What type of poppies for opium

This minimal processing leads to the retention of cellular material and maintains the integrity of the DNA. The challenge for raw opium is in the purification of DNA from inhibiting compounds and the texture of the sample. The first experimental objective was to identify the lowest raw opium sample mass that would provide reliable and robust results.

The absorbance data obtained from the spectrophotometer provides an indication of sample purity, and thus indicates potential PCR performance. The results indicate that the 0. The target mass may vary due to the multitude of different characteristics displayed in an illicit opium sample, e.

The differences in quality of the material dried, gummy or cooked will likely play the most significant role in determining the amount of starting material Supplementary Information: Fig.

A sample that is overly desiccated and exposed to high temperatures cooked will undoubtedly require an increase in the amount of starting material. The second study focused on identifying an efficient DNA extraction method that results in an acceptable yield and purity of DNA.

The Qiagen-based method will likely result in an increasingly pure extract when compared to the Promega method, with values of 1.

The average ratio for the Qiagen-extracted samples improved to 1. The higher moisture content of the opium samples had a negative impact on the quality of results. To mitigate this problem we attempted two approaches: pretreatment with liquid nitrogen and air-drying. The preferred sample pre-processing method included air-drying in a fume hood and, as needed, treatment of the sample with liquid nitrogen followed by physical disruption with a mortar and pestle and bead-beating.

This greatly increased the surface area of the opium sample exposed to the lysis buffers while simultaneously decreasing the activity of any endogenous DNA degrading DNases.

We recommend extracting poppy DNA from 0. The sample quality was more advantageous in the Qiagen extraction method; however, it is also significantly more labor intensive and, when considering the ratio standard deviations, the two methods perform similarly. The DNA isolation method developed for latex is similar to the method developed for opium. Latex samples are commonly resuspended in water for long-term storage; therefore, the opium extraction method was modified to include the concentration of biological material.

Latex samples had a lower DNA yield and low quality values Table 8. Like heroin, we expect this workflow to yield high amounts of amplifiable template DNA. One gram of samples EE uncooked and OpC-1 cooked were extracted with the Qiastool kit; InhibitEX buffer and lysis reaction volumes were doubled to ensure samples were properly lysed and washed. Quantitative PCR was performed using the 2-stage amplification and the N primer set.

The results indicate that DNA originating from both cooked and uncooked opium have been successfully extracted using the Qiastool kit and amplified using the 2-stage amplification method Fig. In contrast, the cooked opium OpC-1 had a low yield exhibiting a Cp of Although both cooked and uncooked opium were successfully amplified, the DNA yield of the cooked opium may be improved through the extraction of higher amounts of starting material; therefore, the current optimal mass of starting material for extractions from cooked opium will be 1.

The N, N and N loci contain microsatellite sequences; therefore it is possible that size polymorphisms exist within the two genomic copies of the DNA in the diploid genome of a poppy plant. Opium sample EE-2 amplified with N and N yielded a single primary allelic peak homozygote , with an amplicon length of The amplification using primer N yielded two primary allelic peaks heterozygote with amplicon sizes of and bases.

The resulting electropherograms also exhibit minimal artifactual noise, such as non-specific allelic peaks caused by electrical spikes or —A peaks caused by the non-template dependent terminal adenylyl-transferase activity inherent to many DNA polymerases, including the Roche Fast Start High Fidelity DNA polymerase. The stutter products correspond to products that are one or two repeat units smaller than the allelic peak. For example, the N locus contains a tetra-nucleotide repeat default n which, when amplified, resulted in the expected allelic peak at bases peak height rfu and a minor stutter peak at bases peak height: rfu.

The results demonstrate that Promega-isolated opium samples are of sufficient quality and quantity to yield amplifiable DNA and interpretable results. Select electropherograms obtained from the PCR amplification and subsequent CE-analysis of raw opium samples top-primer N, middle-primer N, bottom-primer N X-axis is fragment size in bases and Y-axis is relative fluorescent units.

