Osteospermum and dimorphoteca plants having an altered flower phenotype

Description

[0001] The instant invention relates to an altered flower shape in plants belonging to the genera Osteospermum and Dimorphoteca, which is induced by a mutant allele, as well as to the method of breeding Osteospermum and Dimorphoteca plants having this altered flower shape.

[0002] The genus Osteospermum was introduced as a commercial bedding plant in the beginning of the nineties of the last century. Since then this genus has been very successful in the horticultural market. For 2008, worldwide sales were estimated at almost 100 million plants.

[0003] The genus Osteospermum is a South African native and belongs to the plant family of the Asteraceae. It comprises almost 70 different species representing a broad range of either evergreen shrubs or herbaceous plants with growing habits varying from erect to prostrate. The existing Osteospermum cultivars are thought to be interspecific hybrids of the following main species: O. ecklonis, O. barbariae, O. caulescens, O fruticosum, O. jucundum, and O. chrysanthemifolia. The first breeding with Osteospermum was started between 1970 and 1985 by British hobby breeders and later continued mainly by Danish and Japanese breeders (Allavena, A., et al., Genetic engineering of Osteospermum ssp.: a case story, Acta Hort., 508, 129-133 (2000)). According to Faccioli, et al., professional breeders used the British plant material as well as accessions from South Africa to breed new hybrids (Faccioli, P., et al., Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis, Plant Breed., 119, 351-355 (2000)). During further breeding, which was mainly done by professional Danish and German breeding companies, crossings between the existing plants were made to improve the quality. This approach has resulted in a narrow gene pool of the plant material, which is commercially available today. The genera Osteospermum and Dimorphoteca are very closely related and, in some cases even the distinction of both genera or the classification of certain varieties into these two genera is unclear. In the past the genus Osteospermum belonged to the genus Dimorphoteca, but today Dimorphoteca only comprises the annual species, whereas all semi-perennial species fall into the genus Osteospermum. Crossbreeding between both genera is possible and several commercial varieties result from intergeneric hybridisation between an Osteospermum and a Dimorphoteca parent. The different Osteospermum and Dimorphoteca cultivars, breeding lines and wild species represent a broad range of different ploidy levels varying from 2x up to almost 8x, which also shows that during the development of today's cultivars hybridisation between species took place. For commercial production, Osteospermum and Dimorphoteca plants are mostly propagated asexually by cuttings. However, sexual propagation through seeds is also possible and several seed propagated varieties are on the market.

[0004] Commercially available Osteospermum plants flower from early spring to autumn. The typical flower is a capitulum (flower head) with tubular central disc florets surrounded by a ring of ray florets, which gives the flowers the typical daisy shape (Faccioli, P., et al., Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis, Plant Breed., 119: 351-355 (2000)). Colour and shape of ray florets as well as the colour of the disc florets vary. The colour of the upper surface of the ray florets, which in colloquial language are called petals, is determined by two independent metabolic pathways producing carotenoids, visible as yellow-orange-brown colours, and anthocyanins, resulting in white to pink and purple flower colours (Seitz, C., Klonierung und Charakterisierung von Flavonoidgenen aus Osteospermum, Dissertation an der Technischen Universität München (2004)). Intensive breeding work during the past several years has resulted in a broad range of white, pink, purple, yellow, and orange petal colours and new mixes of the carotenoid and anthocyanin colour groups, as well as in colour patterns like eye types or stripes. Similar to the colour range of the upper surface, the colour of the lower surface of the ray florets also varies from light to dark colours in the bluish-pink or yellow-brown colour range. The colour pattern usually is striped with the coloured stripes running parallel to the petal edges. Typically, the colour of the disc florets is darker than the colour of the ray florets and it may vary from grey to blue, violet or purple or from dark yellow to dark brown. The usual shape of the ray floret is obovate, but in some genotypes the petal edges are rolled upwards resulting in so-called spoon or spider types.

[0005] An Osteospermum and Dimorphoteca breeding program was established in 2002 to produce altered flowering Osteospermum and Dimorphoteca plants. Osteospermum plants with unusual inflorescences are desirable, as altered flowers, which display mainly enlarged disc florets, are believed to stay open even in complete darkness, whereas normal flowers close already under low light conditions (less than 2000 Lux).

[0006] Furthermore, for the altered flowering plants the keepability of the flowers is longer both in the field and in the greenhouse compared to the flowers on a normal-flowering plant. This extended flower keepability is believed to result from a reduced seed set due to the limited pollen availability on the altered flowering plants. This limited pollen availability is a direct consequence of the enlarged disc florets which prevent the pollinating insects from reaching the pollen.

[0007] Finally, although most commercially available Osteospermum and Dimorphoteca varieties or assortments are vegetatively propagated by cuttings, several varieties or assortments of the genera Osteospermum and Dimorphoteca such as "Asti" and "Passion Mix" are propagated by seeds. For the production of F₁ hybrids of the seed-propagated varieties, the flowers of the female crossing parent usually have to be emasculated to prevent self-pollination and then are pollinated with pollen of a selected male parent to produce hybrid seeds. To avoid the labour intensive and costly emasculation and hand pollination procedures, a system that inhibits self pollination on the bisexual Osteospermum and Dimorphoteca plants would be highly desirable. In some plant species biological systems like male sterility or self incompatibility can be used in this respect, but these systems are not described for Osteospermum or Dimorphoteca. However, in case of Osteospermum and Dimorphoteca plants, which exhibit the altered flower type with enlarged disc florets, anthers are covered by the enlarged disc florets and hence the pollen is not freely available for pollinating insects. Therefore, hybrid seeds from these plants can be obtained without emasculation by insect pollination, which reduces the costs for F₁ hybrid seed production significantly.

[0008] For the above reasons, it is desirable to develop altered flowering Osteospermum and Dimorphoteca plants which show enlarged disc florets.

[0009] Several approaches such as mutation treatment and interspecific and intergeneric crosses were attempted to achieve an altered flowering trait in the genera Osteospermum and Dimorphoteca.

[0010] Several experiments on induction of mutations by Gamma-irradiation of Osteospermum and Dimorphoteca plant material were performed. Examples of references that illustrate alteration of flower type via mutation are altered flower type in ornamental sweet potato (Bhate, R.H., Chemically Induced Floral Morphological Mutations in Two Cultivars of Ipomoea purpurea (L.) Roth, Scientia Horticulturae. 88: 133-145 (2001)); in Chrysanthemum (Rana, R.S., Radiation-Induced Variation in Ray-Floret Characteristics of Annual Chrysanthemum, Euphytica. 8: 270-322 (1965)); in roses (Teruo, N., Ikegami, Y., Matsuda, Y., and Toyoda, H., Induction of Morphologically Changed Petals from Mutagen-Treated Apical Buds of Rose and Plant Regeneration from Varied Petal-Derived Calli, Plant Biotechnology, 8: 233-236 (2001)); and in plants in general (Krasaechai, A.L.D., et al., Low-Energy Ion Beam Modification of Horticultural Plants for Induction of Mutation, Surface and Coatings Technology, 203: 2525-2530 (2009)). In this regard it is important to mention that the ploidy level of almost all Osteospermum cultivars is tetraploid, whereas the ploidy level of Dimorphoteca cultivars varies from 2x to 6x. This means that in the case of a recessive mutation at least two generations would be necessary for the phenotype of any recessive mutation to become visible. In the case of a dominant mutation the phenotype would become visible in the M0-generation.

[0011] A first set of experiments was performed by the applicant on mature seeds which had been harvested from different Dimorphoteca cultivars. Batches of 30 seeds each were treated with doses of Gamma-irradiation varying from 15 to 40 Gy for periods varying from 5 to 30 minutes. Immediately after this treatment the seeds were soaked in a solution of 10% Polyethylene glycol (PEG) for 4 hours, the solution was washed off and the seeds were sown in standard seedling substrate. Germination started after about one week. Three weeks after sowing, when the first pair of leaves had developed, the seedlings were transplanted. Three weeks after transplanting, the seedlings were planted into 11 cm diameter pots and grown according to standard protocols. First flowering started about 10 weeks after potting. The plant populations were continuously evaluated for effects or mutants caused by the Gamma-irradiation. Depending on the dosage and the period of irradiation fewer seeds germinated and more malformed seedlings appeared, which did not develop further. Alterations of the growing habit as well as altered foliage types were difficult to evaluate, because the seeds originated from crossbreeding and therefore segregation of these characters in the offspring was expected. However, altered flower colours appeared, which resulted from mutation and not from segregation of the parental flower colours. These new colours showed that overall mutations of flower characteristics had successfully been induced by Gamma-irradiation. However, no altered flower shapes were detected in these plant populations.

[0012] A second set of experiments was performed by the applicant on rooted cuttings from different Dimorphoteca cultivars. Cuttings were rooted in standard paper pots within a period of 4 weeks. After successful rooting the cuttings were pinched above the 5th leaf pair and immediately Gamma irradiated. The dosages and irradiation periods corresponded to the previous experiments on seeds. After irradiation the cuttings were planted into 11 cm diameter pots containing a standard growing substrate and cultivated under standard growing conditions. The young plants were pinched back twice over a period of 6 weeks in order to allow mutated cells to develop into shoots. Flowering started about 13 weeks after planting. The plants were continuously evaluated for mutants. Several altered growing habits, foliage shapes, and flower colours were detected. However, altered flower shapes did not appear on any of the irradiated plants.

Interspecific and Intergeneric Crosses

[0013] Representatives from different species of the genera Osteospermum and Dimorphoteca were collected and crossing experiments with commercial Osteospermum cultivars were performed. In all combinations one parent was a commercial variety.

[0014] Occasionally, in Osteospermum seedling progeny individual plants were detected which exhibited an additional whorl of ray florets. These florets, which were located at the base of the main ray florets, were significantly narrower than the main ray florets and orientated vertically to the first whorl. These flowers still produced female organs at the base of the ray florets and were female fertile which was proven by their seed set. This additional whorl of ray florets was not stable and showed significant genotype-environment interaction. The respective plants were self-pollinated as well as crossbred to stabilize this phenotype. However, the trait was not detected in any of the progeny and therefore it is obviously not genetically stable. In summary, interspecific or intergeneric seed set was achieved for only two combinations, which was shown by an intermediate phenotype of the offspring. Among this offspring, as well as in further generations produced from these plants, no stable altered flowering plants were detected.

[0015] Hence, there is still a need for stable altered flowering plants having enlarged disc florets. Accordingly, the invention is defined in the appended claims.

[0016] The present disclosure describes an altered flower phenotype in Osteospermum and Dimorphoteca expressed in the formation of enlarged or converted disc florets. This altered flower type is induced by a mutant allele called the KLEDF allele.

[0017] The disclosure additionally describes new Osteospermum and Dimorphoteca plants characterized by their unique flowers which may be produced by the described methods.

[0018] Furthermore, described are pollen, seed, and sexual, as well as asexual progeny of such plants with altered flowers.

[0019] In addition, described are methods for propagating said plants and to uses of said plants.

[0020] Further, described is a method for producing an Osteospermum plant having an altered flower phenotype, wherein the method comprises crossing an Osteospermum plant of the present invention with a different Osteospermum plant not having an altered flower phenotype.

[0021] Furthermore, described is a method for producing a Dimorphoteca plant having an altered flower phenotype, wherein the method comprises crossing the Dimorphoteca plant of the present invention with a different Dimorphoteca plant not having an altered flower phenotype.

[0022] Also described is a method for producing an intergeneric hybrid plant having an altered flower phenotype is provided, wherein the method comprises crossing the Osteospermum plant of the present invention with the Dimorphoteca plant of the present invention.

[0023] Also described is a method for producing an intergeneric hybrid plant having an altered flower phenotype, wherein the method comprises crossing the Osteospermum plant of the present invention with a Dimorphoteca plant not having an altered flower phenotype.

[0024] Additionally, described is a method for producing an intergeneric hybrid plant having an altered flower phenotype, wherein the method comprises crossing the Dimorphoteca plant of the present invention with an Osteospermum plant not having an altered flower phenotype.

[0025] Described is a method for producing Osteospermum seed comprising crossing a first parent Osteospermum plant with a second parent Osteospermum plant and harvesting the resultant seed, wherein said first and/or second parent Osteospermum plant is the Osteospermum plant of the present invention.

[0026] Described is a method for producing intergeneric seed, wherein the method comprises crossing the Osteospermum plant of the present invention with a Dimorphoteca plant and harvesting the resulting intergeneric seed.

[0027] Further described is a method for producing intergeneric seed is provided, wherein the method comprises crossing the Osteospermum plant of the present invention with a Dimorphoteca plant not having an altered flower phenotype and harvesting the resulting intergeneric seed.

[0028] Further described is a method for producing intergeneric seed, wherein the method comprises crossing the Dimorphoteca plant of the present invention with an Osteospermum plant not having an altered flower phenotype and harvesting the resulting intergeneric seed.

