# Patent

## Title
Papaver somniferum strain with high concentration of thebaine

## Abstract

The present invention is directed to an improved poppy straw, concentrate of poppy straw and opium of Papaver somniferum for the production of thebaine containing little or no oripavine, codeine or morphine. The present invention also provides plants, stands and seeds of Papaver somniferum and methods for the production of thebaine.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application Ser. Nos. 61/034,583 filed Mar. 7, 2008, 61/088,903 filed Aug. 14, 2008 and 61/089,163 filed Aug. 15, 2008, the contents of each of which are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention is directed to the improved production of thebaine. More particularly, the present invention relates to the use of a mutagenized Papaver somniferum poppy plant to produce thebaine in higher yield.
BACKGROUND OF THE INVENTION
The 14-hydroxymorphinans, such as, oxycodone, naloxone, naltrexone, naltrexone methobromide, nalbuphine and nalmefene are important opiate derivatives due to their behavior as potent analgesics and/or narcotic antagonists The most practical synthetic routes to the preparation of these pharmaceuticals have utilized the alkaloid, thebaine, as a starting material. Other important opiate derivatives such as hydrocodone and the ring-C bridged compounds buprenorphine and etorphine are also most practically prepared from thebaine.




In accordance with one conventional process, thebaine is oxidized to 14-hydroxycodeinone by use of m-chloroperbenzoic acid in an acetic acid/trifluoroacetic acid mixture or by a mixture of hydrogen peroxide and formic acid. 14-hydroxycodeinone is catalytically reduced to oxycodone. See Scheme 1 Oxycodone is a product sold for use as an analgesic and its production consumes large amounts of thebaine.




Oxycodone can be, in turn, O-demethylated with boron tribromide to yield oxymorphone. After blocking of the hydroxyl groups with a suitable blocking agent, such as, acetyl groups, the oxymorphone derivative can be reacted with cyanogen bromide in a von Braun demethylation to yield an N-cyanodihydronormorphinone derivative that is thereafter hydrolyzed to 14-hydroxydihydronormorphinone (noroxymorphone). Noroxymorphone can be readily converted to nal-compounds by N-alkylation with appropriate alkyl halide, or acylation with appropriate acyl halide or anhydride, followed by reduction. Another process, converts the oxycodone of the above process to noroxycodone by the von Braun N-demethylation followed by conversion to a 3-O-methyl-nal-compound using N-alkylation with an appropriate alkyl halide, or by alkylation with an appropriate alkyl halide, or acylation with appropriate acyl halide or anhydride, followed by reduction. The 3-O-methyl-nal-compound is converted to a nal-compound by O-demethylation.
A synthesis using thebaine to produce the ring-C bridged opiate, buprenorphine, is shown in Scheme 2.




Another synthesis using thebaine to produce the 14-hydroxymorphinan, naltrexone as representative of the nal-compounds, is shown in Scheme 3.




Another synthesis uses thebaine to produce hydrocodone as set forth in U.S. Pat. No. 3,812,132.
Although these syntheses are effective, the availability of thebaine is limited by its high cost. The high cost of thebaine contributes to the high cost of the 14-hydroxymorphinans derived from it.
One reason for the limited availability of thebaine, and its high cost, is that total synthesis is difficult. U.S. Pat. Nos. 4,613,668 and 4,795,813 discuss the scarcity of thebaine and teach the total synthesis, or alternative synthesis, of the 14-hydroxymorphinans. Yet, the demand for thebaine remains.
A second reason for the limited availability of thebaine, and its high cost, is that the primary source of thebaine is extraction from the poppy plant, Papaver somniferum. Morphine is the major alkaloid that accumulates in capsules of Papaver somniferum. Thus, the supply of thebaine is to a great degree limited to some fraction of the demand for morphine.
Alkaloids are extracted from the poppy capsules of Papaver somniferum by two commercial methods. In one method, the immature capsule is cut and the latex collected from the wound. The air-dried latex is opium which, according to the Merck Index, 11th edition, contains alkaloids in the amounts shown in Table I. In a second method, the mature poppy capsules and the poppy capsule stems are collected, and threshed to remove the seeds and form a straw. When necessary, the straw is dried to a water content below 16%. Solvent or water extraction is employed to remove the alkaloids from the straw. For the varieties of Papaver somniferum normally grown, the straw, on a dry basis, contains alkaloids in the amounts shown in Table 1.










TABLE 1







opium
straw




















morphine, %
10-16
1-3



codeine, %
0.8-2.5
0.05-0.3 



oripavine, %
  0-0.1
  0-0.05



thebaine, %
0.5-2  
0.15-0.65










As can be seen, the yield of thebaine and oripavine is confounded with that of other alkaloids. A poppy producing predominantly thebaine, e.g., as 90% or more of the total alkaloids, would enable a simpler extraction/purification process, resulting in higher yields better quality and throughput and lower costs.
Where solvent or water or super critical fluid, such as CO2, extraction is employed to remove the alkaloids from the straw, such method, as practiced, involves the production of “Concentrate of Poppy Straw”. Concentrate of Poppy Straw (or “CPS”) is described as “The material arising when poppy straw has entered into a process for the concentration of its alkaloids, when such material is made available in trade,” (Multilingual dictionary of narcotic drugs and psychotropic substances under international control, United Nations, New York, 1983). Not inconsistent with the foregoing description, Concentrate of Poppy Straw is described as “the crude extract of poppy straw in either liquid, solid or powder form which contains the phenanthrene alkaloids of the opium poppy,” 45 U.S. Federal Register 77466, Nov. 24, 1980. When in liquid form, the liquid is preferably concentrated before entering into commerce. The generally preferred Concentrate of Poppy Straw is the powder form which results from removing the solvent or water following extraction of the poppy straw. According to the United Nations publication “Narcotic Drugs: Estimated World Requirements for 2007; Statistics for 2005 (E/INCB/2006/2)”, Concentrate of Poppy Straw is the dried residue obtained through the extraction of alkaloids from poppy straw. Until the second half of the 1990s, only concentrate of poppy straw containing morphine as the main alkaloid was manufactured. Since then, concentrate of poppy straw containing mainly thebaine or oripavine has started to be manufactured.
More recently, Fist et al., in U.S. Pat. No. 6,067,749 (the “749 Patent”), U.S. Pat. Nos. 6,376,221 and 6,723,894, disclosed an improved poppy straw of a stably reproducing Papaver somniferum for the extraction of thebaine and/or oripavine (the “Norman” poppy), the threshed straw having thebaine and oripavine constituting about 50% by weight or greater of the alkaloid combination consisting of morphine, codeine, thebaine and oripavine. The Norman poppy straw constituted 1.68% thebaine, 0.74% oripavine, 0.05% codeine and no morphine as a percent by weight of the dry straw. (Gee, column 15, table III of the '749 Patent). While this alleviated the limited availability and high cost of thebaine to some extent, the problem of producing oripavine concurrently with thebaine contributed significantly to the cost of producing thebaine.
For many years, the perennial poppy Papaver bracteatum has been proposed as a source of thebaine. Thebaine is the predominant alkaloid in this species, and in selected strains it can be as high as 98% of the total alkaloids (Palevitch, D and Levy, A 1992 Acta Horticulturae 306, 33-52). Thebaine is present in the roots as well as capsules. Generally two years of growth would be required to obtain a good yield of both roots and capsules. Papaver bracteatum does not alleviate the problem of limited availability and high cost of thebaine because of its slow growth, low capsule yield and the problems with harvesting and processing roots.
SUMMARY OF THE INVENTION
The present invention is directed to a poppy straw comprising a poppy straw of a stably reproducing Papaver somniferum having thebaine constituting about 90% (preferably, about 95%, more preferably, about 97%) by weight or greater of an alkaloid combination, and having oripavine constituting about 10% (preferably, about 5%, more preferably, about 1%, most preferably, about 0.7%) by weight or less of the alkaloid combination, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine; and wherein thebaine constitutes about 3.0% or greater of the poppy straw on a dry weight basis. In a preferred embodiment, thebaine constitutes about 3.5% or greater (preferably, about 4.0% or greater, more preferably, about 4.3% or greater) of the poppy straw on a dry weight basis. In another embodiment, oripavine constitutes about 0.4% or lower (preferably, about 0.2% or lower) of the poppy straw on a dry weight basis. In another embodiment of the invention, oripavine constitutes between 0.01 to 1.0% (preferably, between 0.02% to 0.5%) by weight of the alkaloid combination in the poppy straw.
There is also provided by the present invention a stably reproducing Papaver somniferum having two genetic traits controlling the thebaine-only characteristic of the plant, one trait being that described in U.S. Pat. No. 6,067,749, and the second trait regulating the step between thebaine and oripavine, resulting in substantially no oripavine. In an embodiment of the present invention is a Papaver somniferum plant having a high thebaine content of over 3% (preferably, over 3.5%, more preferably, over 4.0%, most preferably, over 4.3%) in the straw wherein the high thebaine content is provided by two independent traits, one trait controlling the accumulation of thebaine and oripavine compared with morphine and codeine, and the second trait controlling the accumulation of thebaine compared with oripavine. In still another embodiment of the invention is a Papaver somniferum plant having a high thebaine content of over 3% (preferably, over 3.5%, more preferably, over 4.0%, most preferably, over 4.3%) in the straw on a dry weight basis wherein the high thebaine content is provided by two independent genetic changes, one genetic change controlling the accumulation of thebaine and oripavine compared with morphine and codeine, and the second genetic change controlling the accumulation of thebaine compared with oripavine.
There is also provided by the present invention a method to improve the thebaine yield of a stably reproducing Papaver somniferum plant, the method comprising the steps of:
a) exposing at least one poppy seed of Papaver somniferum to a mutagenizing agent,
b) growing at least one poppy seed to produce a plant bearing a leaf or an immature poppy capsule, optionally through multiple self-fertilized generations.
c) sampling the leaf or poppy capsule (or any other latex-containing tissue) for the presence of thebaine, oripavine, morphine and codeine,
d) repeating steps a) to c) until a poppy plant of Papaver somniferum is obtained having thebaine constituting about 90% (preferably, about 95%) by weight or greater, and having oripavine constituting about 10% (preferably, about 5%, more preferably, about 1%) by weight or less, of the alkaloid combination, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine; and
e) collect the seed from the plant obtained in “d” and grow another generation of plants to ensure that the subsequent generation stably re-produces the high thebaine and low oripavine characteristic. In an embodiment of this method, in step d) the poppy plant obtained has thebaine constituting about 3.0% (preferably, about 3.5%, more preferably, about 4.0%, most preferably, about 4.3%) or greater of the poppy straw, on a dry weight basis, and oripavine constituting about 0.4% (preferably, about 0.2%) or lower of the poppy straw, on a dry weight basis.
There is also provided by the present invention a stably reproducing Papaver somniferum plant or a stand of a stably reproducing Papaver somniferum plants, in which the production or activity of the enzyme responsible for the conversion of thebaine to oripavine has been substantially inhibited with the result that upon the harvesting of their poppy capsules, the poppy plants will yield a poppy straw having thebaine constituting about 90% (preferably, about 95%, more preferably, about 97%) by weight or greater of the alkaloid combination, and having oripavine constituting about 10% (preferably, about 5%, more preferably, about 1%) by weight or less of the alkaloid combination, and the poppy plants wilt yield a poppy straw wherein thebaine constitutes about 3.0% or greater (preferably, about 3.5% or greater, more preferably, about 4.0% or greater, most preferably, about 4.3% or greater) of the poppy straw, on a dry weight basis, and oripavine constitutes about 0.4% or lower (preferably, about 0.2% or lower) of the poppy straw, on a dry weight basis.
The present invention also provides a stably reproducing plant of Papaver somniferum, which upon the harvesting of their poppy capsules will yield a poppy straw having thebaine constituting about 90% (preferably, about 95%, more preferably, about 97%) by weight or greater of an alkaloid combination, and having oripavine constituting about 10% (preferably, about 5%, more preferably, about 1%, most preferably, about 0.7%) by weight or less of the alkaloid combination, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine; and wherein thebaine constitutes about 3.0% or greater of the poppy straw on a dry weight basis. The straw of the stably reproducing Papaver somniferum plant of the present invention preferably contains thebaine constituting about 3.5% or greater (preferably, about 4.0% or greater, more preferably, about 4.3% or greater) of the poppy straw, on a dry weight basis, and oripavine constituting about 0.4% or lower (preferably, about 0.2% or lower) of the poppy straw, on a dry weight basis. In another embodiment of the invention, oripavine constitutes between 0.01 to 1.0% (preferably, between 0.02% to 0.5%) by weight of the alkaloid combination in the stably reproducing Papaver somniferum plant.
Also included in the invention is a plant comprising a stably reproducing plant of Papaver somniferum which upon the harvesting of its poppy capsules will yield a poppy straw having thebaine constituting at least 3% (preferably, at least 3.5%) by weight on a dry basis, and oripavine constituting no more than 0.4% by weight on a dry basis of said straw. Preferably, thebaine constitutes at least 4.0% by weight on a dry basis, and oripavine constitutes no more than 0.2% by weight on a dry basis of said straw of the plant.
The present invention also provides a plant comprising a stably reproducing plant of Papaver somniferum which upon the harvesting of its poppy capsules will yield a poppy straw having thebaine constituting at least about 3% (preferably, about 3.5%, more preferably, about 4%, most preferably, about 4.3%) by weight on a dry basis, and oripavine constituting about 0.05 to about 0.5% (preferably, about 0.05 to about 0.2%) by weight on a dry basis of said straw.
Also included in the invention is a seed of any of the stably reproducing poppy plants described above; preferably, the seed is a Papaver somniferum seed which is ATCC PTA-9109.
Also included in the invention is a concentrate of poppy straw for the extraction of thebaine comprising a concentrate of poppy straw of any of the stably reproducing poppy plants described above.
Another aspect of the invention is an opium for the extraction of thebaine comprising an opium of any of the stably reproducing poppy plants described above.
Still another aspect of the invention is a poppy straw comprising a poppy straw of any of the stably reproducing poppy plants described above.
Another embodiment of the invention is a method for the production of thebaine which comprises the steps of:
a) harvesting poppy capsules of any of the stably reproducing poppy plants described above to produce a poppy straw; and
b) chemically extracting the thebaine from the poppy straw.
Still another embodiment of the invention is a method for the production of thebaine which comprises the steps of:
a) collecting and drying the latex of the immature poppy capsules of any of the stably reproducing poppy plants described above to produce opium; and
b) chemically extracting the thebaine from the opium.
Also included in the invention is a stand of any of the stably reproducing Papaver somniferum plants described above.
The present invention also provides a poppy straw comprising a poppy straw of a stably reproducing Papaver somniferum having thebaine constituting at least about 3% (preferably, about 3.5%, more preferably, about 4%, most preferably, about 4.3%) by weight on a dry basis, and oripavine constituting about 0.05 to about 0.5% preferably, about 0.05 to about 0.2%) by weight on a dry basis of said straw.
In the poppy straw, opium, and concentrate of poppy straw of the present invention, thebaine preferably constitutes about 96% by weight or greater of the alkaloid combination and oripavine constitutes about 0.8% by weight or less of the alkaloid combination; more preferably, thebaine constitutes about 97% by weight or greater of the alkaloid combination and oripavine constitutes about 0.7% by weight or less of the alkaloid combination. In the most preferred embodiments of the present invention, there is substantially no oripavine, morphine or codeine in the alkaloid combination. In another embodiment of the instant invention, the alkaloid combination further comprises salutaridine, reticuline, laudanine, papaverine and noscapine.
The present invention also provides Papaver somniferum plants, and methods for producing such plants, having poppy straw with substantially higher thebaine content, and substantially lower oripavine content, such that thebaine contents in commercially grown and harvested crops are about 3.0% or greater, preferably, about 3.5% or greater, more preferably, 4.0% or greater, most preferably, about 4.3% or greater, and oripavine contents are in the order of about 0.4%, preferably, 0.2%, or lower.
Additional embodiments of the present invention provide poppy straw, concentrate of poppy straw and opium, wherein thebaine constitutes about 95% by weight or greater of the alkaloid combination and oripavine constitutes between about 0.01 to about 1.0% by weight (preferably, between about 0.02% to 0.6% by weight, more preferably, between about 0.02% to 0.5% by weight) of the alkaloid combination.
Exemplifying the invention is a method for producing a poppy plant of Papaver somniferum having a stably heritable high thebaine content (that is, the high thebaine content is stably reproducing) and low oripavine content versus morphine and codeine content, the method comprising the steps of:

    
    
        a) exposing at least one poppy seed of Papaver somniferum to a mutagenizing agent,
        b) growing the at least one poppy seed to produce a plant bearing a leaf or an immature poppy capsule, optionally through multiple self-fertilized generations,
        c) sampling the leaf or poppy capsule for the presence of thebaine, oripavine, morphine and codeine, and
        d) repeating steps b) and a) and optionally step (a) until a poppy plant of Papaver somniferum is obtained which yields a poppy straw having thebaine constituting about 90% (preferably, about 95%) by weight or greater of the alkaloid combination consisting of morphine, codeine, thebaine and oripavine, and having oripavine constituting about 10% (preferably, about 5%) by weight or less of the alkaloid combination, and wherein thebaine constitutes about 3.0% (preferably, about 3.5%, more preferably, about 4.0%, most preferably, about 4.3%) or greater of the poppy straw on a dry weight basis.
    
