US 20060218679A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0218679 A1 Bernuetz (43) Pub. Date: Sep. 28, 2006

(54) METHOD OF PRODUCING EUPHORBIA (30) Foreign Application Priority Data INTERSPECIFIC HYBRIO PLANTS BY CUTTING AND THEN CULTURING THE Oct. 9, 2003 (AU)...... 20O3905549 HYBRIDEMIBRYOS Publication Classification (75) Inventor: Andrew Bernuetz, Silverdale (AU) (51) Int. Cl. AOIH II/00 (2006.01) Siko S. viiserieAU, P.A. (52) U.S. Cl...... 800/295; 47/58.1 R 900 SECOND AVENUE SOUTH (57) ABSTRACT SUTE 82O MINNEAPOLIS, MN 55402 (US) A method for producing an interspecific hybrid Euphorbia plant. The method comprises: (a) providing a first plant (73) Assignee: Bonza Botanicals Pty Limited, Winma- which is a Euphorbia pulcherrima plant and a second plant lee (AU) which is a species of Euphorbia selected from the group consisting of Euphorbia Cornastra, Euphorbia radians, (21) Appl. No.: 11/402,370 Euphorbia colorata and Euphorbia fulgens, (b) pollinating a flower of the second plant with pollen from the first plant (22) Filed: Apr. 7, 2006 or a flower of the first plant with pollen from the second plant in a manner which permits formation of an embryo in Related U.S. Application Data at least one ovule of the pollinated plant; (c) cutting the embryo, and (d) culturing the cut embryo by placing the cut (63) Continuation of application No. PCT/AU04/00257, embryo in contact with culture medium to permit growth of filed on Feb. 27, 2003. the embryo to thereby produce a primary plant.

Patent Application Publication Sep. 28, 2006 US 2006/0218679 A1 Figure 1

US 2006/0218679 A1 Sep. 28, 2006

METHOD OF PRODUCING EUPHORBA SUMMARY OF THE INVENTION INTERSPECIFIC HYBRIO PLANTS BY CUTTING AND THEN CULTURING THE HYBRIDEMIBRYOS 0006 The inventor has found that by using a method in which the embryo is cut and the cut portions placed in FIELD OF THE INVENTION culture, the efficiency of viable plants generated from an 0001. The present invention relates to Euphorbia inter interspecific hybrid embryo developed by hybridising specific hybrid plants and methods for making the same. In Euphorbia pulcherrima with other Euphorbia species can be particular, the invention relates to interspecific hybrid plants improved. derived from the cross between Euphorbia pulcherrima and 0007. In a first aspect, the invention provides a method species of Euphorbia other than Euphorbia pulcherrima, for producing an interspecific hybrid Euphorbia plant com and methods for the generation of interspecific variants with prising: altered characteristics to Euphorbia pulcherrima and other plants. 0008 (a) providing a first plant which is a Euphorbia pulcherrima plant and a second plant which is a species of BACKGROUND OF THE INVENTION Euphorbia selected from the group consisting of Euphorbia 0002. A characteristic of some plants is the ability to cornastra, Euphorbia radians, Euphorbia colorata and cross with different species, called interspecific hybridisa Euphorbia fulgens, tion. This results in the transfer of genetic material between 0009 (b) pollinating a flower of the second plant with the species, leading to the production of an entirely new pollen from the first plant or a flower of the first plant with species of plant. Through the transfer of desirable genetic pollen from the second plant in a manner which permits traits, it is possible to obtain improved species of plants formation of an embryo in at least one ovule of the polli which combine the desirable features of each of the parent nated plant; plants. Interspecific hybridisation thus represents a method for producing new species of plants in which the desirable 0010 (c) cutting the embryo; and features of different species are combined. 0011 (d) culturing the cut embryo by placing the cut 0003. However, not all species of plants are capable of embryo in contact with culture medium to permit growth of undergoing interspecific hybridisation, and the ability of a the embryo to thereby produce an interspecific hybrid plant to be hybridised with a different species can vary Euphorbia plant (a primary plant). widely, depending on the species, the chromosome number 0012 Preferably, a flower of the first plant is pollinated of the plant, and the level of homology between the plant with pollen from the second plant. In other words, preferably species to be crossed. a flower of a Euphorbia pulcherrima plant is pollinated with 0004 Interspecific hybridisation would be desirable pollen from a plant selected from the group consisting of between species within the genus Euphorbia. Euphorbia Euphorbia cornastra, Euphorbia radians, Euphorbia colo comprises a vast number of species. Among these, Euphor rata and Euphorbia filgens. bia pulcherrima, also known as poinsettia, is among the 0013 The cutting step may comprise slicing the embryo most popular of ornamental potted plants. It would be into at least two portions. The term “cutting includes desirable to be able to cross other species of Euphorbia with slicing, splitting, breaking, or any other act that separates the Euphorbia pulcherrima to retain the desirable characteris embryo into at least two portions. Preferably, the portions tics of E. pulcherrima while also inheriting the desirable are roughly equal portions. characteristics of other Euphorbia species. Features such as flowering period, flower colour, branch length, plant height, 0014. In one embodiment, the embryo is cut while con branch internode length etcetera may then be improved in tained in the ovule. In a preferred embodiment, the ovule is the new species to provide a more desirable ornamental at least 3 millimetres in length. The ovule may be sliced plant. transverse to the longitudinal axis, or along the longitudinal axis. Preferably, the ovule is sliced along the longitudinal 0005 Methods for generating interspecific hybrids axis of the ovule. between Euphorbia species have met with limited success. For example, interspecific hybridisation has been possible to 0015 Typically, the sliced ovule containing the sliced a limited extent between the poinsettia Euphorbia pulcher embryo is placed in contact with the culture medium. rima and Euphorbia cornastra as described in WO 0016 Preferably, the culturing step employs ovule slice 02/32217. This document discloses an interspecific hybrid culture. Euphorbia plant that was obtained using embryo rescue following pollination of Euphorbia pulcherrima with 0017. In another embodiment, the embryo is cut follow Euphorbia cornastra. Embryo rescue involves growing the ing excision from the ovule. For example, the embryo may embryo to a globular shaped stage of development following be cut following embryo rescue. Preferably, the embryo is pollination, completely excising the embryo (and Suspensor excised from an ovule that is at least 3 millimetres in length. cells) from the ovary, and Subsequently culturing the embryo 0018. In one embodiment, the method comprises the in vitro. However, many of the embryos that are excised further steps of: during embryo rescue do not survive, and/or do not grow into plants in culture. Further, generally it is not possible to 0019 (a) obtaining a cutting from the primary plant; produce more than one plant from a single embryo using embryo rescue. As a consequence, embryo rescue is often 0020 (b) incubating the cutting under conditions suffi inefficient and/or unsuccessful for use in crosses between cient to propagate the primary plant. Euphorbia species. 0021 Preferably, the cutting is a shoot. US 2006/0218679 A1 Sep. 28, 2006

0022. The cutting may be treated to induce root forma 0036) The mutated plant, or a portion of the mutated tion. For example, root formation may be induced by plant, may be propagated by: treating the cutting with a composition containing a hor mone capable of inducing root formation. Preferably, the 0037 (a) obtaining a bract from the mutated plant; hormone is an auxin. Examples of Suitable auxins include 0038 (b) placing the bract in a solution capable of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and disinfesting the bract; C.-napthalene acetic acid (NAA). 0039 (c) washing the bract; and 0023. A free-branching agent may be transmitted to the 0040 (d) cultivating the bract to thereby produce a propa primary plant to provide the primary plant with a free gated mutated plant. branching phenotype. Thus, in one embodiment, the method 0041) Preferably, the solution capable of disinfesting the of the invention comprises the further step of transmitting a bract is bleach. The bleach (NaOCl) is preferably used at a free-branching agent to the primary plant. The free-branch concentration of between 1% and 3. ing agent may be transmitted to the primary plant by any means. For example, the free branching agent may be 0042. The primary plant, or a portion of the primary transmitted to the primary plant by a dodder (a parasitic plant, may be propagated by: plant), by a leafhopper insect or by a graft. 0043 (a) obtaining a bract from the primary plant; 0024 Preferably, the free-branching agent is transmitted 0044 (b) placing the bract in a solution capable of to the primary plant by: disinfesting the bract; 0.025 (a) providing a free-branching plant having a free 0045 (c) washing the bract; and branching agent; 0046 (d) cultivating the bract to thereby propagate the 0026 (b) cutting the primary plant and the free-branching primary plant. plant to expose tissue of the plant; 0047 Preferably, the solution capable of disinfesting the 0027 (c) making a graft union between the tissue of the bract is bleach. The bleach (NaOCl) is preferably used at a free-branching plant and the primary plant, whereby at least concentration of between 1% and 3%. one characteristic of a vegetative shoot arising from said 0048. In a second aspect, the invention provides a plant graft is different from the free-branching plant and the produced according to the method of the first aspect of the primary plant; and invention. 0028 (d) growing said shoot to obtain a grafted primary 0049. In a third aspect, the invention provides part of a plant with at least one altered growth characteristic. plant including, for example, a flower, cutting, a pollen grain, an ovule, a cell, a seed or an embryo, produced 0029. The grafted primary plant preferably differs from according to the method of the first aspect of the invention. the primary plant in that the grafted primary plant has a free-branching phenotype. Thus, at least one altered growth 0050. The plant may be a Euphorbia plant that is clonally characteristic is preferably a free-branching phenotype. propagated. Preferably, the free-branching phenotype is due to the free 0051. In a fourth aspect, the invention provides a method branching agent. for producing an interspecific hybrid Euphorbia plant com prising: 0030 The free-branching agent may be any agent which induces a free-branching phenotype and which can be trans 0052 (a) providing a first plant which is a Euphorbia mitted by a graft, such as a bacterium, virus or phytoplasma. pulcherrima plant and a second plant which is a species of Preferably, the free-branching agent is a phytoplasma. Euphorbia that is not Euphorbia pulcherrima, 0031 Variations in the characteristics of the grafted pri 0053 (b) pollinating a flower of the second plant with mary plant may be introduced by mutagenesis of the plant or pollen from the first plant or a flower of the first plant with parts thereof. In one embodiment, the method further com pollen from the second plant in a manner which permits prises the steps of formation of an embryo in at least one ovule of the polli nated plant; 0032 (a) obtaining a cutting of the primary plant or the grafted primary plant; 0054 (c) cutting the embryo; and 0055 (d) culturing the cut embryo by placing the cut 0033 (b) exposing the cutting to a mutagen; and embryo in contact-with culture medium to permit growth of 0034 (c) cultivating the cutting to produce a mutated the embryo to thereby produce a primary plant. plant. 0056. In a fifth aspect, the invention provides a plant 0035. The mutagen may be any mutagen capable of produced according to the method of the fourth aspect of the mutating plant DNA. The mutagen may be a chemical invention. mutagen Such as ethylmethane Sulfonate, sodium azide, 0057. In a sixth aspect, the invention provides part of a N-nitroso-N-ethylurea or N-nitroso-N-methylurea, a bio plant including, for example, a flower, cutting, a pollen logical mutagen Such as a transposon, or radiation. In one grain, an ovule, a cell, a seed or an embryo, produced embodiment, the mutagen is radiation. Preferably, the radia according to the method of the fourth aspect of the inven tion is gamma radiation. tion. US 2006/0218679 A1 Sep. 28, 2006