[0029] Also described is the use of the primers according to SEQ ID Nos. 1 and 2 for identifying plants with an altered flowering phenotype. Preferably, the plants are plants of the family Asteraceae. More preferably, the plants are Osteospermum or Dimorphoteca plants. Preferably, the primers are used in an AFLP analysis. Preferably, a fragment of about 151 bp is obtained using these primers.

[0030] Also described is the use of the primers according to SEQ ID Nos. 3 and 4 for identifying plants with an altered flowering phenotype. Preferably, the plants are plants of the family Asteraceae. More preferably, the plants are Osteospermum or Dimorphoteca plants. Preferably, the primers are used in an SNP analysis.

[0031] Described herein is an isolated nucleic acid molecule encoding a protein with homology to the CYCLOIDEA-like protein from Senecio vulgaris and comprising a nucleic acid sequence according to SEQ ID No. 5 or 11. In a preferred embodiment, the nucleic acid sequence has a C in a position corresponding to position 73 of SEQ ID No. 5. Further, the present invention refers to an isolated nucleic acid molecule consisting of a nucleic acid sequence according to SEQ ID No. 5 or 11. In a preferred embodiment, the nucleic acid sequence has a C in a position corresponding to position 73 of SEQ ID No. 5. The present invention also refers to an isolated protein encoded by any of the isolated nucleic acid molecules defined above and transgenic plants which have been transformed with any of the nucleic acid molecules defined above and which display an altered flower phenotype. Preferably, the transgenic plants are from the family of Asteraceae.

[0032] Finally, described is a method for transferring the KLEDF mutant allele from one Osteospermum plant into another Osteospermum or Dimorphoteca plant, comprising crossing the Osteospermum plant of the present invention with another Osteospermum or Dimorphoteca plant, collecting F₁ seeds from said cross, selfing or crossing the F₁ plants derived from said F₁ seeds for one or more generations and screening plants derived from said selfing or crossing for the presence of said mutant KLEDF allele.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 

Fig. 1 shows a close-up of a normal flowering Osteospermum plant.

Fig. 2 shows a close-up of an altered flowering Osteospermum plant with enlarged tubular disc florets enclosing the gynoecium and androecium.

Fig. 3 shows a close-up of an altered flowering Osteospermum plant with enlarged open disc floret transformed into ligulate florets.

Fig. 4 shows a drawing of a longitudinal cross-section of an inflorescence of a normal flowering Osteospermum plant.

Fig. 5 shows a drawing of a longitudinal cross-section of an inflorescence of an altered flowering Osteospermum plant with tubular disc florets. The close-up shows that the enlarged tubular disc floret encloses the gynoecium and androecium.

Fig. 6 shows a drawing of a longitudinal cross-section of an inflorescence of an altered flowering Osteospermum plant with open disc florets transformed into ligulate florets.

Fig. 7 shows a whole plant with flowers of genotype OE 2008 248.

Fig. 8 shows a whole plant with flowers of genotype OE 2008 258.

Fig. 9 shows a whole plant with flowers of genotype OE 2008 274.

Fig.10 shows a whole plant with flowers of genotype OE 2008 285.

Fig. 11 shows a whole plant with flowers of genotype OE 2008 384.

Fig. 12 shows a whole plant with flowers of genotype OE 2008 390.

Fig. 13 shows the determination of the disc floret length in a normal flowering Osteospermum plant (a), an altered flowering Osteospermum plant with enlarged tubular disc florets (b) and an altered flowering Osteospermum plant with ligulate florets (c).

Fig. 14 shows in the upper part a normal flowering Osteospermum plant (1), an altered flowering Osteospermum plant with enlarged tubular disc florets (2) and an altered flowering Osteospermum plant with ligulate florets (3) and in the lower part the determination of the disc floret corolla length in these plants (4).

Fig. 15 shows the AFLP analysis of normal flowering Osteospermum plants (lanes 1-4) and altered flowering Osteospermum plants (lanes 5-10). The marker band of 151 bp which is only present in the altered flowering plants of the present invention is marked with an arrow.

DEFINITIONS

[0034] In the description and tables that follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

Adapter. Adapter molecules for the AFLP technique are short nucleic acid molecules of which a part is complementary to the sticky end of the restriction fragment and a part is a double strand of known sequence, usually comprising 10 to 15 bp. Exemplary adapter molecules are depicted in SEQ ID Nos. 6 to 9.

Allele. An allele is any of one or more alternative forms of a gene which relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Altered flower. As used herein "altered," "converted and "enlarged" flower, flowers, flowering, floret, and florets are used interchangeably and refer to Osteospermum or Dimorphoteca plants producing inflorescences with significantly enlarged disc florets. These enlarged disc florets have a length of at least 0.8 cm, whereas the length of disc florets in normal or typical flowering plants is typically less than 0.8 cm. Preferably, the enlarged disc florets have a length of at least 1.3 cm. The enlarged disc florets may still be tubular and enclose the gynoecium and androecium (Fig. 2 and Fig. 5) or the enlarged disc florets may be further extended and open and they may be transformed into ligulate florets (Fig. 3 and Fig. 6) resulting in a double flowering phenotype. All transitions of these phenotypes may occur in plants of the present invention.

AFLP. Amplified fragment length polymorphism (AFLP) is a highly sensitive method for detecting polymorphisms in DNA. Following restriction enzyme digestion of DNA, a subset of DNA fragments is selected for PCR amplification and visualization on a denaturing polyacrylamide gel. The AFLP analysis is described in detail in Vos et al. (1995) Nucleic Acids Res 23: 4407-14. By AFLP analysis of the plants of the present invention compared to normal flowering plants a marker was identified which can be used to to discriminate between normal and altered flowering plants. This marker is a fragment of about 151 nucleotides which is obtained by digesting genomic DNA from the plants with the restriction enzymes EcoRI and MseI, ligating the adapters according to SEQ ID Nos. 6 to 9 and amplifying DNA from the altered flowering plants with the primers according to SEQ ID Nos. 1 and 2. The 151 bp fragment is only present in the altered flowering plants, but not in the normal flowering plants.

Androecium. Male parts of a plant flower (= collectivity of stamens)

Asexual propagation/Asexual reproduction. Asexual propagation or reproduction means every type of plant propagation apart from seeds, e.g., by cuttings, grafting, division, or regeneration in tissue culture.

Average length. The average length of e.g. disc florets on an Osteospermum inflorescence is determined by measuring the smallest and the longest disc floret length and calculating their average which is then taken as average length of the disc floret of that respective inflorescence.

Backcrossing. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F₁ with one of the parental genotypes of the F₁ hybrid.

Bulked Segregant Analysis. A rapid mapping strategy suitable for monogenic qualitative traits. When DNA of ten plants is bulked into one pool, all alleles must be present. Two bulked pools of segregants differing for one trait will differ only at the locus harbouring that trait.

Capitulum. Capitulum refers to an inflorescence in the form of a central disc of sessile flowers called disc florets and an outer ring of petal-like structures called ray florets. The disc florets are generally perfect while the ray florets are generally imperfect.

Cell. Cell as used herein includes a plant cell, whether isolated, in tissue culture, or incorporated in a plant or plant part.

Chimera. A chimera or a chimeric plant is a plant that consists of two or more genetically distinct groups of cells. The genetic distinctness usually originates from a mutation.

Corolla- The collective term for all the petals on a single flower. The single flower in this case may be a disc floret or a ray floret.

CYCLOIDEA-like protein from Senecio vulgaris. A single nucleotide polymorphism was identified in a nucleic acid sequence showing homology to a nucleic acid sequence encoding a CYCLOIDEA-like protein from Senecio vulgaris. The amino acid sequence of the CYCLOIDEA-like protein from Senecio vulgaris and the nucleic acid sequence coding for it are deposited under the accession number FJ356703 at the NCBI web page (http://www.ncbi.nlm.nih.gov).

Daminozide. Is a plant growth regulator having the chemical structure N-(dimethylamino)succinamic acid which is marketed under the name Daminozide, Alar, Kylar or B9.

Dimorphoteca plant. Refers to a plant of the genus Dimorphoteca, for example Dimorphoteca sinuata or Dimorphoteca pluvialis.

Disc floret. One of the small tubular, actinomorphic florets which make up the central part of the flower head in Compositae or Asteraceae plants. The enlarged disc florets of the present invention may still be tubular or be transformed into ligulate florets.

Dominant inheritance. Refers to a mode of inheritance in which the phenotype of a certain characteristic or trait is determined by a dominant allele.

Dominant allele. The phenotype of a dominant allele is visible in a heterozygous genotype.

Double flower. In the Asteraceae or Compositae plant family, the term "Double flower" or "Semi-double flower" refers to inflorescences which have more than one whorl of ray florets. In completely "double-flowering" plants, all disc florets are transferred into ray florets, whereas in "semi-double-flowering" plants, only several whorls of disc florets are mutated into ray florets.

Embryo. The embryo is the small juvenile plant contained within a mature seed.

Embryo rescue. Refers to a technique intended to rescue inherently weak, immature or hybrid embryos to prevent degeneration of the embryos which otherwise would have died in a later stage of development. The rescued embryos are then grown on a suitable medium.

Flower. A flower is the reproductive structure found in flowering plants. Single flowers stand on petioles or maybe arranged in clusters forming an inflorescence standing on a stem.

F₂. The "F₂" symbol denotes a generation resulting from the selfing of the F₁ generation along with selection for type and rogueing of off-types. The "F" number is a term commonly used in genetics, and designates the number of the filial generation. The "F₂" generation denotes the offspring resulting from the selfing or self mating of members of the generation having the next lower "F" number, viz. the F₁ generation.

Gamete. A gamete is a cell that fuses with another gamete during fertilization in organisms that reproduce sexually. In plants the gametes are the ovule and pollen cell.

Gene. As used herein, "gene" refers to a segment of nucleic acid. A gene can be introduced into a genome of a species, whether from a different species or from the same species, using transformation or various breeding methods.

Gene-environment interaction/Genotype-environment interaction. Refers to the phenotypic effect of interactions between genes and the environment.

Genetic transformation. Refers to the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material.

Gene converted (Conversion). Gene converted (conversion) plants refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a plant are recovered in addition to the one or more genes transferred into the plant via the backcrossing technique, genetic engineering or mutation.

Genotype. Refers to the genetic constitution of a cell or organism.

Gynoecium. Ovule-producing parts of a plant flower

Heterozygous. Refers to a genetic constitution in which the corresponding alleles of a certain gene locus are different.

Homozygous. Refers to a genetic constitution in which the corresponding alleles of a certain gene locus are identical.

Inbreeding depression. Inbreeding depression is the reduced fitness in a given population as a result of breeding of close relatives or in plants also resulting from self-pollination.

INDEL. Indel describes a special mutation class, i.e. a mutation resulting in a colocalized insertion and deletion and a net gain or loss in nucleotides.

Inflorescence. A group or cluster of flowers arranged on a stem that is composed of a main branch or an arrangement of branches. In a normal flowering Osteospermum the inflorescence is formed by disc florets surrounded by ray florets.

Intergeneric cross. Intergeneric cross means the sexual hybridization of two individuals, each from a different genus such as Osteospermum and Dimorphoteca.

Intergeneric hybrid. Intergeneric hybrid means a plant of the F₁ generation resulting from an intergeneric cross or a cross between two different genera.

Interspecific cross. Interspecific cross means the sexual hybridization of two individuals, each from different species.

Interspecific hybrid. Interspecific hybrid means a plant of the F₁ generation resulting from an interspecific cross or a cross between two different species.

Keepability. Keepability refers to the period between the complete opening of the flowers, i.e. when the flower buds start showing the color of the petals and the wilting of the flowers, i.e. when two-three petals started showing aged symptoms and flower started appearing dull. Alternatively, the complete opening of the flowers is the time point at which the ray florets are unfolded and the complete loss of the flowers is characterized by the folding of the ray florets and the concomitant bending of the flower stalk. For each genotype the difference between the day the flower opened and the day it wilted is called the flower keepability. The keepability of the plants of the present invention has been determined under green house conditions in Central Europe (e.g. in Stuttgart). Under these conditions, the mean keepability of the normal flower plants was approximately 12 days, whereas the average keepability of the plants of the present invention was at least 15 days, preferably 16 or 17 days. Alternatively, the keepability of the plants of the present invention has been determined in the field in summer in Central Europe (e.g. in Stuttgart), i.e. under long-day conditions with changing environmental conditions. The altered flower plants of the present invention were grown isolated from the plants of the normal flowers to avoid pollination of the altered flower plants by the normal flower plants. Under these conditions, the keepability of the normal flower plants was less than ten days, for example eight or nine days and the keepability of the plants of the present invention was at least 15 days, preferably 16 or 17 days, more preferably 18 or 19 days and most preferably 20 days.

KLEDF allele. The allele which is responsible for the development of the altered flower phenotype of the present invention, i.e. a phenotype wherein at least one inflorescence has at least one disc floret with a length of at least 0.8cm. If the KLEDF allele is present, the plant will show the phenotype of the present invention. The presence of the KLEDF allele can be determined by means of molecular marker technology, e.g. by the AFLP technique. When applying this technique to the plants of the present invention, genomic DNA of a plant of the present invention is digested with EcoRI and MseI, adapters according to SEQ ID Nos. 6 to 9 are ligated to the fragments and the DNA is amplified by PCR using the primers according to SEQ ID No. 1 and 2. This amplification yields a fragment of about 151 nucleotides which is present in the altered flowering plants, but not in the normal flowering plants.