    


Preferably, the Papaver somniferum seed exposed to the mutagenizing agent in step (a) of this method is a Papaver somniferum yielding, upon the harvesting of its poppy capsules, a poppy straw having a thebaine and oripavine content constituting 50% by weight or greater of the alkaloid combination consisting of morphine, codeine, thebaine and oripavine.
In another embodiment of this method, step (b) comprises growing the at least one poppy seed to produce a plant bearing a leaf or an immature poppy capsule, and self-pollinating to produce seed, and taking the seed thereby produced and producing an M2 generation of plants, and step (c) comprises screening the M2 plants and selecting plants which yield a poppy straw having thebaine constituting about 95% by weight or greater of the alkaloid combination consisting of morphine, codeine, thebaine and oripavine, and having oripavine constituting about 5% by weight or less of the alkaloid combination, each on a dry weight basis, and wherein thebaine constitutes about 3.5% (preferably, about 4.0%, more preferably, about 4.3%) or greater of the poppy straw on a dry weight basis.
In another aspect of this method, the Papaver somniferum seed exposed to the mutagenizing agent in step (a) of this method is seed selected from ATCC PTA-9110 or ATCC PTA-9109.
The present invention is also directed to progeny of Papaver somniferum ATCC-9109, said progeny yielding a poppy straw having thebaine constituting about 90% (preferably, about 95%) by weight or greater of an alkaloid combination, and having oripavine constituting about 10% (preferably, about 5%) by weight or less of the alkaloid combination, and wherein thebaine constitutes about 3.0% (preferably, about 3.5%, more preferably, about 4.0%, most preferably, about 4.3%) or greater of the poppy straw on a dry weight basis, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine.
Another example of the present invention is a mutant or variant of Papaver somniferum ATCC PTA-9109 or ATCC PTA-9110, said mutant or variant yielding a poppy straw having thebaine constituting about 90% (preferably, about 95%) by weight or greater of an alkaloid combination, and having oripavine constituting about 10% (preferably, about 5%) by weight or less of the alkaloid combination, each on a dry weight basis, and wherein thebaine constitutes about 3.0% (preferably, about 3.5%, more preferably, about 4.0%, most preferably, about 4.3%) or greater of the poppy straw on a dry weight basis, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 provides a chromatogram showing separation of the alkaloids using the isocratic UPLC method.
FIG. 2 provides a chromatogram of the poppy straw of the M3 generation of FN1-1242-3 ATA indicates thebaine, ori indicates oripavine peak, AMA represents morphine, and ACA represents codeine.



DETAILED DESCRIPTION OF THE INVENTION
Utilizing the mutagenized plants of Papaver somniferum as described herein, persons skilled in the art easily know how to grow them, reproduce them, collect the latex or the dried straw and purify the thebaine. As one enablement of the present invention, seeds to the mutagenized plants of Papaver somniferum (FN1-1242-3), as described herein, have been deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, on Mar. 20, 2008, under ATCC® Patent Deposit Designation PTA-9109, and will be made available upon the maturation of this application into a patent. The availability of these seeds is not to be construed as a license to practice this invention in contravention of rights granted under the authority of any government in accordance with its patent or breeder's rights laws. Regardless of the enablement provided by this deposit, persons skilled in the art of mutagenizing seed, can obtain the seed herein by employing the mutagenesis process as described below.
The production of mutagenized seed is well known in the art. Methods of seed mutagenesis as well as mutagens suitable for use in these methods, such as ethyl methanesulfonate (EMS), are described in the Manual on Mutation Breeding, 2nd ed., I.A.E.A., Vienna 1977 or in Plant Breeding, Principles and Prospects, Chapman and Hall, London 1993. For X-ray mutagenized seeds, hydrated seeds might be treated with 20,000 rads, (30 cm from the source for 45 minutes using a filter). X-ray mutagenesis is described and compared to EMS mutagenesis by Filippetti, A. et al., “Improvement of Seed Yield in Vicia Faba L. By Using Experimental Mutagenesis II Comparison of Gamma-Radiation and Ethyl-Methane-Sulphonate (EMS) in Production of Morphological Mutants”, Euphytica 35 (1986) 49-59. DEB, diepoxybutane, mutagenized seeds might be obtained by soaking the seeds in water overnight, then soaking in 22 mM DEB for 4 hours, followed by extensive washing. Further mutagens include ethyl-2-chloroethyl sulphide, 2-chloroethyl-dimethylamine, ethylene oxide, ethyleneimine, dimethyl sulphonate, diethyl sulphonate, propane sulphone, beta-propiolactone, diazomethane, N-methyl-N-nitrosourethane, acridine orange and sodium azide.
Mutagenesis utilizing EMS is well described in the literature. The Manual on Mutation Breeding, supra, reports a preferred EMS mutagenesis process for barley seeds as practiced by K. Mikaelson. In this preferred process, the seeds are prepared, pre-soaked, treated with the mutagen and post-washed.
U.S. Pat. No. 6,067,749, incorporated by reference herein n its entirety, describes the use of EMS for the preparation of a Papaver somniferum strain with a high concentration of thebaine and oripavine.
Irradiation methods such as fast neutron mutagenesis may also be used to produce mutagenized seed. (See, Li, X. et al., A fast neutron deletion mutagenesis-based reverse genetics system for plants. The Plant Journal 27(3), 235-242 (2001)). Applicants employed and prefer fast neutron mutagenesis (“FNM”) as the mutagen herein.
Fast neutron mutagenesis is described by Kodym and Afza (2003), Physical and Chemical Mutagenesis, pp 189-203, in Methods in Molecular Biology, vol 236: Plant Functional Genomics: Methods and Protocols (Ed. E. Grotewold), Humana Press inc, Totowa, N.J.
Gamma (γ) Rays are electromagnetic waves of very short wavelengths and are obtained by disintegration of radioisotopes Co or Cs. γ sources can be installed in a γ cell, a γ room or γ field. These are shielded by lead or concrete. Most γ sources as suitable for seed irradiation, as long as the size of irradiation space is sufficient and the dose rate allows practical irradiation times.
Fast neutrons are uncharged particles of high kinetic energy and are generated in nuclear reactors or in accelerators. The scientist should assess the feasibility for seed irradiation with the operators, since not all facilities are suitably equipped and can produce fast neutrons at a low degree of contamination with other radiation.
The two radiation types differ in their physical properties and, hence in their mutagenic activity. γ Rays have a lower relative biological effectiveness (RBE) than fast neutrons, which implies that in order to obtain the same biological effect, a higher dose of γ radiation must be given. RBE is mainly a function of the linear energy transfer (LET), which is the transfer of energy along the ionizing track. γ Rays produce a few ionizations per micron of path (low LET) and belong to the category of sparsely ionizing radiation. Fast neutrons (high LET, densely ionizing radiation) impart some of their high kinetic energy via collisions, largely with protons within the material.
When radiation passes through tissue, physical events such as ionizations (ejection of electrons from molecules) and excitations (process of raising electrons to a higher energy state) occur and lead to effects in the DNA, membranes, lipids, enzymes, etc. Secondly, chemical events are induced that start with the formation of activated molecules, so-called free radicals (OH. and H.) that arise from OH− and H+. If oxygen is present, it reacts readily with radiation-induced free radicals to form peroxyradicals. In the case of low LET radiation, the formation of peroxyradicals is favoured. In high LET radiation, the formation of hydrogen peroxide (H2O2) by recombination of free radicals is favoured. All radicals and hydrogen peroxide can react with biological molecules. Primary damage caused by radiation occurs randomly and is both physiological and genetic. Physiological recovery and repair of DNA are possible to some extent, as non-damaged molecules may take over metabolic processes and DNA repair mechanisms are activated.
Before starting any mutation induction studies, it is most crucial to select suitable doses. For mutation induction, it is advisable to use two to three doses along with a control. The applicable doses will depend on the breeding or research objective, the radiation type and the particular plant material. It is known that plant genera and species and, to a lesser extent, cultivars differ in their radiosensitivity. Radiosensitivity (radiation sensitivity) is a relative measure that gives an indication of the quantity of recognizable effects of the radiation exposure on the irradiated object. The radiosensitivity is influenced by biological factors (such as genetic differences, nuclear and interphase chromosome vol) and by environmental modifying factors (oxygen, water content, post-irradiation storage, and temperature).
Modifying factors greatly affect mutagenic efficiency and reproducibility of results. Oxygen is the major modifying factor, while moisture content, temperature and storage appear to be secondary, interacting with the oxygen effect. Oxygen shows synergistic action with sparsely ionizing radiation, but oxygen effects during irradiation and post-irradiation storage can easily be prevented by adjustment of seed water content to 12-14% in cereals and most other seeds. In oilseeds such as poppies, the seed water content should be lower, around 7-8%. The critical region is the embryo, but it can be assumed that the water content of the seed and the embryo of most species will be similar. Environmental factors are less important with densely ionizing radiation thus, for fast neutron radiation, no seed moisture adjustment is necessary.
Unless data on the radiosensitivity of a given plant are already published or known from experience, the mutation induction program should be preceded by a radiosensitivity test. This is done by irradiating the seeds with a range of doses and by growing out the plants under greenhouse conditions. Radiosensitivity is assessed based on criteria such as reduced seedling height, fertility, and survival in the M1 generation. A seedling height reduction of 30-40% is generally assumed to give a high mutation yield. The usefulness of radiation can be judged by mutagenic efficiency, which is the production of desirable changes free from association with undesirable changes A high dose will increase mutation frequency (the frequency at which a specific kind of mutation or mutant is found in a population of cells or individuals), but will be accompanied by negative features, such as sterility. When selecting the doses, it will be necessary to find a treatment regime providing high mutagenic efficiency.
For fast neutron radiation, dosimetric measurements have to be done during each radiation treatment, e.g., by performing the sulphur threshold detector method, since the neutron flux in the seed irradiation unit is not constant.
The Gray (symbol Gy), the SI (Systéme Internationale) unit used to quantify the absorbed dose of radiation (1 Gy=1 J/kg) replaced the old unit rad; 1 Gy 100 rads or 1 krad=10 Gy. The absorbed dose rate (Gy/s or Gy/min) indicates how much energy the irradiated material absorbs during a given unit of time. The length of exposure and the dose rate determines the radiation dose. Exposure during short times (s to a few h) at a high dose rate is referred to as acute and is most applied in irradiation programs.
We used the Atomic Energy Research Institute, Konkoly Thebe ut 29/33, X.epulet, H-1121 Budapest, Hungary to irradiate our seeds.
Fast neutrons have been shown to be a very effective mutagen. Kornneef et al. (1982) found that about 2500 lines treated with fast neutron at a does of 60 Gy are required to inactivate a gene once on average (Koornneef, M., Dellaert, L. W. M. and van der Veen, J. H. (1982) EMS— and radiation-induced mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh. Mulat. Res. 93, 109-123). If the plant genome contains about 25000 genes, it is estimated that about 10 genes are randomly deleted in each line.
FNM offers a number of advantages over using chemical treatment such as EMS. Notably, the treatment is applied to the dried seed, which can be sown at a later date, while with EMS the seed needs either to be sown immediately after treatment, or carefully re-dried for sowing later.
After the seeds have been exposed to the mutagen, the seeds are grown to maturity in controlled conditions and self-pollinated. The seeds from the mature plant are taken and at least one seed is planted to grow an M2 generation. The M2 generation is screened for alkaloid production. Of course, it is possible to screen the M1 generation, but there are several advantages to screening the M2 generation. Firstly, screening the M2 generation insures that the trait resulting from mutagenesis can be inherited. Secondly, by growing the M2 generation, the basic hardiness of the plant is proven before screening. Thirdly, traits resulting from mutagenesis are generally inherited as recessive genes. Typically the mutated gene will be in the heterozygous state in the M1 generation, and thus the mutation will be masked by the dominant (non-mutated) form of the gene. In the M2 generation, however, in a proportion of the plants the gene will be in the homozygous state, and the affect of the mutation apparent. The M2 plants can be grown to produce an immature capsule, but it is possible to save time and labor it the plants are screened at an earlier stage of growth. It is recommended that the plants be screened at a point beginning at the 6 leaf stage, up to the 10 leaf stage. Screening at this early stage allows many plants to be managed in a small space. The screening process itself is the most labor intensive. Thus, to improve return on labor, only plants that appear healthy should be screened.
In the screening process, the objective is to measure each plant for morphine, codeine, thebaine and oripavine content. Additional alkaloids which can also be measured during the screening process include salutaridine, reticuline, laudanine, papaverine and noscapine. This can be accomplished by extracting, for example, a dry leaf into a liquid buffer or by dissolving a latex sample into a buffer. The buffer solutions are placed onto 96 well trays and fed mechanically through any of the high-throughput UPLCs available on the market. In a preferred embodiment, an isocratic Ultra high performance Liquid Chromatography (UPLC) method, as described herein, is utilized which provides very rapid latex screening.
Plants with interesting alkaloid contents are grown further and examined in more detail. According to the procedure herein, a second sample is taken from about 3% of plants to clarify or confirm the results of the initial screen. A more precise gradient UPLC method as described herein is used to obtain more accurate peak identification and quantification. Plants confirmed to have an unusual alkaloid profile are transplanted to 200 mm (approx 8 inch) pots for growing to maturity. Twenty one plants having high thebaine and substantially no oripavine, morphine or codeine were found after screening approximately 34,358 plants.
As used herein, the term “poppy straw” or “straw” shall mean the straw material which results when the mature poppy capsules and the poppy capsule stems of a Papaver somniferum plant are collected, and threshed to remove the seeds to form a straw.
The term “opium”, as used herein, shall refer to the air-dried, milky exudation (i.e., the latex) from incised, unripe poppy capsules of a Papaver somniferum plant.
As used herein, the term “concentrate of poppy straw” or “CPS” shall mean the material arising when poppy straw has entered into a process for the concentration of its alkaloids in either liquid, solid or powder form which contains the phenanthrene alkaloids of the opium poppy.
The phrase “stand of Papaver somniferum” or “stand of stably reproducing Papaver somniferum”, as used herein, refers to a group of two or more Papaver somniferum plants or stably reproducing Papaver somniferum p ants located together.
As used herein, the term “alkaloid combination” shall refer to a combination of alkaloids wherein the alkaloid comprises morphine, codeine, thebaine and oripavine. In another embodiment of the present invention, the alkaloid combination further comprises salutaridine, reticuline, laudanine, papaverine and noscapine in addition to morphine, codeine, thebaine and oripavine.
A “stably reproducing” Papaver somniferum poppy plant as described herein refers to a poppy plant that is stably reproducing as required to plant and harvest seed poppy crop over multiple generations where each generation would be suitable, without seed selection, for commercial planting of a field crop or stand of plants exhibiting the desired alkaloid characteristics. A stably reproducing poppy plant contains the desired alkaloid characteristics as described herein, and when self pollinated, or cross pollinated by a plant with the same genes controlling alkaloid content, produces a subsequent generation of plants which substantially all have seed that when grown produces plants with the same desired alkaloid characteristics as the parent plant. Moreover, in the absence of pollination with pollen from other chemotypes (e.g. conventional morphine accumulating plants), the line will continue to produce similar plants over multiple generations, without the need for selection to maintain the desired alkaloid characteristic. An example of a desired alkaloid characteristic which can be passed on to future generations by a stably reproducing Papaver somniferum poppy plant includes the improved thebaine characteristics (e.g., wherein thebaine constitutes about 90% (preferably, 95%, more preferably, 96% and most preferably, 97%) by weight or greater, and oripavine constitutes about 10% (preferably, 1%, more preferably, 0.8% and most preferably, 0.7%) by weight or less of the alkaloid combination).
As used herein, the “M1 population” is the seeds and resulting plants exposed to a mutagenic agent, while “M2 population” is the progeny of self-pollinated M1 plants, “M3 population” is the progeny of self-pollinated M2 plants, “M4 population” is the progeny of self-pollinated M3 plants, and generally “Mn population” is the progeny of self-pollinated Mn−1 plants.
As stated above, there is obtained by the present invention, a poppy straw, concentrate of poppy straw or opium having thebaine constituting about 90% by weight or greater and oripavine constituting about 10% by weight or less of an alkaloid combination comprising morphine, codeine, thebaine and oripavine. Preferably, thebaine constitutes about 95% (more preferably, about 96% and most preferably, about 97%) by weight or greater, and oripavine constitutes about 1% (more preferably, about 0.8% and most preferably, about 0.7%) by weight or less of the alkaloid combination comprising morphine, codeine, thebaine and oripavine. In a preferred embodiment, the alkaloid combination further comprises salutaridine, reticuline, laudanine, papaverine and noscapine. More preferably, there is substantially no oripavine, morphine or codeine in the alkaloid combination, and most preferably, there is substantially no oripavine, morphine, codeine, salutaridine, reticuline, laudanine, papaverine or noscapine in the alkaloid combination.
As used herein, the term “substantially no” when referring to oripavine content means that oripavine constitutes less than 0.6% by weight, preferably, less than 0.5% by weight, more preferably, less than 0.4% by weiglt, and most preferably, between 0% and 0.2% by weight of the alkaloid combination of the poppy straw, concentrate of poppy straw or opium.
The term “substantially no”, when referring to morphine, codeine, salutaridine, reticuline, laudanine, papaverine or noscapine, as used herein, means that each of the specified alkaloids constitutes less than 1% by weight, preferably less than 0.5% by weight, more preferably, less than 0.3% by weight, and most preferably, between 0% and 0.2% by weight of the alkaloid combination of the poppy straw, concentrate of poppy straw or opium.
The term “trait”, as used herein, mean a distinct heritable phenotypic characteristic. The desired traits, i.e., high thebaine content versus oripavine, morphine or codeine content, once established are highly heritable. To maintain the desired traits, care should be taken to prevent cross-pollination with normal plants unless such cross-pollination is part of a controlled breeding program.
The desired traits can be transferred into poppy lines having other characteristics (e.g. different height, early or late maturity or having disease resistance) by cross pollinating the high thebaine plant with the second parent plant, collecting F1 seed, growing a F1 plant which is allowed to self-pollinate and collect the F2 seed. The F2 seed would then be grown, and individual plants that have the high thebaine characteristic could be selected according to the methods herein, along with the other desired characteristics such as disease resistance. A skilled operator will be able to apply variations to this method as known in plant breeding.
Conducting test crosses with plants of known genotype can provide information regarding the genetic changes introduced through mutation. The characteristics of the F1 generation produced by crossing to a normal (wild type) parent will indicate whether a trait inherits as a recessive or dominant gene. Self pollinating the F1 plants and determining the phenotypes of the subsequent F2 population of plants will provide information regarding the numbers of genes responsible for particular characteristics.
The theory whereby mutagenesis has been found to be capable of raising the thebaine content of Papaver somniferum relative to the oripavine, morphine and codeine content is not capable of a certain or definite explanation at this time. The mutagenesis might have modified the biosynthesis pathway in any number of ways to minimize the production of oripavine. Despite the fact that definite answers are not now available, there are good reasons to believe that the correct answer is known.
Papaver somniferum is postulated to have two biosynthetic pathways from thebaine to morphine as shown in Scheme 4. Pathway A via neopinone, codeinone and codeine was proposed by Parker, H. I., J. Am. Chem. Soc., 94, 1276-1282 (1972). Pathway B via oripavine and morphinone was proposed by Brochmann-Hanssen, E., Planta Med., 50, 343-345 (1984). The enzyme codeinone reductase (NADPH) is believed to be active in both pathways, reducing codeinone to codeine and morphinone to morphine. Further, the TOP1 mutation (Miligate et al., Nature, Vol. 431, 413-414, 2004) affects both pathways, preventing thebaine being converted to neopinone in Pathway A, and preventing oripavine being converted to morphinone in Pathway B. The TOP1 mutation appears to block demethylation of the enol ether which converts thebaine to neopinone, as well as the demethylation of the same enol ether in oripavine.
By the methods herein, plants of Papaver somniferum were obtained having substantially no oripavine, morphine or codeine. Both Pathway A and Pathway B were inoperative to produce morphine in the parent line using the TOP1 mutation. The most probable step that has been affected by mutation is the phenolic O-demethylation step between thebaine and oripavine. Thus, it is believed, for the Papaver somniferum plants described herein, that the production or activity of the phenolic O-demethylase enzyme that converts thebaine to oripavine has been substantially inhibited. Stably reproducing Papaver somniferum in accordance with the present invention may also be obtained by recombinant DNA techniques. In particular, after isolation and sequencing of the gene coding for thebaine demethylase, the gene may be modified or deleted to inhibit or prevent the production of thebaine demethylase. Techniques for modifying gene activity such as RNAi, antisense and other techniques are well known to those skilled in the art. Once the gene coding is established, a TILLING technique may be used to more efficiently recover mutants from populations (Henikoff, S., Till, B. J. and Comai, L. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiology 135, 630-636).
Knowing that there are genetic means of reducing the conversion of thebaine to oripavine in poppies, and that now that we have shown that these poppies are achievable and viable, even conventional breeding approaches may ultimately be used to develop such plants




