3.2.4. Optimization of the Tray Numbers

The tray numbers directly affect the equipment investment cost and energy consumption, so a reasonable number of trays must be used for the minimization of the TAC. First, the number of the HPC trays is fixed, and that of the LPC trays is gradually changed. The corresponding TAC is calculated, and the specific results for each case are listed in Table 2. The flow rate of the recycle stream gradually decreases as the number of trays increases, and the smallest TAC is obtained when the number of the LPC trays is 23.

**Table 2.** Specific results for the tray number of the low pressure column (LPC).

The number of the LPC trays is fixed 23, the number of the HPC trays is changed, and the corresponding TAC is calculated. The specific results for each case can be seen in Table 3. The smallest TAC is obtained when the number of the HPC trays is 17.

**Table 3.** Specific results for the tray number of the high pressure column (HPC).

### 3.2.5. Process Optimization Results

The optimization results of the whole process are shown in Figure 8. The number of the LPC trays is 23, and the acetonitrile–water mixture and the recycle stream are fed at 20th and 16th, respectively, with a reflux ratio of 0.2. The number of the HPC trays is 17, the feeding position is 9th, and the reflux ratio is 0.7. Finally, the acetonitrile content in the wastewater at the bottom of the LPC is less than 0.1%, and the purity of acetonitrile product at the top of the HPC is 99.9%, which meets the separation requirements.

**Figure 8.** Optimized flow diagram of the pressure swing distillation (PSD) with full-heat integration.

#### *3.3. Comparison of Two Technological Processes*

After the optimization of the whole process, the two processes are compared. The results in Table 4 show that the TAC of the PSD without heat integration process is higher than that of the full-heat integration process. The latter can effectively reduce equipment investment and operation cost, thereby reducing the TAC to 515.45 × 103 \$/y. Thirty-two point three nine percent of the TAC of the PSD with full-heat integration is saved compared with that of the process without heat integration.

**Table 4.** Comparison of two technological processes.

#### **4. Conclusions**

PSD was used to separate acetonitrile and water mixture. The sensitivity of the acetonitrile–water azeotrope concentration to the pressure changes indicates the feasibility of PSD. The distillation sequence of the LPC and HPC was determined through a phase diagram. The pressures of the LPC and HPC were set to 1 and 4 atm, respectively. The final purity of the acetonitrile product reached 99.9%, and the content of acetonitrile in wastewater was less than 0.1%, which met the separation requirements.

Based on the principle of the minimum TAC, the design variables of the PSD without heat integration were optimized, and the optimum process parameters were obtained. Then, PSD with full-heat integration was optimized. The two processes were compared, and the results showed that 32.39% of the TAC of the PSD with full-heat integration is saved compared with that of the process without heat integration. Therefore, it is more economical to separate acetonitrile and water mixture by PSD with full-heat integration. This study provides technical support for the separation design of such azeotropes.

**Author Contributions:** J.L. and K.W. conceived and designed this case-study as well as wrote the paper; M.L. and Z.L. reviewed the paper; All authors interpreted the data; T.D. substantively revised the work and contributed the process simulation.

**Funding:** This work is financially supported by Guizhou Province United Fund (Qiankehe J zi LKLS[2013]27), Excellent engineers education training plan (LPSSY zyjypyjh201702), Guizhou Solid Waste Recycling Laboratory of Coal Utilization ([2011]278), Guizhou Province United Fund (Qiankehe LH zi [2015]7608) and Academician Workstation of Liupanshui Normal University (qiankehepingtairencai [2019]5604).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**6. Ongoing Studies**

Current efforts to improve diagnostics of FMD aim to identify screening methods that would have a high sensitivity and specificity for FMD, and be easy to use, cost-effective, and available in the clinical practice. Preliminary analysis of the very high-Frequency Ultrasonography for arterial phenotyping in patients with Cervico-cerebral artery dissection, Hypertension, Spontaneous coronary artery dissection and fibromuscular dysplasIA (FUCHSIA) study showed an eutrophic remodelling of the radial artery wall in 11 hypertensive female FMD patients with a peculiar "blurred" pattern [41]. Similar images have also been identified in 5 female SCAD patients [55]. New information is also expected from the Pathophysiological Mechanisms of Fibromuscular Dysplasia (MeDyA) study (ClinicalTrials.gov Identifier: NCT01935752), a pathophysiology study designed to assess endothelial function in patients with FMD compared to hypertensive and healthy controls, and to identify possible plasmatic biomarkers (circulating microparticles, circulating microRNAs, endothelial proteins), and vascular echo-tracking abnormalities related to the FMD.