Linkage/Genetic Linkage. Describes the tendency of certain genetic loci or alleles to be inherited together. Genetic loci on the same chromosome are physically close to one another and tend to stay together during meiosis, and are thus genetically linked.

Locus. A locus confers one or more traits such as, for example, herbicide tolerance, insect resistance, disease resistance, flower colour, flower shape, plant height, etc. The trait may be, for example, conferred by a naturally occurring gene introduced into the genome of the plant by backcrossing, a natural or induced mutation, or a transgene introduced through genetic transformation techniques. A locus may comprise one or more alleles integrated at a single chromosomal location.

M0. The M0 generation is the generation treated with a mutagen.

Meiosis. Refers to the process of reduction division in which the number of chromosomes per cell is divided in half and which results in the formation of the gametes. Meiosis is essential for the sexual reproduction.

Molecular Marker/Genetic Marker. A molecular marker or genetic marker is a fragment of a DNA sequence that is associated to a part of the genome and linked to a certain phenotype such as the altered flowering phenotype of the present invention. One molecular marker described herein is a fragment of about 151 bp which was identified by the AFLP technique and which is only present in the altered flowering plants, but not in the normal flowering plants.

Monogenic inheritance. Refers to a mode of inheritance in which the phenotype of a certain characteristic or trait is determined by a single gene.

Mutation. Mutations are changes in the DNA sequence of a cell's genome and are caused by mutagens like radiation or chemicals as well as by errors that occur during DNA replication.

Mutation treatment. Refers to any treatment which is intended to introduce mutations in the DNA sequence of a cell's genome, in particular the treatment with radiation such as Gamma-irradiation and the treatment with chemical mutagens such as ethylmethanesulfonate (EMS).

Normal flower. As used herein "normal," "typical," "usual," and "regular" flower, flowers, flowering, floret, and florets are used interchangeably and refer to currently available commercial Osteospermum and Dimorphoteca plants which produce inflorescences with tubular disc florets enclosing but standing below the mature gynoecium and androecium, the disc florets having an average length of less than 0.8 cm (see Fig. 1 and Fig. 4).

Osteospermum plant. Refers to plants of the genus Osteospermum, for example Osteospermum ecklonis or Osteospermum jucundum.

PCR (Polymerase Chain Reaction). PCR describes a molecular biological method for amplifying a nucleic acid molecule, essentially comprising the steps of denaturation of the DNA, annealing of the primers and elongation of the primers with a thermostable DNA polymerase, using the nucleic acid sequence to be amplified as a template.

Phenotype. Refers to any observable characteristic or trait of an organism like flower colour, plant size, etc.

Plant. As used herein, the term "plant" includes reference to an immature or mature whole plant, including a plant from which seed or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.

Plant growth regulator. Refers to a natural or synthetic compound which can regulate plant growth, including antiauxins, auxins, cytokinins, defoliants, ethylene inhibitors, ethylene releasers, gametocides, gibberellins, growth inhibitors, morphactins, growth retardants and growth stimulators. Within the present invention, preferably a growth retardant and more preferably daminozide is used to increase seed yield in an altered flowering plant.

Plant Parts. As used herein, the term "plant parts" (or an Osteospermum or Dimorphoteca plant, or a part thereof) includes, but is not limited to, protoplasts, leaves, stems, roots, root tips, anthers, pistils, seed, embryo, pollen, ovules, cotyledon, hypocotyl, capitulum, ray petal/floret, disc petal/floret, shoot, tissue, petiole, cells, meristematic cells, and the like.

Pollination. Pollination is the process by which pollen is transferred in plants, thereby enabling fertilization and sexual reproduction.

Polymorphism. In plant biology polymorphism occurs when two or more clearly different phenotypes exist in the same plant population, resulting from multiple alleles of a gene within this population.

Primer. A primer is a short, single stranded nucleic acid which serves as the starting point for DNA synthesis, e.g. in PCR.

Progeny. As used herein, includes an F₁ Osteospermum or Dimorphoteca plant or a hybrid plant of Osteospermum or Dimorphoteca produced from the cross of two Osteospermum or Dimorphoteca plants and progeny. It further includes, but is not limited to, subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosses with plants of the same generation.

Protoplast fusion/Somatic fusion. Refers to a method in plants by which protoplasts (i.e., plant cells without cell walls) from two different plants are fused together to form a new hybrid plant with characteristics of both.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.

Ray floret. A ray floret or ligulate floret, is one of the outer, irregular florets in the flower heads of some Compositae or Asteraceae plants. Colloquial in Asteraceae or Compositae plants the ray florets are called petals.

Recessive inheritance. Refers to a mode of inheritance in which the phenotype of a certain characteristic or trait is determined by a recessive allele.

Recessive allele. The phenotype of a recessive allele is visible only in a homozygous genotype.

Regeneration. Regeneration refers to the development of a plant from tissue culture.

Segregating Progeny. Refers to a seedling progeny, in which the phenotypic traits and the respective genes divide onto the single individuals according to Mendelian rules.

Selection. Refers to the process of choosing from among the progeny of a crossing or the plants resulting from transformation, protoplast fusion or embryo rescue those plants which display the desired phenotype and/or genotype. Within the meaning of the present invention, the desired phenotype is the altered flower phenotype as described herein and the desired genotype is characterized by the presence of the KLEDF allele.

Sexual propagation/Sexual reproduction. Refers to the propagation of plants from seeds.

SNP. A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an individual. It has been found that in the altered flowering plants of the present invention the N within the sequence TTTGANAAAG (SEQ ID No. 10) in a DNA fragment amplified with primers according to SEQ ID Nos. 3 and 4 is C, whereas in normal flowering plants N is T. In some altered flowering plants the nucleotide substitution is on position 73 of the nucleic acid sequence according to SEQ ID No. 5.

Transformation. Refers to a process wherein a polynucleotide which is not in its natural context, e.g. linked to a promoter with which it is not naturally linked, or which is isolated from the other parts of the gene with which it is normally linked, is transferred into a plant. Suitable transformation methods include, but are not limited to, Agrobacterium mediated transformation, particle bombardment and electroporation.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The altered flowering plants of the present invention preferably have substantially all altered flowers resulting in a double-flowering phenotype. However, under certain circumstances, only part of the disc florets may be enlarged or only some of the inflorescences are altered inflorescences. This means that the number of enlarged disc florets per inflorescence may vary from only a few to more than 100 per inflorescence. All previously known Osteospermum and Dimorphoteca plants do not have the altered flowers of the present invention. Unexpectedly, the mutant allele of the present invention results in inflorescences having disc florets which are significantly enlarged compared to the disc florets of previously known Osteospermum and Dimorphoteca inflorescences.

[0036] The new altered flowering phenotype of the present invention resulting from a mutant allele does not necessarily eliminate the fertility of the flower. The anthers, which are located in the disc floret may be fertile and produce pollen. However, since the anthers are covered by the enlarged disc florets, the pollen is not visible and not freely available for insects and stigmas are covered by enlarged disc florets and are therefore not easily accessible for pollinating insects. Therefore, in the field plants with the altered flowering phenotype show significantly reduced seed set originating from insect pollination and consequently an extended flower keepability. Seed set usually originates from the gynoecium located at the base of the ray florets, whereas the disc floret gynoecium seems to be degenerated in both normal flowering plants and the new altered flowering plants.

[0037] Described are Osteospermum plants exhibiting an altered flower phenotype and having at least one enlarged disc floret, preferably at least two, five, eight, ten, twelve, 15, 18 or 20 enlarged disc florets, more preferably at least 22, 25, 28, 30, 35, 38 or 40 enlarged disc florets, even more preferably at least 42, 45, 48, 50, 52, 55, 58 or 60 enlarged disc florets, particularly preferably at least 62, 65, 68, 70, 72, 73, 75, 78 or 80 enlarged disc florets and even more preferably 82, 84, 85, 86, 88, 89, 90, 92, 94, 95, 96, 98, 100 or 108 enlarged disc florets. Most preferably, all disc florets of an inflorescence are enlarged.

[0038] The enlarged disc florets as described herein have a length of at least 0.8 cm, 0.9 cm, 0.96 cm, 1.00 cm, 1.01 cm, 1.15 cm, 1.21 cm, 1.26 cm, 1.29 cm, 1.30 cm, 1.31 cm, 1.34 cm, 1.38 cm, 1.40 cm, 1.42 cm, 1.45 cm, 1.49 cm, 1.51 cm, 1.52 cm, 1.55 cm, 1.57 cm, 1.61 cm, 1.63 cm, 1.64 cm, 1.66 cm, 1.69 cm, 1.70 cm, 1.80 cm, 1.90 cm, 2.0 cm, 2.01 cm, 2.1 cm, 2.16 cm, 2.19 cm, 2.20 cm, 2.23 cm, 2.26 cm, 2.28 cm, 2.30 cm, 2.33 cm, 2.36 cm, 2.37 cm, 2.39 cm, 2.4 cm, 2.5 cm, 2.6 cm, 2.7 cm, 2.8 cm, 2.9 cm, 3.0 cm, 3.1 cm, 3.2 cm, 3.3 cm, 3.4 cm, 3.5 cm, 3.6 cm, 3.7 cm, 3.8 cm, 3.9 cm, 4.0 cm, 4.1 cm, 4.2 cm, 4.3 cm, 4.4 cm, 4.5 cm, 4.6 cm, 4.7 cm, 4.8 cm, 4.9 cm, 5.0 cm, 5.1 cm, 5.2 cm, 5.3 cm, 5.4 cm, 5.5 cm, 5.6 cm, 5.7 cm, 5.8 cm, 5.9 cm and 6.0 cm, including all integers and fractions thereof.

[0039] Alternatively or additionally, the average length of the shortest and the longest disc floret on at least one inflorescence of the plant is at least 1.45 cm, 1.49 cm, 1.51 cm, 1.52 cm, 1.55 cm, 1.57 cm, 1.61 cm, 1.63 cm, 1.64 cm, 1.66 cm, 1,69 cm, 1.70 cm, 1.80 cm, 1.90 cm, 2.0 cm, 2.01 cm, 2.1 cm, 2.16 cm, 2.19 cm, 2.20 cm, 2.23 cm, 2.26 cm, 2.28 cm, 2.30 cm, 2.33 cm, 2.36 cm, 2.37 cm, 2.39 cm, 2.4 cm, 2.5 cm, 2.6 cm, 2.7 cm, 2.8 cm, 2.9 cm, 3.0 cm, 3.1 cm, 3.2 cm, 3.3 cm, 3.4 cm, 3.5 cm, 3.6 cm, 3.7 cm, 3.8 cm, 3.9 cm, 4.0 cm, 4.1 cm, 4.2 cm, 4.3 cm, 4.4 cm, 4.5 cm, 4.6 cm, 4.7 cm, 4.8 cm, 4.9 cm, 5.0 cm, 5.1 cm, 5.2 cm, 5.3 cm, 5.4 cm, 5.5 cm, 5.6 cm, 5.7 cm, 5.8 cm, 5.9 cm and 6.0 cm, including all integers and fractions thereof.

[0040] Described herein is an average length of at least one disc floret of between 0.8 cm and 6.0 cm, between 0.8 cm and 5.8 cm, between 0.8 cm and 5.5 cm, between 0.8 cm and 5.2 cm, between 0.8 cm and 5.0 cm, between 0.8 cm and 4.8 cm, between 0.8 cm and 4.5 cm, between 0.8 cm and 4.2 cm, between 0.8 cm and 4.0 cm, between 0.8 cm and 3.8 cm, between 0.8 cm and 3.5 cm, between 0.8 cm and 3.2 cm, between 0.8 cm and 3.0 cm, between 0.8cm and 2.8 cm, between 0.8 cm and 2.5 cm, between 0.8cm and 2.4 cm, between 0.8cm and 2.3 cm, between 0.8 cm and 2.2 cm, between 0.8 cm and 2.1 cm, between 0.8 cm and 2.0 cm, between 0.8 cm and 1.9 cm, between 0.8 cm and 1.8 cm, between 0.8 cm and 1.7 cm, between 0.8 cm and 1.6 cm, between 0.8 cm and 1.4 cm, between 0.8 cm and 1.3 cm or between 0.8 cm and 1.2 cm.