Note, a free-dye artifact is present at approximately bases; this is independent of the sample and can be disregarded from analyses. PCR conditions were optimized in anticipation of the low quality and quantity of DNA encountered in processed opium and heroin samples. The EE opium sample was anticipated to exhibit elevated levels of inhibition and low DNA quantities due to characteristics such as moisture level, color and texture Fig.

The combined enzyme master mix with a PCR buffer pH of 9. The leaf sample displayed consistent amplification C p within each assay group. This was expected and served as a benchmark for the lower quality EE opium sample. To the best of our knowledge, this paper is the first to demonstrate successful DNA extraction of opium poppy DNA from heroin samples. The primary challenges were the low quality and quantities of DNA expected in processed opium and heroin samples coupled with the challenges posed from sample matrix issues and cutting agents.

In that regard, the successful extraction of DNA fragments is very significant. The quantities, as measured through the qPCR crossing point values, were low; however, this was not unexpected. The most critical finding was that we were able clearly distinguish opium poppy amplification plots from negative controls and background noise.

Preliminary data indicate that we have successfully sequenced poppy DNA for some of these samples data not shown. The opium gum or latex contains a mixture of alkaloids, terpenoids, phenols, and various proteins Opium is simply the dried latex that is collected from the opium poppy. It is typically in the raw form only dried or in the cooked form where raw opium is boiled, filtered to remove impurities, and reconstituted for heroin production.

The development of a successful DNA isolation strategy relies heavily on the ability to address the complexities present in latex or opium gum samples. Despite the increased quality metrics, the Qiagen procedure remains more labor intensive and less reliable compared to the automated Promega methods. Despite this, we recommend the use of the Qiagen stool kit as it proved to be more versatile.

In order to improve the DNA yield and overall quality of heroin samples further, we employed a 2-stage amplification reaction using two DNA polymerases. This dual polymerase system attempts to balance the amplification of damaged DNA while maintaining a high fidelity and minimize errors. This method led to more consistent responses than either polymerase alone. The combined effects of exceedingly low levels of damaged template DNA and the presence of inhibitors necessitated the modification of conventional analytical methods to increase sensitivity, for example, the cycle, 2-stage amplification mentioned above.

The combined use of these methods resulted in the successful detection of opium poppy DNA from heroin and opium samples, but it also may have contributed to the low-level activity observed in the no-template controls. The most commonly detected non-specific signals are due to the formation of primer dimers. The primer concentration can be lowered to avoid dimerization. In addition, the use of such a high concentration is likely unnecessary when amplifying low-copy samples like those obtained from heroin extracts.

The activity of the NTC is on average 4. The heroin-positive samples exhibited T m s distinct from the activity detected in the NTC with clear differences in curve morphology Supplementary Information Fig. In addition, the signal obtained from NTC samples are reproducible across amplification reactions and instrument runs.

We conclude that the NTC activity is not responsible for the primary signal obtained from the heroin positive samples. While we believe that our proposed methods perform sufficiently well with high purity heroin samples i. Our inventory of opium and heroin samples included over samples consisting of white and brown powder and black tar heroin, as well as several types of opium. However, these samples were also limited in quantity due to the nature of the seizures in source countries.

In addition, few samples had ample mass to enable replicative comparisons of the methods we evaluated in this study. This limited the depth of our sub-studies and we recommend that the data presented herein be used as a starting point for further refinement of the procedure to extract opium poppy DNA from heroin. For example, our small whole genome amplification sub-study indicated that there was no benefit to the use of these methods.

Due to the nature and type of samples, any research group would face similar limitations to successful identification of poppy DNA caused by sample availability. The ability to obtain an opium poppy DNA profile from heroin opens a host of new avenues for law enforcement. This genetic information, through the use of high throughput sequencing, will likely enable the identification of the source country or region of seized heroin and positive identification of poppy-derived illicit drugs.

This lays the foundation for the information that can then be used for interdiction of the drug trade from a local to a global scale. Source information may provide valuable intelligence leading to the interruption of this terror-funding stream or other illegal activities. Louis, MO. The reagent blanks consist of all reagents used in the extraction and without added DNA and were processed and analyzed quantified and amplified in concert with all experimental samples.