0.058. In a seventh aspect, the invention provides a method such that described in WO O2/32217 because the method for propagating a Euphorbia plant comprising: present method allows more than one plant to be obtained from the same ovule or embryo. As a consequence, effi 0059 (a) obtaining a bract from the plant; ciency of interspecific hybrid production may be improved 0060 (b) placing the bract in a solution capable of using the method of the invention. This is a Surprising result disinfesting the bract; because it was thought that the embryo produced by Euphor bia species was fragile and consequently would not survive 0061 (c) washing the bract; and disruption Such as cutting of the embryo. Further, it was 0062 (d) cultivating the bract to thereby produce a propa thought that an embryo from a Euphorbia interspecific gated mutated plant. hybrid was likely to be even more fragile than an embryo from an intraspecific hybrid. It was thought that cutting the 0063 Preferably, the solution capable of disinfesting the Euphorbia embryo would not only damage the fragile tissue bract is bleach. The bleach (NaOCl) is preferably used at a of the embryo and prevent normal function, but also that concentration of between 1% and 3%. cutting a Euphorbia embryo would disrupt normal devel 0064. In an eighth aspect, the invention provides a plant opment of the embryo resulting in death of the embryo. produced according to the method of the seventh aspect of 0071. In a preferred embodiment, the first plant is a the invention. Euphorbia pulcherrima plant and the second plant is 0065. In a ninth aspect, the invention provides part of a selected from the group consisting of Euphorbia cornastra, plant including, for example, a flower, cutting, a pollen Euphorbia radians, Euphorbia colorata and Euphorbia fill grain, an ovule, a cell, a seed or an embryo, produced gens. according to the method of the seventh aspect of the inven 0072 The Euphorbia pulcherrima plant may be any tion. cultivar of Euphorbia pulcherrima. Examples of suitable cultivars of Euphorbia pulcherrima include, for example, BRIEF DESCRIPTION OF THE DRAWING varieties such as 97/176.2, 97/176.3, 97/24.1, 97/54.1, 0.066 FIG. 1 illustrates the plants resulting from an 97/144, and cultivars such as Angelika (U.S. Plant Pat. No. embodiment of the method of the invention. The parent 5.492), Freedom red (U.S. Plant Pat. No. 7,825) and V10 plants Euphorbia pulcherrima (A) and Euphorbia Cornastra Amy red. The Euphorbia pulcherrima plant may be the (B) are shown, with various interspecific hybrids of these derivative of a cross between two different Euphorbia parent plants in between A and B illustrating the variation in pulcherrima cultivars. For example, cultivar 97/24.1 is the length, leaf size and internodal distance. product of a cross between Euphorbia pulcherrima cV. Freedom white (U.S. Plant Pat. No. 8,772) and cv. V10 Amy DETAILED DESCRIPTION OF THE red, cultivar 97/54.1 is the result of a self pollination of cv. V10 Amy red, 97/144 is the result of a cross between cv. INVENTION Peppermint pink and cv. V1 o Amy red. 0067. Before the present methods are described, it is 0073. As a first step in carrying out the method of the understood that this invention is not limited to the particular invention, the first and second plants are cultivated. As used materials and methods described, as these may vary. It must herein, the term “cultivating” means to expose a plant to be noted that as used herein, the singular forms “a,'an, and conditions which result in production of pollen from the first “the include plural reference unless the context clearly plant and presentation of a receptive stigma from the second dictates otherwise. Thus, for example, a reference to “plant” plant, or in the production of pollen in the second plant and includes a plurality of such plants. Unless defined otherwise, the presentation of a receptive stigma from the first plant. all technical and Scientific terms used herein have the same Preferably, the conditions result in production of a flower in meanings as commonly understood by one of ordinary skill the first and second plants. Methods for cultivation of in the art to which this invention belongs. Although any Euphorbia species are well-known in the art and are materials and methods similar or equivalent to those described in, for example, Ecke, P., Matkin, O. A. and described herein can be used to practice or test the present Hartley, D. E. (1990) The Poinsettia Manual. Paul Ecke invention, the preferred materials and methods are now Poinsettias, Encinitas, Calif., USA. It will be appreciated by described. those skilled in the art that growth conditions for the various 0068 Publications mentioned hereinafter are cited for the Euphorbia plants used in the method of the invention may purpose of describing and disclosing the protocols, reagents vary from species to species, and may depend on growth and vectors which are reported in the publications and which requirements such as photoperiod for each individual spe might be used in connection with the invention. C1GS. 0069. The present invention provides a method of pro 0074 The first or second plant may be emasculated to ducing an interspecific hybrid formed by crossing Euphor avoid self-pollination. This may be achieved by manual bia pulcherrima and a species of Euphorbia selected from removal of the anthers, or by chemical means using game the group consisting of E. cornastra, E. radians, E. colorata tocides such as described in U.S. Pat. No. 4,936,904. and E. fulgens. 0075) The flower of the first plant is then pollinated with 0070 The inventor has found that by cutting the embryo pollen from the second plant, or the flower of the second formed from interspecific pollination, the efficiency of plant is pollinated with pollen from the first plant. Prefer hybrid plant production in culture is improved, and it is often ably, the flower of the first plant is pollinated with the pollen possible to obtain multiple clonal interspecific hybrid plants from the second plant. In other words, in a preferred from a single original embryo. This has advantages over a embodiment, Euphorbia pulcherrima is pollinated with pol US 2006/0218679 A1 Sep. 28, 2006 len from Euphorbia cornastra, Euphorbia radians, Euphor g/L. pH is preferably adjusted to 5.8. The cultured embryo bia colorata or Euphorbia filgens. As used herein, the term preferably is subcultured every 3 to 4 weeks. The embryo "pollinating refers to any method by which pollen is may be subcultured into fresh regeneration medium or may brought into contact with the Stigma of a plant in a manner be subcultured into proliferation medium. An example of which results in formation of an embryo in at least one ovule suitable proliferation medium includes MS salts at 4.42 g/L, of the plant. In the case of interspecific hybridisation, the 0.3 mg/L 6-benzylaminopurine, 1 g/L casein hydrolysate, pollen from one species of Euphorbia is used to pollinate a sucrose preferably at 30 g/L and agar preferably at 7 g/L. pH different species of Euphorbia. Pollinating may be con is preferably adjusted to 5.8. The cultured embryo may be ducted manually using implements such as paintbrushes or subcultured every 3 to 4 weeks onto either the regeneration other methods known in the art for pollinating plants (see, or proliferation medium, whichever promotes growth for for example, Watts, L. (1980) Flower and Vegetable Plant that interspecific hybrid. Breeding. Grower Books, London; Allard, R. W. (1999) 0080. The plantlets that emerge from the cut embryo are Principles of Plant Breeding, John Wiley & Sons, incorpo a primary plant. As used herein, the term “primary plant” rated herein by reference). It will be appreciated by those refers to an interspecific hybrid of a Euphorbia plant pro skilled in the art that the time for pollinating will depend on duced by the method of the first or fourth aspect, and flowering times of the plant, and rate of production of includes the plantlet obtained from culturing the embryo, anthers and pistils. It will also be appreciated by those and plants obtained from incubating cuttings of the plantlet skilled in the art that the times for flowering and obtaining under conditions which result in production of a plant. The pollen will vary from species to species and may be deter primary plant may be propagated by obtaining a cutting and mined empirically using approaches well known in the art. treating the cutting under conditions which stimulate growth Preferably, fresh pollen is collected and applied liberally to of a plant from the cutting. Conditions which stimulate all areas of the receptive stigmatic Surface. growth of a plant from the cutting may be any conditions 0.076 Once the plant is pollinated, embryo development that result in production of a plant, and may include treating is detected, the embryo is cut, and the cut embryos cultured. the cutting, and/or placing the cutting in a particular envi Embryo development is preferably detected by observing ronment. For example, the cutting may be treated with a cyathia with swollen ovaries. The embryo is then cultured composition comprising a hormone capable of inducing root from those ovaries that are swollen. Preferably, the stage at formation. The hormone may be, for example, indole acetic which the embryo is cultured is when an abscission layer is acid (IAA), indole butyric acid (IBA), or napthalene acetic formed on the cyathium pedicel. As used herein, the term acid (NAA), preferably at 2000 mg/L for a period of “cultured’ refers to the process by which one or more between 3 and 20 seconds, preferably 5 seconds. Once the embryos is grown in vitro, or in other words, in culture. The cutting is exposed to the hormone, the cutting is preferably cultured embryo may be in contact with the ovule, or may placed in propagation medium. As used herein, a propaga be free of the ovule. tion medium is a medium which Supports growth of a cutting. An example of a propagation medium may be 0077. In one embodiment, the embryo is cut by removing propagation plugs such as those that are sold under the the ovules from the swollen ovaries, and thereafter slicing trademark Jiffy (Jiffy Products). Preferably, a water fog or the ovule in a manner which cuts the embryo. The ovule may mist is applied to the plants prior to the development of be sliced in any direction which results in cutting of the roots. Primary plants that are planted are preferably planted embryo. For example, the ovule may be sliced along the in Suitable potting mix containing commonly used fertilisers longitudinal axis, transverse of the longitudinal axis or known to persons skilled in the art. diagonal to the longitudinal axis. The ovule is preferably 0081. To establish whether the primary plant is an inter sliced along the longitudinal axis to thereby cut the embryo specific hybrid plant (as opposed to a Euphorbia pulcher along its longitudinal axis. The ovule and embryo is then rima or Euphorbia species self-pollinated plant), techniques placed in culture medium. The ovule may be placed in any known in the art may be employed such as physical char manner which permits growth of the embryo in the culture acteristics, genome size, karyotype analysis, hybridisation medium. Preferably, the cut ovule is placed in culture techniques such as that described by Schwarzacher et al. medium with the cut portion facing upwards. (1989). In situ localisation of parental genomes in a wide 0078. In another embodiment, the embryo is extracted hybrid, Ann. Bot. 64: 315-324, analysis using genotyoping from the ovule and the embryo is subsequently cut prior to methods such as that described in Starman, T. W., Duan, X. placing the cut embryo in culture medium. R. and Abbitt, S. (1999) Nucleic acid scanning techniques distinguish closely related cultivars of poinsettia, Hort 0079 The culture medium may be any medium that Science 34(6): 1119-1122. For example, interspecific permits growth of the embryo in a manner that results in hybrids may be genotyped to determine a representative plantlets being generated from the embryo. For example, the sample of the inherited markers it possesses relative to the culture medium may be tissue culture medium such as that parent plants. Genetic markers are alleles at a single locus. described in, for example, Murashige, T and Skoog. F They are preferably inherited in co-dominant fashion so that (1962). A revised medium for rapid growth and bioassays the presence of both alleles at a diploid locus is readily with tobacco tissue cultures. Physiologica Plantarum detectable, and they are free of environmental variation i.e. 15:473-497. Suitable culture mediums may include regen their heritability is 1. The array of single locus genotypes is eration medium. An example of regeneration medium expressed as a profile of marker alleles, two at each locus. includes Murashige and Skoog (MS) salts (Sigma-Aldrich The marker allele composition of each locus can be either Pty. Ltd. catalogue number M5519) at 4.42 g/L activated homozygous or heterozygous. Homozygosity is a condition charcoal preferably at 1 g/L, casein hydrolysate preferably at where both alleles at a locus are characterised by the same 1 g/L. Sucrose preferably at 40 g/L and agar preferably at 7 nucleotide sequence or size or a repeated sequence. Het US 2006/0218679 A1 Sep. 28, 2006 erozygosity refers to different conditions of the gene at a Such as poinsettia branch-inducing phytoplasma (PoiBI), or locus. A preferred type of genetic marker could be used, for virus such as poinsettia mosaic virus (PnMV) or poinsettia example, restriction length polymorphisms (RFLPs), cryptic virus(PnCV). Poinsettia plants carrying one or more amplified fragment length polymorphism (AFLPs), single of these agents include, for example, cv. V10 Amy red. nucleotide polymorphisms (SNPs), isozymes, etc. to iden However, it will be appreciated by those skilled in the art tify the interspecific hybrid produced by the method of the that the free-branching agent may be transferred to practi invention. cally any Euphorbia pulcherrima of choice and that infected 0082 It will be appreciated by those skilled in the art that Euphorbia pulcherrima of choice may then be used to once a primary plant is produced, it may be propagated for transmit the free-branching agent to the primary plant. an unlimited number of generations. The primary plant may Confirmation that the free-branching agent has been trans be used to produce further plants with characteristics that are mitted to the Euphorbia pulcherrima of choice or the different to that of the primary plant by performing inter primary plant may be achieved by techniques known in the specific or intraspecific hybridisation, by grafting the pri art Such as, for example, morphological examination to note mary plant with other species or cultivars of Euphorbia, or the free branching characteristics of the plant. In addition or by mutating the primary plant or plants produced from the alternatively, the actual free-branching agent may be primary plant. detected using well known techniques such as PCR (poly merase chain reaction), ISEM (immunosorbent electron 0.083 For example, the primary plant may be used to microscopy), etc. produce a plant having free-branching characteristics. As used herein, the expression “free-branching characteristics’ 0086 Examples of free-branching Euphorbia plants that refers to a characteristic in which lateral branching occurs at are suitable for use in the method include, for example, the a higher frequency than that of a plant that is not free Euphorbia pulcherrima varieties Freedom (U.S. Plant Pat. branching. In order to generate a plant having free-branching No. 7,825), Success Red (U.S. Plant Pat. No. 8,773), Red characteristics, a free-branching agent is transmitted to the Velvet (U.S. Plant Pat. No. 11,124), Peterstar (U.S. Plant primary plant. Pat. No. 8,259), Annette Hegg Dark Red (U.S. Plant Pat. No. 3.160), and V-14 Glory (U.S. Plant Pat. No. 4,384). 0084. In one embodiment, a free-branching agent may be transmitted to the primary plant by grafting a plant having a 0087. The characteristics of the plant produced by the free branching agent with the primary plant. The graft used method of the invention may be further altered by exposing may be any graft which results in transmission of the the plant or a part thereof to a mutagen. The plant or part free-branching agent from the free-branching plant to the thereof that is exposed to the mutagen may be any part of the primary plant. Preferably, the grafting method used to trans plant from which a mutated plant can be generated. For mit the agent is an approach grafting method. An approach example, the plant or part of a plant may be the entire plant grafting method involves cutting a section of stem, prefer or a cutting, a shoot, a seed, an embryo, an ovule, a bract, a ably approximately 10 to 30 mm long and sufficiently deep leafor any other part of the plant from which a mutated plant to reach the cambium, in both the primary plant and the can be generated. As used herein, the term “mutagen' refers free-branching plant, and Subsequently maintaining the cut to a compound or process that results in the introduction of portions in contact with each other until transfer of the mutations in the plant genome. Examples of Suitable free-branching agent from the free-branching plant to the mutagens include biological mutagens such as transposons, primary plant has occurred. Cuttings may then be planted chemical mutagens such as N-nitroso-N-ethylurea, N-ni and plants having free-branching characteristics grown from troso-N-methylurea, ethylmethane sulfonate (EMS), sodium the cuttings. In another embodiment, the free-branching azide, radiation, or other mutagens. The radiation may be agent may be transmitted to the primary plant using a ultra-violet radiation, X-ray radiation, gamma radiation, parasitic plant dodder (e.g. Cuscuta sp.). For example, the alpha-radiation, beta-radiation, ion beams such as 'He'" and parasitic dodder may be used to transfer the free-branching H', etc. Preferably, the radiation is gamma radiation. The agent from a free-branching plant to a non-free-branching dose of gamma radiation will vary depending on the inter plant. Suitably, the parasitic dodder may have the free specific hybrid, the size of the plant or part thereof and the branching agent. The use of parasitic dodders for transmit robustness of the interspecific hybrid. Preferably, the dose of ting agents are known in the art and are described in, for gamma radiation is between 1 and 10,000 rads of gamma example, Lee, I-M., Klopmeyer, M., Bartoszyk, I., Gunder radiation. Preferably, the dose is between 1000 and 10000 son-Rindal, D., Chou, T., Thomson, K. and Eisenreich, R. rads. More preferably, the dose is between 2,000 and 8,000 (1997), Phytoplasma induced free-branching in commercial rads. It is also envisaged that other methods of mutagenesis poinsettia cultivars, Nature Biotechnology 15: 178-182. In Such as temperature fluctuation, Somoclonal variation and yet another embodiment, the free-branching agent may be mutagen selection through plant tissue culture may be transmitted through a leafhopper. Use of leafhoppers for employed to generate plants with different characteristics. transmission of agents between plants is known, and 0088. Following mutagenesis, the plant or parts thereof, described in, for example, McCoy, R., Caudwell, A., Chang, preferably shoots, are propagated under protocols well C., Chen, T., Chiykowski, L., Cousin, M., Dale, J. de known in the art for poinsettia propagation and are described Leeuw, G., Golino, D., Hackett, K., Kirkpatrick, B., Mar in, for example, (Ecke et al. 1990. The Poinsettia Manual, witz, R., Petzold, H., Sinha, R., Sugiura, M., Whitcomb, R., Paul Ecke Poinsettias, Encinatis, Calif.). Preferably, the Yang, I., Zhu, B. Seemuller, E. (1989), Plant diseases apical growth point of the growing plant is routinely associated with mycoplasma-like organisms, In: The Myco removed to encourage branching, and further shoots may be plasmas 5: 545-563. removed to encourage branch formation. 0085 Examples of free-branching agents capable of 0089. The primary or mutated plant may be propagated being transmitted to the primary plant include phytoplasmas by culturing a bract of the primary or mutated plant, by US 2006/0218679 A1 Sep. 28, 2006 cutting or flowering shoot propagation as described above, or by any other means Suitable for propagation of the TABLE 1-continued primary or mutated plant. Euphorbia pulcherrima parental germplasm used in 0090. In one embodiment, the primary or mutated plant is experiment propagated by culturing a bract of the primary or mutated Euphorbia plant. A bract may be selected and the bract is preferably pulcherrima placed in a solution capable of disinfesting the bract. As used cultivar Pedigree herein, the expression “solution capable of disinfesting refers to a solution that is able to remove or kill at least a E. pulcherrima Self seed from wild poinsettia 97,176.3 portion of organisms that are located on, or associated with, E. pulcherrima Seedling of unknown parentage the bract. Preferably, the solution capable of disinfesting the cv. V10 Amy red bract is capable of disinfecting the bract. Preferably, the E. pulcherrima cv. Pepride X wild-type breeding line 41 poinsettia X self solution capable of disinfesting the bract is capable of E. pulcherrima cv. Freedom Red X cv. V10 Amy sterilising the bract. Preferably, the solution capable of breeding line 75 Red disinfesting the bract is bleach. Suitably, the bleach solution E. pulcherrima cv. Freedom Marble X cv. is at a concentration of between 1% and 3%. Preferably, the breeding line 82 Freedom Red X wild-type bleach solution is at a concentration of about 2%. Preferably, poinsettia X self the bract is sterilised by the bleach solution. Accordingly, the bract is preferably placed in the bleach solution for sufficient time to sterilise the bract. Following treatment with the 0094 Parental germplasm of Euphorbia cornastra, bleach solution, the bleach is preferably replaced with sterile Euphorbia radians, Euphorbia colorata and Euphorbia fill water to thereby wash the bract. Preferably, multiple sterile gens are shown in Table 2. water washes are carried out. Once the bract is washed, the bract is preferably dissected and placed in contact with TABLE 2 culture medium to promote growth. The culture medium may be any culture medium that is sufficient to Support Euphorbia species germplasm (other than growth of a plant from the bract. For example, adventitious bulcherrima) used in experiment root medium as described in (Roest, S. and Bokelman, G. S. Euphorbia species Initial Propagules Source (1980). Vegetative propagation of poinsettias in test-tubes), Euphorbia Seeds A. LeDuc, Vegetatieve Vermeerdering van poinsettia in kweenbuizen, COficiSira Louisiana State Vakblad voor de Bloemisterij 35(47): 36-37, (As translated University, U.S.A. in: Horticultural Abstracts (1981), 51(6): 430). Euphorbia radians Tubers V. Steinmann, Santa Ana Botanic 0.091 Embodiments of the invention are now described in Garden, California the following Examples which will be understood to merely U.S.A. Euphorbia colorata Tubers V. Steinmann, exemplify and not to limit the scope of the invention. Santa Ana Botanic Garden, California EXAMPLES U.S.A. Euphorbia fittgens Cutting Readily commercially Example 1 available from The Plant Place, Interspecific Hybridisation between Euphorbia Gosford pulcherrima and Euphorbia sp.

