AU2012244129A1 - Scopolamine production - Google Patents

Abstract

The invention relates to duboisia hybrids and uses thereof for tropane alkaloid production

Description

Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Scopolamine production The following statement is a full description of this invention, including the best method of performing it known to us: 2 Scopolamine Production Field of the invention The invention relates to processes for the production of scopolamine and related tropane alkaloids, including use of plants in said processes, breeding, selection and growth of said plants, 5 molecular typing of said plants and extraction of tropane alkaloids therefrom. Background of the invention Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be 10 expected to be ascertained, understood and regarded as relevant by a person skilled in the art. Plant-derived secondary metabolites have long been exploited as medicinal agents. A number of these compounds are too difficult to manufacture by conventional methods of biochemical synthesis and therefore the pharmaceutical industry remains reliant on the extraction of many important drugs directly from plants or from in vitro plant culture. 15 Select genera of the Solanaceae family produce pharmaceutically important secondary metabolites, especially tropane alkaloids. Two such alkaloids, hyoscyamine and scopolamine, are anti-cholinergic agents which act principally on the parasympathetic nervous system. Scopolamine differs from hyoscyamine only by an epoxidation of the tropane ring, a reaction that may be catalysed by either 6p-hydroxyhyoscyamine epoxidase or hyoscyamine 6p 20 hydroxylase. Of the members of the Solanaceae family producing hyoscyamine and scopolamine, many produce significantly higher proportions of hyoscyamine to scopolamine. Furthermore, scopolamine has relatively fewer side-effects and has a higher physiological activity than hyoscyamine. Consequently, the commercial demand for scopolamine is at least 10 fold greater than that for hyoscyamine. 25 There have been a number of studies aimed at generating transgenic Solanaceae plants which produce higher levels of scopolamine compared to their non-transgenic counterparts. For example, WO 02/083888 describes the stimulation of secondary metabolite production in plants by transformation of the plants with expression cassettes, encoding, for instance, transporters 3 associated with alkaloid biosynthetic pathways. Similarly, WO 07/07224 describes methods for increasing nicotinic alkaloid biosynthesis in transgenic plants overexpressing regulatory genes associated with alkaloid biosynthesis. The principal commercial sources of scopolamine are members of the Australian native 5 plant genus Duboisia and interspecific hybrids of these. The hybrids cultivated today are descendants from hybrids obtained from Australian government agencies before 1950 or were obtained through selection from spontaneous interspecific crosses produced accidentally within large Duboisia populations. One problem with use of plants for manufacture of pharmaceutical actives is that there 10 are significant associated input costs. Commercial production requires large amounts of agriculture land, water, energy and labour, and like any agriculture, is susceptible to climate and other risk. Therefore, plants having high active producer phenotypes are highly desirable, as are plants that have physical characteristics that confer properties such as resistance to wind and other weather conditions, resistance to soil pathogens, tolerance to soil pH and/or salt, water 15 deprivation, and ease of harvest. The highest commercial production of scopolamine by Duboisia is described in Australian patent no. 598463. The patent describes the breeding and selection of a cultivar ("H38/3") that was able to produce between 1.4 to 2.3% hyoscine (scopolamine) per dry weight of harvested leaf This production was marginally higher than that observed in the parent clones 20 (("M3/1": 0.94%) and ("H1/25": 2.5%)) and a "standard hybrid" (1 to 2.3%), although significantly higher than Duboisia species found in the wild (0.36%). Accordingly, since this time, it has not been clear whether it would be possible to improve beyond this highest level of scopolamine production, especially without loss of the other physical characteristics required for commercial production. 25 A key problem with conventional plant breeding approaches for scopolamine production is that the relevant phenotype for selection does not arise until the plant has fully differentiated into an adult state. This then requires maintaining a very large number of cultivars for years with the associated input costs before it is possible to field test, assay for alkaloid content and select clones for further breeding.

4 Another problem is that the robustness of the scopolamine production phenotype for selection of suitable Duboisia clones for further breeding is basically not known as the relevant biosynthesis pathway and genes are not currently known. What this means is that selection of a plant on the basis of scopolamine phenotype could result in plants that have different alleles 5 across relevant genes and therefore very different outcomes in terms of progeny. This is another reason why very large numbers of cultivars are required in a breeding program. Accordingly, significant time and money is invested in an individual cultivar before it is established whether it is a "high producer" of scopolamine. These large numbers of cultivars increase the cost and time inputs required in development of a breeding program. [0 It is believed that the costs and risks associated with breeding, selection, and commercial agricultural production are drivers for other approaches to scopolamine production. However, while scopolamine can be produced by chemical synthesis, the yields are low and the process is both lengthy and expensive (Huang et al., 2005) and a more common approach has been to synthesise scopolamine and other tropane alkaloid production through use of transgenic plants 15 and in particular, in transgenic cell culture. Members of the Solanaceae family are extremely diverse in their habit, habitat and morphology. The family includes members of the Solanum tribe (potatoes), Capsicum tribe, the Petunia tribe and the Nicotiana tribe (tobacco plants). Only a few genera within Solanaceae produce high levels of biologically active tropane alkaloids. These include members of the 20 Hyoscyamus tribe (eg. Hyoscyamus niger, Scopolia tangutica and Atropa belladonna), and the Australian endemic Anthocercis tribe (Duboisia sp). Some members of the Solanaceae family produce only hyoscyamine and not scopolamine (eg Solanum toberosum (potato)). Many Solanaceae produce neither hyoscyamine nor scopolamine (eg. Nicotiana). Phylogenetic studies of the Solanaceae family indicate that the closest relatives of 25 Duboisia are members of the Nicotiana tribe, which includes Nicotiana tobacum (tobacco tree; see Figure I herein; Knapp et al., 2004). Interestingly, members of the Nicotiana tribe do not synthesise hyoscamine or scopolamine (Hakkinen et al 2005). Most of the work relating to scopolamine biosynthesis has been conducted in members of the Hyoscyamus tribe (see example 1 herein). Not only do phylogenetic studies demonstrate the 30 evolutionary distance between Hyoscyamus and Duboisia, but the plants of these tribes also 5 differ considerably in their morphology: Hyoscyamus plants are herbaceous and have fruit of a thornless seed capsule with a cover, whereas Duboisia are trees growing up to 14 m in height bearing black berries. The pathway by which hyoscyamine and scopolamine are produced is a complex and 5 poorly understood process. Whilst some of the genes encoding enzymes involved in this pathway have been identified, (Hashimoto et al., 1991), there has been no definitive characterisation of the complete pathway in a single plant species, let alone in Duboisia. For example, in the Kegg pathway (see Figure 3 herein) outlining the steps in the scopolamine biosynthetic pathway, some of the putative key enzymes have only been identified in bacterial species (eg. Cinnamoyl 10 CoA:phenyllactate CoA-transferase from Clostridium botulinum). Furthermore, it is unclear if some enzymes are capable of catalysing multiple steps in the pathway, or if there is redundancy in other steps of the pathway, with two enzymes being capable of catalysing the same steps (E.C 1.14.11.11 and E.C 1.14.11.14). It would be advantageous to provide for plants having a higher scopolamine production, 15 and especially plants having other phenotypic characteristics that enable commercial agricultural production with minimum risk, cost and ease of harvest. It would also be advantageous to provide for methods of assessing the scopolamine producing capacity of individual plants in a more time and cost-efficient manner, or at least for cross checking or correlating a measured scopolamine production phenotype with another trait 20 that is linked with the phenotype. Summary of the invention The invention seeks to at least minimise one or more of the above mentioned problems or limitations and in one embodiment provides an interspecies hybrid of D. myoporoides and D. leichhardii, the hybrid being capable of producing more than 4% (w/w) (scopolamine/hybrid), 25 the hybrid having 1.5 to 9.0 fold more H6H RNA than a wild type D. myoporoides and D. leichhardtii plant. In another embodiment there is provided an interspecies hybrid of D. myoporoides and D. leichhardti, the hybrid being capable of producing more than 4% (w/w) (scopolamine/hybrid), wherein the hybrid has a H6H gene including the nucleotide sequence shown in SEQ ID No:7.