DNA was extracted from opium gum by first mechanically pulverizing via mortar and pestle, either 1. Latex samples are typically suspended in water; therefore, the extraction technique in this method was similar to that used for opium, but it required removal of the aqueous phase.

The remaining procedure is identical to the opium procedure. Black tar heroin is particularly difficult to handle due to the gummy yet dense nature of the sample. Another important alkaloid is codeine which is considered as a prodrug because it is converted into morphine and codeineglucuronide C6G in in vivo [ 12 , 13 ]. Codeine is a natural isomer of morphine and is formulated as 3-methyl morphine.

Codeine is less effective and has lower dependence-liability than morphine [ 13 ]. Similar to all other opioids, continuous use of codeine induces physical dependence and it can be psychologically addictive. However, mild effects are caused due to its withdrawal, so is less addictive than other opiates. Codeine is also used as antitussive drug against coughing and widely used in the treatment of severe diarrhea and diarrhea predominant bowel syndrome.

In addition to analgesic and antitussive effect there are some side effects of codeine which includes euphoria, itching, drowsiness, vomiting, orthostatic hypotension, urinary retention, depression and constipation [ 16 ].

One of the most serious adverse effects includes respiratory depression [ 17 ]. Another alkaloid thebaine is also produced which is non-narcotic in nature can also be used as an analgesic. It is used for the production of oxycodone and other semi-synthetic analgesic opiates [ 18 , 19 ].

Higher doses of thebaine cause convulsions similar to that of strychnine poisoning [ 20 ]. Another important constituent in opium latex is noscapine which is used in relieving cough and headache. Researchers are continuously investigating of its use in treatment of several cancers and hypoxic ischemia in stroke patients.

Early studies in the treatment of prostate cancer are very promising [ 21 ]. Scientists have found a noticeable decrease in mortality in patients treated with noscapine [ 22 ].

Noscapine is non-addictive, widely available, has low incidence of side effect and can be easily administered orally, prompting a huge potential for its use in developing countries. Papaverine is used in the treatment of spasms of the gastrointestinal tract, bile ducts and ureter.

It is also used as a cerebral and coronary vasodilator in subarachnoid hemorrhage combined with balloon angioplasty and coronary artery bypass surgery [ 21 , 23 - 25 ]. Papaverine is also used as an erectile dysfunction drug alone or sometimes in combination with phentolamine [ 26 , 27 ].

During microsurgery, papaverine is used as a smooth muscle relaxant and is directly applied to blood vessels [ 28 , 29 ]. It is also applied in cryopreservation of blood vessels along with other glycosaminoglycans and protein suspensions [ 21 , 30 ].

Papaverine also functions as a vasodilator during cryopreservation when used in conjunction with verapamil, phentolamine, nifedipine, tolazoline or nitroprusside [ 22 , 31 ].

Scientists are continuously investigating for its use as a topical growth factor in tissue expansion with some success [ 23 ]. All these effects of papaverine are attributed to its inhibitory effect on phosphodiesterases [ 32 ].

Though papaverine has such extra ordinary attributes but has some common side effects which include polymorphic ventricular tachycardia, constipation, increased transaminase levels, increased alkaline phosphatase levels, somnolence and vertigo.

The area under poppy cultivation varied according to the total demand of opium put through the United Nation. Keeping in mind, the enormous importance of opium poppy among the diverse array of medicinal plants, researchers were encouraged to work for its genetical improvement. Researchers engaged in opium poppy researches are continuously working to develop designer plants having all specific alkaloids in latex in large quantities.

Previously, both conventional and molecular approaches have been applied to develop varieties rich in specific alkaloids. This chapter deals a detailed account in different subheadings of the conventional breeding techniques applied to upgrade the latex and alkaloid status along with its nutritional content in opium poppy. Since, opium poppy is widely and commonly used for dual purpose i. The different breeding objectives are depicted in following subheadings.

However, they have been able to develop several high opium yielding varieties, but yet it is not able to fulfill the pressure created due to enhance global demand raised as a consequence of population growth. At present our scientists have been able to discover more than 80 alkaloids of immense medicinal importance.