0092. In this experiment, pollen from Euphorbia cornas Growth Conditions tra, Euphorbia radians, Euphorbia colorata and Euphorbia fiulgens was used to pollinate various cultivars of Euphorbia 0095 Plants used for pollinations were grown in two pulcherrima. insect-free environments, namely E1 and E2. 0093 Parental germplasm of Euphorbia pulcherrima 0096 Environment 1 (E1) was a standard environment used in this experiment are detailed in Table 1. used for 15 intraspecific-hybridisation. Temperature was maintained at 21°C.1° C., and a 10 hour photoperiod was TABLE 1. provided in “microclimate' rooms contained within a green Euphorbia pulcherrima parental germplasm used in house. Supplementary light of approximately 300 umolm experiment s' was provided. The species E. cornastra and the E. Euphorbia pulcherrima cultivars/lines 97/144 and 97/54.1 were grown pulcherrima in environment E1. cultivar Pedigree 0097. In Environment 2 (E2), temperature was main E. pulcherrima cv. Freedom white x cv. V10 tained between 21° C. and 24° C., natural daylight was 97.24:1 Amy red provided, and plants were positioned in a northerly aspect to E. pulcherrima cv. V10 Amy red X self 97.54.1 ensure good light intensity. The species E. radians and the E. pulcherrima cv. Pink peppermint X cv. V10 poinsettia lines 97/24.1, 97/176.3, and cv. Freedom red, and 97,144 Amy red cv. V10 Amy red were grown in E2 to facilitate crossing with Euphorbia species and also in E1 for all other crosses. US 2006/0218679 A1 Sep. 28, 2006