6 Typically the hybrid has a sequence that is complementary to the cDNA sequence shown in SEQ ID No: 5. Typically the root tissue of the hybrid has 9.0 fold more H6H RNA than root tissue of the wild type plant. 5 In another embodiment there is provided an interspecies hybrid of D. myoporoides and D. leichhardtii as referred to in the DSMZ statement attached herein referring to the biological material H352/2 received by DSMZ on August 30, 2011. In a further embodiment there is provided a hybrid having approximately half of the genetic content of a hybrid as described above, wherein the hybrid is capable of producing more 10 than 4% (w/w) (scopolamine/hybrid), the hybrid having 1.5 to 6 fold more H6H RNA than a wild type D. myoporoides and D. leichhardtii plant. In further embodiments there is provided a tissue or cutting of a hybrid as described above. In further embodiments there is provided a non viable material harvested from a hybrid 5 as described above,. In further embodiments there is provided an extract formed from a hybrid, tissue, cutting or material as described above, wherein the extract includes scopolamine and/or hyoscyamine. In further embodiments there is provided a peptide having an amino acid sequence of the Duboisia H6H protein, preferably as shown in SEQ ID No: 1. 20 In further embodiments there is provided a nucleic acid having a nucleotide sequence encoding the amino acid sequence of the Duboisia H6H protein, preferably as shown in SEQ ID No:3, 4 or 5. In further embodiments there is provided a plant or material obtained therefrom including - a nucleic acid having a nucleotide sequence shown in SEQ ID No:5. 25 In further embodiments there is provided a process for producing scopolamine including the steps of: 7 a) growing a hybrid as described above in agricultural conditions; b) harvesting plant material from the hybrid; c) forming a scopolamine-containing extract from the harvested material; d) purifying scopolamine from the extract 5 thereby producing scopolamine. Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings 10 Figure 1: Phylogenetic tree of Solanaceae Family. Figure 2: Scopolamine biosynthesis in H. niger. Figure 3: Kegg pathway of putative scopolamine biosynthetic pathway (from http://www.genome.jp/kegg-bin/show_pathway?aly00960) Figure 4: Amino acid sequence alignment of H6H from Hyoscymus niger and Duboisia 15 hybrids. Figure 5: Alignment of h6h cDNA sequences from Hyoscyamus niger and Duboisia hybrids. Figure 6: Graph depicting fluctuation in scopolamine and hyoscyamine content of H352/2 throughout the year due to fluctuations in rainfall and temperature. 20 Figure 7: Variation in scopolamine and hyoscyamine content between the different clones investigated. Figure 8: Alignment of promoter sequences of H6H gene. Hp+ refers to clone 352/2.

8 Detailed description of the embodiments As used herein, except where the context requires otherwise, the term "comprise" and variations of the term. such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. 5 Agricultural Conditions generally refers to one or more of the conditions in which the Duboisia hybrid of the invention may be grown, preferably to obtain improved production of scopolamine. These conditions are discussed in more detail and summarised below: Soil Duboisia cultivation is practiced on Red Ferrosol soils. These soils are preferred as they allow 10 adequate drainage of water, retain adequate moisture, retain adequate nutrient levels and generally have a deep soil profile, which is ideal for the cultivation for Duboisia. Duboisia spp. does not grow well in very wet soils or soils which retain high levels of moisture as found in clay soils. Likewise, soils which do not retain moisture such as sandy soils, do not retain adequate moisture and nutrients due to excessive leaching. 15 Soil pH may be approximately 4.5 to 6.5. One example of a useful soil profile for providing agricultural conditions is shown in Table 13. Salinity Bore water may be used for watering hardened plants (plants with adequate roots). This water has been measured with very low conductivity levels and does not affect plant growth over short 20 periods of irrigation. Irrigation Generally the agricultural conditions do not include irrigation. That is, Duboisia cultivation is regarded as 'dry-land' farming. Climate 9 Severe frost will damage Duboisia plants. Very high humidity and rainfall in summer can also reduce plant survival and/or vigour due to disease and the plants natural resistance to high moisture. Geography 5 Particularly useful agricultural conditions are those occurring at latitude of 26.55 *S; longitude of 151.85 *E. Here, the above described soil conditions may occur. The climatic conditions are described below: Annual mean maximum temperature (1947- 2001): 24.8*C Annual mean minimum temperature (1947- 2001): 11.3*C 10 Annual mean rainfall (1905-2001): 776.2mm Annual mean number of clear days (1957-2001): 118.5 Annual mean number of cloudy days: (1957-2001): 103.1 H6H generally refers to hyoscyamine-6D-hydroxylase, as in E.C 1.14.11.11. Percentage w/w (% w/w) refers to the gram amount of the relevant tropane alkaloid detected per 15 100 g of dry plant tissue weight. For example, a Duboisia hybrid producing 6 % w/w scopolamine has 6 g of detectable scopolamine per 100 g of dry weight plant tissue. Similarly, a Duboisia hybrid producing 3 % hyoscyamine has 3 g of detectable hyoscyamine per 100 g of dry weight tissue. Dry weight tissue typically refers to the weight of plant material (for example but not limited to roots, leaves) which has been dried (either air drying or by other method) and is 20 easily manipulated for grinding or reducing to powder form. As described herein, the inventors have observed a linkage between high scopolamine producing traits of plants studied herein and a molecular trait in the form of the amount of H6H RNA in plant tissue. Specifically, in plants having a high scopolamine production of more than 4% w/w studied herein, a higher amount of H6H RNA was found than in plants having a lower 25 scopolamine production. In a particularly high producer, H352/2, which consistently produces scopolamine in amounts of 6% w/w or more, the amount of H6H RNA in root tissue was seen to be 5 to 6 fold greater than in a low scopolamine producer (M3/1). The results are unanticipated, 10 as at the time of the invention, it was not known whether a high scopolamine phenotype generally arises from high amounts of hyoscyamine (as found in H352/2 and other high scopolamine producing plants studied herein) or whether from some other molecular mechanism controlled by a particular locus. As shown herein, the inventors found that certain loci 5 understood to be active in the Duboisia scopolamine biosynthesis pathway either do not exist, or otherwise are identical in terms of amount RNA as between high and low producing strains. The invention seeks to at least minimise one or more of the above mentioned problems or limitations and in one embodiment provides an interspecies hybrid of D. myoporoides and D. leichhardtii, the hybrid being capable of producing more than 4% (w/w) (scopolamine/hybrid), [0 the hybrid having 9 fold, or 1.5 to 6 fold more H6H RNA than a wild type D. myoporoides and D. leichhardtii plant. Typically the the hybrid has a sequence that is complementary to the cDNA sequence shown in SEQ ID No: 5. Typically the root tissue of the hybrid has 6 fold more H6H RNA than root tissue of the t5 wild type plant. In another embodiment there is an interspecies hybrid of D. myoporoides and D. leichhardtii, wherein, in agricultural conditions, the hybrid has more H6H RNA than a control D. myoporoides and D. leichhardtii interspecies hybrid that produces less than 4% w/w (scopolamine/hybrid) in agricultural conditions. 20 Methods for measuring H6H RNA are discussed further herein. In one embodiment, the hybrid may have from 2 to 6 fold more H6H RNA than the control hybrid in agricultural conditions, preferably from 3 to 5 fold or 4 fold more H6H RNA. In some embodiments, the hybrid may have greater than 6 fold more H6H RNA. Typically, the root tissue of the particular cultivar is examined to identify amount of H6H 25 RNA. In particular, it has been found that in the cultivars studied herein there are large and significant differences as between root tissues of high and low scopolamine producing plants in terms of H6H RNA amount. In one embodiment, the root tissue of the hybrid has 6 fold more H6H RNA than root tissue of the control hybrid in agricultural conditions.