[0041] Described herein is an average length of the shortest and the longest disc floret of at least one inflorescence of between 0.8 cm and 6.0 cm, between 0.8 cm and 5.8 cm, between 0.8 cm and 5.5 cm, between 0.8 cm and 5.2 cm, between 0.8 cm and 5.0 cm, between 0.8 cm and 4.8 cm, between 0.8 cm and 4.5 cm, between 0.8 cm and 4.2 cm, between 0.8 cm and 4.0 cm, between 0.8 cm and 3.8 cm, between 0.8 cm and 3.5 cm, between 0.8 cm and 3.2 cm, between 0.8 cm and 3.0 cm, between 0.8cm and 2.8 cm, between 0.8 cm and 2.5 cm, between 0.8cm and 2.4 cm, between 0.8cm and 2.3 cm, between 0.8 cm and 2.2 cm, between 0.8 cm and 2.1 cm, between 0.8 cm and 2.0 cm, between 0.8 cm and 1.9 cm, between 0.8 cm and 1.8 cm, between 0.8 cm and 1.7 cm, between 0.8 cm and 1.6 cm, between 0.8 cm and 1.4 cm, between 0.8 cm and 1.3 cm or between 0.8 cm and 1.2 cm.

[0042] Described herein is an average length of at least one disc floret of between 1.0 cm and 6.0 cm, between 1.1 cm and 6.0 cm, between 1.2 cm and 6.0 cm, between 1.3 cm and 6.0 cm, between 1.4 cm and 6.0 cm, between 1.5 cm and 6.0 cm, between 1.6 cm and 6.0 cm, between 1.7 cm and 6.0 cm, between 1.8 cm and 6.0 cm, between 1.9 cm and 6.0 cm, between 2.0 cm and 6.0 cm, between 2.1 cm and 6.0 cm, between 2.2 cm and 6.0 cm, between 2.5 cm and 6.0 cm, between 2.7 cm and 6.0 cm, between 3.0 cm and 6.0 cm, between 3.2 cm and 6.0 cm, between 3.5 cm and 6.0 cm, between 3.8 cm and 6.0 cm, between 4.0 cm and 6.0 cm, between 4.2 cm and 6.0 cm, between 4.5 cm and 6.0 cm, between 4.8 cm and 6.0 cm, between 5.0 cm and 6.0 cm, between 5.2 cm and 6.0 cm, between 5.5 cm and 6.0 cm or between 5.8 cm and 6.0 cm.

[0043] Described herein is an average length the longest and the shortest disc floret of at least one inflorescence of between 1.0 cm and 6.0 cm, between 1.1 cm and 6.0 cm, between 1.2 cm and 6.0 cm, between 1.3 cm and 6.0 cm, between 1.4 cm and 6.0 cm, between 1.5 cm and 6.0 cm, between 1.6 cm and 6.0 cm, between 1.7 cm and 6.0 cm, between 1.8 cm and 6.0 cm, between 1.9 cm and 6.0 cm, between 2.0 cm and 6.0 cm, between 2.1 cm and 6.0 cm, between 2.2 cm and 6.0 cm, between 2.5 cm and 6.0 cm, between 2.7 cm and 6.0 cm, between 3.0 cm and 6.0 cm, between 3.2 cm and 6.0 cm, between 3.5 cm and 6.0 cm, between 3.8 cm and 6.0 cm, between 4.0 cm and 6.0 cm, between 4.2 cm and 6.0 cm, between 4.5 cm and 6.0 cm, between 4.8 cm and 6.0 cm, between 5.0 cm and 6.0 cm, between 5.2 cm and 6.0 cm, between 5.5 cm and 6.0 cm or between 5.8 cm and 6.0 cm.

[0044] Described herein is an average length of at least one disc floret is between 1.0 cm and 3.0 cm, between 1.0 cm and 2.9 cm, between 1.0 cm and 2.8 cm, between 1.0 cm and 2.7 cm, between 1.0 cm and 2.6 cm, between 1.0 cm and 2.5 cm, between 1.0 cm and 2.4 cm, between 1.0 cm and 2.3 cm or between 1.0 cm and 2.2 cm.

[0045] Described herein is an average length of the longest and the shortest disc floret of at least one inflorescence of between 1.0 cm and 3.0 cm, between 1.0 cm and 2.9 cm, between 1.0 cm and 2.8 cm, between 1.0 cm and 2.7 cm, between 1.0 cm and 2.6 cm, between 1.0 cm and 2.5 cm, between 1.0 cm and 2.4 cm, between 1.0 cm and 2.3 cm or between 1.0 cm and 2.2 cm.

[0046] Described herein is an average length of the shortest and the longest disc floret of at least one inflorescence of between 1.25 cm and 3.0 cm, between 1.25 cm and 2.9 cm, between 1.25 cm and 2.8 cm, between 1.25 cm and 2.7 cm, between 1.25 cm and 2.6 cm, between 1.25 cm and 2.5 cm, between 1.25 cm and 2.4 cm, between 1.25 cm and 2.3 cm or between 1.25 cm and 2.2 cm. In yet further embodiments of the present invention the average length of the shortest and the longest disc floret of at least one inflorescence is between 1.4 cm and 2.4 cm, between 1.4 cm and 2.2 cm, between 1.4 cm and 2.0 cm, between 1.4 cm and 1.9 cm or between 1.4 cm and 1.8 cm.

[0047] The present invention further comprises altered flowering Osteospermum plants wherein the average length of the longest and the shortest disc floret of at least one inflorescence is 1.45 cm, 1.5 cm, 1.55 cm, 1.6 cm or 1.7 cm.

[0048] The length of the disc florets is determined by removing the disc florets from the inflorescence and measuring the length from the base of the disc florets to the top (see Figures 13 and 14). It is possible to determine the length of all disc florets of an inflorescence, but in cases where the appearance of the disc florets is uniform, it is sufficient to determine the length of e.g. ten disc florets and take the average value of the length. Alternatively, the average value of the smallest and longest disc floret of an inflorescence is determined. In preferred embodiments, the length of the disc floret refers to the length of the disc floret corolla and therefore does not include the length of the anthers which poke out of the corollas in normal flowering plants.

[0049] Further described is a ratio of the average length of the longest and the shortest ray floret of at least one inflorescence to the average length of the longest and the shortest disc floret of at least one inflorescence of less than 2.0, preferably less than 1.95, more preferably less than 1.9 and most preferably less than 1.88. In another embodiment the ratio of the average length of the longest and the shortest ray floret of at least one inflorescence to the average length of the longest and the shortest disc floret of at least one inflorescence is between 1.2 and 2.0, preferably between 1.25 and 1.95, more preferably between 1.3 and 1.9 and most preferably between 1.35 and 1.88.

[0050] Also described is an average length of the longest and the shortest disc floret of an inflorescence of at least 1.25 cm and the ratio of the average length of the longest and the shortest ray floret of at least one inflorescence to the average length of the longest and the shortest disc floret of at least one inflorescence is less than 2.0. Alternatively, the average length of the longest and the shortest disc floret of an inflorescence is between 1.25 cm and 2.0 cm and the ratio of the average length of the longest and the shortest ray floret of at least one inflorescence to the average length of the longest and the shortest disc floret of at least one inflorescence is between 1.2 and 2.0. In a further embodiment, the average length of the longest and the shortest disc floret of at least one inflorescence is between 1.4 cm and 1.8 cm and the ratio of the average length of the longest and the shortest ray floret of at least one inflorescence to the average length of the longest and the shortest disc floret of at least one inflorescence is between 1.35 and 1.88. The plants of the present invention may also be obtained by mutation treatment with Gamma-irradiation dosages up to 500 Gy, incubation periods of up to several hours, repeated irradiation treatments and/or the irradiation of pollen or flower buds. In addition to Gamma-irradiation X ray or UV radiation may also be applied. Furthermore, the plants may be treated with a chemical mutagen, e.g. with ethyl methanesulfonate (EMS), as it is e.g. described for the induction of altered flower colours and flower shapes in Petunia hybrida (Gerats T. and Strommer J. (eds.), Petunia - Evolutionary, Developmental and Physiological Genetics, Springer Life Sciences, 2nd. ed., XXII (2009); Harten van, A.M., Mutation Breeding: Theory and Practical Applications, Cambridge University Press (1998)).

[0051] The new altered flowering Osteospermum plants are genetically stable, as evidenced by the stability of the altered-type phenotype through asexual propagation and the transmission of this trait to the progeny after sexual crosses.

[0052] The evaluation of further segregating progeny combined with the application of molecular marker technology has clarified the inheritance of the new altered flower phenotype in Osteospermum in more detail. To map the mutant KLEDF allele, a segregating F₂ or backcross population of plants showing the altered flower phenotype derived from a cross between a normal and an altered flowering plant was produced. On this segregating population a molecular marker analysis was performed to screen for polymorphism between the parents and the normal flowering genotypes and the altered flowering genotypes, respectively. The aim was to identify markers, which are polymorphic in both the parents and differentiate between the normal and the altered flowering genotypes. For this analysis different molecular marker techniques like the AFLP- and SNP- technique were used. With these polymorphic DNA markers a linkage analysis is performed on the segregating progeny to identify the chromosomal locations of the KLEDF allele.

[0053] The AFLP analysis has shown that a fragment of about 151 nucleotides which is obtained by digesting genomic DNA from the plants with the restriction enzymes EcoRI and MseI, ligating the adapters according to SEQ ID Nos. 6 to 9 and amplifying DNA from the altered flowering plants with the primers according to SEQ ID Nos. 1 and 2 can only be detected in plants with an altered flowering phenotype but not in plants with a normal flowering phenotype.

[0054] Further, the SNP analysis showed that the N within the sequence TTTGANAAAG (SEQ ID No. 10) in a DNA fragment amplified with primers according to SEQ ID Nos. 3 and 4 is C, whereas in normal flowering plants N is T.

[0055] The analysis of the altered flowering plants of the present invention has also shown a reduced seed yield in these plants. However, the seed yield could be increased and the phenotype of the plants could be transformed almost to the normal flowering phenotype by treating the altered flowering plants with a plant growth regulator such as daminozide. The plants treated in this way could be used in crosses. Suitable concentrations of the plant growth regulator, preferably daminozide, are 0.05% to 0.4%, preferably 0.1% to 0.3%, more preferably 0.15% to 0.25% and most preferably 0.2%.

EXAMPLES

Example 1 - Development of an Altered Osteospermum Flower Phenotype

[0056] In spring 2007 in a proprietary population of Osteospermum plants, a single plant was found which showed a slightly modified flower phenotype. A more detailed analysis of the flowers on this plant showed that on some of the flowers, the disc florets were elongated and covered the androecium and the gynoecium, whereas in the normal flowers on the same plant the androecium as well as the gynoecium were standing above the disc florets. The phenotype of this plant was identified as a novelty with a certain potential to be useful for the development of a new flower type within the genus Osteospermum. Therefore, further analyses on this plant were performed.

[0057] Firstly, it had to be shown if this altered phenotype was the result of a genetic mutation or if it was a modification induced by environmental conditions like the extra whorl of ray florets described above. For this purpose, cuttings from this plant were taken repeatedly, rooted, and grown to flowering plants. Among these plant populations, three different groups of individuals were detected. The first group of individuals was comprised of plants which exhibited only normal flowers, whereas the second group of plants exhibited flowers with the altered phenotype. A further third group of plants consisted of plants which exhibited both types of flowers, the normal type as well as the altered type. This result indicated that the altered flower type could be transmitted by asexual propagation, but it further indicated that this plant was a chimera.

[0058] Next, it had to be shown that the new flower type could be transmitted through sexual propagation. Therefore a breeding program with this altered flowering plant was designed. The sexual transmission of the altered phenotype may depend on the genetic background of the respective crossing parent. Therefore these were carefully selected by their phenotypes and also by their genotypes, making use of a genetic distance analysis which had been performed in the framework of a research project by Gawenda and Debener reported in 2009 (Gawenda, I. and Debener, T., Genetic diversity of Osteospermum genotypes analysed by AFLP and chloroplast SSR markers, Europ. J. Hort. Sci., 74 (2), 86-94 (2009)). The normal-flowering Osteospermum genotypes, which were used as crossing parents, were all proprietary assortment varieties or breeding lines.

[0059] A first series of pollinations was performed wherein the plant exhibiting the modified flower type was used as a male as well as a female parent. The flowers of the female crossing parents were emasculated before mature pollen appeared. On average, 2 days after emasculation, the stigmas at the base of the ray florets extend which indicates that they are ready for pollination. At this stage they were pollinated repeatedly with pollen from the respective male parent. The use of the altered flowering plant as the male parent needed specific skills of the pollinators, because the pollen had to be collected carefully at the base of the altered disc florets. About 4 weeks after pollination the seeds were ready for harvesting. In total, 32 crossing combinations had been performed, but only 16 of the combinations produced seeds. In total, almost 5,000 seeds were harvested and sown.

[0060] For sowing the seeds were soaked in a solution of 10% PEG for 4 hours, the solution was washed off, and the seeds were sown in standard seedling substrate. Germination started after about one week. Three weeks after sowing, when the first pair of leaves had developed, the seedlings were transplanted. Three weeks after transplanting the seedlings were planted into 11 cm diameter pots and grown according to standard protocols. First flowering started about 10 weeks after potting.