Crossings hybrid, one 97/54.1xE. cornastra hybrid) and 17 cv. V10 0.098 Pollinations (self and cross) were performed Amy redXE. cornastra hybrids) and two parental controls depending upon flowering times and rate of production of (cv. V10 Amy red and E. cornastra) were placed under short photoperiod (10 hours) conditions in environment E1 after anthers and pistils. Fresh pollen was collected prior to approximately 3 months from deflasking. Plants were placed midday from most plants. Pollen was applied liberally to all at an interpot distance of 300 mm as measured from the areas of the receptive Stigmatic Surface. centre of the pots and drip irrigated. Approximately 7 weeks Embryo Cutting and Culturing afterwards, bract development in relation to controls was 0099 Cyathia with swollen ovaries were deemed to pos noted for these 19 hybrids. sess ovules containing fertilised egg cells and were collected Infection of E. pulcherrimax.E. cornastra Putative Hybrids prior to abortion. This stage was reached when an abscission with PoiBI layer formed on the cyathium pedicel. Ovaries were disin fected for 10 minutes in 4% NaOCL with 1 drop of Tween 0.104 Vegetative cuttings were harvested from all stock 20, then rinsed 3 times in autoclaved distilled water and plants of E. pulcherrimaxE. cornastra hybrids and propa allowed to dry in a lamina flow cabinet. Upon dissection, all gated according to standard practices for poinsettias. Cut ovules were removed. The ovules were bisected into tings of cv. V10 Amy red containing PoiBI without PnMV approximately two halves along their longitudinal axis using were also propagated to enable graft transferral of PoiBI to a sterile scalpel to ensure that the embryo inside the ovule be conducted. After acclimatisation, each cutting of cv. V10 was cut, and placed cut side upwards onto tissue culture Amy red was planted in a 150 mm pot adjacent to a cutting media (regeneration media) containing MS basal salts of a putative hybrid. Standard potting mix was used, slow (Murashige and Skoog 1962), 1 g/l activated charcoal, 1 g/1 release fertiliser was applied at the recommended rate and casein hydrolysate, 40 g/L Sucrose and 7 g/l agar. pH was plants were manually watered and maintained at 25°C.2 adjusted to 5.8. Media were selected based on previous work C. under long photoperiod conditions (light intensity greater (see for example, Roest and Bokelman 1980, Lee et. al. than 2 molm's for 4 hours commencing at 10 pm) for 1997). Primarily plants were cultured at 25°C.i.2° C. under several weeks. When the height of both plants was approxi Crompton 40W RS White fluorescent lights to provide a mately 100 mm, plants were approach grafted. light intensity of approximately 60-70 umol ms' at cul 0105. Approach grafting involved cutting a vertical sec ture container lid level for 16 hrs/day. Developing embryos tion of the stem on both plants of approximately 20 to 30 mm were subcultured onto the above mentioned regeneration in length and deep enough to cut through to the cambium. media or a proliferation media containing MS basal salts, 0.3 The two cut portions were then placed facing each other and mg/l 6-benzyl amino purine, 1 g/l casein hydrolysate, 40 g/L the graft union sealed with parafilm M laboratory film. Upon Sucrose and 7 g/L agar. pH was adjusted to 5.8. Subsequent development of Sufficient growth post-grafting, cuttings Subculturing was performed at approximately 3 to 4 week were removed from grafted putative hybrids and propagated intervals onto fresh media of either composition depending according to standard protocols. Cuttings with roots were upon growth. planted into 150 mm pots and grown in a commercial 0100 Plantlets (primary plants) developed in vitro were greenhouse according to standard poinsettia production deflasked by either planting the regenerated plantlets methodology (from vegetative growth to flowering). emerged directly from embryos, or by cutting and dipping Results developed shoots in 2000 mg/L IBA for 5 seconds, prior to placement in expanded Jiffy R propagation plugs. A constant E. pulcherrimaxEuphorbia sp. Pollinations water fog was initially applied and later gradually reduced to 0106) A total of 1093 pollinations resulted in 3.279 facilitate acclimatisation once plantlets had developed roots. ovules being pollinated. Unequal pollination numbers Plant Growth resulted among crosses due to different rates of cyathia and 0101 Plants developed in in vitro culture (ovule slice anther production. Pollinations between poinsettias and the culture) were planted into 150 mm pots containing standard twelve species yielded at least 1 swollen ovary, from each potting mix with Supplementary Osmocote R plus 4 month cross combination, indicating possible fertilisation. Swollen slow release fertiliser applied to the potting mix Surface at ovaries numbered 243 and contained 689 ovules. All ovules the recommended rate. Pots were placed at approximately were in vitro cultured only ovules greater than 3 mm in 300 mm interpot distance as measured from the centre of the length became organogenic (regenerated Structures such as pots. Water was applied manually and plants were grown callus, embryos or shoots) when in vitro cultured. Ovules under a long photoperiod environment (light intensity greater than 3 mm in length numbered 370 of which, 119 greater than 2 umol mis' for 4 hours commencing at 10 became organogenic. Ovules 3 mm or less were identical in pm) at approximately 25°C. in a greenhouse. size and appearance to unpollinated ovule controls and did not exhibit organogenesis in vitro (when harvested just prior 0102 Putative hybrids from the E. pulcherrimax.E. cor to cyathia abortion). nastra cross (25 hybrids) and two parental controls (cv. V10 Amy red and E. cornastra) were removed from in vitro E. pulcherrima 97/24.1xE. radians and cv. V10 Amy Redx culture. These plants were acclimatised to the greenhouse E. radians environment as described previously and then grown under 0.107 From a total of 146 crosses conducted among E. long photoperiod conditions (light intensity greater than 2 pulcherrima and E. radians, 42 Swollen ovaries containing umol mis' for 4 hours commencing at 10 pm). 126 ovules resulted. From these ovaries, 36 ovules greater 0103) Nineteen (19) putative hybrids from the E. pull than 3 mm in length were obtained. Of these ovules, 4 cherrimax.E. cornastra cross (one 97/144xE. cornastra exhibited organogenesis. US 2006/0218679 A1 Sep. 28, 2006

E. pulcherrimaxE. cornastra 0108. Among 380 crosses conducted between poinsettias TABLE 4 and E. cornastra, 110 Swollen ovaries were observed. From Mean Height and node number of interspecific a total of 177 ovules greater than 3 mm in length cultured in hybrids. vitro, 76 ovules showed organogenesis. The efficiency of production based on the number of plants generated from the Means Hybrid plants number of ovules pollinated ranged from 0 to 27.3%. Higher E. Corna Stra and Standard errors (n = 25, range levels of efficiency were broadly related to fertility of female (n = 3) cv. V10 Amy red (n = 5) of heights) poinsettia parents. For example, 97/24.1 and 97/54.1 pro duced 27.3% and 16.7% plants/ovule pollinated respec Height (mm) 522+f-51 537 +f- 28 199-62O tively. Node Number 26.0 +f- 2.9 33.0+f- 0.71 22- 47 H/N (mm) 20.3 +/- 2.0 16.3 +/- 0.63 741-17.3 0109 Crosses which resulted in ovules that exhibited organogenesis in vitro are Summarised in table 3.