11 It is also possible to measure the amount of H6H RNA in leaf or stem tissue of high and low scopolamine producing plants, as the data herein shows differences exist in these tissues, albeit less significant than observed in root tissue. Thus in one embodiment, the leaf tissue of the hybrid has 1.5 fold more H6H RNA than leaf tissue of the control hybrid in agricultural 5 conditions. One particularly preferred example of hybrid is described herein as "H352/2". Thus in one embodiment, the hybrid has a genotype that is substantially the same as the hybrid deposited in the deposit referred to in the DSMZ statement attached herein referring to the biological material H352/2 received by DSMZ on August 30, 2011, and in which there is more H6H RNA [0 than in a control hybrid in agricultural conditions. One particularly surprising aspect of the invention has been the large increase in scopolamine production in certain examples of interspecies hybrids exemplified in the Examples. In particular, H352/2 has been observed to produce consistently from 5 to 7% w/w scopolamine. This represents more than a doubling of scopolamine production as compared with previous [5 cultivars of the prior art. What is more surprising is that the other physical characteristics of the plant required for commercial cropping are retained, such as resistance to wind, and appropriate branch and leaf structure for harvesting. Thus in one embodiment, there is provided an interspecies hybrid of D. myoporoides and D. leichhardtii, wherein, in agricultural conditions, the hybrid produces more than 4% w/w (scopolamine/hybrid). Preferably, in agricultural 20 conditions, the hybrid produces from 5 to 10 % w/w (scopolamine/hybrid). More preferably, the hybrid of the invention may include, in agricultural conditions, from I to 3% w/w (hyoscyainine/hybrid). Still more preferably, the hybrid may produce, in agricultural conditions, scopolamine and hyoscyamine in a ratio of from 10 (scopolamine) to 1 (hyoscyamine). 25 In further embodiments there is provided an interspecies hybrid of D. myoporoides and D. leichhardtii as deposited in the deposit referred to in the DSMZ statement attached herein referring to the biological material H352/2 received by DSMZ on August 30, 2011. A full description of this hybrid is provided in the Examples.

12 It will be understood that this hybrid has particular utility in the production of progeny by breeding with other Duboisia stock. Thus in one embodiment there is provided a Duboisia cultivar having approximately half of the genetic content of a hybrid, wherein said hybrid has more H6H RNA than a control D. myoporoides and D. leichhardtii interspecies hybrid that 5 produces less than 4% w/w (scopolamine/hybrid) in agricultural conditions. In further embodiments there are provided tissues and cuttings that contain an amount of scopolamine of at leas-: about 4% w/w. Relevant tissues include leaf, branch, stem and root. Cuttings are generally capable of propagation. Processes for producing cuttings are discussed further herein and are also known to those skilled in the art. .0 In one embodiment, the tissues include a polynucleotide that contains one or more of the bases: adenine 07 , thymine 63 0 and cytosine' 0 , as numbered according to the sequence shown in row _4_root-hp_+_assemblyHYSH6H in Figure 5. In one embodiment, the tissues include an amino acid sequence shown in SEQ ID No: 1 or a polynucleotide sequence as shown in SEQ ID No: 5. [5 Particularly useful materials are dried plant materials from which scopolamine containing extracts can be obtained. These may be prepared by any methods known in the art. In one embodiment, the harvested material includes a polynucleotide that contains one or more of the bases: adenine O', thymine610 and cytosine 10, as numbered according to the sequence shown in row _4_roothp_+_assemblyHYSH6H in Figure 5. 20 In one embodiment, the harvested material includes an amino acid sequence shown in SEQ ID No: 1 or a polynucleotide sequence as shown in SEQ ID No: 5. In another embodiment, there is provided a process for producing scopolamine including the steps of: a) growing a hybrid as described above in agricultural conditions; 25 b) harvesting plant material from the hybrid; c) forming a scopolamine-containing extract from the harvested material; 13 d) purifying scopolamine from the extract thereby producing scopolamine. Scopolamine can be extracted from plant material by any relevant method commonly used in the pharmaceutical industry. For example, the extraction of alkaloids has been described 5 by Hamerslag (1950). The two main processes used for extracting compounds from plant material are steam distillation and solvent extraction. Steam distillation typically involves passing steam through plant material and then condensing the steam. Alternatively, steam distillation may involve immersing the plant material in boiling water, boiling the mixture and condensing the steam. In either case, volatile [0 compounds are extracted from the plant material and are condensed with the steam. Typically the extracted compounds are in the form of an oil that is insoluble in the condensed water, and which can be separated from the water by a simple decanting process. Solvent extraction typically involves immersing the plant material in a solvent for a period of time and under conditions suitable for compounds to be extracted from the plant [5 material into the solvent, and then physically separating the solvent from the plant material. The compounds extracted into the solvent are typically then separated from the solvent by heating the solvent containing the extracted compounds to evaporate the solvent, leaving a residue comprising extracted compounds. Solvents used in solvent extraction include alcohols, particularly methanol and ethanol, hydrocarbons, particularly hexane, ketones, particularly 20 acetone, halogenated hydrocarbons, and ethers. In a further embodiment there is provided an extract formed from a hybrid, tissue, cutting or material described above wherein the extract includes scopolamine and/or hyoscyamine. The inventors have for the first time characterised an amino acid sequence of H6H enzyme from Duboisia in the form of an amino acid sequence as shown in SEQ ID NO: 1. The 25 peptide is clearly homologous to H6H enzymes from related plants such as Hyoscyamus niger (SEQ ID No:2) (the sequences are 88% identical). Figure 4 shows an alignment of H. niger H6H with Duboisia H6H. A number of amino acid differences are observed and these include potentially important residues in regions known to be associated with iron-binding and consequently important to catalytic activity.

