**5. Conventional breeding strategies applied for genetic upgradation of opium poppy**

The conventional breeding approaches are a step by step procedure to develop desired plant type. The important steps involved in opium poppy breeding program are described in fol‐ lowing subheadings:-

#### **5.1. Plant Introduction**

Conventional plant breeding programs require distinct plant genotypes with specific charac‐ teristics to initiate any hybridization technique. The distinctness in the base material ensures higher percentage of success through breeding programme. The collection of diverse germ‐ plasm from different geographical regions can be the best approach for initiation of any breeding programme with specific objectives. The foremost step to initiate any crop breed‐ ing program is plant introduction. The procedure of growing a variety or a species into an area where it has not been grown earlier is termed as Plant Introduction. However, bringing plant material from one environmental condition to another within a country or continents is also called as plant introduction. Plant introduction and germplasm collection thus be‐ comes one of the richest sources of creation of variability [37, 38].

In India, researches on opium poppy are confined at some agricultural and scientific insti‐ tutes viz., Central Institute of Medicinal and Aromatic Plants, Lucknow, National Botanical Research Institute, Lucknow, Jahawarlal Nehru Krishi Vishwavidalaya-College of Agricul‐ ture, Jabalpur, Narendra Dev University of Agriculture and Technology, Faizabad, National Bureau for Plant Genetic Resources, New Delhi, Rajasthan Agricultural University, Udaipur. These centers have been working on genetic upgradation of opium poppy for the last four to five decades. Khanna and Singh [39] bought 190 strains from Russia, Hungary, Poland, U.K. and other temperate countries and evaluated these strains at NBRI, Lucknow. They noticed that most of the cultivars belonging to European countries require long photoperiod, hence were unsuitable in Indian climatic conditions. However, the cultivars of Iran were only pos‐ sible to cultivate in India by introduction. Similarly, Prajapati et al. [40] screened capsule husk of a set of 115 Indian land races of opium poppy (*Papaver somniferum* L.) for papaver‐ ine, reticuline, narcotine, thebaine, codeinone, codeine, morphine and oripavine at CIMAP, Lucknow. These germplasms were grouped into four clusters on the basis of alkaloid pro‐ file. Based on the study of alkaloid profiles of these germplasm and correlations between al‐ kaloids in all the four groups of accessions, they concluded that in Indian genetic resources of *P*. *somniferum* (a) morphine is synthesized from codeine rather than oripavine, (b) net al‐ kaloid content was low under narcotine deficiency, and (c) accumulation of morphine and codeine was in limited upstream of codeinone and morphinone. It was also depicted from their study that the accessions identified based on alkaloid profiles, harboring genetic blocks in phenanthrene and benzylisoquinoline biosynthetic pathways can be useful for under‐ standing the genetic control of secondary metabolism in opium poppy.

the latex oozes out. The mature green capsule is lanced with an instrument called "Nastar" having 3-4 small blades designed to ensure uniformity in depth of incisions. Generally 3-4 lancing is done in each capsule with parallel longitudinal cuts which is performed after mid day and allow the latex to remain overnight on the capsules for coagulation. In the follow‐ ing morning the latex is collected from the capsule walls with blunt edge of small iron scoop. The opium is kept in small plastic box or earthen pot or copper bowls. The latex col‐ our varies from dark to light brown to pink based on the variety. The depth of incision should not be more than 1.2 mm. After collection of opium, lanced capsules are left to dry

**5. Conventional breeding strategies applied for genetic upgradation of**

The conventional breeding approaches are a step by step procedure to develop desired plant type. The important steps involved in opium poppy breeding program are described in fol‐

Conventional plant breeding programs require distinct plant genotypes with specific charac‐ teristics to initiate any hybridization technique. The distinctness in the base material ensures higher percentage of success through breeding programme. The collection of diverse germ‐ plasm from different geographical regions can be the best approach for initiation of any breeding programme with specific objectives. The foremost step to initiate any crop breed‐ ing program is plant introduction. The procedure of growing a variety or a species into an area where it has not been grown earlier is termed as Plant Introduction. However, bringing plant material from one environmental condition to another within a country or continents is also called as plant introduction. Plant introduction and germplasm collection thus be‐

In India, researches on opium poppy are confined at some agricultural and scientific insti‐ tutes viz., Central Institute of Medicinal and Aromatic Plants, Lucknow, National Botanical Research Institute, Lucknow, Jahawarlal Nehru Krishi Vishwavidalaya-College of Agricul‐ ture, Jabalpur, Narendra Dev University of Agriculture and Technology, Faizabad, National Bureau for Plant Genetic Resources, New Delhi, Rajasthan Agricultural University, Udaipur. These centers have been working on genetic upgradation of opium poppy for the last four to five decades. Khanna and Singh [39] bought 190 strains from Russia, Hungary, Poland, U.K. and other temperate countries and evaluated these strains at NBRI, Lucknow. They noticed that most of the cultivars belonging to European countries require long photoperiod, hence were unsuitable in Indian climatic conditions. However, the cultivars of Iran were only pos‐ sible to cultivate in India by introduction. Similarly, Prajapati et al. [40] screened capsule husk of a set of 115 Indian land races of opium poppy (*Papaver somniferum* L.) for papaver‐ ine, reticuline, narcotine, thebaine, codeinone, codeine, morphine and oripavine at CIMAP,

over plants for next 15-20 days for harvesting of seeds.

comes one of the richest sources of creation of variability [37, 38].