Despite of their best possible efforts to identify more and more alkaloids, the demand for five major alkaloids i. The importance of these five major alkaloids has been discussed earlier. Previously, morphine being the main pain killer was in high demand, for which our scientist made great success in development of high morphine containing varieties.

But now a days, the demand for specific alkaloids i. The scientists are now trying to develop varieties with specific alkaloid in opium latex through conventional and molecular techniques.

Opium poppy is a narcotic crop, due to the presence of morphine narcotic constituent in major proportion of opium latex. In recent days, scientists are working to develop low morphine or morphine less varieties to check its illegal cultivation. The development of low morphine or morphine less varieties can also help Narcotics Department, as it will not require issuing license for growing opium poppy to the cultivators.

Globally, different group of researchers are engaged in this direction using both conventional and molecular approaches. Poppy seeds having high nutritive values are also in high demand and major emphasis has been given for the development of food grade poppy which can only be possible, if opium-less poppy varieties can be developed.

The development of such varieties can assist opium cultivators to grow food grade poppy without any restriction or permission in form of license. Seeds of opium poppy have high value in global market which puts a great pressure on plant breeders to develop high seed yielding varieties that can substantiate the ever increasing global demands.

The importance of poppy seeds has been described earlier in details. However, many high seed yielding varieties have been developed but since global population is increasing at an enormous rate, plant breeders are continuously putting their best possible efforts to capture this ever increasing demand. Diseases are major problem in cultivation of any crop.

The development of multiple disease resistant varieties is in need from very long time in opium poppy. A number of diseases occur which ruins the entire crop and ultimately the opium products. Several researchers especially plant breeders have faced many challenges during specific breeding objectives due to severe disease in opium poppy.

Our scientists have put their best possible efforts and continuously trying to develop such varieties resistant to major diseases through molecular and conventional tools. One of the major hindrances in any successful breeding program is the prevalence of certain fungal, bacterial, insect borne diseases etc. Opium poppy crop is highly susceptible to certain diseases but the most contagious diseases are caused by fungus results high losses in yield.

Some commonly found fungal, bacterial, viral and pest related diseases in opium poppy are summarized below Downy Mildew: The causal agent for this most serious and widely spread disease of opium poppy is Peronospora arborescens. The symptoms include hypertrophy and curvature of the stem and flower stalks. The infection starts spreading upwards from the lower leaves and the entire leaf surface gets covered by brown powder.

The plants dies prematurely as the stem, branches and even capsules are also attacked by this causal organism. In India, the disease appears annually on the crop from seedling to maturity stage mainly in the areas of Madhya Pradesh, Uttar Pradesh and Rajasthan.

Capsule formation is also adversely affected due to infection causing significant reduction in opium yield. The primary inoculum of the pathogen is oospore which is present in infested soil and leaf debris introgresses through underground plant parts and infects the plant giving rise to stunting and chlorotic syndrome etc in the fields of opium poppy [ 34 ].

The major control measures of the disease include disinfection and spraying of the seed beds with 0. Some other control measures include use of Bisdithane 0. Powdery Mildew: This disease is caused by Erysiphae polygoni and causes severe losses in opium production. It caused severe damage to poppy in Rajasthan in The symptoms appear in late stages of plant growth with white powder on the surface of leaves and capsules.

The control measures include field sanitation along with spray of Spersul 0. Decline in seed yield, premature death of infected plant appears with the progress of disease in plants [ 35 ]. Seed borne diseases: Seed borne diseases are also a curse to opium poppy crop both in terms of production and yield. The major effect of seed borne disease is on capsules and seeds only, which results reduction in germination percentage and seedling delays.

Some commonly spreading seed borne diseases have been discussed. Leaf Blight causal agent - Pleospora calvescens : Symptoms include defused yellow spots followed by premature drying of infected leaves. During the course of pathogenesis, toxins are released by the parasites enabling it to assimilate the requisite nutrient.