Recovering thebaine from either the dried straw or from the opium of Papaver somniferum is a process well established in the art. Until now, thebaine has been extracted from this plant species either as a part of the process of extracting morphine and codeine, or more recently as part of the process of extracting thebaine and oripavine.
In one process, the straw is treated with a small amount of lime and water to soften the capsules and to form a free base of the alkaloids. Countercurrent extraction of the softened straw with methanol, ethanol or other suitable solvent forms a solvent/water extract or “miscella” containing the alkaloids, with morphine at a concentration of about 1 g/L where the straw is from standard Papaver somniferum. The volume of the miscella is reduced about 30× under vacuum to produce an aqueous concentrate. Thebaine is extracted from aqueous concentrate using a liquid/liquid extraction with toluene, adjusting pH for the best separation of thebaine. The thebaine is recovered from the toluene. Of course, recovering thebaine from the improved Papaver somniferum provided herein will be facilitated by the fact that the concentration of the thebaine in the miscella will be much higher than that of other alkaloids and thus can be more easily collected by precipitation. Also, in the substantial absence of oripavine, morphine and codeine, the thebaine might be directly extracted from the straw using toluene, xylene or other organic solvent in which thebaine has solubility.
The following Examples are set forth to aid in the understanding of the invention, and are not intended and should not be construed to limit in any way the invention set forth in the claims which follow thereafter.
EXAMPLE 1
A selection of Papaver somniferum poppy, WF03-0802 is used as the starting material. This line contains the TOP1 mutation and therefore has the characteristics of containing thebaine and oripavine in its poppy straw and opium, and is substantially free of morphine and codeine. Seeds of WF03-0802 have been deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 2010-2209, on Mar. 20, 2008, under ATCC Patent Deposit Designation PTA-9110, and will be made available upon the maturation of this application into a patent. The availability of these seeds is not to be construed as a license to practice this invention in contravention of rights granted under the authority of any government in accordance with its patent or breeder's rights laws.
Six seed samples each of 10 g were prepared. One sample was retained as a control. After obtaining the necessary inspections and permits, the 5 samples were sent to the Atomic Energy Research Institute, Budapest, Hungary for irradiation. At the Institute, the samples were removed from their vials, packed into plastic bags and cadmium holders and irradiated with fast neutrons. The dose rates and exposure times were as follows:

    Treatment 1 10 Gy 13 minutes 17 seconds
    Treatment 2 20 Gy 26 minutes 30 seconds
    Treatment 3 25 Gy 33 minutes 16 seconds
    Treatment 4 35 Gy 46 minutes 27 seconds
    Treatment 5 50 Gy 66 minutes 13 seconds


The reported parameters of the irradiation were as follows:

    
    
        Irradiation geometry at BIF of BRR at AERI: 2Y/Cd, rotated
        Monitored by U-235, Th-232 fission chambers and GM counter.
        The dose homogeneity within one package is better than 1%
        The overall dose uncertainty is less than 5%.
        Total surface gamma activity of samples after irradiation:  ̃695 BGND
        Total surface gamma activity of samples on 20 Oct. 2005; <2 BGND
        (1 BGND (background) is  ̃90nGy/h)
    
    


The seeds were returned to applicants by the institute, reweighed, and found to have lost an average mass of 1.1%. This is compatible with being transferred into and out of plastic bags, as well as losing some moisture during irradiation.
Samples of the seeds were placed on damp filter paper in a Petri dish. After 3 and 7 days, the seeds were examined. Treatments 1 to 4 germinated well, while treatment 5 had very short radicals and only a small percentage of plants with shoots.
A pot trial was grown to evaluate the effect of the FNM treatment on plant growth. Two pots were sown with 10 seeds from each of treatments 1, 2, 3, 5 and control. These pots were observed for emergence and plant growth. Irradiation reduced plant emergence and survival from a mean of 9 plants per pot in the control treatment to 8.5 plants in Treatment 1, 7.5 plants in treatment 2, 7 plants in treatment 3, and 4 plants in treatment 5. Development was also delayed with increasing dose, and plant height was reduced by about 10% in treatment 1, 20% in treatment 2, and 40% in treatment 3.
The seeds from the treatments 2 and 3 were sown into 200 mm pots filled with potting mix from Forestry Commission Nursery at Perth, Tasmania. 10 seeds were sown per pot, and were covered with vermiculite. The plants were grown through to maturity in a greenhouse. All flowers were self pollinated by transferring pollen from the anthers onto the stigmatic disc. The mature capsules were harvested into large paper bags, labeled with the treatment number, keeping the different treatments separate. Where there were 2 or more capsules on one plant, these were picked into a paper bag so they stayed together. Distinctive plants were harvested into separate bags and notes made on their appearance. The harvested capsules were stored for a week or so to ensure that the seed was air dry.
The seed was separated from the capsules in the laboratory, and weighed into paper envelopes labeled with FN1-X where FN1 refers to Fast Neutron experiment 1, and X is the sequence number of the seed sample. The seed from multiple capsules from the same plant was combined into the one sample.
The seed from a total of 8,495 plants was harvested. 7,280 of these were from radiation treatment 2. The median weight of seeds harvested per M1 plant was 0.41 g.
Growth and Screening of M2 Generation
Plant Growth
M2 plants were grown in a greenhouse in trays each with 288 cells. 12 cells were sown with seed from each M1 plant. Two seeds were sown in each cell, and thinned to one plant per cell after 1-2 weeks. The plants were sown in batches of 4-17 trays each week to spread the workload over 17 weeks.
Leaf Sampling
When the plants were approximately 6 weeks old, they were analysed for alkaloid profile using the latex from the youngest fully expanded leaf (YFEL). 240 μL of latex extraction solution (23 g NH4H2PO4 dissolved in 800 mL deionized (Dl) water, made up to 1 L with ethanol) was added to the wells of 96 well filter plates (Pall AcroPrepTM 96 Filter Plate 0.2 μm GHP membrane, natural housing, 350 μL PN S5045, (Pall Corporation, East Hills, N.Y.)). The tip of the YFEL was removed from each plant and placed in a well of the filter plate using fine forceps. Three filter plates were required to sample the plants in one tray. The plates were allowed to stand for about 30 minutes after sampling to allow the latex to bleed out of the leaves into the extraction solution. The solution was then filtered into a 96 well collection plate, which was sealed with an ABgene® Adhesive PCR foil seals (Abgene, part of ThermoFisher Scientific, Rockford, Ill.) to eliminate evaporation.
UPLC Method
The UPLC method used for the first screening stage is described in Example 2. Peak areas were exported to a Excel file (Microsoft Corporation, Seattle, Wash.) for data analysis. No correction was applied for differing UV absorption between the alkaloid peaks. The relative absorption of oripavine and thebaine, the main peaks of interest, were in any case very similar at the wavelength used.
Data Analysis
Relative peak areas were calculated for all identified alkaloids. The Excel data files were then sorted to identify plants having high thebaine content and low oripavine content relative to all identified alkaloids extracted. The chromatograms of plants identified as being of interest were reviewed to ensure that the peaks of interest were correctly integrated.
Confirmation
The plants identified in the first screening were then resampled to provide confirmation of the alkaloid profile, and to ensure that the correct plant was located prior to transplanting. The selected plants were marked with a plastic coated wire when retested so that they could be identified reliably for transplanting. A gradient UPLC system with a 2.5 minute run time (described in Example 2) was used in the confirmation testing in order to obtain more accurate peak identification and integration.
Transplanting
Plants confirmed as being of interest were transplanted into 200 mm pots, and labelled with a code, indicating the M1 seed line from which they were derived. For instance, if two selections were made from the M1 seed sample labeled FN1-1234, these selections were labelled FN1-1234-1, and FN1-1234-2. Up to 5 plants were transplanted into each pot.
Table 2, below, shows the number of plants analysed, the number of selections made, and the number of selections confirmed. Over the project, 34,358 M2 plants (from 4,176 M1 lines) were tested, and 1,049 were selected for further testing. 549 of these were confirmed and transplanted into pots. Of the 549 transplanted, 366 were selected on the basis of high thebaine and low oripavine content.