TABLE 3 Types of in vitro regeneration from interspecific Euphorbia crosses. Morphological Plant Female Plants classification production parent Male parent Regeneration* regenerated of hybridity efficiency (%) 97,144 E. Cornastra 3 (E) 2 (1 died) Hybrids 1.0 (2/198) 97,176.3 E. Cornastra 3 (E) 3 Hybrids 10.0 (3/30) 97.24.1 E. Cornastra 10 (E) 9 (1 died) Hybrids 27.3 (9/33) 97.54.1 E. Cornastra 5 (E) 5 (1 died) Hybrids 16.7 (5/30) 1 (G) cv. Angelika E. Cornastra 1 (E) 1 Hybri 2.4 (1.f42) cv. Freedom E. Cornastra 1 (E) 1 Hybri 1.0 (1/102) red cv. V10 Amy E. cornastra 47 (E) 44 (3 died) Hybrids 12.3 (44/357) red 97.24.1 E. radians.1 1 (E) 1 (1 died) Hybri 1.8 (2/111) 2 (G) cv. V10 Amy E. radians.1 1 (C) 1 Hybri 0.5 (1/183) red cv. V10 Amy E. colorata 5 (c) Lost in fire Hybri Destroyed in red shoots fire prior to developed deflasking cv. 10 Amy E. fittgens 2 (c) Lost in fire Hybri Destroyed in red 2 (E) shoots fire prior to developed deflasking 97.24.1 E. fittgens 1 (c) Lost in fire Hybri Destroyed in 2 (E) shoots fire prior to developed deflasking * Method of regeneration from ovules: (E) = somatic embryos from Zygote; (c) = callus; (G) = direct germination.

Characterisation of Putative F1 Progeny from E. pulcher 0.111 Characterisation of 19 putative hybrids (one rimaxEuphorbia Species Pollinations 97/144xE. cornastra hybrid, one 97/54.1xE. cornastra hybrid and seventeen cv. V10 Amy redxE. cornastra E. pulcherrimaxE. cornastra hybrids) grown under a 10 hour short photoperiod environ ment showed that 17 exhibited earlier bract development 0110 All generated F1 plants displayed characteristics of compared to cv. V10 Amy red. The male parent E. cornastra both poinsettia and E. cornastra parents. In visual compari was the earliest to flower, but exhibited rapid bract loss after son to the female poinsettia parents, leaf size was Smaller, reaching anthesis. This species also produced numerous stem diameter was reduced and internode length was shorter seeds, due to self-pollination. Seed production during long (see FIG. 1 and Table 4). Of the 25 lines characterised for photoperiod conditions was not observed for this species, vegetative growth (21 were hybrids between V10 Amy red although it did flower under such conditions. and E. cornastra), 23 exhibited reduced height (less than E. cornastra) and 15 increased node number, when compared 0112 All putative hybrids exhibit pink bract colour with to cv. V10 Amy red (see Table 4). The remaining 4 lines that varying degrees of colour intensity. Of the 19 putative were not hybrids with cv. V10 Amy red were from crosses hybrid lines observed, 9 exhibited male and female sterility with lines 97/144, 97/54.1 and 97/176.3 and were similar in (lack of reproductive structures) and the remaining 10 appearance to the other plants. possessed stamens only. US 2006/0218679 A1 Sep. 28, 2006

0113 Considering E. cornastra flowered under long pho toperiod conditions, hybrid plants were observed under TABLE 5-continued artificial long photoperiods for 18 months to determine if they would flower. Some lines appeared to commence long Characteristics of an interspecific hybrid photoperiod flowering as indicated by partial colour devel designated 98EC-18.4/5. opment of leaves/bracts, but complete development was 98EC-18.4fS never observed. When approach grafted to cv. V10 Amy red 137A). Hairs are present on the underside of leaves and the leaf colour containing PoiBI, putative hybrids developed swollen buds is lighter green (RHS 137C). Leaf followed by branches within 2 to 3 weeks of grafting. margins are entire, with some serration Cuttings harvested from these free-branching plants pos generally closer to the leaf base. sessed increased branching and improved ornamental Bract Transitional bracts (bracts with some characteristics: green and some pink/white colour) appearance when grown under commercial conditions indi numbered from 1–3 and these are found cating transmission of PoiBI. Profuse branching occurred between the last leaf produced and the from all nodes regardless of the number of nodes remaining first bract produced on each branch. on the primary stem after an apical decapitation (from 6 to Their shape was lanceolate with some Serration. The upper colour of 12. nodes). Cuttings produced roots rapidly under standard transitional bracts was pink with some propagation conditions suitable for poinsettias, and plants green sections. The lower colour was grew vigorously. white with some green sections. Length was from 70–90 mm and width from 35–45 mm. 0114. The characteristics of some examples of the plants The first true bracts produced were from that were produced using the method are illustrated in Tables 80–110 mm in length and 40–60 mm in diameter. Bract length and width 4 to 8 below. decreased as they were produced closer to the cyathia. Bracts directly TABLE 5 Subtending the cyathia were from 20–40 mm long and 10–20 mm wide. Their shape was Characteristics of an interspecific hybrid symmetrical about the mid rib and designated 98EC-18.4/5. similar to the leaves, but lacking 98EC-18.4fS Serration. Upper bract colour was an intense pink (RHS N57D) and faded as Origin: An interspecific hybrid created by bracts aged. The underside bract colour pollinating a poinsettia plant known as was a light pink/white (close to RHS cultivar V10 Amy Red with pollen from a 65C). On plants kept for an extended Euphorbia connastra plant developed from period of time the upper surface of seed older bracts became almost white (close Classification: Euphorbia X hybrid to RHS 155C). Form: Soft wooded shrub Flower Plants can be forced to flower by Infection Infected with Poinsettia Branch Inducing induction: placing under a short photoperiod of Status: Phytoplasma (PoiBI) 10 hours. They will naturally flower Growth Habit: Plants were grown according to standard under decreasing day length conditions, commercial production procedures for similar to a poinsettia. poinsettias. Finished plant height was Flowers: Generally, cyathia numbered from 20–30 approx. 350 mm and width approx. 550 mm. at maturity. Stamens were present on the The plant had an upright but mounded more mature cyathia. No pistils were habit. The average bract diameter of a produced. Mature cyathia were approx. flowering branch was approx. 250 mm. 8 mm long and 5 mm wide and their colour Branches developing from the main stem was green. terminate in an inflorescence. If the main shoot tip is removed prior to flower induction the plant forms branches from all nodes present on the 0115 Colour descriptions are based on RHS colour charts main stem radiating in all directions ue to the removal of apical dominance. used from 2001 (Royal Horticultural Society, London, 2001) The described plant had 11 nodes remaining after apical decapitation and TABLE 6 hus 11 branches. Each branch had from 6–7 leaves. Characteristics of an interspecific hybrid Growth Rate: Cuttings propagated under mis designated 507.1. (according to standard practices for 507.1 poinsettias) produce roots from 10–21 ays. Cuttings can form roots at lower Origin: An interspecific hybrid created by temperatures than standard poinsettia pollinating poinsettia breeding line 82 cultivars. Plants have medium vigour, (pedigree: Freedom Marble X Freedom producing one leaf approximately every 5 Red X wild-type poinsettia X self) ays (depending on environmental with bulk pollen from several Euphorbia conditions). cornastra plants developed from seed Leaf Leaves are symmetrical about the mid rib (via self pollination) characteristics: and have a lanceolate appearance. Leaf Classification: Euphorbia X hybrid length is typically 80–100 mm and width Form: Soft wooded shrub 30–50 mm. leaf petioles are green with a Infection Not infected with Poinsettia Branch slight red tinge on the upper Surface Status: Inducing Phytoplasma (PoiBI) and are typically 20–40 mm long and Growth Habit: Plants were grown according to standard approx. 2 mm diameter. The upper leaf commercial production procedures for Surface is lacking of hairs and slightly poinsettias. Finished plant height was wrinkled, it is mid green in colour (RHS approx. 450 mm. The plant had an upright US 2006/0218679 A1 Sep. 28, 2006 10