From a genetic and pathophysiologic point of view, the Defining the Basis of Fibromuscular Dysplasia (DEFINE) study (ClinicalTrials.gov Identifier: NCT01967511) is in the process of recruitment. The researcher's aim is to establish functional, molecular and genetic profiles of fibroblasts from FMD, SCAD and CeAD patients as compared to matched control subjects, in order to dissect the core biologic mechanisms underlying these disorders. Such data should allow to identify similarities and differences in pathophysiology, which could help stratify patients by using biological markers.


**8. Screening and evaluation for oil and fatty acids**

phologic and agricultural traits and morphine content in husk in relation to ideotype, which in these indicators represents 100% of the value. They observed lower values of morphine in husk of white coloured seeds while high morphine in blue to grey seeds. They observed that these cultivars achieved very good values in the morphological indicators and average value in the economic indicators. Based on their results they concluded that these results will be used in selection and classification of suitable genetic resources of poppy as industrial forms. Nemeth-Zambori et al. [56] conducted a hybridization experiment between five pa‐ rents with different chemotypes namely Minoan, Medea, Korona, Przemko and Kozmosz and studied the alkaloid profile for F1 to F3 generations. They observed that in some cross combinations with high alkaloid containing parents, the content of total alkaloid, morphine and thebaine showed significant increase in hybrid generation which persisted upto F3 gen‐ eration. However, the concentration of narcotine was lesser than mid parent value and also showed decreasing trend over generations. As a matter of fact, homogenous strains started to accumulate at F3 generation. In contrast to the high alkaloid parents, the cross combina‐ tions with low alkaloid parents exhibited considerable heterosis for total alkaloid content in F1 while low alkaloid containing recessive individuals segregated in F2 and stabilized in F3 generation. They finally concluded that their experiment reflected well with the effects of genetic regulation at three levels of enzymatic processes during the alkaloid biosynthesis. The morphinans and narcotine was controlled by complex polygenic effects so, the selection for fixing of very low content of narcotine may be effective in early F2 generation as narco‐ tine was found lesser than mid parent value. However, selection for morphinane alkaloids which are in major proportion is not worthy before F3 generation. Yadav et al. [57] investi‐ gated inheritance pattern for different quantitative traits through generation mean analysis using five parameter model on five cross combinations with five generations i.e. parents, F1s, F2s, and F3s selected from an extensive hybridization programme carried out in partial mating design. They found that additive x additive and dominance x dominance was higher in magnitude than combined main effect of additive and dominance effect for all the traits in all five crosses. However, dominance x dominance effect was predominant over additive x additive for all the traits except for few. They also observed substantial amount of realized heterosis, residual heterosis and high broad sense heritability with moderate genetic ad‐ vance and significant correlation among important traits in positive direction. Based on their study they finally advised selective diallel mating and biparental mating in early genera‐ tions followed by recurrent selection which can be used for genetic upgradation of opium poppy. Kumar and Patra [58] undertook a study to understand the gene action involved in the inheritance of opium yield and its component traits (plant height, leaves per plant, pe‐ duncle length, capsule index, seed and straw yield per plant and morphine content) in two families viz., VG26 x VG20 and SG35II x VE01 of opium poppy. They found significant addi‐ tive, dominance and epistatic genetic components for the inheritance of different traits and concluded that biparental mating followed by recurrent selection involving desired re‐ combinants may be utilized for genetic upgradation of opium poppy through components

222 Plant Breeding from Laboratories to Fields

traits.

