doc 1/11/01