TABLE 6-continued TABLE 7-continued Characteristics of an interspecific hybrid Characteristics of an interspecific hybrid esignated 507.1. designated 474.2. 507.1 474.2

habit. The average bract diameter of a Status: inducing Phytoplasma (PoiBI) flowering branch was from 160–180 mm. Growth Habit: Plants were grown according to standard Branches developing from the main stem commercial production procedures for terminate in an inflorescence. poinsettias. Finished plant height was Growth Rate: Cuttings propagated under mist approx. 300 mm. The plant had an upright (according to standard practices for habit. The average bract diameter of a poinsettias) produce roots from 10–21 flowering branch was from 130–140 mm. days. Plants have a comparatively Branches developing from the main stem average rate of growth. erminate in an inflorescence. Leaf Leaves are symmetrical about the mid rib Growth Rate: Cuttings propagated under mist characteristics: and have an oakleaf appearance with 4 (according to standard practices for lobes. Leaf length is typically 80–100 mm poinsettias) produce roots from 10–21 and width 65–80 mm. Leaf petioles were days. Plants have a comparatively slow typically 20 mm long and approx. 2 mm growth rate. diameter. The upper leaf surface is Leaf Leaves are symmetrical about the mid rib green in colour (RHS 139A). The characteristics: and have a semi-oakleaf appearance (2 underside of the leaves is green in obes). Leaf length is typically 65–80 mm colour (RHS 138A). and width 45–60 mm. Leaf petioles were Bract Transitional bracts (bracts with some typically 15–20 mm long and approx. 2-3 mm characteristics: green and some pink colour) numbered diameter. The upper leaf surface is from 2–4 and these were found between green in colour (RHS 137A). The leaf the last leaf produced and the first underside colour is (RHS 137C). bract produced on each branch. Their Bract Transitional bracts (bracts with some shape was oakleaf with four lobes. The characteristics: green and some pink/white colour) upper colour of transitional bracts was numbered from 3–5 and these are found pink with some green sections. The lower between the last leaf produced and the colour was mostly light green. Length first bract produced on each branch. was from 70–90 mm and width from 50–70 mm. Their shape was semi-oaklef with 2 The first true bracts produced were from lobes. The upper colour of transitional 55-75 mm in length and 35–50 mm in bracts was light pink with some green diameter. Bract length and width Sections. The lower colour was lime decreased as they were produce closer green with some white sections. to the cyathia. Bracts directly Transitional bract length was from 60–70 mm Subtending the cyathia were from 20–30 mm and width from 35–50 mm. The first long and 10–15 mm wide. Their shape was true bracts produced were from 50–60 mm lanceolate. Upper bract colour was a in length and 20–35 mm in diameter. Bract dark pink (RHS N66A). The underside length and width decreased as they were bract colour was pink (RHS N57C). produced closer to the cyathia. Bracts Flower Plants can be forced to flower by directly subtending the cyathia were induction: placing under a short photoperiod of from 25–35 mm long and 15–20 mm wide. 10 hours. They will naturally flower Their shape was lanceolate. Upper bract under decreasing day length conditions, colour was a light pink (RHS 65A). The similar to a poinsettia. underside bract colour was a lighter Flowers: Generally, cyathia numbered from 14–20 pink (RHS 65C). at maturity. Stamens were not present on Flower Plants can be forced to flower by mature cyathia. No pistils were induction: placing under a short photoperiod of produced. Mature cyathia were approx. 10 hours. They will naturally flower 9 mm long and 6 mm wide and their colour under decreasing day length conditions, was green becoming red near the apex. similar to a poinsettia. Flowers: Generally, cyathia numbered from 16–18 at maturity. Stamens were present on the more mature cyathia. No pistils were 0116 Colour descriptions are based on RHS colour charts produced. Mature cyathia were approx. 7 mm long, 6 mm wide and their colour was used from 2001 (Royal Horticultural Society, London, 2001) green. TABLE 7 Characteristics of an interspecific hybrid 0.117 Colour descriptions are based on RHS colour charts designated 474.2. 474.2 used from 2001 (Royal Horticultural Society, London, 2001) Origin: An interspecific hybrid created by TABLE 8 pollinating a poinsettia plant known as poinsettia breeding line 75 (Freedom Red XV10 Characteristics of an interspecific hybrid Amy Red) with bulk pollen from designated 892.1. several Euphorbia connastra plants 892.1 developed from seed (via self pollination). Origin: An interspecific hybrid created by Classification: Euphorbia X hybrid pollinating a poinsettia plant known as Form: Soft wooded shrub Poinsettia breeding line 41 (Pedigree: Infection Not infected with Poinsettia Branch Pepride X wild-type poinsettia X self) US 2006/0218679 A1 Sep. 28, 2006 11

TABLE 8-continued TABLE 9 Characteristics of an interspecific hybrid Characteristics of an interspecific hybrid designated 892.1. designated 674.3. 892.1 674.3 with bulk pollen from several Euphorbia Origin: An interspecific hybrid created by cornastra plants developed from seed pollinating a poinsettia plant known as (via self pollination) cultivar V10 Amy Red with pollen from Classification: Euphorbia X hybrid several Euphorbia connastra plants Form: Soft wooded shrub developed from seed (via self Infection Not infected with Poinsettia Branch pollination) Status: inducing Phytoplasma (PoiBI) Classification: Euphorbia X hybrid Growth Habit: Plants were grown according to standard Form: Soft wooded shrub commercial production procedures for Infection Not infected with Poinsettia Branch poinsettias. Finished plant height was Status: inducing Phytoplasma (PoiBI) rom 350–400 mm. The plant had an upright Growth Habit: Plants were grown according to standard habit. The average brac diameter of a commercial production procedures for flowering branch was approx. 90–110 mm. poinsettias. Finished plant height was Branches developing from the main stem approx. 800-850 mm. The plant had an erminate in an inflorescence. upright habit. The average bract Growth Rate: Cuttings propagated under mist diameter of a flowering branch was 130–150 mm. (according to standard practices for Branches developing from the main poinsettias) produce roots from 10–21 stem terminate in an inflorescence. days. Plants have a comparatively Growth Rate: Cuttings propagated under mist average rate of growth. (according to standard practices for Leaf Leaves are symmetrical about he mid rib poinsettias) produce roots from 10–21 characteristics: and have a semi-oakleaf appearance (2 days. Plants have a comparatively fast obes). Leaf length is typically 90–110 mm rate of growth. and width 50–60 mm. Leaf petioles Leaf Leaves are symmetrical about the mid rib were typically 20–25 mm long and approx. characteristics: and have a lanceolate appearance. Leaf 2 mm iameter. The upper lea Surface is ength is typically 140–160 mm and width 60–90 mm. Leaf petioles are typically 40–45 mm green in colour (RHS 39B). The ong and approx. 2 mm diameter. The underside leaf colour is green (RHS upper leaf Surface is green in colour 139C). (RHS 137B). The underside leaf colour is Bract Transitional bracts (bracts with some ighter green (RHS 137C). characteristics: green and some pink/white co our) Bract Transitional bracts (bracts with some numbered from 3–4 and these are found characteristics: green and some pink/white colour) between the last leaf produce and the numbered from 4–6 and these are found first bract produced on each branch. between the last leaf produced and the Their shape was lanceolate. T he upper first bract produced on each branch. colour of transitional bracts w as pink heir shape was lanceolate. The upper with some green sections. The lower colour of transitional bracts was pink colour was green with some very pale with some green sections. The lower pink sections. Transitional bract ength colour was very pale green with some was from 40–60 mm and width rom 25–35 mm. white sections. Transitional bract The first true bracts produced were from length was from 80–100 mm and width from 35–45 mm in length and 20–35 mm in width. 35–45 mm. The first true bracts produced Bract length and width decrease as they were from 45–60 mm in length and 15–25 mm were produced closer to the cya hia. in diameter. Bract length and width Bracts directly subtending the cyathia decreased as they were produced closer were from 20–25 mm long and 10–20 mm wide. to the cyathia. Bracts directly Their shape was lanceolate. Upper bract Subtending the cyathia were from 20–35 mm colour was medium pink (RHS N57D). The long and 10–15 mm wide. Their shape was underside bract colour was a lighter symmetrical about the mid rib and pink (RHS 65C). similar to the leaves. Upper bract Flower Plants can be forced to flower by colour was a light pink (RHS 63C). The induction: placing under a short photoperiod of underside bract colour was a lighter 10 hours. They will naturally flower pink (RHS 65D). under decreasing day length conditions, Flower Plants can be forced to flower by similar to a poinsettia. induction: placing under a short photoperiod of Flowers: Generally, cyathia numbered from 11–16 10 hours. They will naturally flower at maturity. Stamens were present on the under decreasing day length conditions, more mature cyathia. No pistils were similar to a poinsettia. produced. Mature cyathia were approx. Flowers: Generally, cyathia numbered from 20–65 8 mm long and 5 mm wide and their colour at maturity. Stamens were not present on was green becoming pink near the apex. mature cyathia. No pistils were produced. Mature cyathia were approx. 10 mm long and 9 mm wide and their colour was green. 0118 Colour descriptions are based on RHS colour charts used from 2001 (Royal Horticultural Society, London, 2001) US 2006/0218679 A1 Sep. 28, 2006

0119) Colour descriptions are based on RHS colour charts Results used from 2001 (Royal Horticultural Society, London, 2001). 0.129 Results are summarised in Table 11. 0120. It will be understood by those skilled in the art that TABLE 11 plant characteristics are variable under different environ mental conditions and thus results obtained in the above Irradiation results. table 5 to 9 may vary somewhat under different growing Treatment No. Surviving conditions. Cutting time after after 8 Conclusions No. Dose (Rads) (minutes) 1 week % weeks % 55 O (control) O 55 1OO 55 100 0121 The use of ovule slice culture to culture the grow 102 2000 (T1) 43 102 1OO 96 94 ing embryo has been used to produce crosses between a 102 3000 (T2) 64 102 1OO 95 93 number of different Euphorbia species. 102 4000 (T3) 8O 102 1OO 72 70 102 8000 (T4) 171 102 1OO 5 5 Example 2 Irradiation of Interspecific Hybrids to Produce New 0.130. At 3 months post-irradiation (4 weeks past pot Varieties ting), shoots were apically decapitated to promote branch ing. All plants except controls had speckled green and light 0122) The aim of this experiment was to determine whether plants could be produced having variation in char green patches on leaves from radiation. acteristics from the primary plants by irradiating E. pulcher 0.131. At 6 months post-irradiation, shoots of all plants rimaxE. cornastra F hybrids. were further apically decapitated to promote branching. Materials and Methods: 0.132. After approximately 6 months, all plants were 0123. One interspecific hybrid (98EC-18.4/5) was taken to a short photoperiod environment (10 hour day selected. The characteristics of this interspecific hybrid are length) to flower. listed in Table 5 above. 0.133 After 7.5 months, plants were flowering. Selections 0124 Cuttings were obtained from plant 98EC-18.4/5. were made for phenotypic attributes such as bract colour and The cuttings were made to obtain healthy and uniform tissue shape, leaf variegation and plant habit. Subsequently all having a high node number. Unnecessary leaves were branches on selected plants except the mutated branches removed with a scalpel. were removed. 0125 Irradiation of cuttings was performed as shown in 0.134. After 8.5 months, the best plants were reselected Table 10. All cuttings were covered with plastic freezerbags. from primary selections. The Gamma Source used emitted irradiation at approxi mately 500 rads per 10 minutes. 0.135). After 9 months, all remaining stock (unselected) was cutback and allowed to reflower. The first batch of 0126 Five experimental treatments for the cuttings (con flowering shoot cuttings were taken from selected, mutated trol and T1 to T4) were set up. The details of each of the branches. Flowering shoots containing mutations were treatments are shown in Table 10. propagated following standard propagation procedures for TABLE 10 Poinsettia (Ecke 1990). 0.136 Interspecific hybrid mutants were selected and Irradiation treatment of cuttings. propagated in propagation media under standard conditions Treatment type Dose (Rads) Treatment Time (minutes) for propagation of poinsettia cuttings, and in tissue culture C O (control) O (leave in box) via bract culture (see Example 3) Results are summarised in T1 2OOO 43 Table 12 which shows the number of plants with visible, T2 3OOO 64 useful mutations. T3 4000 8O T4 8OOO 171 TABLE 12 Summary of interspecific hybrid mutagenesis.