High temperature and heavy rainfall favors the disease. Seedling Blight causal agents - Phytium ultimum and Phytium mamimmatum : Few studies undertaken on characterization of the disease revealed that the disease affects physiological process in poppy. However, no control measures could be found with total control effects. Leaf Spots causal agent - Helminthosporium sps. The disease is not of much importance, but due to correlation between opium alkaloids and leaf spot, it may be considered harmful.

Several control measures to control the disease include seed disinfection or spraying of seed beds with 0. The infection appears at the stem base followed by damping of roots. The disease causes reduction in opium yield and can be controlled only by the removal of infected plants. Diseases caused by bacteria: It would be worthwhile if there is a lack of description of bacterial disease in opium poppy.

Since the bacteria are ubiquitous in nature, opium poppy is also not left by bacterial infection where heavy losses occur. Systemic infection prevails with the entry of bacterium through stomata and aquapores in later stage of growth.

Multiplication of the bacterium starts in vascular system. Seeds are malformed and discolored as a result of infection. Plant parts are also damaged due to bacterial infection. Diseases caused by viruses: There are certain viral diseases in opium poppy which are caused by Cabbage ring spot virus, beat yellow virus etc. The symptoms include yellowing of plants, elongation of stem, irregular chlorotic bands along the veins, stunting etc.

These viral disease cause heavy losses to poppy crop in terms of seed and opium yield and sometimes the whole plant dies results total loss of crop. Diseases caused by insect and pests: Apart from different diseases caused by fungus, bacteria, viruses etc. The most common among them are those damaging roots i. Root Weevil, damaging leaf and stem i. Aphids, floral damage i. A brief description of these are summarized here. Root weevil causal agent - Sternocarus fuliginosus : This pest is known for maximum damage to poppy crop by boring into upper parts of the roots which ultimately turns blackish and leaves wither due to chlorosis while the larva mines the leaf lamina.

However, the application of lindane 1. Cutworm causal agent - Agrotis spp. It mostly targets young plants destroying basal part of the stem while the adult, brown in color and dark color spots on wings also destroys the crop severely.

Additionally, poison bait with rice bran, jiggery and carbonyl can also be used as preventive measures. Aphid causal agent - Myzus persicae : This is also another major pest of opium poppy crop. The nymphs and adults suck the leaf sap results damage of leaf and consequently whole plant.

The adults are yellowish green rarely reddish. The control measures include spray of neem oil 0. However, natural enemies like coccinellid beetle can also be encouraged. Capsule borer causal agent - Helicoverpa armigera : The capsule borer is also a serious pest in opium poppy which harms capsule to a maximum extent.

It destroys whole capsule eating up the floral head and seeds. Saunders, N. The poppy. Oneworld Publications, London. A long history of medicinal value The flowers of the field poppy have long been used as a painkiller for soothing mild aches and pains e.

In Flanders fields the poppies blow. Between the crosses, row on row But not all scars are visible. Epilogue Many millions died in wars before the field poppy became the symbol of remembrance; many more will do so. References Bimonte, S. Potter, J. Seven flowers and how they shaped our world.

Small drops of white, milky juice then begin to flow from the incision, and exposed to the air solidify and grow darker.

The incision must be neither too shallow nor too deep. If it is too shallow, the number of latex vessels affected may be too small and too little juice will drip out; if the incision is too deep, so that the capsule is entirely cut through, the latex will flow into the capsule and the whole yield will be lost.

The incisions are made with special knives or with special tools, consisting of wooden handles fitted with sharp pieces of iron often razor blades. The size of the blade automatically regulates the depth of the incision. Tools with two or more blades are also used, to allow two or three cuts with a single motion.

As soon as the incision is made, the latex begins to drip. In order to avoid brushing against the capsules, the harvesters making incisions must walk backwards. Unlike the procedure in certain Far Eastern countries the incisions are never repeated. At dawn the following day, the gathering of the raw opium begins; this consists of collecting the solidified drops of latex which have gathered overnight around the incision in the capsule.