TABLE 2








Irradiation

Number
Selections
Confirmations
















Treatment
Tray
plants

%

% of
% of


Batch
No.
numbers
analysed
No.
selns
No.
plants
selections


















1
2
1-4
927
51
5.5
23
2.5
45


2
2
5-8
856
15
1.8
12
1.4
80


3
2
 9-12
914
29
3.2
23
2.5
79


4
2
13-16
976
37
3.8
27
2.8
73


5
2
17-20
924
40
4.3
14
1.5
35


6
2
21-28
1900
47
2.5
21
1.1
45


7
2
29-35
1670
27
1.6
16
1.0
59


8
2
36-43
1746
85
4.9
30
1.7
35


9
2
44-52
2134
39
1.8
39
1.8
100


10
2
53-60
1890
31
1.6
31
1.6
100


11
2
61-77
3524
104
3.0
51
1.4
49


12
2
78-91
2737
86
3.1
29
1.1
34


13
2
 92-111
4591
119
2.6
58
1.3
49


14
3
297-313
2808
109
3.9
74
2.6
68


15
2
129-145
2108
117
5.6
44
2.1
38


16
2
146-162
2467
56
2.3
34
1.4
61


17
2
163-174
2186
57
2.6
23
1.1
40


Totals:


34358
1049
3.1
549
1.60
52.3









Table 3, below, lists the 366 selections made on the basis of high thebaine and low oripavine content in latex from leaf samples. The alkaloid profile is based on peak area, not alkaloid concentration.












TABLE 3








M1


Alkaloid profile (percentage of area under peaks)





















Seln
Seed

Plate/
Mor-
Ori-
Saluta-






Selection


No.
Line
Tray
Position
phine
pavine
ridine
Codeine
Reticuline
Laudanine
Papaverine
Thebaine
Noscapine
name