One of the important aspects of breeding programmes is selection which is based on several factors and requires experience and command to observe. Selection can be based on maturi‐ ty period, disease resistance, lodging, withering and yield etc. Investigation about oil yield, fatty acid compositions and total protein content of three varieties of Turkish poppy were done by Azcan et al. [59] who found that solvent extraction of yellow seed gave highest oil yield upto 49.2%, while white seed had 36.8% and blue seed 33.6% which was considerably low. Fatty acid compositions of oils were determined by GC/MS in which major components were of linoleic (56.4–69.2%), oleic (16.1–19.4%), and palmitic (10.6–16.3%) acid depending on the color of the seeds. Similar investigation on volatile compounds of several seed oil samples from *Papaver somniferum* L. using solid phase micro extraction (SPME) with DVB/ Carboxen/PDMS Stable-Flex fiber was done by Krist et al. [60]. They identified 1-Pentanol (3.3-4.9%), 1-hexanal (10.9-30.9%), 1-hexanol (5.3-33.7%), 2-pentylfuran (7.2-10.0%), and cap‐ roic acid (2.9-11.5%) as the main volatile compounds in all examined poppy seed oil sam‐ ples. Furthermore, the TAG (Triglyceride) composition of these oils was analyzed by MALDI-TOF and ESI-IT-MS/MS. The predominant TAG components were found to be com‐ posed of linoleic, oleic and palmitic acid, comprising 70% of the oil. Similarly, Ozcan and Atalay [61] investigated physical and chemical properties of seven poppy varieties. Weight of 1000 seeds, moisture, crude protein, crude ash, crude fibre, HCl-insoluble ash, crude ener‐ gy and crude oil content of all seven varieties of poppy seeds were 0.29-0.429 g, 3.39-4.76 %, 11.94-13.58 %, 4.92-6.25 %, 22.63-30.08 %, 0.72-1.68 %, 6367.0-6740.5 kcal/100g and 32.43-45.52 % respectively. The poppy seed oil contained an appreciable amount of beta-to‐ copherol (309.5 ppm-567.3 ppm). Poppy seed oil also contained stearic, palmitic, oleic, lino‐ leic and linolenic acid as the main constituent of fatty acids. Linoleic acid was established as the dominant fatty acid in all varieties. Similar investigation were also done by Hakan et al. [62] who investigated fatty acid, tocopherol and sterol content of the oil of several poppy seeds. They found that the main fatty acids in poppy seed oil were linoleic, oleic and palmit‐ ic acid while oil contained an appreciable amount of gamma-tocopherol and alpha-toco‐ pherol. The concentrations of total sterol ranged from 1099.84 mg kg-1 to 4816.10 mg kg-1. The major sterols were beta-sitosterol ranged from 663.91 to 3244.39 mg kg-1; campesterol ranged from 228.59 to 736.50 mg kg-1 and delta (5)-avenasterol ranged from 103.90 to 425.02 mg kg-1.

## **9. Stability analysis for identification of stable and adaptable varieties**

The analysis of genotype x environmental interaction, which indicates the stability of geno‐ types has always been part of plant breeding programmes before release of any variety for commercial cultivation. To study the GxE interaction, several methods have been proposed to analyze it i.e. univariate methods such as Francis and Kannenberg's coefficient of variabil‐ ity [63], Plaisted and Peterson's mean variance component for pair-wise GE interactions [64], Wricke's ecovalence [65], Shukla's stability variance [66], Finlay and Wilkinson's regression coefficient [67], Perkins and Jinks's regression coefficient [68] and Eberhart and Russell's sum of square deviations from regression [69]. Simultaneously, two other stability models based on graphical representation of the genotypes in different environments are available i.e. Yan's GGE Biplot model and AMMI model. Yadav et al. [70] investigated stability for seed yield, opium yield and morphine content in 11 advanced breeding lines over five years in opium poppy. Combined ANOVA showed that both main effects and interactions were significant, indicating the presence of genotype x environment interactions. Yadav et al. [19, 36] studied phenotypic and genotypic variability, broad sense heritability, genetic advance under selection and interrelationship of traits in 74 and 122 accessions of opium poppy re‐ spectively. They found high variations among the accessions along with broad sense herita‐ bility and genetic advance. Genetic correlation analysis revealed negative correlation between opium yield and morphine and papaverine content while other alkaloids showed positive correlation. The 11 genotypes of opium poppy were evaluated on the basis of nonparametric model by Yadav et al. [71] for opium yield and morphine content over 5 environ‐ ments to identify stable and promising genotypes which can sustain adverse environmental conditions. Several of the evaluated genotypes were found to be stable in all the environ‐ mental conditions and were stable for both the traits i.e. opium yield and morphine content. Yadav et al. [72] evaluated 22 strains of opium poppy to find out variability and suitable se‐ lection indices for opium and seed yield. The discriminant functions based on single charac‐ ter were less efficient while on the basis of combination it was in general more efficient. The comparison of different functions revealed that capsule weight/plant, capsule length, plant height were major yield components and thus practicing selection for attainment of high opium and seed yielding lines, maximum weightage should be given to these characters. The positive association of opium and seed yield suggested that by adopting suitable com‐ ponent breeding and selection, a dual-purpose variety (opium and seed yield) can be devel‐ oped. Singh et al. [44] investigated the extent of genetic variability, heritability, correlation and path analysis for opium yield, seed yield and eight component traits in a group of 101 germplasm lines of different ecogeographical origins. They noticed high heritability coupled with high genetic advance and coefficient of variability for most of the traits. Path coefficient analysis indicated that capsule per plant had high direct path towards opium yield followed by four other traits.