[0061] The seedling populations were evaluated over a period of 4 weeks mainly focusing on the selection of plants exhibiting the altered flower-type. The progeny comprised 2,459 flowering plants in total. In all progeny plants were selected which showed an altered flowering phenotype, meaning that these plants had at least one elongated disc floret. The number of altered flowering plants compared to the total number of seedlings varied among the different progeny. Since the first altered flowering plant, which was used as a breeding parent, was a chimera, meaning it produced altered flowers as well as normal flowers on one plant, the segregation pattern was unpredictable, although only altered flowers had been used for pollination. Table 1 summarises the number of altered flowering and normal flowering-plants for 9 progeny, which comprised enough seedlings for a segregation analysis. Unexpectedly, the frequency of seedlings exhibiting altered flowers was in all combinations far from that corresponding to a 1:1-segregation, the expected segregation in case of a dominant inheritance, which is shown by the respective χ², values (Table 1). Also unexpectedly none of the progeny exhibited exclusively normal-flowering plants, which would be expected in the case of a recessive inheritance. Furthermore, the data in Table 1 show that the altered flowering trait is not linked to flower colour.

[0062] In Table 1, column 1 shows the colour of the ray florets of the respective crossing partners exhibiting normal flower types. Columns 2 through 5 show the number of plants producing normal inflorescences for each colour and the total number of plants. Columns 6 through 9 show the number of plants producing altered inflorescences for each colour and the total number of plants with altered inflorescences. Columns 10 through 13 show the total number of plants for each colour as well as the total number of evaluated seedlings per progeny. Finally, column 14 shows the χ²-square values for each row which correspond to a 1:1 segregation of altered flower phenotype to normal-flower phenotype.

[0063] After sexual propagation by crossbreeding into different Osteospermum cultivars and breeding lines, stable altered flowering seedlings were found among the F₁ progeny. The successful transmission of the altered flower trait into sexual progeny shows that this trait is genetically stable. However, the segregation pattern of the altered flower trait in different F₁ progeny does not explain the mode of inheritance of this mutation.

[0064] To further investigate the segregation pattern of the mutation leading to the altered flowering Osteospermum phenotype, four stable altered flowering genotypes were crossed with four normal flowering Osteospermum genotypes and five altered flowering genotypes were crossed among each other. The breeding parents were again selected according to their genetic distance based on the analysis of Gawenda and Debener (Gawenda, I. and Debener, T., Genetic diversity of Osteospermum genotypes analysed by AFLP and chloroplast SSR markers, Europ. J. Hort. Sci., 74 (2), 86-94 (2009)). Since the altered flowering seedlings were not chimeric anymore, the segregation ratio in their offspring for normal-flowering genotypes should correspond to a 1:1 ratio as in the case of dominant inheritance. Pollination, seed harvest, and sowing as well as cultivation of the seedlings were performed as described above for the F₁ progeny. The progeny were evaluated for their flower types. Results showed that all progeny resulting from a cross of an altered and a normal flowering parent correspond to a segregation ratio of 1:1. In case of progeny from crosses of two altered flowering parents three out of four populations corresponded to a segregation ratio of 3:1. These ratios observed in this analysis confirm a monogenic dominance inheritance of the mutation responsible for the altered flowering Osteospermum phenotype.

[0065] In the upper half of Table 2, column 1 shows the number of plants having the altered flowering phenotype resulting from crosses between an altered flowering F₁ plant with a normal flower plant, while column 2 shows the number of plants having the normal flowering phenotype and column 3 shows the total number of plants resulting from the crosses. Column 4 shows the χ² square values corresponding to the expected 1:1 segregation of altered flowering phenotypes to normal flowering phenotypes for each row. In the lower half of Table 2, the same analysis was performed for crosses between two plants having the altered flowering phenotype.

Example 2 - Molecular Marker analysis of flower type in Osteospermum: Identification of an AFLP marker linked to the KLEDF allele

[0066] The aim of this project was the identification of markers linked to the altered flowering trait in Osteospermum by using a Bulked Segregant Analysis (BSA) approach. The markers were identified by using BSA in which proprietary F2 populations 511, 499 and 503 derived from a cross between an altered flowering Osteospermum parent and a normal flowering Osteospermum parent (Table 2) were used to detect the DNA markers linked to the locus controlling the altered flower phenotype. AFLP marker screening was carried out on these populations including their parents (n = 279).

DNA extraction and AFLP analysis:
Genomic DNA was isolated from the leaf material using a modified CTAB protocol (Stewart and Via (1993) Biotechniques 14(5): 748-750). The AFLP analysis was conducted according to the standard protocol described by Vos et al. (1995) Nucl. Acids Res. 23:4407-4414 with the following details:

EcoRI adapter:

5'-CTCGTAGACTGCGTACC (SEQ ID No. 6)

3'-CATCTGACGCATGGTTAA-5' (as 5' to 3': SEQ ID No. 7)

EcoRI adapter oligonucleotides:

5'-CTCGTAGACTGCGTACC-3' (SEQ ID No. 6)

5'-AATTGGTACGCAGTCTAC-3' (SEQ ID No. 7)

MseI adapter:

5'-GACGATGAGTCCTGAG-3' (SEQ ID No. 8)

3'-TACTCAGGACTCAT-5' (as 5' to 3': SEQ ID No. 9)

Msel adapter oligonucleotides:

5'-GACGATGAGTCCTGAG-3' (SEQ ID No. 8)

5'-TACTCAGGACTCAT-3' (SEQ ID No. 9)

Pre-amplification primers:

E01L: 5'-AGACTGCGTACCAATTCA-3' (SEQ ID No. 10)

M02: 5'-GATGAGTCCTGAGTAAC-3' (SEQ ID No. 11)

Final AFLP PCR oligos:

E40: 5'-GACTGCGTACCAATTCAGC-3' (SEQ ID No. 1)

M54: 5'-GATGAGTCCTGAGTAACCT-3' (SEQ ID No. 2)

Results:

[0067] An AFLP marker was found (E40/M54-151) which could clearly discriminate between the normal and altered flowering phenotype in Osteospermum. After separation on denaturing polyacrylamide gel this marker showed a band of size approximately 151 base pairs in altered flowering individuals, which was absent in all normal flowering ones (see Figure 15). This AFLP marker therefore could immediately be used in breeding for identifying genotypes having an altered flowering phenotype.

Example 3 - Molecular Marker analysis of flower type in Osteospermum: Identification of SNP marker linked to the KLEDF allele

[0068] To identify molecular markers linked to the altered flowering phenotype in Osteospermum a candidate gene (CG) approach was used as an alternative to the AFLP marker approach. The working hypothesis assumed that single nucleotide polymorphisms (SNP) or insertion/deletions (Indel) within the candidate gene sequence are associated with the phenotype of interest. To our knowledge there has been no study involving genes responsible for alteration of floral morphology in Osteospermum. Among other members of Asteraceae such as sunflower and Gerbera phenotypes showing altered floral morphology have been reported. Several important genes which are assumed to be involved in change of floral morphology have been reported (Kotilainen et al. (2000) Plant Cell 12: 1893-1902; Theißen (2001) Current Opinion in Plant Biology 4: 75-85; Teeri et al. (2002) In: Cronck QCB, Bateman RM, Hawkins JA, eds. Developmental genetics and plant evolution. London: Taylor & Francis, 220-232; Fambrini et al. (2003) Genesis 36: 25-33; Uimari et al. (2004) Proc. Natl. Acad. Sci. USA 101: 15817-15822; Broholm et al. (2008) Proc. Natl. Acad. Sci. USA 105: 9117-9122). From these studies we chose the genes GAGA1, CYCLODEA and GRCD2 as possible candidates causing floral morphology change in Osteospermum.

[0069] Since there has been no report on these genes being studied in Osteospermum, Gerbera sequences for these three genes were used for designing primers for Osteospermum homologs of these genes. Genomic DNA was extracted from the leaf material by using a standard extraction protocol. At the end of this experiment only one gene (CYCLODEA) homolog in Osteospermum could successfully be amplified and sequenced. In this fragment (approx.330 bp long) a SNP (nucleotide T for normal to C for altered flowering plant) was identified which shows a strong linkage with the altered flowering Osteospermum genotypes.

[0070] Primer sequence and PCR conditions:

Forward primer:
CYC2f6- 5'-AAGATCGACACAGCTCACGG-3' (SEQ ID No. 3)

Reverse primer:
CYC2r7- 5'-TCTGCCCTTGACTGATTCAC-3' (SEQ ID No.4)

[0071] Polymerase chain reaction amplifications were performed in 25 µL reaction volumes consisting of 2 µl (25-30ng/ µl) genomic DNA, 5 µl 5x GoTaq-buffer, 1,5 µl dNTPs (25µM), 0,5 µl primer CYC2f6 (10µM), 0,5 µl primer CYC2r7 (10µM), 0,2 µl GoTaq (5U/µl), and 15,3 µl H₂0. The amplification protocol consisted of 35 cycles with an initial denaturing step of 5 min at 94°C followed by 34 cycles at 94°C for 1 min for denaturation, followed by primer-specific annealing 60 °C for 1 min and extension at 72°C for 2 min. After 35 cycles, there was a final extension step of 10 min at 72°C.

Analysis of the Polymorphism in CYC2

[0072] All sequence data for individuals involved in the analysis were assembled, edited and prepared for SNP screening. In the gene CYC2 sequence fragment we found a SNP with nucleotide base C which is tightly linked to the altered flowering phenotype. In the normal flowering individuals there is a T at this position. The SNP position within SEQ ID No. 10 is shown below in Table 3.

Table 3: SNP position within the sequence TTTGANAAAG (SEQ ID No. 10)

Flower type	Adjacent bases 5' of the SNP	SNP	Adjacent bases 3' of the SNP
Normal	TTTGA	T	AAAG
Altered	TTTGA	C	AAAG

[0073] Example: Below a sequence alignment of the CYC2 gene fragment of a normal Osteospermum flowering plant (SEQ ID No. 11) with that of an altered flowering plant (SEQ ID No. 5) is shown and the position of the SNP is indicated in bold letters. The sequence alignment was performed using the EMBOSS Pairwise Alignment Algorithms available under: http://www.ebi.ac.uk/Tools/emboss/align/.

[0074] This result was confirmed for population 511 as well as for population 503 and for other individuals involved in this study. This SNP marker could thus immediately be used in breeding for identifying genotypes having altered flowering phenotype.

Example 4 - Further characterization of the altered flowering plants

[0075] The altered flowering trait was crossbred into different genetic backgrounds representing the available range of growing habits and flower colours in Osteospermum. The segregation of plant characteristics like flower colour, flower size, earliness, branching, vigour, and foliage quality in the offspring was according to the segregation patterns in normal-flowering offspring. Surprisingly, these plant characteristics do not seem to be linked to the altered flowering trait, as is also indicated by the results presented in Table 4. Furthermore, all individuals which were selected from the different progeny and analysed further performed similarly to typical-flowering Osteospermum plants in terms of rooting, cutting production, growing habit and disease/pest resistance.

[0076] Table 4, column 1 shows the code for each line, column 2 shows whether the line has the altered flowering phenotype (AF) or the normal-flowering phenotype (NF), columns 3 through 6 show the number of ray florets, the length and width in centimeters of the ray florets and the upper surface colours of the ray florets, respectively. For those plants, which are bicoloured or exhibit playing or fading colours, more than one RHS number according to the colour chart available from the Royal Horticultural Society (available under http://www.rhs.org.uk/Plants/RHS-Publications/RHS-colour-charts) is listed. Columns 7 through 9 show the number of disc florets and the length in centimeters of the disc florets as well as the average length, columns 10 and 11 show the length and width in centimeters of the immature leaves, columns 12 and 13 show the length and width in centimeters of the mature foliage, column 14 shows the colour of the mature foliage, and column 15 shows the number of basal shoots for each line.

[0077] By the described method 6 genotypes were developed which stably exhibit the new altered flower phenotype (see Fig. 7 through Fig. 12). Table 4 summarizes the comparison of certain plant characteristics in these altered flowering genotypes compared to normal-flowering Osteospermum plants. As shown in Table 4, unexpectedly, the altered flowering group consistently differs from the normal-flowering ones in the length of the disc florets, whereas all further plant characteristics show the same variation in the altered flowering genotypes as in the normal-flowering ones.

[0078] From the progeny described previously, 10 altered flowering Osteospermum genotypes were selected and evaluated under different environmental conditions in North and South Europe, East Africa, and the United States covering a broad temperature range and different light regimes. The plants were grown in the greenhouse as well as in the field. The altered flowering trait was always expressed, whereas the expression level as well as the expression of the different phenotypes described above varied depending on the growing conditions. These observations indicate that the mutant KLEDF allele of the present invention is genetically stable.