29
105
7
P20 B10
2
1.7
1
0
0
1
0
94
0
FN1-105-2


41
165
9
P27 B8
0
4.9
1
0
1
2
0
90
1
FN1-165-1


42
165
9
P27 B9
0
4.4
2
0
2
2
0
90
0
FN1-165-2


46
183
10
P29 C6
0
6.5
1
0
1
1
0
91
0
FN1-183-1


54
223
12
P34 B1
0
5.7
2
0
0
2
0
91
0
FN1-223-1


64
269
13
P39 D9
0
3.7
1
0
1
1
0
92
0
FN1-269-1


65
270
13
P39 E3
0
4.5
2
0
1
1
0
90
0
FN1-270-1


66
272
13
P39 G8
0
2.3
1
0
2
1
0
92
0
FN1-272-1


68
291
14
P42 B12
0
2.2
2
0
1
1
0
93
0
FN1-291-1


72
300
15
P43 C10
0
6.3
1
0
1
1
0
90
1
FN1-300-2


74
317
15
P45 D2
0
2.4
1
0
0
0
0
96
0
FN1-317-1


77
329
16
P46 H2
0
4.9
1
0
1
1
0
92
0
FN1-329-1


78
333
16
P47 D10
0
5.4
1
0
1
0
0
93
0
FN1-333-1


79
340
16
P48 C1
0
5.8
0
0
1
1
0
91
1
FN1-340-1


80
340
16
P48 C2
0
6.1
0
0
1
1
0
91
1
FN1-340-2


83
340
16
P48 C10
1
6.4
1
0
1
1
0
90
0
FN1-340-5


84
345
16
P48 H1
0
2.6
1
0
2
1
0
92
1
FN1-345-1


85
345
16
P48 H5
0
4.9
1
0
2
1
0
91
1
FN1-345-2


94
399
19
P55 F9
0
1.5
0
0
1
2
0
92
1
FN1-399-3


95
399
19
P55 F11
0
2.1
0
0
1
0
3
90
0
FN1-399-4


96
404
19
P56 C5
0
1.5
0
0
1
1
0
93
0
FN1-404-1


100
461
21
P63 D6
0
3.6
1
0
1
1
1
92
0
FN1-461-1


103
486
22
P66 D1
0
3.5
1
0
2
2
0
91
1
FN1-486-1


106
557
25
P75 C1
0
1.1
0
0
0
0
0
98
0
FN1-557-1


107
593
27
P79 G5
0
3.9
0
0
1
1
0
94
1
FN1-593-1


110
601
27
P80 G6
0
0.4
0
0
1
0
0
97
1
FN1-601-1


111
607
27
P81 E12
0
3.5
0
0
1
0
0
95
0
FN1-607-1


114
622
28
P83 D9
0
4.3
0
0
1
1
0
93
1
FN1-622-1


115
625
28
P83 G12
0
3.6
0
0
1
1
0
94
0
FN1-625-1


117
630
28
P84 D11
0
4.4
0
0
0
0
0
96
0
FN1-630-1


119
633
28
P84 G9
0
4.3
0
0
2
1
0
92
1
FN1-633-1


120
503
23
P68 E8
0
3.3
1
0
1
0
0
94
0
FN1-503-1


122
640
29
P85 F8
0
4.0
0
0
1
1
0
93
1
FN1-640-1


123
658
29
P87 H4
0
5.8
0
0
2
1
0
90
1
FN1-658-1


124
674
30
P89 H7
0
2.8
0
0
1
1
0
95
1
FN1-674-1


125
675
30
P90 A10
0
1.5
0
0
1
2
0
94
1
FN1-675-1


127
736
33
P97 F8
0
3.9
0
0
1
1
0
93
1
FN1-736-1


128
743
33
P98 E8
0
4.2
0
0
3
1
0
91
1
FN1-743-1


129
748
33
P99 B7
0
4.8
0
0
2
1
0
91
1
FN1-748-1


130
754
33
P99 H8
0
5.6
0
0
2
1
0
91
1
FN1-754-1


131
754
33
P99 H12
0
4.1
0
0
1
1
0
93
1
FN1-754-2


132
775
34
P102 E8
0
3.9
0
0
2
1
0
93
0
FN1-775-1


134
695
31
P92 E4
0
9.0
0
0
0
0
0
90
1
FN1-695-2


139
809
36
P106 G1
0
1.7
0
0
0
8
0
91
0
FN1-809-1


140
809
36
P106 G2
0
0.0
0
0
0
0
4
96
0
FN1-809-2


143
841
37
P110 G10
4
2.1
0
0
0
0
0
94
0
FN1-841-1


144
846
37
P111 D1
1
2.0
0
0
1
1
0
95
0
FN1-846-1


145
846
37
P111 D2
0
0.0
0
0
0
0
0
100
0
FN1-846-2


146
846
37
P111 D12
0
1.1
0
0
2
0
0
97
0
FN1-846-3


147
874
38
P114 H5
0
1.6
0
0
0
0
0
98
0
FN1-874-1


148
874
38
P114 H6
0
3.2
0
0
0
0
0
97
0
FN1-874-2


149
875
39
P115 A5
0
0.0
0
0
0
0
0
100
0
FN1-875-1


150
884
39
P116 B9
3
2.0
0
0
0
0
0
95
0
FN1-884-1


153
900
40
P118 B1
0
0.0
0
0
0
0
0
98
1
FN1-900-1


154
900
40
P118 B3
0
0.5
0
0
0
1
0
97
1
FN1-900-2


155
900
40
P118 B6
0
0.3
0
0
0
0
0
97
1
FN1-900-3


156
900
40
P118 B8
0
0.0
0
0
0
0
0
98
1
FN1-900-4


157
900
40
P118 B12
0
0.0
0
0
1
1
0
98
0
FN1-900-5


158
902
40
P118 D3
0
1.6
0
0
0
0
0
97
0
FN1-902-1


159
912
40
P119 F4
0
1.8
0
0
1
0
1
92
1
FN1-912-1


160
915
40
P120 A6
0
1.9
0
0
0
1
0
93
2
FN1-915-1


161
916
40
P120 B12
0
1.2
0
0
0
1
0
98
0
FN1-916-1


162
945
41
P123 G8
0
2.5
0
0
0
0
0
94
1
FN1-945-1


163
945
41
P123 G11
0
1.6
0
0
0
0
0
96
0
FN1-945-2


167
998
44
P130 D6
0
0.8
0
0
0
0
0
99
0
FN1-998-1


168
998
44
P130 D8
0
2.1
0
0
0
0
0
97
0
FN1-998-2


172
1027
45
P134 A4
0
0.9
0
0
0
0
0
98
0
FN1-1027-1


173
1027
45
P134 A10
0
1.4
0
0
0
0
0
98
0
FN1-1027-2


174
1050
46
P136 H5
0
1.6
0
0
0
0
0
98
0
FN1-1050-1


175
1050
46
P136 H11
0
2.5
0
0
0
1
0
96
0
FN1-1050-2


176
1085
47
P141 C5
0
2.7
0
0
0
0
0
96
0
FN1-1085-1


178
1108
48
P144 B,3
0
0.0
3
0
0
0
0
97
0
FN1-1108-1


179
1116
49
P145 B,9
0
3.6
0
0
0
0
0
94
1
FN1-1116-1


180
1123
49
P146 A,11
0
3.6
0
0
0
0
0
95
0
FN1-1123-1


181
1133
49
P147 C,9
0
1.3
0
0
0
1
0
93
2
FN1-1133-1


183
1139
50
P148 A,5
0
2.9
0
0
0
0
1
95
1
FN1-1139-1


184
1139
50
P148 A,8
0
3.8
0
1
0
0
2
92
0
FN1-1139-2


185
1141
50
P148 C,6
0
0.0
1
1
0
0
0
95
1
FN1-1141-1


187
1149
50
P149 C,3
0
1.3
0
0
0
0
0
97
2
FN1-1149-1


188
1153
50
P149 G,4
0
0.4
0
0
0
0
1
97
0
FN1-1153-1


192
1170
51
P151 H,2
0
3.6
0
0
0
0
0
94
1
FN1-1170-1


194
1176
51
P152 F,12
0
3.9
0
0
0
0
0
93
1
FN1-1176-1


195
1180
51
P153 B,7
0
0.7
0
2
0
1
0
92
2
FN1-1180-1


196
1180
51
P153 B,10
0
2.5
0
0
0
0
0
97
0
FN1-1180-2


197
1183
51
P153 E,9
0
0.9
0
0
0
0
2
95
1
FN1-1183-1


198
1183
51
P153 E,11
0
0.8
0
0
0
0
0
94
3
FN1-1183-2


199
1185
51
P153 G,8
0
0.4
0
1
0
0
0
98
0
FN1-1185-1


200
1186
51
P153 H,9
0
2.2
0
0
0
0
0
95
1
FN1-1186-1


203
1201
52
P155 G,3
0
3.5
0
0
1
0
0
93
1
FN1-1201-1


204
1204
52
P156 B,1
0
3.0
0
0
0
0
0
95
1
FN1-1204-1


205
1211
52
P156 G,2
0
2.5
0
0
0
0
0
95
0
FN1-1211-1


208
1242
54
P160 E1
0
0.0
0
0
0
0
0
100
0
FN1-1242-1


209
1242
54
P160 E8
0
0.0
0
0
0
0
0
100
0
FN1-1242-2


210
1242
54
P160 E9
0
0.0
0
0
0
0
0
100
0
FN1-1242-3


213
1270
55
P164 A11
0
0.0
0
0
0
0
0
100
0
FN1-1270-1


214
1272
55
P164 C6
0
0.0
0
0
0
0
0
100
0
FN1-1272-1


216
1303
56
P168 B,2
0
4.3
0
0
1
1
0
92
1
FN1-1303-1


217
1313
57
P169 D,8
0
5.2
0
0
0
0
1
94
0
FN1-1313-1


218
1326
57
P171 A,5
0
3.9
0
0
0
0
0
93
1
FN1-1326-1


219
1326
57
P171 A,11
0
2.5
0
1
0
0
1
94
1
FN1-1326-2


220
1331
57
P171 F,7
0
2.3
0
0
0
0
0
96
1
FN1-1331-1


224
1366
59
P176 3:A,11
0
1.9
0
0
0
0
0
98
0
FN1-1366-1


225
1370
59
P176 3:E,6
0
2.2
0
0
0
0
0
97
0
FN1-1370-1


226
1373
59
P176 3:H,11
0
0.5
0
0
0
0
0
99
0
FN1-1373-1


227
1376
59
P177 4:C,4
0
0.0
0
0
0
0
0
98
0
FN1-1376-1


229
1381
59
P177 4:H,11
0
2.2
0
0
0
0
0
97
1
FN1-1381-1


230
1387
60
P178 5:F,1
0
1.2
0
0
0
0
0
97
0
FN1-1387-1


232
1401
60
P180 7:D,11
0
0.0
0
0
0
0
0
95
1
FN1-1401-1


233
1402
60
P180 7:E,12
0
0.0
0
0
0
0
0
96
3
FN1-1402-1


234
1403
60
P180 7:F,1
0
1.2
0
0
0
0
0
95
1
FN1-1403-1


235
1405
60
P180 7:H,5
0
0.0
0
0
0
0
0
96
2
FN1-1405-1


236
1405
60
P180 7:H,6
0
0.0
0
0
0
0
1
97
0
FN1-1405-2


237
1413
61
P181 H4
0
0.0
0
0
0
0
0
100
0
FN1-1413-1


238
1419
61
P182 F2
0
0.0
0
0
0
0
0
100
0
FN1-1419-1


239
1432
62
P184 C3
0
0.0
0
0
0
0
0
100
0
FN1-1432-1


240
1444
62
P185 G1
0
0.0
0
0
0
0
0
100
0
FN1-1444-1


241
1447
62
P186 B3
0
0.0
0
0
0
0
0
100
0
FN1-1447-1


242
1474
63
P189 E7
0
0.0
0
0
0
0
0
100
0
FN1-1474-1


245
1519
65
P195 B6
0
0.0
0
0
0
0
0
100
0
FN1-1519-1


246
1519
65
P195 B7
0
0.0
0
0
0
0
0
100
0
FN1-1519-2


248
1533
66
P196 H12
0
0.0
0
0
0
0
0
100
0
FN1-1533-1


249
1534
66
P197 A5
0
0.0
0
0
0
0
0
100
0
FN1-1534-1


250
1534
66
P197 A6
0
0.0
0
0
0
0
0
100
0
FN1-1534-2


251
1535
66
P197 B11
0
0.0
0
0
0
0
0
100
0
FN1-1535-1


252
1536
66
P197 C7
0
1.7
0
0
0
0
0
98
0
FN1-1536-1


254
1567
67
P201 B7
0
0.0
0
0
0
0
0
100
0
FN1-1567-1


255
1571
67
P201 F10
0
0.0
0
0
0
0
0
100
0
FN1-1571-1


256
1571
67
P201 F11
0
0.0
0
0
0
0
0
100
0
FN1-1571-2


257
1571
67
P201 F12
0
0.0
0
0
0
2
0
98
0
FN1-1571-3


258
1571
67
P201 F2
0
0.0
0
0
0
0
0
100
0
FN1-1571-4


259
1571
67
P201 F3
0
0.0
0
0
0
0
0
100
0
FN1-1571-5


260
1571
67
P201 F4
0
0.0
0
0
0
0
0
100
0
FN1-1571-6


261
1571
67
P201 F5
0
0.0
0
0
0
0
0
100
0
FN1-1571-7


262
1571
67
P201 F7
0
0.0
0
0
0
0
0
100
0
FN1-1571-8


263
1571
67
P201 F8
0
0.0
0
0
0
0
0
100
0
FN1-1571-9


264
1571
67
P201 F9
0
0.0
0
0
0
0
0
100
0
FN1-1571-10


265
1573
67
P201 H12
0
0.0
0
0
0
0
0
100
0
FN1-1573-1


268
1600
69
P205 C11
0
0.0
0
0
0
0
0
100
0
FN1-1600-1


269
1621
69
P207 H4
0
0.0
0
0
0
0
0
100
0
FN1-1621-1


270
1625
70
P208 D2
0
1.9
2
0
1
2
0
94
0
FN1-1625-1


271
1659
71
P212 F8
0
1.4
0
0
2
0
0
97
0
FN1-1659-1


272
1660
71
P212 G8
0
1.9
0
0
1
0
0
97
0
FN1-1660-1


273
1662
71
P213 A11
0
1.8
1
0
1
0
0
96
0
FN1-1662-1


274
1662
71
P213 A12
0
1.5
0
0
1
0
0
97
0
FN1-1662-2


275
1701
73
P217 H2
0
1.0
0
0
0
0
0
99
0
FN1-1701-1


276
1702
73
P218 A11
0
2.0
0
0
0
0
0
97
1
FN1-1702-1


277
1703
73
P218 B2
0
1.3
0
0
0
0
0
99
0
FN1-1703-1


279
1719
74
P220 B1
0
0.0
0
0
0
0
0
100
0
FN1-1719-1


280
1741
74
P222 H8
0
1.5
0
0
0
0
0
98
0
FN1-1741-1


281
1741
74
P222 H12
0
0.0
0
0
0
0
0
100
0
FN1-1741-2


282
1744
75
P223 C9
0
0.0
0
0
0
0
0
100
0
FN1-1744-1


283
1763
75
P225 F8
0
0.0
0
0
0
0
0
100
0
FN1-1763-1


284
1771
76
P226 F2
0
0.8
1
0
0
0
0
98
0
FN1-1771-1


285
1771
76
P226 F3
0
0.0
0
0
0
0
0
100
0
FN1-1771-2


287
1813
77
P231 H11
0
1.8
1
0
1
0
0
94
0
FN1-1813-1


290
1835
78
P234 C9
0
1.9
0
0
3
3
0
92
0
FN1-1835-2


291
1835
78
P234 C11
0
1.8
0
0
4
2
0
92
0
FN1-1835-3


292
1841
79
P235 D8
0
0.0
0
0
0
0
0
100
0
FN1-1841-1


295
1869
80
P238 H5
0
0.9
0
0
0
0
0
99
0
FN1-1869-1


296
1869
80
P238 H8
0
1.4
0
0
0
0
0
98
0
FN1-1869-2


298
1898
81
P242 E5
0
1.3
0
0
1
1
0
97
0
FN1-1898-1


299
1913
82
P244 D4
0
0.0
0
0
0
0
0
100
0
FN1-1913-1


301
1944
83
P248 C1
0
1.6
0
0
1
0
0
97
0
FN1-1944-1


304
2017
86
P257 C12
0
1.0
1
0
0
0
0
98
0
FN1-2017-1


307
2085
89
P265 G1
0
2.3
0
0
0
0
0
96
0
FN1-2085-1


308
2085
89
P265 G2
0
1.4
0
0
0
0
0
99
0
FN1-2085-2


309
2103
89
P267 H2
0
1.2
0
0
0
0
0
99
0
FN1-2103-1


310
2114
90
P269 C2
0
0.8
1
0
1
0
0
97
0
FN1-2114-1


311
2115
90
P269 D5
0
1.0
0
0
0
0
0
99
0
FN1-2115-1


312
2152
91
P273 H3
0
0.8
0
0
0
0
0
99
0
FN1-2152-1


313
2152
91
P273 H7
0
0.0
0
0
0
0
0
100
0
FN1-2152-2


314
2152
91
P273 H9
0
0.8
0
0
0
0
0
99
0
FN1-2152-3


315
2152
91
P273 H11
0
0.0
0
0
0
0
0
100
0
FN1-2152-4


317
2154
92
P274 B4
0
4.1
0
0
0
0
4
92
0
FN1-2154-1


318
2172
92
P276 D7
0
0.0
0
0
0
0
0
100
0
FN1-2172-1


319
2175
92
P276 G8
0
0.0
0
0
0
0
0
100
0
FN1-2175-1


321
2186
93
P278 B6
0
2.0
0
0
0
0
0
98
0
FN1-2186-1


322
2186
93
P278 B11
0
0.0
0
0
0
0
0
98
0
FN1-2186-2


324
2196
93
P279 D5
0
1.3
0
1
1
3
0
94
0
FN1-2196-1


325
2199
93
P279 G3
0
0.0
0
0
0
0
0
100
0
FN1-2199-1


326
2199
93
P279 G10
0
0.0
0
0
0
0
0
100
0
FN1-2199-2


327
2200
93
P279 H1
0
0.0
0
0
0
0
0
97
0
FN1-2200-1


330
2215
94
P281 G2
0
0.0
0
0
0
0
3
91
0
FN1-2215-1


331
2215
94
P281 G6
0
0.0
0
0
0
0
0
97
0
FN1-2215-2


332
2219
94
P282 B2
0
0.0
0
0
0
0
0
100
0
FN1-2219-1


333
2219
94
P282 B6
0
0.0
0
0
0
0
0
100
0
FN1-2219-2


335
2221
94
P282 D8
0
0.0
4
0
0
0
4
93
0
FN1-2221-1


336
2224
94
P282 G4
0
0.0
0
0
0
0
0
100
0
FN1-2224-1


337
2231
95
P283 F5
0
0.0
0
0
0
0
0
100
0
FN1-2231-1


338
2231
95
P283 F12
0
0.0
0
0
0
0
0
100
0
FN1-2231-2


339
2233
95
P283 H7
0
3.0
0
0
0
0
0
97
0
FN1-2233-1


340
2241
95
P284 H1
0
0.0
0
0
0
0
0
100
0
FN1-2241-1


341
2241
95
P284 H4
0
2.7
0
0
0
0
0
97
0
FN1-2241-2


342
2243
95
P285 B9
0
0.0
0
0
0
0
0
96
0
FN1-2243-1


343
2245
95
P285 C5
0
0.0
0
0
0
0
0
98
0
FN1-2245-1


344
2245
95
P285 C10
0
0.0
0
0
0
0
0
100
0
FN1-2245-2


345
2255
96
P286 E9
0
0.0
0
0
0
0
0
100
0
FN1-2255-1


346
2267
96
P288 A10
1
0.0
1
0
0
0
0
99
0
FN1-2267-1


349
2280
97
P289 F11
0
0.0
0
0
0
5
0
91
4
FN1-2280-1


354
2288
97
P290 F9
0
0.9
0
0
1
1
0
96
1
FN1-2288-1


355
2325
99
P295 C5
0
0.0
0
0
0
0
0
100
0
FN1-2325-1


356
2329
99
P295 G4
0
0.0
0
0
2
2
0
97
0
FN1-2329-1


357
2365
100
P300 C9
0
0.0
0
0
0
2
0
98
0
FN1-2365-1


358
2372
101
P301 B1
0
0.0
0
0
0
0
0
100
0
FN1-2372-1


361
2412
102
P306 B2
0
0.7
0
0
0
0
0
99
0
FN1-2412-1


362
2425
103
P307 G8
0
0.0
0
0
0
0
0
100
0
FN1-2425-1


363
2427
103
P308 A6
0
0.0
0
0
0
0
0
100
0
FN1-2427-1


364
2437
103
P309 C12
0
0.0
0
0
0
0
0
100
0
FN1-2437-1


365
2443
104
P310 A1
0
0.7
0
0
1
0
0
97
0
FN1-2443-1


367
2492
106
P316 A5
0
0.0
0
0
0
1
0
99
0
FN1-2492-1


374
2604
110
P330 A6
0
0.0
0
0
0
1
0
99
0
FN1-2604-1


376
7299
298
P892 C2
0
0.0
0
0
0
3
0
97
0
FN1-7299-2


377
7299
298
P892 C3
0
0.0
0
0
0
2
0
98
0
FN1-7299-3


378
7299
298
P892 C4
0
0.5
0
0
0
1
0
98
0
FN1-7299-4


379
7299
298
P892 C10
0
0.7
0
0
0
2
0
98
0
FN1-7299-5


380
7299
298
P892 C12
0
0.0
0
0
0
3
0
97
0
FN1-7299-6


381
7303
298
P892 G10
0
0.0
0
0
0
0
0
100
0
FN1-7303-1


382
7304
298
P892 H2
0
0.0
0
0
0
0
0