#### **10. Mutation breeding approaches**

Besides, different hybridization programs, mutation breeding program was also flourished and encouraging results were obtained all over the world. An era of mutation breeding came into existence due to significant achievements obtained in many crops of pharmaceuti‐ cal, industrial and food interest. In opium poppy also scientists obtained fascinating results. A mutation breeding experiment was carried out using physical and chemical mutagens to develop non-narcotic opium poppy from narcotic crop [73]. They isolated two families con‐ taining twenty latex less/opium-less and twelve partial latex bearing plants in M1 generation which gave similar observations in M2 generations also. The best mutant genotype, LL-34 of family C1-Comb-113-2 with 5.66 g seeds/capsule had 52.6% oil was designated as cv. 'Sujata'. This was the world's first opiumless and alkaloid free seed poppy cultivar, offers a cheap and permanent (fundamental) solution to the global problem of opium-linked social abuse. Simultaneously, it serves as a food grade crop with proteinacious seeds along with healthy unsaturated seed oil. Similarly Chatterjee et al. [74] studied induced mutation through gam‐ ma rays, EMS and their combined doses in two varieties of opium poppy (NBRI-1 and NBRI-5) to create new genetic variability for isolation of high yielding genotypes along with specific alkaloids. The genetic coefficient of variability (GCV), heritability and genetic ad‐ vance was noticed higher for opium and seed yield and capsule weight for all the doses in both the varieties with some exception. They finally concluded that the criteria for selection of plants should be based on capsule weight and capsule number which can provide ideal plant type with enhanced yield potential. Chatterjee et al. [75] also found a variant plant of opium poppy (*Papaver somniferum* L.) having high thebaine content. The M2 seeds of variant plant were subjected to *in vitro* studies to investigate the prospects of thebaine production through tissue culture. Consequently, alkaloid profile of variants showed higher thebaine in stem followed by leaf callus, stem callus and cotyledons. From the same mutation breeding experiment Chatterjee et al. [76] made an effort of identify appropriate dose of the mutagens for the enhancement of specific alkaloid especially thebaine and also studied correlation be‐ tween cytological aberrations and their effects on alkaloid quantity in two stable high yield‐ ing varieties of opium poppy i.e. NBRI-1 and NBRI-5. They found that NBRI-1 was more sensitive than NBRI-5 and that the mutagen EMS was most potent in creating chromosomal abnormalities. They concluded that two doses i.e. kR 10 + 0.2% EMS and 0.2% EMS was most effective for getting fruitful results. The dose kR 10 + 0.2% EMS possessed high chiasms frequency while 0.2% EMS in combinations with all doses of gamma was effective in enhancing the total alkaloid as well as specific alkaloids. In continuation of their study Chatterjee et al. [77] also tried to broaden the genetic variability and to evaluate the advance generations for different agronomic and chemotypic traits in the experimental high yielding varieties i.e. NBRI-1 and NBRI-5 through induced mutations. Here, they noticed that the dose kR30 and kR10 + 0.4% EMS gave highest positive results for genotypic coefficient of variability, heritability and genetic advance (%) for seven traits in NBRI-1 and ten traits in NBRI-5 respectively. They further concluded that their study confirmed that the morphinan and phthalideisoquinilone pathway bifurcated at lower combined doses i.e. kR30 and kR10 + 0.4% EMS which was effective in causing micromutation in morphinan and phthalideiso‐ quinilone pathways respectively.