Example 5 - Genotype OE 2008 248

[0079] Through the breeding process described above, Osteospermum genotype OE 2008 248 was developed. OE 2008 248 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 5

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 248
PLANT:
Ploidy level:  4x
Number of basal shoots: 6
Internode length: 0.05 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 3.5 cm
Width: 0.9 cm
Mature leaves:
Length: 4.8 cm to 5.0 cm
Width: 1.6 cm to 1.9 cm
Colour: RHS 146A

RAY FLORETS:
Number: 14 to 16
Length: 3.0 cm
Width: 0.7 cm
Colour of upper surface: RHS 92D
Colour of lower surface: RHS 85A

DISC FLORETS:
Number: 80 to 85
Length: 1.3 cm to 2.1 cm
Average length: 1.7 cm
Colour of inner surface: RHS 85D
Colour of outer surface: RHS 91A

Example 6 - Genotype OE 2008 258

[0080] Through the breeding process described above, Osteospermum genotype OE 2008 258 was developed. OE 2008 258 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 6

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 258
PLANT:
Ploidy level: 4x
Number of basal shoots: 5
Internode length: 0.5 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 3.2 cm
Width: 0.8 cm
Mature leaves:
Length: 5.2 cm
Width: 1.9 cm to 2.0 cm
Colour: RHS 144A

RAY FLORETS:
Number: 17 to 18
Length: 2.6 cm to 2.7 cm
Width: 0.7 cm to 0.8 cm
Colour of upper surface: RHS 92D
Colour of lower surface: RHS 76A

DISC FLORETS:
Number: 73 to 89
Length: 1.0 cm to 1.9 cm
Average length: 1.45 cm
Colour of inner surface: RHS 85D
Colour of outer surface: RHS 76C

Example 7 - Genotype OE 2008 274

[0081] Through the breeding process described above, Osteospermum genotype OE 2008 274 was developed. OE 2008 274 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 7

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 274
PLANT:
Ploidy level: 4x
Number of basal shoots: 5
Internode length: 0.5 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 3.2 cm
Width: 0.8 cm to 0.9 cm
Mature leaves:
Length: 4.5 cm to 4.9 cm
Width: 2.0 cm
Colour: RHS 146A

RAY FLORETS:
Number: 25
Length: 2.6 cm to 2.9 cm
Width: 0.7 cm to 0.9 cm
Colour of upper surface: RHS 78B
Colour of lower surface: RHS 82A

DISC FLORETS:
Number: 95 to 96
Length: 1.3 cm to 1.7 cm
Average length: 1.5 cm
Colour of inner surface: RHS 78A
Colour of outer surface: RHS 82D

Example 8 - Genotype OE 2008 285

[0082] Through the breeding process described above, Osteospermum genotype OE 2008 285 was developed. OE 2008 285 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 8

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 285
PLANT:
Ploidy level: 4x
Number of basal shoots: 5
Internode length: 0.5 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 4.0 cm to 4.5 cm
Width: 1.2 cm to 1.6 cm
Mature leaves:
Length: 7.0 cm to 7.5 cm
Width: 2.5 cm to 3.5 cm
Colour: RHS 137A

RAY FLORETS:
Number: 22 to 25
Length: 2.6 cm to 3.0 cm
Width: 0.6 cm to 0.8 cm
Colour of upper surface: RHS N74B
Colour of lower surface: RHS 84A

DISC FLORETS:
Number: 78 to 85
Length: 0.8 cm to 2.2 cm
Average length: 1.5 cm
Colour of inner surface: RHS 72A
Colour of outer surface: RHS 84A

Example 9 - Genotype OE 2008 384

[0083] Through the breeding process described above, Osteospermum genotype OE 2008 384 was developed. OE 2008 384 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 9

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 384
PLANT:
Ploidy level: 4x
Number of basal shoots: 4
Internode length: 0.5 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 3.4 cm
Width: 1.1 cm
Mature leaves:
Length: 4.1 cm
Width: 2.1 cm
Colour of upper surface: RHS 146A

RAY FLORETS:
Number: 23 to 27
Length: 2.1 cm
Width: 0.6 cm to 0.7 cm
Colour of upper surface: RHS 72A
Colour of lower surface: RHS N80D

DISC FLORETS:
Number: 94 to 108
Length: 1.4 cm to 1.7 cm
Average length: 1.55 cm
Colour of inner surface: RHS 72A
Colour of outer surface: RHS 84B

Example 10 - Genotype OE 2008 390

[0084] Through the breeding process described above, Osteospermum genotype OE 2008 390 was developed. OE 2008 390 displays the altered flower phenotype of the present invention having enlarged disc florets.

TABLE 10

PHENOTYPIC DESCRIPTION OF THE GENOTYPE OE 2008 390
PLANT:
Ploidy level: 4x
Number of basal shoots: 5
Internode length: 0.5 cm to 1.0 cm

FOLIAGE:
Immature leaves:
Length: 3.3 cm
Width: 0.9 cm to 1.1 cm
Mature leaves:
Length: 6.5 cm
Width: 2.0 cm to 2.5 cm
Colour of upper surface: RHS 146A

RAY FLORETS:
Number: 19 to 22
Length: 2.6 cm
Width: 0.8 cm to 0.9 cm
Colour of upper surface: RHS 77c and RHS 75D
Colour of lower surface: RHS 85A

DISC FLORETS:
Number: 68 to 100
Length: 1.4 cm to 1.8 cm
Average length: 1.6 cm
Colour of inner surface: RHS 77C
Colour of outer surface: RHS 85A

Example 11 - Incorporating the Mutant Allele KLEDF into Osteospermum plants

[0085] The altered flowering cultivars having the mutant allele named KLEDF of the present invention maintain functional female and male organs. Therefore, the altered flowering trait can be incorporated into Osteospermum cultivars through conventional breeding, although the execution/implementation of these crosses requires specific skills of the respective pollinators. By crossbreeding, the KLEDF allele of the present invention can be incorporated into a broad range of Osteospermum plants having different flower colours and shapes (e.g., spider types) as well as into different foliage types. Furthermore, the mutant allele can be incorporated into plants having different growing habits, e.g., prostrate or hanging types can be developed besides erect or semi-erect types.

[0086] Using conventional breeding methods, an altered flowering Osteospermum plant having the mutant allele KLEDF of the present invention is crossed with a normal flowering Osteospermum plant lacking the mutant allele of the present invention. The resulting seeds are sown and the seedlings are grown according to conventional methods. The flowering F₁ progeny are then scored for altered flowering plants. Selected F₁ plants are further crossbred or can be crossed back to their altered flowering or to their normal-flowering parent in order to combine the altered flowering phenotype with further desirable plant characteristics. However, depending on the genetic distance between the parents, inbreeding-depression might occur in this backcross progeny. Alternatively, selected plants from this F₁ progeny can be outcrossed to selected plants from a different F₁ progeny or to another cultivar, which is far related to the respective F₁ progeny.

Example 12 - Incorporating the Mutant Allele KLEDF into other Osteospermum Species to Create Interspecific Hybrids

[0087] The mutant allele KLEDF of the present invention can be introduced into an interspecific hybrid made between one species of Osteospermum having the mutant allele of the present invention and a different Osteospermum species. A selected Osteospermum plant having the mutant allele KLEDF is crossed, using conventional methods, as either a male or a female parent, to a selected genotype of any further Osteospermum species. Depending on the Osteospermum species from which the crossing parent is selected, seeds will set easily, e.g., in crossings with O. jucundum. For crosses with plants from less closely related species, specific techniques like bud pollination, removal of the stigma and pollination of the remaining style, GA₃-treatments of the pollinated stigmas, and/or embryo rescue of the immature embryo may be necessary. The seeds resulting from the cross are sown and the seedlings are grown according to conventional methods. The flowering F₁ progeny are then scored for altered flowering plants. The further breeding procedure is similar to the procedure described in Example 8.

Example 13 - Incorporating the Mutant Allele KLEDF into Dimorphoteca Plants to Create Intergeneric Hybrids

[0088] Furthermore, the mutant KLEDF allele of the present invention can also be introduced into an intergeneric hybrid through crosses between a selected Osteospermum plant having the mutant allele KLEDF of the present invention and a Dimorphoteca plant lacking the mutant allele of the present invention. The methods to be used to realize these hybrids correspond to the methods which have been described for interspecific crosses, meaning that specific techniques like bud pollination, removal of the stigma and pollination of the remaining style, GA₃-treatments of the pollinated stigmas, and/or embryo rescue of the immature embryo may be necessary to realize these hybrid seedlings.

Example 14 - Creating Plants of the Present Invention with the Assistance of Molecular Markers

[0089] The incorporation of the mutant allele KLEDF of the present invention into a different genetic background requires repeated crossbreeding or backcrossing, meaning that the gene of interest has to be followed over several generations in the respective progeny. Molecular markers are a very powerful tool to make the selection more efficient and to accelerate the breeding process. Debener (Debener, T., Molecular markers as a tool for analysis of genetic relatedness and selection in ornamentals, Breeding for Ornamentals: Classical and Molecular Approaches, 329-345, Kluwer Academic Publishers (2002)) have described several examples of successful marker-assisted breeding in ornamentals.

[0090] We have found one AFLP and one SNP marker which clearly identify altered flowering genotypes. This is of particular importance to accelerate a breeding program through marker-assisted selection when identification of genotypes having an altered flowering phenotype is required at an early stage.

[0091] The application of these techniques will enable molecular mapping of the described Osteospermum KLEDF mutant allele of the present invention.

Example 15 - Creating Plants of the Present Invention Using Protoplast Fusion

[0092] In some plant species protoplast fusion is a powerful technique to combine the genes of two different species instead of performing crosses between plants of the respective species (Horita, M., Morohashi, H., and Komai, F., Production of fertile somatic hybrid plants between oriental hybrid lily and Lilium x formolongi, Planta, 597-601 (2003); Griesbach, R.J., Recent advances in the protoplast biology of flower crops, Scientia Horticulturae, 37, 247-256 (1988); Kumar, A. and Cocking, E.C., Protoplast Fusion: A Novel Approach to Organelle Genetics in Higher Plants, American Journal of Botany, 741, 1289-1303 (1987)). Besides the addition of two complete genomes, parts of both genomes can be combined. Prerequisite is an efficient protocol for the regeneration of plants from single protoplasts. Even the transmission of just single chromosomes of one partner into the genome of the second partner or the incorporation of the genome of one partner into the cytoplasm of the second partner, as well as a patch-work cytoplasm can be achieved through protoplast fusion (see, for example, Lössl A., Adler, N., Horn, R., Frei, U., and Wenzel, G., Chondriome-type characterization of potato: Mta, β, γ, δ, ε_and novel plastid mitochondrial configurations in somatic hybrids, Theoretical and Applied Genetics, 99: 1-10 (1999)).

[0093] Protoplast fusion has been described in the genus Dimorphoteca (J. S. Al-Atabee, and J. B. Power. (1987) Plant Cell Reports 6:414-416.). However, since plants of both the Osteospermum and the Dimorphoteca genus are routinely propagated in tissue culture and can easily be regenerated from leaf explants, protoplast regeneration, as well as protoplast fusion might as well be possible in the genus Osteospermum (Allavena, A. et al.(2000) Acta Hort. 508: 129-133; Giovannini, A. et al. (1999) In Vitro Cell. Dev. Biol. Plant 35: 70-75). Fusion of protoplasts from an Osteospermum plant having the mutant allele KLEDF of the present invention with protoplasts from either an Osteospermum or a Dimorphoteca plant lacking the mutant allele of the present invention, regeneration of plants thereof, and selection of altered flower individuals among these regenerated fusion products, enable the transmission of the mutant allele of the present invention into new genetic backgrounds.

Example 16 - Using Mutagens on Plants of the Present Invention to Create Altered Plants

[0094] Osteospermum and Dimorphoteca plants having the mutant allele KLEDF of the present invention can be used to induce further mutations, leading to further altered flower shapes or new flower colors, altered growing habits, foliage characteristics, etc. Mutants may appear spontaneously or mutations can be induced with Gamma irradiation or through treatment with certain chemical agents like ethyl methanesulfonate (EMS) (Broertjes, C. and van Harten, A.M., Applied mutation breeding for vegetatively propagated crops, Developments in Crop Science 12, Elsevier Science Publishers B.V. (1988)). Whereas these treatments mainly induce point mutations or chromosome mutations, genome mutations such as doubling of chromosome numbers can be produced, e.g., by treatment with colchicine. Even tissue culture can induce mutations, which are generally described as somaclonal variation (Chen, W.H., Chen, T.M., Fu, Y.M., and Hsieh, R.M., Studies on somaclonal variation in Phalaenopsis, Plant Cell Rep, 18, 7-13 (1998)).

Example 17 - Using Transformation on the Plants of the Present Invention

[0095] Within the past decades genetic transformation has been a very powerful technique to transfer single genes from one plant into another regardless of crossing barriers. Besides genes for single structural proteins, which might modify flower color or induce specific biotic resistances, genes encoding transcription factors, which manipulate a broader range of complex plant characters, have been successfully transferred even between different plant families or organisms.

[0096] A protocol for genetic transformation of Osteospermum has been developed and several genes, including the marker gene ß-glucuronidase as well as the rolB-gene, have been introduced into this plant species by Agrobacterium tumefaciens-mediated gene transfer. The following protocol has been developed, briefly: Leaf segments from Osteospermum tissue culture plants are incubated with a disarmed Agrobacterium tumefaciens strain which carries a vector with the gene of interest and a gene encoding a specific selectable marker, e.g., nptII for Kanamycin selection. By regeneration under selection pressure according to the selectable marker, transgenic plants can be regenerated from single transformed cells (Allavena, A. et al. (2000) Acta Hort. 508: 129-133; Giovannini, A. et al. (1999) In Vitro Cell. Dev. Biol. Plant 35: 70-75). The application of alternative transformation techniques like particle bombardment has not been described yet for Osteospermum or Dimorphoteca, but may be possible.