100
0
FN1-7304-1


383
7304
298
P892 H5
0
0.0
0
0
1
1
0
98
0
FN1-7304-2


384
7306
298
P893 B6
0
0.0
0
0
0
1
0
98
1
FN1-7306-1


385
7325
299
P895 E10
0
0.0
1
0
1
2
0
97
0
FN1-7325-1


386
7329
299
P896 A8
0
0.0
0
0
0
0
0
100
0
FN1-7329-1


387
7329
299
P896 A10
0
0.0
0
0
0
1
0
99
0
FN1-7329-2


388
7332
299
P896 D3
0
0.0
0
0
0
1
0
99
0
FN1-7332-1


389
7341
299
P897 E3
0
0.0
0
0
0
0
0
100
0
FN1-7341-1


390
7342
299
P897 F1
0
0.0
0
1
0
2
0
97
0
FN1-7342-1


391
7342
299
P897 F9
0
0.0
0
1
0
2
0
96
1
FN1-7342-2


393
7348
300
P898 D5
0
1.1
0
0
0
0
0
99
0
FN1-7348-1


394
7353
300
P898 H1
0
0.6
0
0
0
0
0
99
0
FN1-7353-1


395
7354
300
P899 A3
0
0.0
0
0
0
1
0
99
0
FN1-7354-1


396
7354
300
P899 A8
0
0.0
0
0
0
1
0
99
0
FN1-7354-2


397
7355
300
P899 B2
0
0.0
0
0
0
0
0
100
0
FN1-7355-1


398
7355
300
P899 B5
0
0.0
0
0
0
1
0
99
0
FN1-7355-2


399
7373
301
P901 C10
0
1.0
0
0
0
0
0
99
0
FN1-7373-1


400
7378
301
P901 H1
0
0.0
0
0
1
0
0
99
0
FN1-7378-1


401
7380
301
P902 B9
0
0.0
0
0
0
0
0
100
0
FN1-7380-1


402
7386
301
P902 H6
0
0.0
0
0
1
1
0
98
0
FN1-7386-1


403
7386
301
P902 H9
0
0.0
0
0
0
0
0
100
0
FN1-7386-2


404
7397
302
P904 B1
0
0.0
0
0
0
0
0
100
0
FN1-7397-1


405
7399
302
P904 D3
0
0.0
0
0
0
3
0
97
0
FN1-7399-1


406
7400
302
P904 E12
0
0.0
0
0
0
0
0
100
0
FN1-7400-1


407
7445
303
P909 H12
0
0.9
0
0
0
1
0
98
0
FN1-7445-1


408
7447
304
P910 B9
0
0.7
0
0
0
0
0
98
0
FN1-7447-1


409
7448
304
P910 C6
0
0.0
0
0
0
0
0
98
2
FN1-7448-1


410
7452
304
P910 G1
0
0.0
0
0
1
1
0
98
0
FN1-7452-1


411
7462
304
P911 H6
0
0.9
0
0
0
0
1
99
0
FN1-7462-1


412
7467
304
P912 C1
0
0.6
0
0
0
1
0
97
0
FN1-7467-1


413
7467
304
P912 C2
0
0.4
1
0
0
2
0
97
0
FN1-7467-2


414
7471
304
P912 G7
0
1.0
0
0
0
0
0
98
1
FN1-7471-1


415
7472
304
P912 H2
0
0.0
0
0
0
0
0
95
5
FN1-7472-1


416
7491
305
P914 G8
0
0.8
0
0
0
0
0
99
0
FN1-7491-1


417
7499
305
P915 F2
0
1.2
0
0
0
0
0
97
0
FN1-7499-1


418
7506
306
P916 B6
0
1.1
0
0
1
0
1
97
0
FN1-7506-1


419
7509
306
P916 D1
0
0.0
0
0
0
0
0
100
0
FN1-7509-1


420
7513
306
P916 H5
0
1.0
0
0
1
1
0
96
1
FN1-7513-1


421
7525
306
P918 C10
0
1.3
0
0
1
1
0
95
1
FN1-7525-1


422
7529
306
P918 G11
0
0.0
0
0
0
0
0
100
0
FN1-7529-1


423
7535
307
P919 E8
0
0.0
0
0
0
0
0
97
3
FN1-7535-1


424
7536
307
P919 F1
0
1.2
0
0
2
1
0
96
0
FN1-7536-1


426
7551
307
P921 C5
0
0.0
0
0
0
0
0
100
0
FN1-7551-1


427
7557
308
P922 A12
0
0.0
0
0
0
0
0
100
0
FN1-7557-1


428
7558
308
P922 B7
0
0.0
0
0
0
0
0
100
0
FN1-7558-1


429
7560
308
P922 D8
0
1.2
0
0
0
1
0
98
0
FN1-7560-1


430
7566
308
P923 B9
0
0.0
0
0
0
0
2
98
0
FN1-7566-1


431
7579
308
P924 F3
0
0.8
0
0
3
0
2
92
2
FN1-7579-1


432
7584
309
P925 B3
0
0.0
0
0
1
1
0
98
0
FN1-7584-1


433
7587
309
P925 E7
0
0.0
0
0
0
0
0
100
0
FN1-7587-1


434
7592
309
P926 B2
0
0.0
0
0
0
0
0
100
0
FN1-7592-1


435
7592
309
P926 B4
0
0.7
0
0
1
1
0
97
0
FN1-7592-2


436
7598
309
P926 H3
0
0.7
0
0
1
2
0
95
1
FN1-7598-1


437
7598
309
P926 H10
0
0.7
1
0
1
1
0
96
1
FN1-7598-2


438
7600
309
P927 B1
0
0.0
0
0
0
0
0
100
0
FN1-7600-1


439
7600
309
P927 B5
0
0.0
0
0
0
0
0
100
0
FN1-7600-2


440
7629
310
P930 C8
0
1.1
0
1
0
0
0
95
1
FN1-7629-1


441
7640
311
P931 B4
0
0.0
0
1
0
0
0
96
1
FN1-7640-1


442
7647
311
P931 F1
0
0.0
0
3
0
0
0
96
0
FN1-7647-1


443
7656
311
P932 F3
0
1.7
0
1
0
0
0
94
1
FN1-7656-1


444
7666
311
P933 G8
0
1.0
0
1
0
0
0
95
1
FN1-7666-1


445
7674
312
P934 F3
0
1.4
0
1
0
0
0
96
1
FN1-7674-1


446
7686
312
P936 A6
0
0.0
0
0
0
0
0
100
0
FN1-7686-1


447
7702
313
P937 H8
0
0.9
0
0
0
0
0
98
1
FN1-7702-1


448
7718
313
P939 G6
0
0.0
0
0
0
1
0
99
0
FN1-7718-1


450
3122
132
P394 E2
0
0.0
0
0
0
5
0
95
0
FN1-3122-1


451
3123
132
P394 F12
0
0.0
0
0
0
5
0
95
0
FN1-3123-1


452
3132
132
P395 G11
0
0.0
0
0
0
6
0
94
0
FN1-3132-1


453
3141
132
P396 G1
0
0.8
0
0
1
1
0
96
1
FN1-3141-1


454
3141
132
P396 G10
0
0.0
0
0
0
0
4
96
0
FN1-3141-2


455
3158
133
P398 G6
0
0.0
0
0
0
3
0
97
0
FN1-3158-1


456
3176
134
P400 G1
0
0.9
0
0
3
1
0
95
0
FN1-3176-1


457
3206
135
P404 E3
0
0.0
0
0
0
0
4
96
0
FN1-3206-1


458
3209
135
P404 H11
0
0.0
0
0
1
0
2
96
1
FN1-3209-1


459
3215
135
P405 F5
0
1.0
0
0
1
0
2
95
1
FN1-3215-1


460
3228
136
P407 C8
0
0.0
0
0
1
2
0
97
0
FN1-3228-1


461
3258
137
P410 G4
0
0.0
0
0
1
0
4
94
1
FN1-3258-1


462
3270
138
P412 B3
0
0.0
0
0
0
5
0
95
0
FN1-3270-1


463
3288
138
P414 C7
0
0.0
0
0
0
0
3
97
0
FN1-3288-1


464
3295
139
P415 A2
0
0.0
0
0
2
4
0
94
0
FN1-3295-1


465
3296
139
P415 B10
0
0.0
0
0
0
3
0
97
0
FN1-3296-1


466
3296
139
P415 B12
0
1.0
0
0
0
2
0
97
0
FN1-3296-2


467
3297
139
P415 C12
0
0.0
0
0
1
0
3
95
0
FN1-3297-1


468
3299
139
P415 E1
0
0.0
0
0
0
0
3
96
1
FN1-3299-1


469
3300
139
P415 F2
0
0.0
0
0
1
0
3
95
0
FN1-3300-1


472
3310
139
P416 H4
0
0.0
0
0
0
2
0
96
1
FN1-3310-1


473
3320
140
P418 B10
0
0.6
0
0
1
2
0
96
1
FN1-3320-1


474
3326
140
P418 H2
0
0.0
0
0
0
2
0
98
0
FN1-3326-1


475
3328
140
P419 B2
0
0.0
0
0
1
1
0
98
1
FN1-3328-1


476
3328
140
P419 B7
0
0.0
0
0
0
0
7
91
0
FN1-3328-2


477
3306
139
P416 D7
0
3.9
0
0
0
0
1
94
1
FN1-3306-1


478
3365
141
P423 F3
0
0.0
0
0
0
7
0
93
0
FN1-3365-1


479
3368
141
P423 H10
0
0.0
0
0
0
0
0
100
0
FN1-3368-1


480
3376
142
P424 H9
0
0.0
0
0
0
2
0
98
0
FN1-3376-1


481
3376
142
P424 H11
0
0.0
0
0
0
2
0
98
0
FN1-3376-2


482
3383
142
P425 G9
0
0.0
0
0
0
0
0
100
0
FN1-3383-1


483
3386
142
P426 B9
0
0.0
0
0
0
2
0
98
0
FN1-3386-1


484
3387
142
P426 C5
0
0.0
0
0
1
1
0
98
0
FN1-3387-1


485
3387
142
P426 C7
0
0.0
0
0
0
0
0
98
2
FN1-3387-2


486
3388
142
P426 D7
0
0.0
0
0
0
1
0
97
1
FN1-3388-1


487
3406
143
P428 F12
0
0.0
0
0
0
0
0
100
0
FN1-3406-1


488
3408
143
P428 H11
0
1.0
0
0
1
1
0
97
0
FN1-3408-1


489
3413
143
P429 E2
0
0.0
0
0
2
0
0
98
0
FN1-3413-1


491
3444
145
P433 C12
0
0.0
0
0
0
0
0
100
0
FN1-3444-1


493
3488
146
P438 F4
0
0.0
0
0
2
0
0
98
0
FN1-3488-1


494
3492
147
P439 B4
0
0.0
0
0
4
2
0
93
0
FN1-3492-1


495
3497
147
P439 G3
0
0.0
0
0
0
0
0
100
0
FN1-3497-1


496
3497
147
P439 G4
0
0.0
0
0
0
0
0
100
0
FN1-3497-2


497
3497
147
P439 G11
0
0.0
0
0
0
0
0
100
0
FN1-3497-3


498
3531
148
P443 H1
0
0.0
0
0
0
0
0
100
0
FN1-3531-1


500
3608
151
P453 C8
0
1.1
0
0
1
0
1
97
0
FN1-3608-1


501
3612
151
P453 G6
0
0.0
0
0
0
0
0
100
0
FN1-3612-1


503
3635
152
P456 F1
0
1.1
0
0
1
1
0
96
1
FN1-3635-1


504
3635
152
P456 F2
0
0.0
0
0
0
0
0
98
0
FN1-3635-2


505
3635
152
P456 F4
0
0.7
0
0
0
0
0
95
0
FN1-3635-3


506
3635
152
P456 F9
0
0.5
0
0
0
0
0
99
0
FN1-3635-4


507
3635
152
P456 F10
0
0.0
0
0
0
0
0
100
0
FN1-3635-5


508
3635
152
P456 F11
0
0.2
0
0
0
0
0
99
0
FN1-3635-6


509
3679
154
P461 G5
0
0.6
0
0
0
1
0
98
0
FN1-3679-1


510
3708
155
P465 D2
0
0.0
0
0
0
0
0
100
0
FN1-3708-1


511
3710
155
P465 F5
0
0.0
0
0
0
0
0
100
0
FN1-3710-1


512
3718
156
P466 F5
0
0.0
0
0
0
0
0
100
0
FN1-3718-1


513
3718
156
P466 F7
0
1.0
0
0
0
0
0
98
1
FN1-3718-2


514
3770
158
P473 B8
0
1.6
0
0
1
0
0
97
0
FN1-3770-1


516
3794
159
P476 A7
0
0.4
0
0
0
0
0
100
0
FN1-3794-1


517
3799
159
P476 F4
0
0.9
0
0
1
0
0
97
1
FN1-3799-1


518
3803
159
P477 B9
0
0.9
0
0
1
0
0
98
1
FN1-3803-1


519
3804
159
P477 C6
0
1.0
0
0
1
0
0
96
1
FN1-3804-1


520
3805
159
P477 D12
0
1.0
0
0
1
0
0
98
1
FN1-3805-1


521
3817
160
P478 H11
0
0.0
0
0
0
0
0
100
0
FN1-3817-1


522
3821
160
P479 D1
0
1.1
0
0
1
0
0
98
0
FN1-3821-1


523
3821
160
P479 D3
0
1.4
0
0
0
0
0
98
1
FN1-3821-2


524
3821
160
P479 D10
0
0.0
0
0
0
0
0
100
0
FN1-3821-3


525
3827
160
P480 B6
0
0.7
0
0
0
0
0
99
0
FN1-3827-1


526
3841
161
P481 H3
0
0.0
0
0
3
0
0
96
1
FN1-3841-1


528
3978
166
P498 H2
0
0.0
0
0
0
0
0
100
0
FN1-3978-1


529
3995
167
P501 A5
0
0.0
0
0
0
0
0
100
0
FN1-3995-1


531
4027
169
P505 A6
0
0.0
0
0
0
0
0
100
0
FN1-4027-1


532
4027
169
P505 A8
0
0.6
0
0
0
0
0
99
0
FN1-4027-2


534
4050
169
P507 H3
0
0.4
0
0
1
0
0
99
0
FN1-4050-1


535
4053
170
P508 C9
0
0.7
0
0
0
0
0
99
0
FN1-4053-1


537
4093
171
P513 C5
0
1.0
0
0
2
1
0
94
1
FN1-4093-1


541
4121
172
P516 G9
0
1.2
0
0
0
0
0
97
1
FN1-4121-1


542
4124
173
P517 B2
0
0.7
0
0
1
1
0
97
1
FN1-4124-1


543
4128
173
P517 F6
1
1.3
0
0
1
0
0
94
0
FN1-4128-1


546
4144
173
P519 F12
0
0.9
0
0
0
0
0
97
0
FN1-4144-1


547
4145
173
P519 G7
0
0.7
0
0
0
0
0
99
0
FN1-4145-1


548
4145
173
P519 G11
0
0.5
0
0
0
0
0
97
0
FN1-4145-2


549
4148
174
P520 B7
0
0.7
0
0
1
1
0
98
0
FN1-4148-1









Testing of Poppy Straw Alkaloid Content in M2 Generation

Capsules were harvested from the greenhouse as they matured. Seed was removed and weighed into seed envelopes. The poppy straw was placed into 50 mL BD FalconTM tubes (BD Biosciences, San Jose, Calif.) without grinding and dried either on the lab bench for several days at room temperature or in the laboratory oven at 50° C. for 3 hours. Where capsules were large, only a portion of the capsule was used for analysis, the rest being discarded.
For analysis, the poppy straw was weighed, and either 5 mL or 10 mL acid extractant (5% ethanol (“EtOH”), 0.17% phosphoric acid) added, depending on whether the straw samples weighed less or more than 0.2 g respectively. The samples were agitated with a Ratek orbital shaker (Ratek Instruments, Boronia, Victoria, Australia) for 3 hours. The liquid phase was then filtered using Pall AcroPrepTM 96 filter plates (PN S5045), and the filtrate was analysed for alkaloids using a Waters Acquity UPLC® system (Waters Corporation, Milford, Mass.). The UPLC method used was the same 2.5 minute method as used for leaf samples. Additional extractant was transferred to 1.2 mL wells in 96 well plates, sealed and frozen in case further analysis was required.
The alkaloid contents and profiles were calculated from the UPLC results and weight data. Where the weight was <0.1 g, the weight was deemed as 0.1 g.
The seeds were allowed to dry on the laboratory bench, catalogued and stored.
Of the 549 M2 plants originally selected, 434 survived to produce at least one capsule for harvest (79%). A further 6 mislabeled capsules were harvested, so their M1 parents were not known. 395 plants produced some seed, although 58 had <0.06 g seed, and only 171 plants produced more than 1 g seed.
Due to the potential lack of accuracy with the alkaloid content data (due to low capsule weights and large particle sizes), this data was analysed further only after conversion to alkaloid profiles: i.e. the alkaloid contents in comparison with the total alkaloid content.
Where multiple capsules were harvested from one plant the mean was determined using Minitab 14 statistical software (Minitab Inc., State College, Pa.).
Table 4, below, shows the results for 133 M2 selections from 92 independent M1 plants that were high in thebaine (>90% of alkaloids) and low in oripavine (<10%). Thirty six M2 selections from 27 independent M1 plants had more than 95% of the alkaloid in the straw as thebaine. Twenty one selections from 16 independent M1 plants had more than 96% of the alkaloid in the straw as thebaine. Eight selections from 6 independent M1 plants had more than 97% a of the alkaloid in the straw as thebaine. One selection, FN1-900-5, was identified that had more than 98% of its alkaloid present as thebaine. It can be seen that the oripavine content in the straw of these plants was very low, with several selections having less than 1% of the alkaloid combination, and some with less than 0.5% of the alkaloid combination. All “thebaine-only” plants however contained a small proportion of oripavine in their poppy straw.







TABLE 4







Alkaloid profiles (based on alkaloid concentrations in poppy straw) of M2 plants


selected for high thebaine and low oripavine. Means are shown where the


number of capsules (caps) was more than one.


















Seln













no.
Seln name
Caps
Morphine
Oripavine
Codeine
Salutaridine
Reticuline
Laudanine
Papaverine
Thebaine
Noscapine





