14 Also provided is a nucleic acid having a nucleotide sequence encoding the Duboisia H6H enzyme described herein. Typically the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 3, 4 or 5. Also provided are nucleic acids having a nucleotide sequence that is capable of forming 5 Watson-Crick base pairing with a nucleic acid of the invention. These are particularly useful for detecting the amount of H6H RNA in a cultivar, especially as according to a method described further below. These acids may take the form of a fragment of a nucleic acid having a length of about 8 to 50 nucleotides. In further embodiments there are provided methods of determining whether an 10 interspecies hybrid of.D. myoporoides and D. leichhardtii is capable of producing more than 4% w/w (scopolamine/hybrid) in agricultural conditions including the following steps: a) growing an interspecies hybrid of D. myoporoides and D. leichhardtii in agricultural conditions; b) measuring the amount of H6H RNA in root tissue of the hybrid; 15 c) comparing the measured amount of H6H RNA with the amount of H6H RNA in a control D. myoporoides and D. leichhardtii interspecies hybrid that produces less than 4% w/w (scopolamine'hybrid) in agricultural conditions; d) determining that the interspecies hybrid of a) is capable of producing more than 4% w/w (scopolanine/hybrid) in agricultural conditions where the hybrid has more H6H 20 RNA than the control in agricultural conditions. In one embodiment, the hybrid is at a seedling stage of growth. In other embodiments the hybrid is at a later stage of the lifecycle, including, intermediate and fully grown. A variety of technologies are available for measuring H6H RNA. These include quantitative RT-PCR and northern blotting. 25 In another embodiment, the invention provides for an antibody which is selective for Duboisia H6H. The antibody can be produced by any of a number of methods known in the art including generating peptides derived from recombinant Duboisia H6H for use as antigens in the 15 generation of anti-sera against H6H. In a further embodiment, the antibody can be generated using the full-length Duboisia H6H protein, derived by recombinant techniques. Generally, the antibody will be selective for Duboisia H6H and will not bind to H6H from other species. The antibody may be monoclonal or polyclonal. 5 In yet another aspect of the invention, there is provided a method for detecting whether a plant is a high scopolamine producer by using an antibody which is selective for Duboisia H6H. For example, whole protein extracts from cultivars of interest can be extracted by conventional methods, and subjected to conventional Western blotting techniques of which the skilled person would be familiar. High levels of H6H protein in plant extracts would be indicative of high h6h 10 expression and consequently, high scopolamine production. Examples Example 1: Scopolamine biosynthetic Pathway To date, the scopolamine biosynthetic pathway has principally been investigated in members of the Solanaceae family belonging to the Hyoscyamus tribe (see Fig 2, Zhang et al 15 2004). There has been no published investigation regarding the scopolamine biosynthetic pathway in members of the Anthocercis tribe, of which Duboisia is a member. The closest relatives to the Anthocercis, members of the Nicotiana tribe, do not synthesise hyoscyamine nor scopolamine. There are a number of distinct proposed enzymatic steps in the biosynthesis of 20 scopolamine. Figure 3 provides a general overview of enzymes hypothesised to be involved in various steps associated with tropane alkaloid pathway. Only a few functional genes of the tropane alkaloid pathway have been characterised and not all of them are plant enzymes. For example, some of the putative key enzymes have only been identified in bacterial species (eg. Cinnamoyl-CoA:phenyllactate CoA-transferase from Clostridium botulinum). 25 As depicted in Figure 2, there are any number of enzyme candidates which may be important in the synthesis of scopolamine. However, given the phylogenetic distance of Duboisia from other members of the Solanaceae family, there is no reason to suspect that a) Duboisia expresses the same genes found in other members of the family, or b) that those genes 16 will be regulated in such a way as to be primarily responsible for Duboisia's enhanced scopolamine production relative to other plants. Example 2: Analysis of transcriptomes from various Duboisia clones Total RNA was extracted from cuttings or roots from 3 month old plants. Plant material 5 was washed thoroughly in RNase free water before commencing extraction. Plant material was frozen in liquid nitrogen and then ground to a fine powder in a mortar and pestle. Total RNA was further isolated from the powder using commercially available kits e.g. RNeasy Plant Kit from Qiagen (Hilden, Germany). The quality and yield of total RNA was assessed by performing an RNA 6000 Nano .0 Assay using the Agilent 2100 BioanalyzerTM. mRNA was enriched and purified using the TruSeq RNA Sample Prep Kit (Illumina, Inc). Synthesis of a cDNA library was by conventional laboratory methods (for example, conducting first strand synthesis using a reverse transcriptase with random hexamers, followed by second strand synthesis end repair and adapter ligation with TruSeq (Illumina)). Quantification of the cDNA library was performed using the High .5 Sensitivity DNA Assay on an Agilent 2100 BioanalyzerTM and dsDNA HS Assay on a Qbit@ 2.0 (Invitrogen). For cluster generation a TruSeq PE cluster Kit v2 and a flow cell type v1.0 (Illumina, Inc) were used. Sequencing on HiSeqTM 2000 was done using TruSeq SBS kit-HS (200). Table 1: Number of reads to map; whole genome Lane Sample Number of reads to map S_2 Leaf clone M3/1 (WT) 83,958,853 S_3 Root clone M3/1 (WT) 103,814,220 S_4 Root clone 352 (hp+) 88,693,251 S_5 Leaf clone 352 (hp+) 112,586,737 S_6 Leaf clone 329 (hp) 81,012,838 17 S_7 Root clone 329 (hp) 94,891,153 We next compared the transcriptomes of wild type Duboisia myoporoides (low scopolamine producers) with high scopolamine-producing Duboisia hybrids derived from several years of crossing and breeding. RNA isolated from leaves and roots of plants was used to 5 sequence the transcriptome as described below in Example 3. We referred to the previously known scopolamine biosynthetic pathway (Figure 3) to identify genes which might be differentially expressed in low as compared with high scopolamine producers. For example, the KEGG pathway suggested that enzyme EC 1.14.11.14 (6 beta-hydroxyhyoscyamine epoxidase) was a likely candidate to be involved in increased 0 scopolamine production. We therefore used known cDNA sequences encoding this enzyme (from the ncbi.nlm.nih.gov non-redundant database) to perform BLASTn searches against the sequenced reads. Only those search results covering at least 60 % of the read length were considered. These steps were performed using the shell script blastingpb.sh. BLAST results were further analyzed using the java class BLASTAssemblyMappingParser to generate a final L5 output which quantified the total number of reads mapped. We were surprised that our BLAST results did not return any reads mapping to this enzyme. This suggested that Duboisia do not express the gene encoding 6 beta hydroxyhyoscyamine epoxidase (see Table 2 below). We next investigated whether the gene encoding enzyme EC 2.8.3.17 (cinnamoyl 20 CoA:phenyllactate CoA-transferase) was differentially expressed in high and low producers. EC 2.8.3.17 catalyses the conversion of phenyllactate to littorine, the rate limiting step in the production of the scopolamine precursor, hyoscamine. Again, we could not detect any transcripts for this enzyme in roots or leaves of any Duboisia plants analysed. EC 1.1.1.206 (tropine dehydrogenase) acts further upstream in the scopolamine 25 biosynthetic pathway. It is responsible for the conversion of tropinone to tropine, an alkaloid which reacts with phenyllactate CoA to ultimately produce hyoscamine. Again, we referred to known cDNA sequences encoding this enzyme (eg, from Datura straminium, jimson weed) to perform BLAST searches against the transcriptomes of high and low producing Duboisia clones.