[0097] The use of genetic transformation is imaginable for both directions: foreign genes can be transferred into an Osteospermum or a Dimorphoteca plant having the mutant allele KLEDF of the present invention and lead to a plant with completely new characteristics. Alternatively, the mutant KLEDF allele of the present invention or its respective cDNA can be transferred into foreign genetic backgrounds and induce altered flowers in the resulting transgenic plants.

Example 18 - Incorporating the Mutant Allele KLEDF into Dimorphoteca Plants

[0098] Through intergeneric hybridization the mutant allele named KLEDF of the present invention can be introduced into the genus Dimorphoteca. Furthermore, the altered flowering trait can be incorporated into a broad range of Dimorphoteca cultivars through conventional breeding. By crossbreeding, the KLEDF allele of the present invention can be incorporated into Dimporphoteca cultivars having different flower colours and shapes as well as different foliage types. Furthermore, the mutant allele can be incorporated into plants having different growing habits, e.g., prostrate or hanging types can be developed besides erect or semi-erect types.

[0099] Using conventional breeding methods, a Dimorphoteca plant having the mutant allele KLEDF of the present invention is crossed with a Dimorphoteca plant lacking the mutant allele of the present invention. The resulting seeds are sown and the seedlings are grown according to conventional methods. The flowering F₁ progeny are then scored for altered flowering plants. Selected F₁ plants are further crossbred or they can be crossed back to their altered flowering or to their normal-flowering parent in order to combine the altered flowering phenotype with further desirable plant characteristics. However, depending on the genetic distance between the parents, inbreeding-depression might occur in this backcross progeny. Alternatively, selected plants from this F₁ progeny can be outcrossed to selected plants from a different F₁ progeny or to another cultivar, which is far related to the respective F₁ progeny.

Example 19 - Incorporating the Mutant Allele KLEDF into Dimorphoteca Plants to Create Interspecific Hybrids

[0100] The mutant allele KLEDF of the present invention can be introduced into an interspecific hybrid made between one species of Dimorphoteca having the mutant allele of the present invention and a different Dimorphoteca species. A selected Dimorphoteca plant having the mutant allele KLEDF is crossed, using conventional methods, as either a male or a female parent, to a selected genotype of any further Dimorphoteca species. For crosses with plants from less closely related species, specific techniques like bud pollination, removal of the stigma and pollination of the remaining style, GA₃- treatments of the pollinated stigmas, and/or embryo rescue of the immature embryo may be necessary. The seeds resulting from the cross are sown and the seedlings are grown according to conventional methods. The flowering F₁ progeny are then scored for altered flowering plants. Further breeding procedure is similar to the procedure described in Example 18. However, since the genus Dimorphoteca is representing species with different ploidy levels ranging from 2x to 6x, the resulting hybrids might for example be triploid and not produce viable seeds. Therefore, for further breeding the chromosome number of these plants must e.g. be doubled through treatment with colchicine.

Example 20 - Incorporating the Mutant Allele KLEDF into Osteospermum Plants to Create Intergeneric Hybrids

[0101] Furthermore, the mutant KLEDF allele of the present invention can also be introduced into an intergeneric hybrid through crosses between a selected Dimorphoteca plant having the mutant allele KLEDF of the present invention and an Osteospermum plant lacking the mutant allele of the present invention. The methods to be used to realize these hybrids correspond to the methods which have been described for interspecific crosses, meaning that specific techniques like bud pollination, removal of the stigma and pollination of the remaining style, GA₃-treatments of the pollinated stigmas, and/or embryo rescue of the immature embryo as well as chromosome doubling with colchicine may be necessary to realize these hybrid seedlings.

Example 21 - Comparison of seed yield of altered and normal flowering Osteospermum genotypes.

[0102] During the course of altered flowering Osteospermum breeding work it was observed that normal flowering Osteospermum have a higher seed yield than altered flowering Osteospermum. To confirm this, ten normal and ten altered flowering Osteospermum genotypes were planted on the beds in open fields without any isolation barrier between them. Four plants per genotype were planted in one plot. Furthermore altered flowering Osteospermum genotypes were planted in isolation as well. Matured seeds were collected from each plant of each genotype and counted. It was observed that there was no difference in the seed yield between altered flowering Osteospermum planted alongside normal flowering types and those planted in isolation (mean seed yield /plant = 21). However, the statistical analysis of the seed data clearly showed that under both conditions the altered flowering Osteospermum genotypes had significantly lower seed yields than normal flowering Osteospermum genotypes.

Table 11: Seed yields of the normal and altered Osteospermum genotypes

Flower Type	N	Mean number of seeds	Standard error of mean	t	Significance
Normal	10	233.22	44.36	4.76	0.001
Altered	10	20.44	5.4

Example 22 - Comparison of flower keepability of altered and normal flowering Osteospermum genotypes.

[0103] It was observed that altered flowering Osteospermum flowers have a longer keepability than normal flowering Osteospermum plants. In order to confirm this observation, we planted ten plants each of four normal and four altered flowering Osteospermum genotypes in the greenhouse (August-December 2010). The plants were not pinched and were thus allowed to grow and bear flower on the central stem. These flowers were regularly marked as, i) opened: when the flower buds start showing the color of the petals and were marked as ii) wilted: when two-three petals started showing aged symptoms and flower started appearing dull. For each genotype the difference between the day flower opened and the day it wilted was taken as flower keepability.

Table 12: Comparison of flower keepability of the normal and altered flowering Osteospermum genotypes

Flower Type	N	Mean (days)	t	Significance
Normal flowering	4	11.98	-2.575	0.05
Altered flowering	4	15.30

Example 23 - Plant growth regulator treatment of altered flowering Osteospermum

[0104] Spraying an altered flowering Osteospermum rooted cutting weekly with a 0.2 % solution of plant growth regulator Daminozide (sold under brand name Alar) transformed altered flowering Osteospermum flowers back to phenotypically almost normal appearing flowers. On plants of an altered flowering Osteospermum which were sprayed this way sometimes a part of and sometimes all elongated disc florets disappeared, so that the disc florets resembled the disc florets on a normal flowering Osteospermum plant In an altered flowering Osterospermum flower pollen is usually sparse which seriously limits the seed yield resulting from the crosses involving an altered flowering male parent. In contrast, flowers of a plant sprayed with Alar had almost comparable quantities of pollen as seen on a plant having normal type of flowers. This makes the crossing procedure convenient, as there is ample of pollen on plants with altered flowers which are genetically altered, but exhibit the normal type of flowers. Therefore, an altered flowering plant treated with Daminozide may easily and quickly be crossed with a normal flowering plant and with another altered flowering plant as well.

DEPOSIT INFORMATION

[0105] Osteospermum seeds containing the KLEDF mutant allele of this invention and capable of displaying the altered flower phenotype of the present invention have been placed on deposit under the Budapest Treaty with National Collections of Industrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom under NCIMB Accession No. 41698. The date of deposit was February 26, 2010.

TABLE 1. Segregation of Flower Type and Flower Colour in F₁ Progeny

Petal colour of the normal flowering crossing partner	# of Normal Flowering Plants				# of Altered Flowering Plants				Total # of Plants				χ2 Values
	White	Pink	Purple	Total	White	Pink	Purple	Total	White	Pink	Purple	Total	Corresponding to a 1:1 segregation for flower type
White	174	23	41	238	46	5	44	95	220	28	85	333	*61.4
Purple	3	8	263	274	3	3	68	74	6	11	331	348	*114.8
Pink	3	217	33	253	1	7	6	14	4	224	39	267	*213.94
White	59	28	35	122	23	7	21	51	82	35	56	173	*29.14
White-purple bicoloured	16	12	0	28	3	12	0	15	19	24	0	43	*3.94
Purple	0	9	61	70	0	2	9	11	0	11	70	81	*42.98
Pink	57	59	24	140	5	15	3	23	62	74	27	163	*83.98
Pink	6	47	41	94	0	8	7	15	6	55	48	109	*57.26
Ivory	110	0	0	110	23	0	0	23	133	0	0	133	*56.90

*Respective progeny deviate significantly from a 1:1 segregation at χ2 = 3.84

TABLE 2 Segregation of the Altered Flower-Type in F₂ Progeny

	Crosses between altered flowering F1 plants and normal-flowering plants
Population code	# of Altered flowering plants	# of Normal flowering plants	Total # of Plants	χ2 Values corresponding to a 1:1 segregation pattern
468	64	54	118	0.84
469	65	89	154	3.74
471	83	90	173	0.28
499	52	41	93	1.30
501	48	44	92	0.18
503	35	45	80	1.25
504	74	72	146	0.16
506	38	44	82	0.44
511	107	96	203	0.58
476	46	43	89	0.10

	Crosses between two altered flower-type F1 plants
Population code	# of Altered flowering plants	# of Normal flowering plants	Total # of Plants	χ2 Values corresponding to a 3:1 segregation pattern
8	129	44	173	0.01
9	66	18	84	0.84
3	15	52	67	0.24

* Respective combinations deviate significantly from the expected 3:1 segregation at χ2=3.84

TABLE 4. Comparison of Certain Plant Characteristics between Normal- and Altered Flowering Osteospermum Genotypes

Code	Type	Ray florets				Disc florets			Foliage - Immature		Foliage - Mature			No. basal shoots
		No.	Length (cm)	Width (cm)	Colour (RHS)	No.	Length (cm)	Average Length (cm)	Length (cm)	Width (cm)	Length (cm)	Width (cm)	Colour (RHS)
OE 2008248	AF	14-16	3.0	0.7	92D	80-85	1.3-2.1	1.7	3.5	0.9	4.8-5.0	1.6-1.9	146A	6
OE 2008258	AF	17-18	2.6-2.7	0.7-0.8	92D	73-89	1.0-1.9	1.45	3.2	0.8	5.2	1.9-2.0	144A	5
OE 2008274	AF	25	2.6-2.9	0.7-0.9	78B	95-96	1.3-1. 7	1.5	3.2	0.8-0.9	4.5-4.9	2.0	146A	5
OE 2008285	AF	22-25	2.6-3.0	0.6-0.8	N74B	78-85	0.8-2.2	1.5	4.0-4.5	1.2-1.6	7.0-7.5	2.5-3.5	137A	5
OE 2008384	AF	23-27	2.1	0.6-0.7	72A	94-108	1.4-1. 7	1.55	3.4	1.1	4.1	2.1	146A	4
OE 2008390	AF	19-22	2.6	0.8-0.9	77C+75D	68-100	1.4-1.8	1.6	3.3	0.9-1.1	6.5	2.0-2.5	146A	5
A-5-43	NF	17-20	3.2-3.3	1.0	155D	65-90	0.5-0.6	0.55	3.3-3.9	1.0-1.2	6.0-7.0	2.3-2.5	146A	4
V 78	NF	19-22	3.3-3.4	1.1-1.2	155D	81-89	0.4-0.6	0.5	3.5-4.0	0.9-1.2	6.2-7.3	2.1-2.4	N137B	5
W 113	NF	18-21	2.4-2.9	0.7-0.9	157C	40-45	0.4-0.5	0.45	3.2-3.6	0.8-1.0	5.5-6.0	1.6-1.8	146A	6
A-48-24	NF	20-23	2.6	0.6-0.8	71A/N81A	77-96	0.5-0.6	0.55	2.4-2.9	0.8-10.	5.5-6.5	2.0-3.0	146A	4
X 95	NF	23-25	3.4-3.7	0.9	N78A/83	65-79	0.5-0.6	0.55	3.1-3.5	1.0-1.1	5.9-6.4	2.5	147A	4
A-69-1	NF	21-25	2.7	0.8	72A/N78A	93-107	0.6	0.6	2.8	0.7-1.1	6.3-6.6	2.5	147A	4
V 34	NF	19-20	2.6-2.8	0.8-0.9	70B/N82A	72-81	0.4-0.5	0.45	2.5-2.7	0.6	6.1-6.4	2.6	N137B	5
W 42	NF	16-20	2.4-2.6	0.7	78B/78C/ 80C	58-75	0.5-0.6	0.55	2.8	0.9-1.0	4.3-4.6	1.6-1.8	146A	5
A-46-1	NF	19-20			N74C+155D/ N82B	63-70	0.5	0.5	2.3-2.8	0.7-0.8	5.8-6.3	2.5-2.7	147A	4

SEQUENCE LISTING

[0106] 