68
FN1-291-1
15
0.4
4.7
0
0.2
0.9
0.1
0.2
93.3
0.2


106
FN1-557-1
1
0
2.8
0
0.3
6.2
0
0
90.7
0


110
FN1-601-1
1
0.1
0.4
0
0
1.5
0.2
0
97.6
0.1


139
FN1-809-1
5
0
1.6
0.1
0.6
4.9
0
0
92.7
0


140
FN1-809-2
2
0
1.7
0.1
0.3
2.1
0
0
95.6
0.2


144
FN1-846-1
1
0
0.1
0
0
2.5
0.1
0
97.2
0


145
FN1-846-2
3
2.6
0.4
0
0
3.1
0.2
0
93.5
0.1


149
FN1-875-1
3
1.8
3.6
0
0.1
1.9
0.1
0
92.4
0.1


153
FN1-900-1
3
0
0.6
0
0.1
1.5
0.2
0
97.5
0.1


154
FN1-900-2
3
0
0.6
0
0.2
1.8
0.2
0
97.1
0.1


155
FN1-900-3
3
0.8
0.5
0
0
2.3
0.2
0
96.1
0.1


156
FN1-900-4
7
2.3
0.6
0
0.2
1.5
0.2
0
94.9
0.3


157
FN1-900-5
1
0
0.6
0
0.2
1
0.1
0
98
0.1


161
FN1-916-1
4
0.6
5.5
0
0.1
1.3
0.1
0
92.3
0.1


167
FN1-998-1
1
1.5
1.1
0.6
0
2.4
0
0
91.4
3.1


168
FN1-998-2
2
0
1
0.1
0.3
2.4
0
0
96.3
0


172
FN1-1027-1
1
0
2.2
0
0
3.1
0
0
94.7
0


173
FN1-1027-2
4
0.3
2.6
0
0.3
2
0.1
0
94.6
0.2


174
FN1-1050-1
8
3.5
1
0
0.4
1.3
0
0
93.7
0


175
FN1-1050-2
2
0.2
3
0
0.1
1.7
0
0
94.8
0.2


180
FN1-1123-1
2
0
1
0
0.4
3.6
0
0
95.2
0


184
FN1-1139-2
1
0
5.7
0
0.1
1.3
0.2
0
92.5
0.1


188
FN1-1153-1
10
0.1
7.9
0
0
1.3
0.1
0
90.6
0


199
FN1-1185-1
3
0
2.9
0
0.1
2.7
0.2
0
93.9
0.2


208
FN1-1242-1
3
1.2
0.6
0
0.1
2.8
0.2
0
95
0.1


209
FN1-1242-2
2
0
0.3
0
0
2.8
0.2
0
96.7
0.1


210
FN1-1242-3
3
0
0.4
0
0.1
1.7
0.1
0
97.5
0.1


219
FN1-1326-2
4
0.5
6.9
0
0.1
1.8
0.1
0
90.6
0.1


240
FN1-1444-1
4
0
5.2
0
0
1.4
0.2
0
93.2
0.2


249
FN1-1534-1
6
0.1
0.7
0
0.5
2.6
0
0
96.1
0


250
FN1-1534-2
2
0.5
1.5
0
0.5
4.9
0
0
92.5
0.2


255
FN1-1571-1
2
0.4
1.5
0
0.5
5.7
0
0
92
0


256
FN1-1571-2
2
0.5
1.8
0
0.7
4.2
0
0
93
0


257
FN1-1571-3
3
0.6
1.5
0
0.5
2.5
0
0
94.7
0


258
FN1-1571-4
2
0.7
1.9
0
0.4
6.9
0.1
0
90.2
0


259
FN1-1571-5
1
0
1.5
0
0.7
3.4
0
0
94.3
0


260
FN1-1571-6
4
0.1
1.2
0.1
0.5
4.9
0
0
93.2
0


261
FN1-1571-7
3
0
1.3
0
0.3
5
0
0
93.3
0


262
FN1-1571-8
1
1
1.7
0
0.8
2.3
0
0
94
0.2


263
FN1-1571-9
3
0.6
1.8
0
0.8
4.6
0
0.1
92.1
0


264
FN1-1571-
4
0.2
1.4
0.1
0.4
5
0
0
92.9
0



10












270
FN1-1625-1
1
0
2.3
0
0.3
1.2
0.3
0
95.7
0.1


273
FN1-1662-1
1
0
4.6
0
0.1
1.4
0
0.6
93.1
0.3


275
FN1-1701-1
2
0
2.9
0.1
0.1
2.2
0.2
0
94.6
0.1


279
FN1-1719-1
4
0.2
0.3
0
0
2.6
0.1
0
96.6
0.1


280
FN1-1741-1
1
0
3.1
0
0
3.4
0.2
0
93
0.3


284
FN1-1771-1
3
0
2
0
0.3
1.1
0.1
0
96.6
0.1


285
FN1-1771-2
2
0
4.8
0
0.2
1.6
0
0
93.4
0.1


292
FN1-1841-1
1
0
4.7
0
0
1.3
0.3
0
93.7
0


296
FN1-1869-2
1
0
2.2
0
0.4
1.7
0.1
0
95.5
0.1


299
FN1-1913-1
3
0
3
0
0
1.8
0
0
95.1
0


307
FN1-2085-1
1
0
4
0
0
2.8
0.1
0
93
0.1


309
FN1-2103-1
5
0.1
6.8
0
0.1
2.1
0.2
0
90.7
0


312
FN1-2152-1
3
0
2.1
0
0.1
5.6
0
0
92.3
0


313
FN1-2152-2
2
0
0.9
0.2
0.6
3.6
0.1
0
94.8
0


314
FN1-2152-3
3
0
1.1
0
0.7
3.4
0
0.1
94.7
0


315
FN1-2152-4
2
0
1.2
0
0.5
3.3
0
0
95
0


318
FN1-2172-1
1
0
4
0
0
4.9
0.2
0
90.8
0.1


319
FN1-2175-1
1
0
8
0
0
1.2
0.1
0
90.4
0.3


321
FN1-2186-1
2
0
3.5
0.1
0.4
1.6
0
0.3
94.1
0.1


325
FN1-2199-1
3
0
0.4
0
0.1
4.3
0.2
0
95
0


326
FN1-2199-2
2
0
0.3
0
0.1
3.6
0.1
0
95.9
0.2


339
FN1-2233-1
1
0
2.8
0
0.1
1.4
0
0
95.7
0


343
FN1-2245-1
2
0
6.1
0
0.1
1.5
0.1
0
92.1
0.2


364
FN1-2437-1
3
0
0.5
0
0.1
3.1
0.2
0
96.2
0


367
FN1-2492-1
1
0
5.5
0
0.1
2.2
0.1
0
92.1
0.1


374
FN1-2604-1
3
0
0.3
0.1
0.1
5.4
0.2
0
93.7
0.2


377
FN1-7299-3
2
0
2.4
0.1
0.2
1.3
0.1
0
95.8
0.2


378
FN1-7299-4
2
0
3.3
0.1
0.2
1.7
0.2
0
94.6
0.2


379
FN1-7299-5
1
0
2.7
0.1
0.4
1.6
0.2
0
94.7
0.3


380
FN1-7299-6
2
0
2
0
0.1
1
0.1
0
96.8
0.1


384
FN1-7306-1
1
0
6.8
0
0
0
0
0
93.1
0.1


396
FN1-7354-2
1
0
0.6
0
1.2
4.3
0
0
93.9
0


400
FN1-7378-1
1
0
4.4
0
0.1
1.7
0
0
93.5
0.3


404
FN1-7397-1
1
0
8
0
0
1
0
0
91
0


407
FN1-7445-1
1
0
5.6
0
0
2.3
0.2
0
91.9
0


408
FN1-7447-1
1
0
3.3
0
0.1
3.5
0.1
0
92.8
0.2


409
FN1-7448-1
1
0
5.2
0
0.1
2.7
0.1
0
91.8
0.2


414
FN1-7471-1
1
0
3.7
0.1
0
3.2
0.2
0
92.3
0.4


416
FN1-7491-1
1
0
6.9
0
0.1
1.1
0.2
0
91.8
0


417
FN1-7499-1
1
0
5.4
0
0
1.6
0.2
0
92.6
0.3


421
FN1-7525-1
2
0
5.5
0
0.1
1.6
0
0.4
92.5
0.1


422
FN1-7529-1
1
0
6.7
0
0.1
2.2
0
0.9
90
0.2


426
FN1-7551-1
1
0
4.6
0
0.1
2.3
0.1
0
92.8
0.1


433
FN1-7587-1
1
0
8.4
0
0
0.6
0.1
0
90.8
0


434
FN1-7592-1
1
0.5
2
0
0.6
0.5
0
0
96.3
0


436
FN1-7598-1
1
0
5.7
0
0.1
2.2
0.1
0
91.7
0.2


437
FN1-7598-2
1
0
4.5
0
0
1.8
0
0
93.5
0.3


443
FN1-7656-1
1
0
7.7
0
0.1
1.4
0
0.5
90.2
0.2


445
FN1-7674-1
5
0
2.4
0
0
2.6
0.1
0.2
94.5
0.2


446
FN1-7686-1
2
0
1.5
0
0.4
4.9
0.1
0
93.3
0


447
FN1-7702-1
1
0
3.2
0
0.1
2
0
0
94.6
0


453
FN1-3141-1
1
0
4.6
0
0.1
2.3
0.2
0
92.7
0.1


454
FN1-3141-2
1
0
8.1
0
0
1.6
0.2
0
90.1
0


455
FN1-3158-1
2
0
8
0
0.1
1.6
0.2
0
90.2
0.1


458
FN1-3209-1
1
0
5
0
0
2
0.2
0
92.5
0.2


461
FN1-3258-1
1
0.3
6.1
0
0
1.8
0.2
0
91.6
0


463
FN1-3288-1
1
0
5.8
0
0.1
1.8
0.2
0
92.1
0.1


465
FN1-3296-1
1
0
8.7
0
0.1
1.1
0.1
0
90.1
0


466
FN1-3296-2
1
0
2.2
0.4
0.2
1.8
0.2
0
95.2
0.1


474
FN1-3326-1
1
0
7.2
0
0.1
1.6
0.2
0
90.9
0.1


475
FN1-3328-1
2
0
0.4
0
0.2
6.5
0.3
0
92.7
0


476
FN1-3328-2
1
0
0.4
0
0.1
2.9
0.2
0
96.3
0.1


477
FN1-3306-1
2
0
2.8
0.1
0.1
2.5
0.1
0
94.3
0.3


480
FN1-3376-1
1
0
5
0
0
1.4
0.3
0
93.3
0


483
FN1-3386-1
1
0
4.6
0.1
0.1
2.6
0
1.1
91.2
0.4


486
FN1-3388-1
1
0
5.5
0
0.1
3.1
0.2
0
90.8
0.3


489
FN1-3413-1
1
0
6.3
0
0.1
2.8
0.1
0
90.6
0.2


495
FN1-3497-1
1
0
1.4
0
0
4
0
0
94.6
0


496
FN1-3497-2
2
0
1.2
0
0.2
2.8
0
0
96
0


497
FN1-3497-3
2
0
1.2
0
0.2
4.7
0
0
94
0


503
FN1-3635-1
2
0
1.2
0
0.3
4
0.1
0
94.3
0.2


504
FN1-3635-2
1
0
1.3
0
0
2.8
0
0
95.9
0


506
FN1-3635-4
1
0
1.2
0
0.4
3.6
0
0
94.8
0


507
FN1-3635-5
1
0
1
0
0
1.9
0
0
97.1
0


508
FN1-3635-6
1
0
1.3
0
0
2.2
0
0
96.5
0


509
FN1-3679-1
1
0
3.2
0.2
0.1
4.6
0
0
91.7
0.3


511
FN1-3710-1
1
0
8.1
0
0
1.3
0.2
0
90.5
0


512
FN1-3718-1
1
0
4
0
0.1
1.5
0.2
0
94.2
0.1


516
FN1-3794-1
2
0
0.1
0
0.1
4
0.2
0
95.7
0.1


521
FN1-3817-1
1
0
7
0
0
1.5
0.1
0
91.4
0


522
FN1-3821-1
1
0.9
5.9
0
0
2.5
0.2
0
90.3
0.3


523
FN1-3821-2
1
0
3.3
0
0.1
3.9
0.2
0
92
0.4


524
FN1-3821-3
1
0
6.7
0
0
2.3
0.1
0
90.9
0


525
FN1-3827-1
1
0
4.7
0
0
1
0.2
0
94.1
0


528
FN1-3978-1
1
0
3
0
0
2.7
0.3
0
94
0


529
FN1-3995-1
2
0
0.8
0
0.1
3.4
0.2
0
95.5
0.2


534
FN1-4050-1
2
0
0.1
0
0.1
2.7
0.1
0
97
0.1


535
FN1-4053-1
3
0
5.1
0
0
1.7
0.2
0
93.1
0


541
FN1-4121-1
1
0
2.2
0
0
1.6
0.3
0
96
0


542
FN1-4124-1
2
0
6.9
0
0
1.4
0.1
0
91.8
0


546
FN1-4144-1
1
0
4
0
0
2.9
0.3
0
92.7
0


548
FN1-4145-2
1
0
1.9
0
0.5
4.1
0
0
93.5
0









Growing and Evaluation of M3 Generation

Two of the highest thebaine lines were selected for increase in a greenhouse over winter 2007 to provide data to confirm their alkaloid composition and genetic stability. Thus, the plants grown in this experiment were the M3 generation.
The pots were sown in the greenhouse on 11 Apr. 2007 in double rows, with 120 pots in each double row. Each pot was thinned to 6 plants. Greenhouse conditions were as used previously except that high intensity lights were used to maintain light intensities of approximately 9900 lux for 12 hours per day.
At green capsule stage, latex samples were taken from 24 randomly chosen plants from each line. The samples were obtained from the stigmatic discs using the ray-pluck technique. A stigmatic ray was removed from each plant and dropped into acid extraction solution (5% EtOH 0.17% H3PO4) in a filter plate (Pall AcroPrepTM 96 Filter Plate 0.2 μm GHP, NTRL, 350 μL). The rest of the procedure was the same as for leaf latex tests. A separate trial established that there was no significant difference in thebaine or oripavine results attributable to using acid extraction solution instead of latex extraction solution as used previously.
Table 5 shows the results of the latex testing at the green capsule stage for the M3 Generation. The number of plants randomly sampled and tested is shown as “N”. All plants tested had the same alkaloid profile of high thebaine and substantially no oripavine or morphine.







TABLE 5







Results of the green capsule stage testing. The table shows the percentage of area under chromatogram peaks.
















The-
Oripa-
Mor-
Co-
Papa-
Nosca-
Thebaine




baine
vine
phine
deine
verine
pine
ratio
Norman ratio
























Selection
N
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE





FN1-900-1
23
98.0
0.1
0.5
0.1
0.2
0.0
0.7
0.0
0.6
0.0
0.0
0.0
0.995
0.001
0.99
0.00


FN1-1242-3
24
98.6
0.0
0.2
0.0
0.1
0.0
0.6
0.0
0.5
0.0
0.0
0.0
0.998
0.000
0.99
0.00





Thebaine ratio is the thebaine area divided by the total of the thebaine and oripavine areas


Norman ratio is the sum of the thebaine at oripavine areas divided by the sum of morphine, oripavine, codeine and thebaine areas






When the capsules were dry, the plants were harvested by hand. The harvested capsules were weighed, and then threshed and sieved to separate seed and straw. The straw was sub-sampled and ground to 2 mm.
The straw was extracted using acid extraction solution (5% EtOH, 0.17% H3O4), and analysed using Waters Acquity ULPCC® for alkaloid content against standard alkaloid solutions on a dry weight basis. The loss on drying (LOD) of the straw was determined by heating a sample at 88° C. for 9 minutes using an infrared (IR) balance (A&D Company Ltd Model AD4717, Japan).
Peak area data was used to calculate alkaloid concentration in the straw according to the following calculation:




  
    Alkaloid
    ⁢
    
      
    
    ⁢
    
      content
      ⁡
      
        (
        %
        )
      
    
  
  =
  
    
      
        0.1
        ×
        
          
            SPLA
            ×
            STDC
          
          STDA
        
        ×
        
          (
          
            EV
            +
            
              
                LOD
                ⁢
                
                  
                
                ⁢
                %
                ⁢
                
                  
                
                ×
                SW
              
              100
            
          
          )
        
      
      
        SW
        ×
        
          
            (
            
              100
              -
              
                LOD
                ⁢
                
                  
                
                ⁢
                %
              
            
            )
          
          100
        
      
    
    ×
    
      STDI
      SPLI
    
  




where SPLA is the area under the sample peak of interest
STDC is the concentration of the standard alkaloid in mg/mL
STDA as the area under the standard peak
EV is extractant volume in mL.
LOD % is the loss on drying of the straw, expressed as a percentage
SW is straw weight extracted in grams.
STDI is the volume of standard injected in microlitres
SPLI is the volume of sample injected in microlitres
FN1-900-1 had noticeably low vigour compared with the other lines. The vigour of FN1-1242-3 appeared to be normal. The vigour differences became apparent well after establishment, indicating that it was not a seed quality effect.
Table 6 shows the loss on drying of straw and the mean alkaloid content of the duplicate straw samples as determined by UPLC.







TABLE 6







Amount of capsule, straw and seed harvested, loss on drying (LOD) of straw, and alkaloid content


determined by UPLC.













Yield (kg/line) air
Straw/
Straw
Alkaloid content (dry weight basis)
















dry basis
Capsule
LOD
Thebaine %
Oripavine %

Thebaine
















Selection
Capsule
Seed
Straw
ratio
(%)
(DWB)
(DWB)
Total %
ratio





FN1-900-1
2.91
1.27
1.64
0.56
7.3
3.07
0.01
3.08
1.00


FN1-1242-3
3.33
1.22
2.11
0.63
7.8
3.23
0.02
3.25
0.99





Thebaine ratio is the thebaine content divided by the sum of thebaine content and oripavine content.