coefficient [67], Perkins and Jinks's regression coefficient [68] and Eberhart and Russell's sum of square deviations from regression [69]. Simultaneously, two other stability models based on graphical representation of the genotypes in different environments are available i.e. Yan's GGE Biplot model and AMMI model. Yadav et al. [70] investigated stability for seed yield, opium yield and morphine content in 11 advanced breeding lines over five years in opium poppy. Combined ANOVA showed that both main effects and interactions were significant, indicating the presence of genotype x environment interactions. Yadav et al. [19, 36] studied phenotypic and genotypic variability, broad sense heritability, genetic advance under selection and interrelationship of traits in 74 and 122 accessions of opium poppy re‐ spectively. They found high variations among the accessions along with broad sense herita‐ bility and genetic advance. Genetic correlation analysis revealed negative correlation between opium yield and morphine and papaverine content while other alkaloids showed positive correlation. The 11 genotypes of opium poppy were evaluated on the basis of nonparametric model by Yadav et al. [71] for opium yield and morphine content over 5 environ‐ ments to identify stable and promising genotypes which can sustain adverse environmental conditions. Several of the evaluated genotypes were found to be stable in all the environ‐ mental conditions and were stable for both the traits i.e. opium yield and morphine content. Yadav et al. [72] evaluated 22 strains of opium poppy to find out variability and suitable se‐ lection indices for opium and seed yield. The discriminant functions based on single charac‐ ter were less efficient while on the basis of combination it was in general more efficient. The comparison of different functions revealed that capsule weight/plant, capsule length, plant height were major yield components and thus practicing selection for attainment of high opium and seed yielding lines, maximum weightage should be given to these characters. The positive association of opium and seed yield suggested that by adopting suitable com‐ ponent breeding and selection, a dual-purpose variety (opium and seed yield) can be devel‐ oped. Singh et al. [44] investigated the extent of genetic variability, heritability, correlation and path analysis for opium yield, seed yield and eight component traits in a group of 101 germplasm lines of different ecogeographical origins. They noticed high heritability coupled with high genetic advance and coefficient of variability for most of the traits. Path coefficient analysis indicated that capsule per plant had high direct path towards opium yield followed

Besides, different hybridization programs, mutation breeding program was also flourished and encouraging results were obtained all over the world. An era of mutation breeding came into existence due to significant achievements obtained in many crops of pharmaceuti‐ cal, industrial and food interest. In opium poppy also scientists obtained fascinating results. A mutation breeding experiment was carried out using physical and chemical mutagens to develop non-narcotic opium poppy from narcotic crop [73]. They isolated two families con‐ taining twenty latex less/opium-less and twelve partial latex bearing plants in M1 generation which gave similar observations in M2 generations also. The best mutant genotype, LL-34 of

by four other traits.

224 Plant Breeding from Laboratories to Fields

**10. Mutation breeding approaches**

A mutant variety known as 'TOP 1' ('thebaine oripavine poppy 1') in opium poppy (*Papaver somniferum*) was developed by Tasmania Company. In this mutant the morphinan pathway is blocked at thebaine results in absence of codeine and morphine. The major loss of this blockage is on the end product i.e. morphine which is absent in this mutant [84]. This mu‐ tant was developed by a mutagen treatment to seeds of commercial poppy cultivar (*P. som‐ niferum*). Phenotypically the mutation is visible in the form of pigmented latex than normal white. In TOP 1 mutant, one possibility is that the gene responsible for an enzyme 6-O de‐ methylase which act on thebaine and oripavine might be affected at its transcriptional level or modified protein structure. It may be possible that there is an alteration occurs in trans‐ port component that blocks the entry of substrates (thebaine and oripavine) of the enzyme to the subcellular compartment for 6-O demethylation. These mutant plants are very impor‐ tant since the production of thebaine is only amenable which can help in checking of drug trafficking. However, identification of the candidate genes which has been blocked can be identified and characterized. The complex mechanism involved in morphinane biosynthesis can also be elucidated. These morphine free plants can be beneficial for the treatment of opioid addiction. But there is a slight risk with this mutant for licit to illicit uses (by conver‐ sion of non-narcotic alkaloids to narcotic alkaloids). The Tasmania drug industry has been using TOP 1 mutants since 1998 for production of various analgesic drugs viz. buprenor‐ phine, oxycodone, naloxone and naltrexone.