18 As depicted in Table 2, the gene encoding tropine dehydrogenase is expressed in Duboisia. However, no differences in the expression levels of this gene were observed between the three clones of Duboisia analysed (see Table 2). We lastly performed BLAST searches using the known cDNA sequence encoding EC 5 1.14.11.11 (hyoscamine beta-hydroxylase, H6H) from Hyoscyamus niger. This enzyme has been reported in other organisms to be involved in the conversion of hyoscamine to scopolamine. However, given that we had not detected any expression of 6 beta-hydroxyhyoscyamine epoxidase, an enzyme catalysing the same step in the scopolamine biosynthetic pathway, we were uncertain whether we would detect any transcript. L0 As the results in Table 2 demonstrate, the gene encoding H6H (EC 1.14.11.11), h6h was highly expressed in all Duboisia clones. However, we observed significantly higher reads mapping to this transcript in the roots of Duboisia clones producing higher levels of scopolamine compared to wild-type. A similar observation was made for transcripts in leaves. Table 2: Number of reads mapping to Duboisia genes encoding scopolamine [5 biosynthesis enzymes Sample EC 1.14.11.14 EC 2.8.3.17 EC 1.1.1.206 EC 1.14.11.11 Root hp+ 0 0 5,362 /5,416 555,411 / 551,874 Root hp 0 0 7,467 /7,292 420,594 / 418,578 Root WT 0 0 6,768 / 6,884 87,360 / 86,908 Leaf hp+ 0 0 1219/1253 27939 /27745 Leaf hp 0 0 1915/1800 37320/36794 Leaf WT 0 0 726/764 14689/14709 readsforward napping / readsreverse mapping Another way of analysing the expression levels of the transcripts investigated is by reference to reads per KB per million reads (RPKM). RPKM was calculated using the method of 19 Mortazavi et al. (RPKM = number of reads mapped/((total number of reads x I E-6) x (bp of gene x 1E-3)) The RPKM results for EC 1.14.11.11 and EC 1.1.1.206 transcripts are shown in Table 3, below. 5 Table 3: RPKM and reads mapped for EC 1.1.1.206 and EC 1.14.11.11 Sample EC 1.1.1.206 EC 1.14.11.11 readsmapped RPKM readsmapped RPKM Root hp+ 5,362 52.66 555,411 4,554.30 Root hp 7,467 68.55 420,594 3,223.55 Root WT 6,768 56.79 87,360 612 Leaf hp+ 1219 9.43 27939 180.48 Leaf hp 1915 20.59 37320 335.03 Leaf WT 726 7.53 14689 127.24 The RPKM values of EC 1.14.11.11 in all tissues are generally much higher in comparison to EC 1.1.1.206. The expression levels of the gene encoding EC 1.14.11.11 in roots was approximately 10-20 times that in leaves. 10 The RPKM values of the high scopolamine content clones (hp+ and hp) were at least five time higher than that of the wild type clone (wt). The RPKM value of the hp+ clone was at least eight times higher than that of the wild type clone. The data demonstrate a correlation between high scopolamine content of plants and the expression levels of the h6h gene in the clones studied here. 15 Example 3: Isolation and characterization of the Duboisia h6h gene 20 We have determined the first ever sequence of a gene encoding H6H in Duboisia sp. Figure 5 shows an alignment of h6h cDNAs from three Duboisia clones (wild-type, high and very high producers) and h6h cDNA from H. niger. Figure 4 shows an amino acid alignment of H6H from the same plants. 5 As previously discussed H. niger is distantly related to Duboisia and similarly belongs to the Solanceae family. The amino acid sequence of Duboisia H6H is 88 % identical to the H6H sequence from H. niger. The length of the proteins is the same in both species. Most of the amino acid differences between H. niger H6H and Duboisia H6H are located in variable regions of the polypeptide. However, a phenylalanine to leucine substitution at residue 169 is in a region .0 previously reported to be involved in iron-binding. Interestingly, the amino acid sequences of H6H from the three Duboisia clones are identical. This indicates that the differences in scopolamine production are not attributable to alterations in the H6H activity, per se. Three single nucleotide polymorphisms (SNPs) were identified, distinguishing wild-type L5 Duboisia h6h cDNA fiom the cDNA of the higher-scopolamine producing clones (see Figure 5). These were located at nucleotides 507, 630 and 1024 (hp + h6h cDNA numbering). An adenine at nucleotide 507, thymine at base 630 and cytosine at base 1024 is characteristic of the genotype of clones producing high levels of scopolamine that are studied here, including H 352/2. The bases at the corresponding positions in wild-type scopolamine producers studied here are 20 cytosine, adenine and adenine, respectively (see figure 5). None of the SNPs result in changes to the amino acids sequence of H6H. However, it is possible that these SNPs contribute to the increased relative expression of the transcript, through epigenetic mechanisms. Example 4: Screeninf' for high scopolamine producers on the basis of H6H RNA trait Using conventional real-time PCR techniques, known to the skilled person, the quantity 25 of H6H RNA can be measured to predict the likelihood of a Duboisia clone studied here producing a comparatively high level of scopolamine. For example, real-time primers can be designed to any suitable region in the Duboisia H6H cDNA sequence (see figure 5). Appropriate primers can be designed manually, or by using 21 software designed to maximise the success of real-time PCR, for example Primer Express (Life Technologies, Carlsbad, CA). cDNA can be isolated from individual plants using conventional techniques, including those referred to in example 2, above. Briefly, RNA can be extracted from plant tissue which 5 includes but is not limited to roots and leaves, using commercially available kits (eg: RNEasy, Qiagen). cDNA can be generated from total RNA using commercially available first strand synthesis kits, such as Superscript First Strand Synthesis (Life Technologies, Carlsbad CA). Amplification and quantification of H6H RNA can be performed using, for example, SybrGreen quantitative PCR on a conventional real-time PCR machine (for example, ABI 7900 HT Real [0 time PCR system) by reference to a constitutively expressed standard. The levels of h6h expression should initially be compared to those of wild-type Duboisia. H352/2 has been demonstrated to exhibit at least 5-7 fold the levels of h6h expression compared to wild-type in roots. That is, by comparing the signal intensity of real-time PCR product in Duboisia clones compared to wild-type (following normalisation to the constitutively expressed [5 standard), the levels of expression can be compared. For example, if the normalised signal intensities following amplification of h6h cDNA are the same, then the fold change is 1. If the signal intensity is twice that of the wild-type intensity, the fold change is 2, and so forth. Worded differently, if there is a 2-fold increase in expression, the levels of h6h cDNA can be said to be 200% that of the wild-type sample. 20 As is well known in the art, normalisation allows one to control for the amount of total RNA which may be different from sample to sample. Typically normalisation is done by reference to a constitutively expressed gene such as actin. The table below shows the comparison of normalised amounts of H6H cDNA from wild type and hp+ (H352/2) hybrids. Table 4: H6H RNA expression in hybrids Hybrid H6H normalised against actin WT 1.0 hp+ 8.5 25 22 Example 5: Screening for high scopolamine producers on the basis of genotype It is also possible to determine the likelihood that a seedling studied here will develop into a high-producing plant by genotyping without the need to destroy the plant, as would be the case in Example 4. The genotype at three nucleotide positions within the h6h coding region were 5 demonstrated to be associated with Duboisia clones studied here producing high levels of scopolamine compared to wild-type and moderate-producers (see Example 3). Genomic DNA can be obtained from plant leaf discs as small as 0.5 cm, for example, using conventional kit~s such as Extract-N-Amp (Sigma Aldrich) or 100 mg wet weight using DNeasy (Qiagen). Preferably, genomic DNA would be isolated from a plant at the earliest 10 possible stage in its development. A suitable method for determining genotype at the relevant SNP position could include, but is not limited to a TaqMan SNP Genotyping Custom assay (ABI, Life Technologies). Alternatively, a region approximating 400 bp can be amplified containing the region of interest, purified and sequenced. using conventional techniques.