The results show that the poppy straw in the two lines FN1-900-1 and FN1-1242-3 are very high in thebaine content, and very low (0.01% and 0.02%, respectively, in oripavine content). There were no other alkaloids (i.e., morphine, codeine, salutaridine, reticuline, laudanine, papaverine and noscapine) detected using the method described.
Growing and Evaluation of M4 Generation
Using seed harvested from the M3 generation, 2 large field plots of FN1-1242-3 and one of FN1-900-1 were grown using commercial equipment and methods. No growth regulator sprays were used. Table 7 summarises the alkaloid contents achieved in these crops. At the first site (Elphinstone, Circular Head district of Tasmania), both selections were grown, along with the Norman parent line, WF03-0802. Hand-picked samples taken from the plots assayed at over 5.0% thebaine, and 0.02% oripavine. Machine harvested samples assayed at 4.65% thebaine and 0.02% oripavine (FN1 lines combined). The Norman parent line in the same paddock assayed at 3.05% thebaine and 0.89% oripavine.
At Roebucks (Merseylea district of Tasmania), the FN1-1242-3 crop assayed at 4.36% thebaine and 0.02% oripavine, whilst the Norman parent line WF03-0802 assayed at an average of 2.08% thebaine and 0.75% oripavine. This data shows that the thebaine-only trait has been inherited into the M4 generation, and that it enables the plants to accumulate very high contents of thebaine.
The method used for the straw analysis is as follows. Ten gram samples of ground straw were extracted with 100 mL extraction solution. The extraction solution comprised 30% ethanol and 10% glacial acetic acid. The samples were shaken for 25 minutes and then filtered through Whatman No. 6 filter paper. The solutions were analysed using a Waters Alliance UPLC system fitted with a Alltech Platinum C18 column, 7 mm×53 mm, with 3 micron packing.
The mobile phase consisted of 8 mL triethylamine, 125 acetonitrile and 950 mL MilliQ water, adjusted to pH 4.1 with phosphoric acid. The flow rate was 3 mL/minute and the column was maintained at 40 C. The alkaloids were detected using a UV detector at 284 nm.
The loss on drying (LOD) of the straw was determined by heating a sample at 88° C. for 9 minutes using an infrared (IR) balance (A&D Company Ltd Model AD4717, Japan).
Alkaloid concentrations were determined by comparison with standard solutions, and results calculated on a dry weight basis. The thebaine and oripavine peaks in these samples accounted for over 98%, and morphine and codeine accounted for less than 1%, of the alkaloid peak area, indicating that there was substantially no morphine and codeine in these samples.







TABLE 7







Summary of commercial results for thebaine-only lines (M4 generation), compared with


parent line.












Grower/Load No
Description
Line
Thebaine %
Oripavine %
Total %
















Elphinstone
1273
Hand
FN1-1242-3
5.10
0.02
5.12



1274
picked
FN1-900-1
5.25
0.02
5.27




samples







1302
Parent line
WF03-0802
3.05
0.89
3.94




control








(machine








harvested)







1312
FN1 lines
FN1-1242-3
4.65
0.02
4.67




combined
FN1-900-1







(machine








harvested)






Roebuck
1047
Parent line
WF03-0802
2.07
0.77
2.84




control







1060
Parent line
WF03-0802
2.09
0.73
2.82




control







1115
FN1 line
FN1-1242-3
4.36
0.02
4.38









Growing and Evaluation of M5 Generation

Seeds of the M5 generation of FN1-1242-3 were sown in a field trial at Gawler, Tasmania on 29 Aug. 2008, along with seeds of the parent line WF03-0802. Both lines were sown in three blocked replications in plots 5 m long by 1.6 m wide. Standard commercial practices were used to grow the trial. No growth regulator sprays were used. The trial was harvested on 19 Feb. 2009 by hand picking all the capsules within 2 m2 quadrats within each plot. The samples were threshed and the poppy straw weighed. After grinding to <2 mm, the poppy straw was extracted and analysed using the same method as used for the M3 generation (described above). Table 8 shows the mean alkaloid contents in the straw and the alkaloid yields per hectare. In this example, the thebaine content of the straw of FN1-1242-3 is 96.3% of the total, and oripavine content is 1.47% of the total, where the total is the sum of morphine, codeine, thebaine and oripavine content of the poppy straw







TABLE 8







Summary of trial results for thebaine-only line FN1-1242-3 (M5 generation),


compared with parent line.












Alkaloid yield




Alkaloid content % (dry weight basis)
(kg/hectare)
Thebaine















Line
Morphine
Codeine
Thebaine
Oripavine
Total
Thebaine
Oripavine
ratio


















WF03-0802
0.13
0.00
2.54
1.71
4.38
29.0
19.6
0.60


FN1-1242-3
0.09
0.00
3.92
0.06
4.07
58.6
0.95
0.98





Total is the sum of the contents of morphine, codeine, thebaine and oripavine.


Thebaine ratio is the thebaine content divided by the sum of thebaine content and oripavine content.


Alkaloid yield is obtained by multiplying the alkaloid content by the straw yield.






EXAMPLE 2
Latex Extraction
Reagent
Latex Extraction Buffer: 23 g of ammonium dihydrogen phosphate was dissolved in approximately 750 mL deionised water and 200 mL of ethanol added, and made up to 1 L with deionised water.
Method
Isocratic Method:
A Pall AcroPrepTM 96 well, 0.2 μm GHP filter plate was placed on a 96 well, 350 μL collection plate. Both filter and collection plate were labeled and 280 μL of buffer pipetted into each well of the filter plate using a multipipette. Using forceps, a leaf tip approx 5 mm×5 mm was torn off from the plant to be tested and added to the extractant. The latex will bleed into the solution over time.
The sample was allowed to incubate at room temperature for at least 30 minutes. The sample was filtered using a vacuum manifold (Pall Corporation product No. 5017). The collection plate was covered with Abgene® adhesive PCR sealing foil (Cat #: AB-0626) to prevent evaporation. The collection plate can be stored in the refrigerator or freezer pending analysis.
Analysis Method
Instrument:

    
    
        Waters Acquity UPLC®, with Sample Organiser and Tunable Ultra Violet (TUV) detector
        Waters Column, Bridged Ethyl Hybrid (BEH) particles, C18, 1.7 μm, 2.1×50 mm
        TUV detector, wavelength 284 nm
    
    


Reagents:
Mobile Phase A—9% methanol, 0.1% formic acid, adjusted with ammonia to pH=9.6
Mobile phase B—91% methanol, 0.1% formic acid, adjusted with ammonia to pH=9.6
Weak Wash—10% methanol
Strong Wash—100% methanol
The Sample Manager option “Load Ahead” was used to save time between samples. With this option, each sample was aspirated ready for injection while the previous one was running.







TABLE 9







Mobile phase settings












Time
Flow Rate





(min)
(mL/min)
% A
% B







0-0.8
0.7
35.0
65.0










The samples were automatically injected (injection volume 2.0 or 3.5 μL) and chromatographed by the Acquity UPLC® along with standard reference alkaloids. After the sample set has been run by the Acquity UPLC®, the peaks were identified by comparison with the standards that were run in the sample set. Typical retention times were as follows:














Alkaloid
Retention time (minutes)









Morphine
0.24



Oripavine
0.27



Codeine
0.31



Papaverine
0.38



Thebaine
0.42



Noscapine
0.68










The separations obtained using this method are shown in FIG. 1. Although the peak shapes and separations are not perfect, they are quite adequate for a very rapid screening method.
Empower software (Waters Corporation, Milford, Mass.) was used to identify peaks and calculate peak areas. The data was then exported to an Excel spreadsheet where peak area data was used to determine which poppies had unusual alkaloid profiles.
Gradient Method:
For more accurate repeat analysis of samples, a 2.5 minute gradient UPLC method was used. It is the same as described above, except that the following gradient conditions were used. FIG. 2 provides a chromatogram of the poppy straw of the M3 generation of FN1-1242-3. The injection volume of the sample in FIG. 2 was 2.0 μL. ATA indicates thebaine, ori indicates oripavine peak, AMA represents morphine, and ACA represents codeine.







TABLE 10







Instrument method details for 2.5 min gradient method











Time
Flow Rate





(min)
(mL/min)
% A
% B
Curve














0
0.8
75.0
25.0



1.4
0.8
1.0
99.0
6


2.5
0.8
75.0
25.0
1









EXAMPLE 3
Determination of Genetics of Thebaine-only Trait
Crosses were conducted between FN1 lines with low content of oripavine (<2% of combination of morphine, codeine, oripavine and thebaine) and normal poppy lines containing morphine. Plants of the first F1 generation all contained morphine indicating that the genes responsible for the thebaine-only characteristic are recessive. The F1 plants were self pollinated. Seeds were collected from the F1 plants, and sown in trays. When the plants were at the 6-leaf stage, latex testing was conducted to determine the chemotypes of the individual F2 plants.
Latex testing was done according to the method of Example 4 by removing the tip of the youngest fully expanded leaf and placing it in acid extractant buffer in a Pall filter plate. After allowing time for the latex to bleed from the leaf into the buffer, the extractant was vacuum filtered into a 96 well plate and sealed. The samples were analysed by UPLC using the method shown in Example 4.
The alkaloid concentrations were calculated from the peak areas by reference to standard alkaloid solutions, and each alkaloid was converted to a percentage of total morphinan alkaloid (morphine, codeine, thebaine and oripavine) in order to determine the chemotype of the plant. A set of rules incorporated in nested “IF” statements was established to determine chemotype. The rules, applied sequentially, were as follows:
If total concentration of morphine, codeine, thebaine and oripavine is less than 5 ug/mL in injected solution, no result.
If Noscapine percentage >15, chemotype=Noscapine.
If Thebaine percentage >98, chemotype=Thebaine-only.
If Thebaine+Oripavine percentage >95, chemotype=Norman.
If Thebaine+Codeine percentage >96, chemotype=Codeine.
If Morphine percentage >2, chemotype=Morphine.
Otherwise, the chemotype was classified as OCT, which indicates that the plant contained oripavine, codeine and thebaine.
Four chemotypes were identified in the populations:
Morphine: morphine present typically with thebaine and codeine
Norman: thebaine and oripavine both present, substantially no morphine
Thebaine-only: thebaine present, substantially no oripavine and morphine
Codeine: thebaine and codeine present, substantially no morphine
The occasional plant was identified as OCT. These generally were very small plants that fitted into one of the four categories as they further developed. Overall, 0.18% of plants were classified as OCT and 0.03% were classified as noscapine and were ignored in calculation of ratios for this analysis.
Chi square tests were conducted to determine if the observed segregation patterns differed significantly from a 9:3:3:1 segregation of chemotypes (Morphine: codeine: Norman: Thebaine-only respectively).
Table 11 shows the results of the 9 populations derived from FN1 parents having a thebaine-only chemotype. Five of these fit a 9:3:3:1 ratio (P<0.05). Of those that didn't fit the expected ratio, FN1-1242 is notable, in that it had more than the expected number of plants with the codeine chemotype, whilst all other lines that did not fit the ratio had less than expected codeine plants. Further work was conducted with progeny of FN1-1242 in which plants were grown to hook stage, and leaf latex was collected and classified into chemotypes. The distribution was 96:41:42:11, which gives a Chi square test of 3.1 which indicates that there is no significant difference from the expected ratio.
The segregation into 4 chemotypes indicates that two separate genes are involved in the thebaine-only chemotype. One of these is the Gene associated with the Norman poppy mutation (described in U.S. Pat. No. 6,067,749). The second gene is responsible for the new low-oripavine or thebaine-only trait, which is responsible for blocking the pathway between thebaine and oripavine. These two genetic changes work together in lines described herein to provide the poppy plants with a high thebaine content and a low oripavine content.







TABLE 11







Segregation patterns for populations using FN1 lines with <2% oripavine in alkaloid profile.













Chi square test



Alkaloid content in straw of FN1

result



M2 plant as percentage of sum of

(ns indicates no



morphine codeine, oripavine and
Observed ratio
significant



thebaine
Morphine:codeine:Norman:thebaine-
difference from











FN1 line
Oripavine %
Thebaine %
only
9:3:3:1 ratio)





FN1-846-2
0.24
98.4
39:14:11:4
0.39 ns


FN1-1719-1
0.31
99.5
45:10:1:0
17.9 <0.001


FN1-2199
0.38
99.6
59:25:18:6
1.5 ns


FN1-1242
0.41
99.2
271:112:81:16
12.81 <0.01


FN1-900
0.58
98.8
191:73:55:19
2.79 ns


FN1-2152
1.38
98.6
73:2:28:14
26.7 <0.001


FN1-1571
1.63
97.9
604:58:248:69
114.7 <0.001









EXAMPLE 4
Leaf Latex Analysis
Reagent
Acid Extractant, A 1 L measuring cylinder was half filled with deionised water. 1 mL of conc. phosphoric acid and 50 mL ethanol were added and the volume made up to 1 L with deionised water.
Method
A Pall AcroPrepTM 96 well, 0.2 μm GHP filter plate was placed on a 96 well 350 μL collection plate. Both filter and collection plate were labeled and 280 μL of acid extractant pipetted into each well of the filter plate using a multipipette. A tip of the youngest fully expanded leaf was torn off each plant to be tested and added to the extractant. The latex bleeds into the solution over time.
The samples were allowed to incubate at room temperature for at least 30 minutes. The samples were filtered using a vacuum manifold (Pall Corporation product No. 5017). The collection plate was covered with ABgene® adhesive PCR sealing foil (Cat #: AB-0626) to prevent evaporation. The collection plate can be stored in the refrigerator or freezer pending analysis.
Analysis Method
The samples were analyzed using the same instrument, reagents and instrument method details (see Table 10) as described for the gradient method in Example 2.
The samples were automatically injected and chromatographed by the Acquity UPLC® along with standard reference alkaloids. After the sample set has been run by the Acquity UPLC®, the peaks were identified by comparison with the standards that were run in the sample set. Empower software (Waters Corporation, Milford, Mass.) was used to identify peaks and calculate peak areas. The data was then exported to an Excel spreadsheet where peak area data was used to determine alkaloid profiles.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the following claims and their equivalents.

## Claims

1. A poppy plant of Papaver somniferum having a stably heritable thebaine-only chemotype which plant upon the harvesting of its poppy capsules will yield a poppy straw having thebaine constituting about 95% by weight or greater of an alkaloid combination, and having oripavine constituting about 5% by weight or less of the alkaloid combination, wherein the alkaloid combination comprises morphine, codeine, thebaine and oripavine; and wherein thebaine constitutes about 3.0% or greater of the poppy straw on a dry weight basis,
wherein the thebaine-only chemotype is provided by two independent genes, the first preventing thebaine being converted to neopinone, and preventing oripavine being converted into morphinone, and the second blocking the pathway between thebaine and oripavine.

2. The plant of claim 1, wherein thebaine constitutes about 3.5% or greater of the poppy straw on a dry weight basis.

3. The plant of claim 1, wherein thebaine constitutes about 97% by weight or greater of the alkaloid combination and oripavine constitutes about 0.7% by weight or less of the alkaloid combination.

4. The plant of claim 1 wherein there is substantially no morphine or codeine in the alkaloid combination.

5. The plant of claim 4 wherein there is substantially no oripavine in the alkaloid combination.

6. The plant of claim 1, wherein oripavine constitutes about 0.4% or lower of the poppy straw on a dry weight basis.

7. The plant of claim 6, wherein oripavine constitutes about 0.2% or lower of the poppy straw on a dry weight basis.

8. The plant of claim 1, wherein oripavine constitutes between 0.02% to 0.5% by weight of the alkaloid combination.

9. A seed of the plant of claim 1.

10. An opium for the extraction of thebaine comprising an opium of the plant of claim 1.

11. A concentrate of poppy straw for the extraction of thebaine comprising a concentrate of poppy straw of the plant of claim 1.

12. A method for the production of thebaine which comprises the steps of:
a) harvesting poppy capsules of the plant of claim 1 to produce a poppy straw; and
b) chemically extracting the thebaine from the poppy straw.

13. A method for the production of thebaine which comprises the steps of:
a) collecting and drying the latex of the immature poppy capsules of the plant of claim 1 to produce opium; and
b) chemically extracting the thebaine from the opium.

14. A stand of stably reproducing Papaver somniferum plants of claim 1.