0127 Cuttings were set up in 6x20 cm diameter vials and Treatment No. of % mutant rotated at 15 cm from the gamma Source. Once treated, Cutting Time No. mutant production cuttings were rewrapped in fresh moistened paper and O. Dose (Rads) (minutes) potted selections efficiency placed back into polystyrene boxes. 55 O (control) O 55 O O 0128 Irradiated (and control) cuttings were placed into 102 2000 (T1) 43 96 51 SO% 102 3000 (T2) 64 95 38 37% Oasis R cubes (Smithers-Oasis) and propagated via standard 102 4000 (T3) 8O 72 46 45% commercial practice, under mist with bottom heat (22° C.) 102 8000 (T4) 171 5 11 11% (Ecke et al. 1990). After 8 weeks all plants with roots were potted following standard practices (Ecke et al. 1990). US 2006/0218679 A1 Sep. 28, 2006 13

0137 The resulting mutants produced by the mutagenesis experiment are shown in Table 13.

TABLE 13 Mutants produced by gamma irradiation of interspecific hybrid Euphorbia 9 months post-irradiation.

New Selected Shoots % bract Treatment shoot Cuttings in RHS 88 level number taken Details culture Description colour (approx) 1 2 stable 2 white, plk veins 155C A. 2 O no white 4 white 155C A. 3 3 2S 1NS white N155D A 4 NS 1 white 69D A. 5 white 155C

TABLE 13-continued Mutants produced by gamma irradiation of interspecific hybrid Euphorbia 9 months post-irradiation.

New Selected Shoots % bract Treatment shoot Cuttings in RHS 88 level number taken Details culture Description colour (approx)

T1. 46 1 NS white with few ful bright pink flecks T1. 47 1 S pale pink speckle 34 T1. 48 1 NS white with pink ful speckle mixed T1. 49 1 NS jingle bell white 34 speckles T1. 50 1 S pale pink ful T1. 51 1 S bright pink white 62A ful flecks pink cyathia T2. 4 stable pk w wh patches ful (ingle bells type) T2. 4 pk, variegated A. grey T2. 1 brightest pk N66A /4 T2. 1 1 pk/wh patches A. T2. 1 2stable babyiv It pk N155C A. 1NS T2. normal? T2. 2 S Brightest pk T2. O missing wh, slipk T2. normal T2. white

T2. 32 T2. 33 S pale pink T2. 34 S white T2. 35 S pale pink T2. 36 S bright pink white 68A speckle T2. 37 S whitefvery pale l pink T2. 38 S pale pinkpink veins US 2006/0218679 A1 Sep. 28, 2006 15

TABLE 13-continued Mutants produced by gamma irradiation of interspecific hybrid Euphorbia 9 months post-irradiation.

New Selected Shoots % bract Treatment shoot Cuttings in RHS 88 level number taken Details culture Description colour (approx) T3. 1 1 S wh, plk veins 155C A. T3. 2 2 S t pk, variegated 34 wh edges T3. 3 2 S pk, variegated wh full edges pk? wh on trans bracts T3. /4 T3. 5 wh, v It pk A. T3. pk, variegated, full whilt green edges, distorted T3. missing pk, wh edged A. variegation T3. 8 missing pk w whblotches 34 T3. S pk, variegated /8 grey, jagged edge T3. 10 S baby pk A. T3. 11 ugly variegated pk T3. 12 1S 1NS wh A. T3. 13 NS wh A. T3. 14 NS pk/lt pk blotches T3. 15 broken t baby pk A. i4O Off T3. 16 NS pkw dk pk speckle T3. 17 s missing wk, pk new bracts T3. 18 missing K, grey A. ariegated T3. 19 NS K, grey ariegated T3. 1S baby 2 + 1 aby pk and v It /3 and 1Slight & A. 1NS T3. 21 1 light pk variegated w 69A Wh edge, jagged T3. 22 S hotpk T3. 23 S pk, variegated, wh edge T3. 24 NS mid pink T3. 25 baby pk T3. 26 S hotpk T3. 27 S baby pk and hot plk T3. 28 nil hotpk cuttings T3. 29 S T3. 30 T3. 31 S T3. 32 no change tpk T3. 33 S pk, variegated 65A T3. 34 Small variegated eaf, wh edge T3. 35 pk, wh speckle on eaves like LD initn T3. 36 bright pk /3 T3. 37 baby pk, variegated A. varieg T3. 38 pale pk wh T3. 39 white T3. 40 bright pk with A. speckles Serrated bracts T3. 41 white T3. 42 pale pink T3. 43 bright pink with speckles T3. 44 pale pink l T3. 45 white l US 2006/0218679 A1 Sep. 28, 2006 16

TABLE 13-continued Mutants produced by gamma irradiation of interspecific hybrid Euphorbia 9 months post-irradiation.

New Selected Shoots % bract Treatment shoot Cuttings in RHS 88 level number taken Details culture Description colour (approx) T3. 46 2 NS jingle bells brt 63B pk T4. 1 O broken jingle bells type full T4. 2 3 NS 1 medium pk N66A /3 T4. 3 5 S pk w bright A. patches T4. 4 4 1S 3NS 1 baby pk full T4. 5 O baby pk, full, variegated, bit A. distorted varieg T4 6 3 S pk, variegated, A. distorted T4 7 2 S pk, Small leaf full variegated w wh edge T4 8 1 S tpk and baby pk 62A /3 and A. T4 9 3 S 1 wh, distorted fol full and bracts T4 10 pk, wh speckle on /4 eaves like LD initn T4 11 1 S white with cream 3/4 yellow tinge *Colours include, standard or normal pink (same as control), brightest pink (br pk), hot pink (hot plk), light pink (It pk), baby pink (baby pk), white (wh), white jingle bells (white with pink fleck), pink jingle bells (pink with white fleck), pink centre (white with Small new bracts pink). No tissue culture except where stated. v = very. w = with. NS = not stable, S = stable, FS = fairly stable

Conclusion Example 4 0138) Irradiation of interspecific hybrids produced from a Bract Tissue Culture Experiment for Interepecific cross between Euphorbia pulcherrima and Euphorbia cor Euphorbia Hybrid nastra resulted in a variety of plants with altered character 0140 Many of the plants produced from the irradiation istics including colour and variegation. experiment had sections or portions of the plant which exhibited desirable characteristics. In other words, the plants Example 3 were chimeric. The differences in the characteristics of these portions were presumably due to Somatic mutations result Stabilisation of Chimeric Mutants ing from the irradiation. This example describes the devel opment of a tissue culture system in which the mutant 0.139. Once a desirable mutation was observed, a cutting portions can be cultured to produce non-chimeric shoots was taken of the flowering shoot. It will be appreciated by from desirable mutated sections of a chimeric plant. Such as those skilled in the art that the greater the mutated section of those with novel colours arising from gamma irradiation the shoot, the greater the chance of stabilising the phenotype treatment. in the next cutting generation. It is desirable for the cutting 01.41 Eighteen bracts and leaves of medium size and age to have most of its bracts removed and to possess at least two were collected with petioles intact from the interspecific green leaves. The cuttings can be propagated following hybrid designated 98EC18.4/5. The following concentra standard procedures suitable for poinsettias. The cuttings are tions of bleach (NaOCl Zixo brand 4.5% active) were then grown to produce flowering plants following standard prepared: procedures for poinsettias. At this stage, shoots that have the desirable mutant phenotype are selected and propagated. 0142) 1% bleach The cycle continues until the desirable mutant is stable 0143 2% bleach (non-chimeric). 0144) 3% bleach US 2006/0218679 A1 Sep. 28, 2006 17

0145 The bracts and leaves were divided into 6 treatment growth was much less than other explants. Where callus was groups listed in table 14 and 15. A separate conical flask was produced this was evident from 2-3 weeks after initiation. used for each treatment/time combination (=6 for bracts and Interspecific hybrid bracts turned from pink to a greenish 6 for leaves). colour and initiated callus in particular where they contacted the media. TABLE 1.4 0151. All of the surviving bracts produced shoots on Treatment groups for bract culturing. ASM medium after about 4 weeks. Shoots arose from the callus. Shooting clumps were then cut and planted onto Treatment group multiplication medium to multiply for several weeks and then shoots were cut and planted into a greenhouse propa 1 2 3 4 5 6 gation facility following standard procedures used for poin Bleach (%) 196 2% 3% settias. Time (min) 5 10 5 10 5 10 Bract no. 3 3 3 3 3 3 TABLE 16 Results of leaf and bract culture of Euphorbia 0146) interspecific hybrid 98EC18.45 3.5 and 4.5 weeks after in vitro initiation TABLE 1.5 Results (3.5 wks) Results (4.5 wks) Treatment groups for leaf culturing. Treatment (3 jars per (3 jars per Explant (see Tables treatment, 5 treatment, 5 Treatment group type 14 and 15) explants perjar) explants perjar)