## **11. Polyploidy approaches**

The event of polyploidization has been observed long back as most of our cultivated and wild species are polyploids as a result of diploidization or cross pollination among various ploidy levels. This has been an important aspect in conventional breeding programs. Basically, poly‐ ploidy is of two types i.e. auto- and allo- ploidy, wherein the auto polyploids arises due to du‐ plication in same genomic content in a species whereas in case of allopolyploidy, there is chromosome complementation i.e. two different chromosomal content from different species combine to form allopolyploid. Few studies on polyploidization in opium poppy have been undertaken so far. Polyploids are beneficial in many aspects viz., organism can resort to high‐ er number of genes and higher number of allelic variants which may lead to substantial in‐ crease in the ultimate product. One of the recent study was undertaken on ploidy aspect of opium poppy [78]. They aimed to understand the phenotypic, genetic and genomic conse‐ quences of induced polyploidy and to enhance total alkaloid content along with specific alka‐ loid using colchicine. They observed that the induced auto-tetraploidy did not show any significant differences in phenotypic level while stomatal and chromosomal studies con‐ firmed the tetraploidy. They also noticed differential gene expression of the diploids and autotetraploids which led to the elucidation of dosage regulated gene expression leading significant enhancement in morphine content in tetraploid plants. Their study in auto-tetra‐ ploids opens avenues towards the development of hexaploids and amphidiploids which can give multifold increase in specific alkaloids. This study also opens a new vista towards under‐ standing of ploidy level changes in term of phenotypic, genetic and genomic and a better un‐ derstanding of the complex mechanism involved in polyploidization.

#### **12. Other conventional approaches**

Apart from different conventional breeding strategies applied for genetic upgradation of opium poppy, several researchers with similar aim carried out several studies in opium poppy. A unique study was carried out on honey bees foraging on plant flowers [79]. They noticed significantly higher foraging response of honeybees (*Apis mellifera*) manifesting hon‐ eybee's preference towards specific plant morphotypes in genetically divergent plant of opi‐ um poppy (*Papaver somniferum*). Furthermore the genotype specific for foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honey for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops. Since the seed of opium poppy is widely used as food in almost all parts of the world, several researchers tried different ways to develop plants producing nutritionally rich seeds. Losak and Richter [80] studied the ef‐ fect of nitrogen supplementation to cultivar 'Opal' of opium poppy plant in a pot experi‐ ment. They applied ammonium nitrate in single dose at two stages of plant growth i.e. at the beginning of growing season and at the stage of flowering. They observed that the increas‐ ing dose of nitrogen increased number of capsules per plant, morphine content and the cap‐ sule volume irregularly. However, an optimum dose of nitrogen i.e. 0.9 g N/pot showed statistically significant positive effect on seed yield. The effect of varying concentration of CO2 (300, 400, 500, 600 μ mol mol-1) was examined on various morphological traits such as number of capsules, capsule weight and latex-yield in *Papaver setigerum*. A significant posi‐ tive effect of increasing CO2 concentration on various morphological traits was noticed with an increase of 3.6, 3.0 and 3.7 times, respectively on per plant basis. Significant and positive response of secondary metabolites especially morphine, codeine, narcotine and papaverine was also noticed to CO2 enrichment. However, the major alkaloid i.e. morphine was signifi‐ cantly increased by 10.4, 11.7, 12.9 and 12.4%, respectively at each dose (300, 400, 500 and 600 μmol mol−1) of CO2 [81]. Szabo et al. [82] investigated the effects of water stress on the alkaloid production and content at three different developmental stages i.e. Rosette, Flower‐ ing and Lancing in opium poppy. They used four types of water conditions i.e. control, withdrawal, 50% water supply and inundation and found that leaves responded significant‐ ly to water stress conditions. They further concluded that constant water supply is beneficial for the accumulation of alkaloids in poppy capsules. In many parts of the world, seeds of opium poppy are widely used as food and efforts are continuously made to develop nutri‐ tionally rich poppy seeds. In Central European countries, the content of selenium is very low in poppy seeds. Hence, with the aim of supplementing opium poppy plants with seleni‐ um (a trace element), Skarpa and Richter [83] tried to explore the effect of foliar application of this element on seed yield, selenium content in seeds and its uptake by the roots. They applied a single dose of selenium of 300 g/ha at two different stages i.e. during the stage of the end of elongation growth and after the fall of blossoms. They found that seed yield was reduced by 11.5% and 11.8% after both stages of application respectively but the content of selenium increased significantly from 139 μg/kg to 757 μg/kg of seeds. However, the uptake of selenium also increased significantly upto 4.8 times.