23 Example 6: Description of the KS H352/2 clone The species Duboisia myoporoides and Duboisia leichhardtii are well distinguishable morphologically, while their hybrids may show slight differences only with respect to the shape of flowers and leaves making visual distinction difficult. 5 Interspecific hybrids D myoporoides X D.leichhardtii have the following characteristics: e Tall shrub or small tree, glabrous, bark thick and corky towards the base. Leaves; Leaves are slightly discolorous to concolorous. * Leaf form- Simple * Leaf arrangement- Alternate 10 * Leaf shape- Oblanceolate, Ovate to Obovate. (Leaf Shape in H352/2 is Obovate) * Leaf Margin- Entire * LeaF tip- Acuminate to Obtuse, 4-15cm long, 0.7-4cm wide. (Leaf tip in H352/2 - Acuminate. Mean length of 9.85cm. Mean length of 2.81cm). 15 9 Leaf base- Tapering, almost sessile or with petiole to 3 cm long. (Leaf base in H352/2 - Mean petiole length of 1cm). Inflorescence; Indefinate, panicle shaped, broadly pyramidal, terminal, leafy. " Flowers are bisexual, slightly zygomorphic, subtended by pairs of opposite bracts 0.5-11mm long; pedicals 2-16mm long. Calyx regular, campanulate, 5 20 lobed, 1-3mm long, the lobed on-fifth to half as long as tube. " Corolla campanulate, white with purple striations in 4.5mm diameter at apex; limb 5-lobed, lobes 3-8mm long, round to acute, volutive in bud. Stamens 4, sometimes 5. Didynamous, inserted at base of corolla tube, 1.5-4mm long. A staminode sometimes present, anthers unilocular, not cohering, dehiscing by a 24 terminal, semicircular slit. Ovary bilocular, style 1.5-4mm long, about as long as upper stamens. Stigma capitate, very shortly bilobed. * Fruit a succulent berry, globose, rarely ellipsoid to ovoid, 4-8mm long, purple black, fruiting pedicels 8-18cm long. Seeds reniform, 2.5-3mm long, n=30. 5 Duboisia hybrid flowers are intermediate between those of D myoporoides and D leichhardtii, the differences being gradual ones only so that they are difficult to describe unmistakeably for identification purposes. Leaf shape, however can be measured physically, and there are stable, statistically significant differences between natural species and hybrid clones. 10 Developmental characteristics From planting to 1st harvest takes approximately one year if there are no special plant husbandry techniques required to strengthen the tree during this first year, for each year after it can be expected that a harvest can be carried out at 12 month intervals. Flowering can be expected to commence in late July August and through to October in Australia, this can vary 15 depending on the intensity of winter and spring. Chemotypical characteristics Average scopolamine and hyoscyamine contents throughout a year can be seen to fluctuate due to the effects of temperature, light intensity and available moisture. Data indicates that June in Australia will be the month of least scopolamine production due to short days, low 20 temperatures and low rainfall. From July to January the contents will rise and then begin to fall around February (see Figure 6). Amount and proportion of tropane alkaloids especially scopolamine compared to wt and high producers Figure 7 depicts the variation of alkaloid content when compared with other trial clones. 25 The improvement in alkaloid content can be seen to increase with the clone numbers. The average alkaloid content/ha of a plantation does decrease over time however this is mostly attributed to the reduced vigour of plants as they physiologically age.

25 Other environmental factors influencing scopolamine content Factors influencing scopolamine content range from, soil moisture, temperature, light intensity, soil nutrition, soil type, pH and disease pressures. The most variable influencing factor is plant available soil moisture. A Duboisia plant 5 which is "water stressed" will produce a higher alkaloid content than one immersed in luxury amounts of water, a p1.ant suffering prolonged exposure to high soil moisture will be under high disease pressures and will have difficulty with aerobic respiration in the root zone, resulting in poor photosynthetic rate, poor biosynthesis of alkaloids and overall poor growth. Light intensity will over time effect the average daily temperature and through a series of 0 biochemical processes affect the photosynthetic rate of Duboisia, which in turn will increase the biosynthesis of alkaloids found in the leaves. It is commonly found that the average alkaloid content found in Duboisia decreases during the cooler months of the year when the sun is tracking in the Northern horizon. Example 7: Methods relevant to the duboisia breeding program: 1 5 Overview of Duboisia trial program: Seedlings were classified as the 'A' Trial (eg. T1/2010A), with the trial number for that year (1), and the year (2010). Selections were made and cuttings taken and propagated to become the 'B' Trial or selection trial (eg. TI/2010B), with the trial number (1) and the year it commenced remaining the 20 same (2010). A clone number was provided according to its parental crossing (eg, H352/2), with H = hybrid and parental crossing number 352, and selection number 2. A monthly characteristic analysis was taken for each clone from the 'B' trail. Leaf analysis and growth characteristics data was collected and further selections were made for the 25 next stage, Mother Plant Quarter, also known as a 'C' trial. Cuttings were again taken and planted. Regular leaf samples and inspections for change in growth were regularly conducted.

26 A clone could then become a commercial clone for production purposes, and plant numbers increased via clonal propagation. Example 8: Field Trial Data Relating to Duboisia Hybrid H352/2: a) Seed Treatment 5 1057 seeds from 18 parent crosses for Trial 2/2002 were obtained. b) Sowing Trial 2/2002 Of the total of 1057 seeds sown, 635 seedlings were later planted into the field- a germination rate of 60.08% (see Table 4). There were 20 seeds sown of parents H222/1 x H262/1 (K5). 17 seeds struck with all 10 seedlings surviving until planting- germination rate of 85%. c) Field Planting Trial 2/2002 Seedlings were then planted in the field by hand in furrows created by the mechanical planter. Records of planting details and maps were created.

27 CL * -w1-0 0 l m oCl 0 l Z cnc Z N co C~5o &2 v -N 6 oo c a& ; oo Z ) n.6.c6 00 N Il U C V ( 0000 0- 04CQ U) z o co, 0, Z0 N w .C (L_ l 0 1

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0 0~) O nN o2o cor- ( NP 0 Z- 0 0 o l 00) L0'j - 0C) O O 0 00000 ca 0 c T C4Q c. to > 0) L oo Lo - cI x. Col nC o O x (~ n D x x ) - - x xx to ~ Cj ' ' C. . ILf 0, I- C, En Z z 28 d) Selection Process of Seedling Trials ('A' Trials): 84 seedlings were selected, identified and tagged with Identification number, and leaf samples taken (40 leaves per tree). 5 plants were selected from parent cross H222/1 x H262/1. Only 1 plant (K5), was further 5 selected as having >4% scopolamine. A description of this plant was noted, with all selections meeting the standard criteria outlined in the methods of selection for Duboisia 'A' trials. Such criteria included greater than 4% scopolamine content of the sampled leaves, and desirable physical characteristics required for Duboisia leaf production. It was noted that this individual (K5) was a short tree and had large, wide leaves. 10 A total of 17 plants were selected from the original 84 as displaying >4% scopolamine. Leaf alkaloid analysis completed using HLPC. Seedling with Identification 'K5', of parents H222/1 x H262/1; resulted in a scopolamine level of 5.91%. Parent crosses planted in descending order begin with identification number of 'A'; 15 therefore 'K' is the 1 Ith parent cross with successful germination, for the Trial 2/2002. The number represents the seedling selection number within the parent cross. For example, '5' is the 5t selection for parent cross, 'K' (H222/1 x H262/1). Of the 17 individual plants selected from Trial 2/2002, 8 plants were selected for cuttings to create a 'B' trail (Trial 2/2002B).