1 2 3 4 5 6 (8WCS 1 very few with dead explants callus, nil dead recorded: 0.5, Bleach (%) 196 2% 3% 0/5, 2/5 Time (min) 5 10 5 10 5 10 (8WCS 2 few with callus O/5, O/5, 2/5 dead Leaf no. 3 3 3 3 3 3 on edge, nil dead (8WCS 3 more callus on some damage edge, some leaf damage 0147 Bracts and leaves were placed into labelled sepa (8WCS 4 no callus, some 4/5, 2/5, 2/5 dead rate flasks. One drop of tween 20 was then added to each eaf damage flask and approximately 50 mL of the appropriate concen (8WCS 5 some callus on 1/5, 3/5, 2/5 dead tration of bleach was added. The flasks were then sealed with edge, especially near vein, Some a stopper (ensuring that plant material was covered with the eaf damage bleach solution) and shaken vigorously to ensure all areas (8WCS 6 dead. Heavy 4/5, 4/5, 4/5 dead inside the flask were covered in bleach solution. The plant eaf damage, no callus material was allowed to settle, and was re-shaken about once bracts 1 all contaminated all contaminated or twice per minute. Following the required time, the bleach bracts all contaminated all contaminated was poured off and 200 mL of autoclaved water was added bracts 3 jar remains, 3 3 callus + shoots to the plant material in the flask. The flask was then shaken greenfred callus 2/5 dead explants once per minute for 5 minutes. The water was Subsequently edges bracts 4 jar remains, 3 3 callus + shoots poured off and a further 200 ml added. This was repeated greenfred callus 2/5 dead explants twice for a total of three rinses of 5 min each. After the last edges rinse with water, more water was added and the material was bracts 5 jar remains, 1 1 callus + shoots allowed to stay in the flasks until required for culturing. piece green 35 dead callus edges 0148. Following bleach treatment, bleach affected bracts 6 all contaminated all contaminated (white) areas of the bracts or leaves were removed. The remaining bracts and leaves were then dissected into irregu lar sized fragments of 1-2 cm diameter and placed into jars Conclusion: containing adventitious shoot media (ASM) (Roest and Bokelman, 1980) containing 4.42g/LMS salts, 3% sucrose, 0152 The use of 2% bleach for 10 minutes for treating 0.7% agar, pH 5.8, 2g/L Myo-inositol, 1.0 mg/L 6-Benzy bracts is recommended for disinfestation resulting in optimal laminopurine (BAP), 0.1 mg/L 1-Napthaleneacetic acid Success for explant introduction to culture. (NAA). Five fragments were placed in each jar and the 0.153 Shoots can be produced from callus derived from fragments incubated at 25° C.2° C. under 40W white interspecific hybrid bracts after several weeks in culture flugrescent lights providing an intensity of 60-70 umol growing on ASM media. ms' at culture container lid level for 16 h/day. Results 0154 Following this experiment bracts from mutated interspecific hybrid selections (Table 13) were introduced 0149 Results after 4.5 weeks indicated a bleach treat into culture following the protocols described above, and ment of 1% for 10 minto 2% for 10 min produced the least plants were regenerated. The plants were grown in a green contaminated cultures with the least amount of over-bleach house and then placed under floral inducing conditions (10 ing. hr photoperiod) to allow them to flower. Upon flowering it 0150 Callus was produced from bracts. Callus was pro was observed that one line treatment T2, shoot number 5 duced from the mutated interspecific hybrid leaves, but its from Table 13 (mutant T2.5) which was labelled very light US 2006/0218679 A1 Sep. 28, 2006

pink had become non-chimeric (stable) and retained the very 18. The method of claim 1 wherein the free branching light pink colour. The mutant non-chimeric plants obtained agent is transmitted to the primary plant by a dodder (a from bract culture of mutant T2.5 appeared phenotypically parasitic plant), by a leafhopper insect or by a graft. very similar to the primary plant 98EC18.4/5 (RHS colour 19. The method of claim 1 wherein the free-branching N57D, pink), except that bract colour was close to RHS agent is transmitted to the primary plant by: N155C, very light pink to almost white. Several plants (a) providing a free-branching plant having a free-branch grown of this line were all non-chimeric. ing agent; 1. A method for producing an interspecific hybrid Euphor (b) cutting the primary plant and the free-branching plant bia plant comprising: to expose tissue of the plant; (a) providing a first plant which is a Euphorbia pulcher (c) making a graft union between the tissue of the free rima plant and a second plant which is a species of branching plant and the primary plant, whereby at least Euphorbia selected from the group consisting of one characteristic of a vegetative shoot arising from Euphorbia cornastra, Euphorbia radians, Euphorbia said graft is different from the free-branching plant and colorata and Euphorbia fulgens, the primary plant; and (b) pollinating a flower of the second plant with pollen (d) growing said shoot to obtain a grafted primary plant from the first plant or a flower of the first plant with pollen from the second plant in a manner which permits with at least one altered growth characteristic. formation of an embryo in at least one ovule of the 20. The method of claim 19 wherein the free-branching pollinated plant; agent is a phytoplasma. 21. The method of claim 1 further comprising the steps of: (c) cutting the embryo; and (a) obtaining a cutting of the primary plant or the grafted (d) culturing the cut embryo by placing the cut embryo in primary plant; contact with culture medium to permit growth of the embryo to thereby produce a primary plant. (b) exposing the cutting to a mutagen; and 2. The method of claim 1 wherein a flower of the first (c) cultivating the cutting to produce a mutated plant. plant is pollinated with pollen from the second plant. 22. The method of claim 21 wherein the mutagen is a 3. The method of claim 2 wherein the second plant is chemical mutagen. selected from the group consisting of Euphorbia cornastra, 23. The method of claim 21 wherein the mutagen is Euphorbia radians, Euphorbia colorata and Euphorbia full radiation. gens. 24. The method of claim 23 wherein the radiation is 4. The method of claim 1 wherein the embryo is cut while gamma radiation. contained in the ovule. 25. (canceled) 5. The method of claim 1 wherein the ovule is at least 3 26. A part of a plant as in claim 35, selected from the millimetres in length. group consisting of a flower, cutting, a pollen grain, an 6. The method of claim 1 wherein the ovule is sliced ovule, a cell, a seed or an embryo. transverse to the longitudinal axis, or along the longitudinal 27. A method for producing an interspecific hybrid aX1S. Euphorbia plant comprising: 7. The method of claim 6 wherein the ovule is sliced along the longitudinal axis of the ovule. (a) providing a first plant which is a Euphorbia pulcher 8. The method of claim 1 wherein the culturing step rima plant and a second plant which is a species of employs ovule slice culture. Euphorbia that is not Euphorbia pulcherrima, 9. The method of claim 1 wherein the embryo is cut (b) pollinating a flower of the second plant with pollen following excision from the ovule. from the first plant or a flower of the first plant with 10. The method of claim 1 wherein the embryo is cut pollen from the second plant in a manner which permits following embryo rescue. formation of an embryo in at least one ovule of the 11. The method of claim 1 wherein the embryo is excised pollinated plant; from an ovule that is at least 3 millimetres in length. 12. The method of claim 1 further comprising the steps of: (c) cutting the embryo; and (a) obtaining a cutting from the primary plant; (d) culturing the cut embryo by placing the cut embryo in contact with culture medium to permit growth of the (b) incubating the cutting under conditions Sufficient to embryo to thereby produce a primary plant. propagate the primary plant. 28. A plant produced according to the method of claim 27. 13. The method of claim 1 wherein the cutting is a shoot. 29. A part of a plant produced according to the method of 14. The method of claim 1 wherein the cutting is treated claim 27. to induce root formation. 30. The method of claim 21 wherein the mutated plant or 15. The method of claim 14 wherein the root formation is a portion of the mutated plant is propagated by: induced by treating the cutting with a composition contain ing a hormone capable of inducing root formation. (a) obtaining a bract from the mutated plant; 16. The method of claim 15 wherein the hormone is an auxin. (b) placing the bract in a solution capable of disinfesting 17. The method of claim 1 comprising the further step of the bract; transmitting a free-branching agent to the primary plant. (c) washing the bract; and US 2006/0218679 A1 Sep. 28, 2006 19

(d) cultivating the bract to thereby produce a propagated (a) obtaining a bract from the mutated plant; mutated plant. (b) placing the bract in a solution capable of disinfesting 31. The method of claim 1 wherein the primary plant, or a portion of the primary plant, may be propagated by: the bract; (c) washing the bract; and (a) obtaining a bract from the primary plant; (d) cultivating the bract to thereby produce a propagated (b) placing the bract in a solution capable of disinfesting mutated plant. the bract; 37. The method of claim 23 wherein the mutated plant or a portion of the mutated plant is propagated by: (c) washing the bract; and (a) obtaining a bract from the mutated plant; (d) cultivating the bract to thereby propagate the primary (b) placing the bract in a solution capable of disinfesting plant. the bract; 32. A method for propagating a Euphorbia plant com prising: (c) washing the bract; and (d) cultivating the bract to thereby produce a propagated (a) obtaining a bract from the plant; mutated plant. (b) placing the bract in a solution capable of disinfesting 38. The method of claim 24 wherein the mutated plant or the bract; a portion of the mutated plant is propagated by: (c) washing the bract; and (a) obtaining a bract from the mutated plant; (b) placing the bract in a solution capable of disinfesting (d) cultivating the bract to thereby produce a propagated the bract; mutated plant. 33. The method of claim 32 wherein the solution capable (c) washing the bract; and of disinfesting the bract is bleach. (d) cultivating the bract to thereby produce a propagated 34. The method of claims 33 wherein the bleach is used mutated plant. at a concentration of between 1% and 3%. 39. The method of claim 30 wherein the solution capable of disinfesting the bract is bleach. 35. A plant or part of a plant produced according to the 40. The method of claim 31 wherein the solution capable method of claim 1. of disinfesting the bract is bleach. 36. The method of claim 22 wherein the mutated plant or a portion of the mutated plant is propagated by: k k k k k