The opium is stripped from the capsule with a special blunt knife and is collected in a conical vessel attached to the harvester's belt or into a rumex or poppy leaf. It is then kneaded until it becomes dark, dried for a short time and thereupon is formed into to 1. That is the form in which it is delivered by the producers. It has been proved that two, three or more incisions yield more opium, but each subsequent incision produces opium with a lower morphine content.

In countries which used to supply the opium smoking market, manifold incisions of the capsules were profitable, since more opium could thus be obtained.

This opium is at the same time more suitable for smoking because of its lower morphine content. Yugoslav opium has an unpleasant taste and a high morphine content and has therefore never been in demand on the smokers' market nor exported for this purpose. It is intended only for the Western market, where morphine content is in demand.

Accordingly, manifold incision of capsules is not profitable. The fact that the capsules are cut only once accounts for the considerably greater output of opium per hectare in Far Eastern countries than in Yugoslavia.

Between 0. The output per hectare varies between 4 and 15 kg, according to the place and year. About three weeks after the opium harvest, the plant becomes fully ripe and the capsules are collected and broken up for the seeds, which are used to manufacture oil. Between and kg of seeds are obtained per hectare, according to the place and year. The cultivation of the opium poppy is highly profitable and, at the same time, very risky for the producer. One advantage is that nearly the whole plant can be used.

Opium and poppy seeds are commodities for which there is always a market. The relatively high value of these products in relation to their volume is a highly favourable factor for producers in parts of the country with poor communications. Continuous contact with the outlet, which is essential, for example, in market gardening, is not required in this case, and opium can be kept for a long time without any danger of deterioration in quality storage leads to a decrease in the moisture, but the amount of morphine remains unchanged.

The seed cakes left after the oil has been extracted are used as cattle food, the dry capsules are sold as raw material for the production of alkaloids and the remaining straw is used as litter and as fuel to replace wood a large proportion of the opium region is poor in timber. The relatively quick growth of the plant often makes it possible to use the soil for yet another crop in the same season, while the early harvesting of opium gives the producer his first income of the year.

The main cultivation operations take place at a time when no other agricultural work has to be done. Finally, some kinds of soil are suitable only for cultivating the opium poppy and yield considerably less of any other crop. On the other hand, this is one of the agricultural crops exposed to the greatest risks, which persist throughout the growing period of the plant. Sowing takes place in the autumn, after the first autumn rains moisten the earth.

If these rains fall too late, the plant is insufficiently developed by the winter, and is therefore sensitive to cold and may be destroyed even in a mild winter.

Another risk of crop failure is that of a hard winter, especially without snow. An unduly mild winter is also unsuitable, for the plants can develop too fast and the opium gathering period may occur too early, when rains are more frequent. A wet spring can promote the development of plant diseases to which the poppy is very sensitive and too much moisture in the ground makes the latex too thin and causes it to trickle out of the capsule on to the ground after incision.

The chief danger of the producer, however, is rain at the time of the harvest, as the harvest can be potsponed for only a few days.

Rain during the night following the incising of the capsules may entirely wash away the drops of latex and completely destroy the crop. Even dew can cause serious damage at that period. In the case of spring cultivation, there is the further risk of dry weather setting in before the plants are sufficiently developed, so that they may wither without yielding either opium or seeds.

The opium poppy is one of the most intensive agricultural cultivations and requires a great deal of manpower; the poppy needs constant care throughout its period of growth. Approximately 30 days of team work and manpower days including collection of opium and seeds are required to cultivate one hectare.

A shortage of specialized labour at harvest-time is yet another risk to which the cultivation of the opium poppy is exposed. The hazards of opium poppy cultivation may best be seen from table 1.

While 70, kg were produced in , only 2, kg were produced in The mechanization of agriculture cannot make the production of opium much cheaper. Apart from ploughing, sowing and winnowing the seeds with ploughs, seeders and winnowers the remaining operations can be performed only by human hand.

Labour takes up from 80 to 90 per cent of the production costs. From above it is evident that opium is not suitable for large state or co-operative farms, using modern agricultural techniques, nor for large areas in general.

However, the opium poppy is being cultivated also on state and co-operative farms, but over, relatively small areas, as well as on individual farms.