29 Table 5: Scopolamine and Hyoscyamine results of selections from Trial 2/2002; Plant No. Cross Id. No. Cross- breeding partner Plant Id. Height (m) Scop % Hyosc % Mother X Father 1 1 H210/1 X H216/1 Al 1.3 3.53 0.12 2 A2 1.6 5.02 1.34 3 A3 1.5 3.62 0.08 4 A4 1.5 3.35 0.07 5 2 H210/1 X H222/1 B1 1.6 4.65 0.13 6 B2 1.7 4.14 0.05 7 B3 2 3.06 0.02 8 B4 1.2 3.48 0.06 9 B5 1.7 3.48 0.12 10 B6 1.4 3.13 0.08 11 B7 1.7 3.88 0.13 12 B8 1.5 4.04 0.98 13 B9 1.5 3.55 0.06 14 B10 2.1 3.41 0.09 15 B11 1.6 3.85 1.39 16 B12 1.6 1.92 1.16 17 B13 1.8 2.72 1.22 18 B14 1.4 3.88 0.09 19 B15 1.5 4.28 0.08 20 B16 1.9 3.46 0.06 21 B17 1.7 3.81 0.05 22 B18 1.8 3.49 0.09 23 B19 1.5 3.52 0.05 24 B20 1.9 2.59 0.04 25 3 H210/1 X H238/3 C1 1.6 4.22 1.67 26 C2 1.7 2.98 0 27 C3 1.8 3.67 0.95 28 C4 2.1 3.52 1.35 29 C5 2.2 3.76 0.1 30 C6 1.8 4.21 1.13 31 4 H210/1 XH251/1 D1 1.5 3.29 0 32 D2 1.7 3.47 0.13 33 D3 1.9 3.83 0.19 34 D4 2.2 3.44 0.12 35 D5 1.6 3.18 0.12 36 D6 1.8 3.37 0.07 37 5 H210/1 X H255/3 E1 1.5 3.26 0 38 E2 1.6 3.75 0.06 39 E3 2 2.55 0.05 40 E4 1.7 3.47 0.8 41 E5 1.6 2.39 0.02 42 E6 1.5 4.74 0.91 43 E7 1.8 2.75 0.08 44, E8 2.4 3.33 0.13 451 1 E9 1.8 2.96 0.12 30 Plant No. Cross Id. No. Cross- breeding partner Plant Id. Height (m) Scop % Hyosc % Mother X Father 46 E10 1.7 2.92 0 47 Eli 1.7 2.97 0.05 48 E12 1.8 2.88 0.09 49 E13 1.8 2.88 0.17 50 E14 1.4 3.6 0.57 51 6 H210/1 X H261/2 Fl 1.6 3.2 0.04 52 F2 2.1 3.01 0.07 53 F3 1.7 3.6 0.06 54 7 H212/1 X H216/1 G1 1.5 3.56 0.02 55 8 H212/2 X H255/3 Hi 1.8 3.43 0.05 56 H2 1.6 4.14 0.78 57 H3 1.5 4.57 0.07 58 H4 1.6 3.25 0.1 59 9 H222/1 X 118/2 11 2 2.69 0.02 60 12 1.6 3.15 0.03 61 13 2 3.12 1.38 62 14 1.7 1.72 0.1 63 10 H222/1 X 254/1 J1 1.8 3.52 0.03 64 J2 1.8 3.33 0.97 65 12 H222/1 X H262/1 Ki 1.6 3.21 0.08 66 K2 1.5 3.42 0.04 67 K3 1.5 3.17 0.09 68 K4 1.7 3.27 0.1 69 K5 1.5 5.91 1.89 70 13 H237/6 X H252/1 Li 1.9 3.25 0.12 71 14 H238/3 X H216/1 Ml 1.8 3.61 0.14 72 15 H238/1 X H222/1 Ni 1.7 4.34 0.09 73 N2 1.9 4.05 1.72 74 N3 2 4.01 1.21 75 16 H238/3 X H254/1 01 1.8 3.31 0.14 76 02 1.7 4.54 0.08 77 03 1.6 3.53 1.77 78 04 1.5 3.53 0.74 79 05 1.8 4.18 1.44 80 17 H238/3 X H255/3 P1 1.5 3.57 0.09 81 P2 1.5 4.45 0.07 82 18 H238/3 X M3/1 Q1 1.5 2.03 0.13 83 Q2 1.6 2.22 1.77 84 Q3 2 1.91 2.59 Average: 1.70 3.46 0.41 -- -,-- - - - 31 0, 00 C0 . 0A 000) 0) (U cn C)M L A-J r- r4 - LI 0 0 . Z 0

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33 e) Propagation of 'K5' (H352/2) cuttings for Selection 'B' Trial (Trial 2/2002B): Cuttings were taken from selected trees in Trial 2/2002. 7 cuttings were taken from selection 'K5'. Cuttings were then sorted with all 7 cuttings struck. All cuttings appeared healthy and had 5 very good roots protruding through the sides of the 'Fertil' pots. Of the 8 selections made from individuals in Trial 2/2002, only 7 were successful in developing roots for the 'B' Trial. A total of 63 cuttings were taken, however, 54 cuttings were 'struck' and used for planting in the 'B' trial- a strike rate of 85.71%. 10 All 'struck' plants placed in the shade house for hardening and awaiting planting. f) Planting of 'K5' (H352/2) cuttings for Selection 'B' Trial (Trial 2/2002B): The K5 cuttings where then planted with buffer rows established by other Duboisia clones. Table 8: Cuttings taken from selections for Trial 2/2002B; 15 Plant No. No. cuttings No. cuttings Scop % Hyosc% taken after sorting A2 10 10 5.05 1.34 B1 6 0 4.65 0.13 C1 7 6 4.22 1.67 C6 12 12 4.21 1.13 E6 7 7 4.74 0.91 H2 7 7 4.14 0.78 K5 7 7 5.91 1.89 02 7 5 4.54 0.88 TOTAL 63 54 20 34 Table 9: Cuttings taken for Trial 2/2002B; Selection Clone No. plants Parent Cross A2 H344/2 10 H210/1 x H216/1 C1 H371/1 6 H210/1 x H238/3 C6 H371/2 12 H210/1 x H238/3 E6 H346/4 7 H210/1 x H255/3 H2 H329/2 7 H212/2 x H255/3 K5 H352/2 7 H222/1 x H262/1 02 H372/1 5 H238/3 x H254/1 5 g) Selection process of 'K5' (H352/2) cuttings for Selection 'B' Trial (Trial 2/2002B): Monthly characteristic analysis and leaf sample were measured using HPLC on consecutive months. An average of 6.20% scopolamine was recorded during the monthly 10 analysis for the selection K5 (H352/2) of parents, H222/1 x H262/1. An average of 1.56% Hyoscyamine was recorded during this time. 2 clone selections were made for Mother Plant Quarter cuttings (Trial 2/2002C). These were A2 (H210/1 x H216/1), and K5 (H222/1 x H262/1). Cuttings taken from all plants of the 2 clones selected in Trial 2/2002B.

35 - o; I- C oL 01- 8 l - -im IV r 0.a) -- -- cpr- (D 0; nt Lr-L)a Dc CD: D R C1400 (D D N r-~ -0 o 6( 6L C Dir I ( t6L c6 4 > > 0~ 0 o EI E - - ID o l -( ! 0 10(100 iD C-j) CD R ) CD C 1 Cr ) C1 C 04 C%) 1:0co0 4 00 ou : 4 0 )CDc CU 0- ce~i ) 0 o rl 4.' .14 u -0c)U U 0C -' M- 4D c D r -r L __( o( D r - .o cIo CO c iO 0) CLC l i .i 36 h) Planting of 'K5' (R352/2) cuttings for MPQ (Trial 2/2002C): 42 plants of clone, 'K5' were planted in Block, X; Contour 6, along with 53 plants of clone 'A2'. Regular leaf samples of A2 and KS were taken and data was recorded into trial database. Clone numbers were then provided for 'A2' and 'K5'; A2- H344/2, and K5- H352/2. 5 Both clones, H344/2 and H352/2 were further increased via cutting propagation to repeat MPQ. 529 cuttings were taken from H352/2 in block, X, contour 6, and propagated in the nursery. 444 H352/2 plants were planted in block Cl; contour 5; rows 3 and 4 with a strike rate of 83.9%. A total of 14070 cuttings taken from H352/2 in 2007 from block Cl; contour 5, rows 3 and 4.