port component that blocks the entry of substrates (thebaine and oripavine) of the enzyme to the subcellular compartment for 6-O demethylation. These mutant plants are very impor‐ tant since the production of thebaine is only amenable which can help in checking of drug trafficking. However, identification of the candidate genes which has been blocked can be identified and characterized. The complex mechanism involved in morphinane biosynthesis can also be elucidated. These morphine free plants can be beneficial for the treatment of opioid addiction. But there is a slight risk with this mutant for licit to illicit uses (by conver‐ sion of non-narcotic alkaloids to narcotic alkaloids). The Tasmania drug industry has been using TOP 1 mutants since 1998 for production of various analgesic drugs viz. buprenor‐

The event of polyploidization has been observed long back as most of our cultivated and wild species are polyploids as a result of diploidization or cross pollination among various ploidy levels. This has been an important aspect in conventional breeding programs. Basically, poly‐ ploidy is of two types i.e. auto- and allo- ploidy, wherein the auto polyploids arises due to du‐ plication in same genomic content in a species whereas in case of allopolyploidy, there is chromosome complementation i.e. two different chromosomal content from different species combine to form allopolyploid. Few studies on polyploidization in opium poppy have been undertaken so far. Polyploids are beneficial in many aspects viz., organism can resort to high‐ er number of genes and higher number of allelic variants which may lead to substantial in‐ crease in the ultimate product. One of the recent study was undertaken on ploidy aspect of opium poppy [78]. They aimed to understand the phenotypic, genetic and genomic conse‐ quences of induced polyploidy and to enhance total alkaloid content along with specific alka‐ loid using colchicine. They observed that the induced auto-tetraploidy did not show any significant differences in phenotypic level while stomatal and chromosomal studies con‐ firmed the tetraploidy. They also noticed differential gene expression of the diploids and autotetraploids which led to the elucidation of dosage regulated gene expression leading significant enhancement in morphine content in tetraploid plants. Their study in auto-tetra‐ ploids opens avenues towards the development of hexaploids and amphidiploids which can give multifold increase in specific alkaloids. This study also opens a new vista towards under‐ standing of ploidy level changes in term of phenotypic, genetic and genomic and a better un‐

derstanding of the complex mechanism involved in polyploidization.

Apart from different conventional breeding strategies applied for genetic upgradation of opium poppy, several researchers with similar aim carried out several studies in opium poppy. A unique study was carried out on honey bees foraging on plant flowers [79]. They noticed significantly higher foraging response of honeybees (*Apis mellifera*) manifesting hon‐

**12. Other conventional approaches**

phine, oxycodone, naloxone and naltrexone.

**11. Polyploidy approaches**

226 Plant Breeding from Laboratories to Fields

Since for the last few decades, scientific researchers have been continuously contributing for the genetic upgradation of opium poppy through various approaches including convention‐ al breeding methodologies, mutation breeding and molecular techniques with breeding and it is a matter of high enthusiasm for the development of varieties, hybrids, synthetics and GMO in opium poppy till date. At present few varieties have been developed through con‐ ventional approaches that can be grouped as-

#### **12.1. Varieties developed by National Botanical Research Institute, Lucknow**

In due course of time many breeding approaches have been applied in opium poppy for the development of new high yielding and disease resistant varieties. These varieties are now stabilized and suited for different agro-climatic conditions. A brief characteristic description of the varieties are given below -

**Figure 3.** Field view of developed high yielding variety "Madakini".

**12.1. Varieties developed by National Botanical Research Institute, Lucknow**

of the varieties are given below -

228 Plant Breeding from Laboratories to Fields

can be cultivated mainly in Northern Indian plains.

can be cultivated mainly in Northern Central India.

part of India is recommended for its cultivation.

ern Indian Plains are recommended as most suitable.

its cultivation.