<110> Klemm & sohn GmbH & Co. KG

<120> Osteospermum and Dimorphoteca plants having an altered flower phenotype

<130> S 10007/RN

<150> EP10158786.3
<151> 2010-03-31

<150> US 12/750,965
<151> 2010-03-31

<160> 11

<170> PatentIn version 3.5

<210> 1
<211> 19
<212> DNA
<213> artificial sequence

<220>
<223> primer E40

<400> 1
gactgcgtac caattcagc   19

<210> 2
<211> 19
<212> DNA
<213> artificial

<220>
<223> primer M54

<400> 2
gatgagtcct gagtaacct 19

<210> 3
<211> 20
<212> DNA
<213> artificial

<220>
<223> primer CYC2f6

<400> 3
aagatcgaca cagctcacgg   20

<210> 4
<211> 20
<212> DNA
<213> artificial

<220>
<223> primer CYC2r7

<400> 4
tctgcccttg actgattcac   20

<210> 5
<211> 330
<212> DNA
<213> artificial

<220>
<223> PCR product of SEQ ID Nos. 3 and 4

<220>
<221> misc-feature
<222> (2)..(3)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (10)..(10)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (38)..(39)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, or t

<220>
<221> misc-feature
<222> (79)..(79)
<223> n is a, c, g, or t

<400> 5

<210> 6
<211> 17
<212> DNA
<213> artificial

<220>
<223> EcoRI adapter 1

<400> 6
ctcgtagact gcgtacc   17

<210> 7
<211> 18
<212> DNA
<213> artificial

<220>
<223> EcoRI adapter 2

<400> 7
aattggtacg cagtctac   18

<210> 8
<211> 16
<212> DNA
<213> artificial

<220>
<223> MseI adapter 1

<400> 8
gacgatgagt cctgag   16

<210> 9
<211> 14
<212> DNA
<213> artificial

<220>
<223> MseI adapter 2

<400> 9
tactcaggac tcat   14

<210> 10
<211> 10
<212> DNA
<213> artificial

<220>
<223> sequence with SNP

<220>
<221> misc_feature
<222> (6)..(6)
<223> n is c, or t

<400> 10
tttganaaag 10

<210> 11
<211> 330
<212> DNA
<213> artificial

<220>
<223> PCR product of SEQ ID Nos. 3 and 4 in normal flowering plants

<220>
<221> misc_feature
<222> (1)..(3)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (6)..(7)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (16)..(18)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (42)..(43)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, or t

<220>
<221> misc_feature
<222> (83)..(83)
<223> n is a, c, g, or t

<400> 11

Claims

1. An Osteospermum plant, having an altered flower phenotype, wherein at least one inflorescence has at least one disc floret with a length of the disc floret corolla of at least 1.7 cm, wherein the average length of the longest and the shortest disc floret corolla of at least one inflorescence is at least 1.45 cm and having the anthers being located in said disc floret corolla, the altered flower phenotype being caused by a mutant allele contained in seed which has been deposited under NCIMB Accession No. 41698, the plant comprising a DNA sequence, which after restriction digestion with EcoRI and Msel and annealing of the adapters according to SEQ ID Nos. 6 to 9 is a template for amplification of a DNA fragment of about 151 nucleotides with the primers according to SEQ ID Nos. 1 and 2.

2. The Osteospermum plant of claim 1, wherein the average length is between 1.45 cm and 1.7 cm.

3. The Osteospermum plant of any of the preceding claims, comprising a DNA sequence which upon amplification with the primers according to SEQ ID Nos. 3 and 4 yields a fragment comprising a sequence TTTGANAAAG (SEQ ID No. 10) wherein N is C.

4. A cell, seed or protoplast produced from the plant of any of the preceding claims or a tissue culture of regenerable cells produced from the plant of any of the preceding claims, where said cells of the tissue culture are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, stem, petiole, ray floret, and disc floret, the cell, seed or protoplast comprising the mutant allele of the plant specified in claim 1.

5. An Osteospermum seed containing the mutant allele of the plant specified in claim 1 which is responsible for the development of the altered flower phenotype wherein at least one inflorescence has at least one disc floret corolla with a length of at least 1.7 cm.

6. An Osteospermum plant, produced by growing the seed of claim 5.

7. A tissue culture of regenerable cells produced from the plant of claim 6, where said cells of the tissue culture are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, protoplast, root, root tip, pistil, anther, stem, petiole, ray floret, and disc floret, the cells comprising the mutant allele of the plant specified in claim 1.

8. An Osteospermum plant regenerated from said tissue culture of claim 7.

9. Intergeneric hybrid plant of Osteospermum and Dimorphoteca, wherein at least one parent of said hybrid plant is the Osteospermum plant of any of claims 1 to 3 and wherein at least one inflorescence of the hybrid plant has at least one disc floret corolla with a length of at least 1.7 cm, wherein the average length of the longest and the shortest disc floret corolla of at least one inflorescence is at least 1.45 cm and having the anthers being located in said disc floret corolla, the altered flower phenotype being caused by a mutant allele of the plant specified in claim 1.

10. The hybrid plant of claim 9 which is obtained by fusion of protoplasts from the Osteospermum plant of claim 1 and a Dimorphoteca plant.

Ansprüche

1. Osteospermum-Pflanze mit einem veränderten Blütenphänotyp, wobei mindestens ein Blütenstand mindestens eine Röhrenblüte mit einer Länge der Blütenkrohnröhre von mindestens 1,7 cm aufweist, wobei die durchschnittliche Länge der längsten und der kürzesten Blütenkrohnröhre mindestens eines Blütenstandes mindestens 1,45 cm beträgt und die Staubbeutel in der Blütenkrohnröhre angeordnet sind, wobei der veränderte Blütenphänotyp durch ein mutiertes Allel verursacht wird, das in Saatgut enthalten ist, das unter der NCIMB-Hinterlegungsnummer 41698 hinterlegt wurde, wobei die Pflanze eine DNA-Sequenz umfasst, die nach Restriktionsverdau mit EcoRI und Msel und Anhaftung der Adapter gemäß SEQ ID Nr. 6 bis 9 eine Matrize für die Amplifikation eines DNA-Fragments von etwa 151 Nukleotiden mit den Primern gemäß SEQ ID Nr. 1 und 2 ist.

2. Osteospermum-Pflanze nach Anspruch 1, wobei die durchschnittliche Länge zwischen 1,45 cm und 1,7 cm liegt.

3. Osteospermum-Pflanze nach einem der vorhergehenden Ansprüche, umfassend eine DNA-Sequenz, die nach Amplifikation mit den Primern gemäß SEQ ID Nr. 3 und 4 ein Fragment ergibt, das eine Sequenz TTTGANAAAG (SEQ ID Nr. 10) umfasst, worin N C ist.

4. Zelle, Samen oder Protoplast, hergestellt aus der Pflanze nach einem der vorhergehenden Ansprüche, oder eine Gewebekultur von regenerierbaren Zellen, hergestellt aus der Pflanze nach einem der vorhergehenden Ansprüche, wobei die Zellen der Gewebekultur aus einem Pflanzenteil hergestellt sind, ausgewählt aus der Gruppe bestehend aus Blatt, Pollen, Embryo, Keimblatt, Hypokotyl, meristematische Zelle, Protoplast, Wurzel, Wurzelspitze, Stempel, Staubbeutel, Stängel, Blattstiel, Strahlenblüte und Röhrenblüte, wobei die Zelle, der Samen oder der Protoplast das mutierte Allel der in Anspruch 1 angegebenen Pflanze umfasst.

5. Osteospermum-Samen, der das mutierte Allel der in Anspruch 1 angegebenen Pflanze enthält, das für die Entwicklung des veränderten Blütenphänotyps verantwortlich ist, wobei mindestens ein Blütenstand mindestens eine Scheibenblumenkrone mit einer Länge von mindestens 1,7 cm aufweist.

6. Osteospermum-Pflanze, hergestellt durch Züchtung des Samens nach Anspruch 5.

7. Gewebekultur aus regenerierbaren Zellen, hergestellt aus der Pflanze nach Anspruch 6, wobei die Zellen der Gewebekultur aus einem Pflanzenteil hergestellt sind, der aus der Gruppe ausgewählt ist, die aus Blatt, Pollen, Embryo, Keimblatt, Hypokotyl, meristematischer Zelle, Protoplast, Wurzel, Wurzelspitze, Stempel, Staubbeutel, Stängel, Blattstiel, Strahlenblüte und Röhrenblüte besteht, wobei die Zellen das mutierte Allel der in Anspruch 1 angegebenen Pflanze umfassen.

8. Osteospermum-Pflanze, die aus der Gewebekultur nach Anspruch 7 regeneriert wurde.

9. Intergenerische Hybridpflanze von Osteospermum und Dimorphoteca, wobei mindestens ein Elternteil der Hybridpflanze die Osteospermum-Pflanze nach einem der Ansprüche 1 bis 3 ist und wobei mindestens ein Blütenstand der Hybridpflanze mindestens eine Blütenkrohnröhre mit einer Länge von mindestens 1,7 cm aufweist, wobei die durchschnittliche Länge der längsten und kürzesten Blütenkrohnröhre mindestens eines Blütenstandes mindestens 1,45 cm beträgt und die Staubbeutel in der Blütenkrohnröhre angeordnet sind, wobei der veränderte Blütenphänotyp durch ein mutiertes Allel der in Anspruch 1 angegebenen Pflanze verursacht wird.

10. Hybridpflanze nach Anspruch 9, die durch Fusion von Protoplasten aus der Osteospermum-Pflanze nach Anspruch 1 und einer Dimorphoteca-Pflanze erhalten wird.

Revendications

1. Plante Osteospermum, ayant un phénotype de fleur altéré, dans laquelle au moins une inflorescence a au moins un fleuron discal avec une longueur de la corolle de fleuron discal d'au moins 1,7 cm, dans laquelle la longueur moyenne de la corolle de fleuron discal la plus longue et la plus courte d'au moins une inflorescence est d'au moins 1,45 cm et ayant les anthères situées dans ladite corolle de fleuron discal, le phénotype de fleur altéré étant causé par un allèle mutant contenu dans la graine qui a été déposée sous le numéro d'accession NCIMB 41698, la plante comprenant une séquence d'ADN qui, après digestion par restriction avec EcoRI et Msel et hybridation des adaptateurs selon les SEQ ID n° 6 à 9, est une matrice pour l'amplification d'un fragment d'ADN d'environ 151 nucléotides avec les amorces selon les SEQ ID n° 1 et 2.

2. Plante Osteospermum selon la revendication 1, dans laquelle la longueur moyenne est comprise entre 1,45 cm et 1,7 cm.

3. Plante Osteospermum de l'une quelconque des revendications précédentes, comprenant une séquence d'ADN qui, après amplification avec les amorces selon les SEQ ID N° 3 et 4, donne un fragment comprenant une séquence TTTGANAAAG (SEQ ID N° 10) dans laquelle N est C.

4. Cellule, graine ou protoplaste produit à partir de la plante de l'une quelconque des revendications précédentes ou culture tissulaire de cellules régénérables produite à partir de la plante de l'une quelconque des revendications précédentes, où lesdites cellules de la culture tissulaire sont produites à partir d'une partie de plante choisie dans le groupe constitué par la feuille, pollen, embryon, cotylédon, hypocotyle, cellule méristématique, protoplaste, racine, extrémité de racine, pistil, anthère, tige, pétiole, fleuron radié et fleuron discal, la cellule, la graine ou le protoplaste comprenant l'allèle mutant de la plante spécifiée dans la revendication 1.

5. Graine d'Osteospermum contenant l'allèle mutant de la plante spécifiée dans la revendication 1 qui est responsable du développement du phénotype de fleur altéré, dans laquelle au moins une inflorescence a au moins une corolle à fleur de disque avec une longueur d'au moins 1,7 cm.

6. Plante Osteospermum, produite par la culture de la graine de la revendication 5.

7. Culture tissulaire de cellules régénérables produites à partir de la plante de la revendication 6, où lesdites cellules de la culture tissulaire sont produites à partir d'une partie de plante choisie dans le groupe constitué par la feuille, le pollen, l'embryon, le cotylédon, l'hypocotyle, la cellule méristématique, le protoplaste, la racine, l'extrémité de la racine, le pistil, l'anthère, la tige, le pétiole, le fleuron radié et le fleuron discal, les cellules comprenant l'allèle mutant de la plante spécifiée dans la revendication 1.

8. Plante Osteospermum régénérée à partir de ladite culture de tissu de la revendication 7.

9. Plante hybride intergénérique d'Osteospermum et de Dimorphoteca, dans laquelle au moins un parent de ladite plante hybride est la plante Osteospermum de l'une quelconque des revendications 1 à 3 et dans laquelle au moins une inflorescence de la plante hybride a au moins une corolle de fleuron discal avec une longueur d'au moins 1. 7 cm, où la longueur moyenne de la corolle de fleuron discal la plus longue et la plus courte d'au moins une inflorescence est d'au moins 1,45 cm et où les anthères sont situées dans ladite corolle de fleuron discal, le phénotype de fleur altéré étant causé par un allèle mutant de la plante spécifiée dans la revendication 1.

10. Plante hybride selon la revendication 9, qui est obtenue par fusion de protoplastes provenant de la plante Osteospermum selon la revendication 1 et d'une plante Dimorphoteca.

Drawing