37 C'J 0 . -: .: I ii 0 a' a IL; 06 Cu 2- n Iob 38 -D 9 9 CO C00 00CDCD <N - - r- I- r- r v n L toN 0 MCN 0 co ~L r- C)) 0n(D U) ' LONr- v 0v tor )c 0F co.-c.,C14 N Nl c) ( 0 U CJ r) c co(0 N'CJ N

-

r too) cm)C) 00 0000000 00 C) 6L 6. & -, uO 0) 0z:D 0 U W Q cv) CN 4~ - - -Nq N i z U) N CJ 06 ' Z 06 N dC(D C w v ~ J z _ D Co) 00 N C14 N 0ON (D CO 00 CN LO - 0 ( Cl .- r- qt 0 L C)0 0 v~ Ce) c D m Cl) 0) LA N1 N - CNNN 0 C) C) cn ~C'N C 0 z T (O~D I, C 0CO 00)'o 0)a00o N z DCON~ r--)NCD W N N CNI N N N N N N N N N N N N N N z. N N N N N N i C4i-C4C4 N C- : -l-4C4- N N N: ~l o U) LA) Lr LO U) LA VA VA LA VA LA LA LA LA LA LA LA) a' C) C'. C> a" C>) C>) C) C) )C" C)C)C)C)C)C) toC' q. 0 " C') C)L LA A'c'c"C" !#) (1 0 ~ 1 39 9'~~4 Y 0 0 0 N oo 0 0000 N 0 m idc 23 4m 0~I 3 E1 C e 'o ~ ~ 3' E 40 Example 9: Extraction of alkaloids The process below exemplifies an assay for measurement of scopolamine content. Reagents: Acetonitrile (IPLC grade) 5 Water 0.05 m Phosporic acid Heptanesulphonic acid sodium salt Phosphoric acid 0.5 % (w/v) Equivalent grades of reagents may be used 10 Standard solution: Accurately weight about 20 mg scopolamine hydrobromide and about 10 mg hyoscyamine into a 100 ml volumetric flask, dissolve, dilute to volume with phosphoric acid 0.5% and mix well. 15 A. Analysis for determination of optimum harvest time Transfer 1.0 g of dried, crumbled raw material into a 250 ml glass container with a twist off closure and put in 200 ml phosphoric acid 0.5 %. Close the container and shake briefly. Extract the substances by leaving the containing to stand for at least 12 hours. Then shake the container again and filter a portion of the extract. 20 B. Analysis for production control Transfer 1.0 g of fine powdered plant material into a 250 ml glass container with a twist off closure and put in 200 ml phosphoric acid 0.5 %. Close the container and shake briefly. Extract the substances by leaving the container to stand 5 hours. Then shake the container again and filter a portion of the extract. Prepare this solution twice.

41 Chromatographic conditions Column: Merck steel cartridge (Cat no 16052) 125-4 mm with Superspher 60 RP 8, 4 urn Mobile phase: 600 ml solution A 5 225 ml acetonitrile Solution A: Dissolve 7.5 g heptansulfonic acid sodium salt in 2500 ml water, add 21 ml 0.05 phosphoric acid and mix well (pH 3.20-3.25) Detection: UV detector, wavelength 190 nm Temperature: Room temperature [0 Flow rate: 1.1 ml/minute Injection volume: 10 jtl Retention times: Scopolamine: about 6 minutes 15 Hyoscyamine about 9 minutes System suitability reference date: Four quantities of standard solution must be injected. The coefficient of variation (relative standard deviation) for all the standard injections should not exceed 2 %, based on 20 measurement of scopolamine.

42 Procedure: Sample A: Inj ect one quantity of sample preparation A and calculate the numbers of integrator units of scopolamine and hyoscyamine extraction. Sample B: Inject one quantity of each sample preparation B and calculate the mean 5 number of integrator units for each scopolamine and hyoscamine extraction. Evaluation: Integrate the peaks with the aid of a suitable laboratory data system. Program the system to perform the calculation by the external standard method. It will be understood that the invention disclosed and defined in this specification extends 0 to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

43 References Huang F, Dai XD, Hu YL, Chen CY and Zhu GZ (2005) Progress in the synthesis of tropane alkaloids. Chem Reagent 27, 141-144 Hakkinen ST, Moyano E, Cusido RM., Palazon J, Pinol MT and Oksman-Caldentey K-M 5 (2005) Enhanced secretion of tropane alkaloids in Nicotiana tabacum hairy roots expression heterologous hyoscyainine-6p-hydroxylase. JExp Bot 420, 2611-2618 Hamerslap FE (1950) The technology and chemistry of alkaloids. D. van Nostrand Comp. Inc., Toronto, New York, London Hashimoto T, Hayashi A, Amano Y, Kohno J, Iwanari H, Usuda S and Yamada Y (1991) 10 Hyoscyamine 6p-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localised at the pericycle of the root. JBiol Chem 266, 4648-4653 Knapp S, Bohs L, Nee M and Spooner DM (2004) Solanaceae - a model for linking genomics with biodiversity. Comp Funct Genom 5, 285-291 Mortazavi,A., Williams,B.A., McCue,K., Schaeffer,L. and Wold,B. (2008) Mapping and 15 quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods, 5, 621-628 Zhang L, Ding R, Chai Y et al (2004) Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc Nat Acad Sci 101, 6786-6791

Claims (22)

1. An interspecies hybrid of D. myoporoides and D. leichhardtii, the hybrid being capable of producing more than 4% (w/w) (scopolamine/hybrid), the hybrid having 1.5 to 9.0 fold more H6H RNA than a wild type D. myoporoides and D. leichhardtii plant. 5

2. An interspecies hybrid of D. myoporoides and D. leichhardtii, the hybrid being capable of producing more than 4% (w/w) (scopolamine/hybrid), the hybrid having a H6H gene including the nucleotide sequence shown in SEQ ID No:7.

3. The hybrid of claim 1 or 2 wherein the H6H RNA of the hybrid has a sequence that is complementary to the cDNA sequence shown in SEQ ID No: 5. 0

4. The hybrid of claim 3 wherein the root tissue of the hybrid has 9.0 fold more H6H RNA than root tissue of the wild type plant.

5. The hybrid of any one of the preceding claims wherein the hybrid has a genotype that is substantially the same as the hybrid deposited in the deposit having accession number:

6. An interspecies hybrid of D. myoporoides and D. leichhardtii as deposited in the 5 deposit having accession no:

7. An interspecies hybrid of D. myoporoides and D. leichhardtii, wherein, in agricultural conditions, the hybrid produces more than 4% w/w (scopolamine/hybrid).

8. The hybrid of claim 7 wherein, in agricultural conditions, the hybrid produces from 5 to 10 % w/w (scopolamine/hybrid). 20

9. The hybrid of claim 8 wherein, in agricultural conditions, the hybrid produces from I to 3 % w/w (hyoscyamine/hybrid).

10. The hybrid of claim 9 wherein, in agricultural conditions, the hybrid produces scopolamine and hyoseyamine in a ratio of from 10 (scopolamine) to 1 (hyoscyamine).

11. A hybrid having approximately half of the genetic content of a hybrid according 25 to any one of the preceding claims, the hybrid being capable of producing more than 4% (w/w) 45 (scopolamine/hybrid), the hybrid having 1.5 to 6 fold more H6H RNA than a wild type D. myoporoides and D. leichhardtii plant.

12. A tissue of a hybrid according to any one of the preceding claims.

13. The tissue of claim 12 wherein the tissue is a leaf, branch or stem. 5

14. A cutting of a hybrid according to any one of the preceding claims wherein said cutting is capable of propagation.

15. A non viable material harvested from a hybrid of any one of the preceding claims.

16. An extract formed from a hybrid, tissue, cutting or material of any one of the preceding claims wherein the extract includes scopolamine and/or hyoscyamine. t0

17. A peptide having an amino acid sequence as shown in SEQ ID No: 1.

18. A nucleic acid having a nucleotide sequence encoding a peptide of claim 17.

19. The nucleic acid of claim 18 wherein the nucleic acid has a nucleotide sequence as shown in SEQ ID No: 5.

20. A nucleic acid having a nucleotide sequence that is capable of forming Watson 15 Crick base pairing with a nucleic acid of claim 19.

21. A fragment of a nucleic acid of any one of the preceding claims having a length of about 8 to 50 nucleotides.

22. A process for producing scopolamine including the steps of: a) growing a hybrid of any one of the preceding claims in agricultural 20 conditions; b) harvesting plant material from the hybrid; c) forming a scopolamine-containing extract from the harvested material; d) purifying scopolamine from the extract' 46 thereby producing scopolamine.