In due course of time many breeding approaches have been applied in opium poppy for the development of new high yielding and disease resistant varieties. These varieties are now stabilized and suited for different agro-climatic conditions. A brief characteristic description

**• BROP-1**:- In this variety the plants are medium sized having 3-4 capsules/plant and cap‐ sules are of three types viz. oily, parrot coloured and black peduncle. Flowers are white. It is a synthetic variety stabilized after hybridization/intermating between three high yield‐ ing cultivars viz. kali dandi (black peduncle), suga pankhi (parrot color) and sufaid dandi (white peduncle) followed by selection. The average opium yield, seed yield and mor‐ phine content are up to 54kg/ha, 1000-1200kg/ha and 13% respectively. Geographically it

**• NBRI-1**:- This variety is developed through selection. The plants are medium tall having large fringed leaves and white flowers. The average opium yield, seed yield and mor‐ phine content are up to 52kg/ha, 1000kg/ha and 12-13% respectively. Geographically it

**• NBRI-2**:- This variety has intermediate tall plants, thick stem, broad leaves, long peduncle with big capsules and flowers are white. This variety is also developed through selection amongst local collection having above characters. Average opium yield, seed yield and morphine content are up to 52 kg/ha, 1200kg/ha and 15%respectively. Northern Central

**• NBRI-6**:- In this variety, plants are medium tall with narrow leaves and white flowers. It is developed by hybridization between two germplasm lines BR007 and BR008 (BR007 x BR008) followed by rigorous selection generation after generation up to eight generations. Average opium yield, seed yield and morphine content are up to 55kg/ha, 1200kg/ha and 13-14% respectively. Geographically Northern Indian plains are mainly recommended for

**• NBRI-9**:- In this variety, plants are intermediate sized with white flowers and large cap‐ sules. The variety is high yielding (seed yield) and is developed by hybridization between germplasm lines S-10 x S-18 followed by rigorous selection until the variety is stabilized (up to eight generations). It's average opium yield, seed yield and morphine content is upto 52kg/ha, 1400kg/ha and 12% respectively. For the cultivation of the variety, North‐

**• NBRI-10**:- The plants in this variety are medium tall, having dark green leaves and white flowers. The development of variety was done through hybridization germplasm lines (IC-30 x S-10) followed by rigorous selection up to eight generations. Average opium yield, seed yield and morphine content are up to 50kg/ha, 1200kg/ha and 12% respective‐

ly. Geographically Northern Central plains are recommended for its cultivation.

#### **12.2. Varieties developed by Central Institute of Medicinal and Aromatic Plants, Lucknow**

**• Rakshit**:-It is a disease resistant and morphine rich variety in CPS (concentrated poppy straw). The plants are 106-112 cm tall with 20-26 cm long green peduncle and oblong cap‐ sules with waxy surface. The variety is developed by hybridization and selection genera‐ tion after generation up to eight generations. Average seed yield and straw yield of the variety are up to 1200-1400kg/ha and 900-1100kg/ha respectively.

**Figure 4.** Field view of developed high thebaine lines.

**Narendra Dev University of Agriculture and Technology, Faizabad** has developed a downy mildew resistant variety by selection and named Kirtiman (NOP-4). The plants of this variety are quite tall having white flowers and 1-2 oval capsules. Opium yield, seed yield and morphine content of the variety ranges 35-46kg/ha, 900-1100kg/ha and up to 12% respectively. Eastern U.P. region is best suited for its cultivation.

**National Bureau of Plant Genetic Resources, New Delhi** developed a variety Trishna (IC 42) for resistance to frost, root rot and downy mildew through inbreeding and selection. The plants of the variety are tall with 5-7 capsules/plant and pink flowers. Opium yield, seed yield and morphine content ranges up to 49-53kg/ha, 1000kg/ha and 12-14.78% respectively.

**Rajasthan Agricultural University, Udaipur** has developed a resistant variety to disease and lodging and named it as **Chetak (UO 285)**. The plants of this variety are average tall with big capsules. Flowers are white with smooth petals. Opium yield, seed yield and mor‐ phine content ranges up to 54kg/ha, 1000-1200kg/ha and 12% respectively. Geographically Rajasthan is most suitable for its cultivation.

**Jawaharlal Nehru Krishi Vishwavidalaya, College of Agriculture, Jabalpur (M.P.)** has also succeeded in developing a downy mildew resistant variety by pure line selection and named as Jawahar Aphim 16 (JA-16). Plants of this variety are tall having white flowers and 1-3 big capsules/plant. The variety has opium yield, seed yield and morphine content up to 45-54kg/ha, 900-1000kg/ha and 12% respectively. Madhya Pradesh is geographically recom‐ mended for its cultivation.