**opium poppy**

218 Plant Breeding from Laboratories to Fields

lowing subheadings:-

**5.1. Plant Introduction**

In continuation of plant introduction, Shukla et al. [41] studied alkaloid spectrum in 1470 in‐ dividual plants belonging to 98 germplasm which has been collected from different sources and maintained at NBRI, Lucknow for several years. Based on alkaloid profiles, the content of different alkaloids were categorized into class interval exhibiting maximum number of plants and accessions for morphine fall in group of 10–15% followed by 15–20%, for codeine in group of 2–4% followed by 4–6%, for thebaine in 1–2% followed by 2–4%, for narcotine in 5–10% followed by 10–15% and for papaverine content 0–2%, while 24 germplasm lines had morphine content above 16.0%. Based on distinctness in morphological and agronomical characteristics, 1,000 distinct poppy germplasm lines were provided by Agriculture faculty, Ankara University from which 99 poppy lines were evaluated in terms of alkaloid analysis in *in vitro* [42]. They observed the range of different alkaloids in poppy husk (CPS) viz., mor‐ phine, thebaine, codeine, papaverine and noscapine from 0.110 to 1.140%, 0.005 to 0.134%, 0.005 to 0.27%, 0.001 to 0.440% and 0.006 to 0.418%, respectively. Dittbrenner et al. [43] eval‐ uated 300 accessions of opium poppy for 35 morphological and agronomic traits collected from all over the world at IPK Gene Bank, Gatersleben, Germany. Based on their study on five major alkaloids taken for two years, they concluded highly significant correlation be‐ tween total alkaloid content and morphine. However, four other major alkaloids i.e. co‐ deine, thebaine, noscapine and papaverine did not show any correlation between them or with total alkaloid content. Additionally they also noticed that there is no important correla‐ tion between morphological traits and alkaloid content. They also determined the chromo‐ some number in each accession and found that the subspecies *setigerum* was natural tetraploid while the rest of the subspecies were diploid. They finally concluded that none of the studied morphological traits could be used for prediction of alkaloid content which may give erroneous information in breeding programmes.

## **6. Diversity analysis through conventional tools**

One of the foremost steps in the genetical improvement of any crop through conventional breeding program is to study the genetic diversity available in the introduced plant/crop material. To conduct any breeding program judiciously, diversity analysis based on mor‐ phological and biochemical traits is prerequisite. In opium poppy, several of the exotic col‐ lections at different research institutes have been evaluated for genetic diversity. Few studies on genetic diversity undertaken so far in opium poppy are summarized here. Singh et al. [44] studied genetic divergence using 101 germplasm lines of different ecogeographical origin for seed and opium yield per plant and its 8 component traits following multivariate and canonical analysis. They grouped the germplasm into 13 clusters on the basis of multi‐ variate analysis which was also confirmed by canonical analysis. 68% genotypes were found genetically close to each other and grouped in 6 clusters while apparent diversity was no‐ ticed for 32 percent of the genotypes who diverged into rest 7 clusters. They concluded that the genotypes in clusters IX, X, XI and XII had greater potential as breeding stock by virtue of high mean values of one or more component characters and high statistical distances among them. Yadav et al. [45] made an effort to study the genetic divergence in a genetically distinct new stock of opium poppy using cluster and principle component analysis. They found that a large amount of variability exists among the accessions and formed 8 clusters from which some accessions were recommended which can be used in hybridization pro‐ gramme to get desirable transgressive segregants. Similarly, Yadav et al. [46] assessed genet‐ ic divergence in 110 population (20 parents and 90 F1 hybrids) using multivariate analysis. All the entries were grouped into 14 clusters which indicated substantial diversity among parental genotypes which had potential to release considerable variation in their crosses. Similarly, Brezinova et al. [47] evaluated 404 genotypes of poppy from world collection to assess genetic diversity over the selected traits based on their morphological characteristic to create a digitalized visual documentation. On the basis of morphometric analysis, the im‐ portant diversity in observed traits were recognized in agro-climatic conditions of Slovakia, documented by statistical characteristics and by digitalized documentation of accessions. Di‐ versity based on alkaloid spectrum in 122 accessions of indigenous opium poppy was un‐ dertaken by Shukla et al. [48]. They obtained 11 clusters based on extent of correlation between five major alkaloids i.e. morphine, codeine, thebaine, narcotine and papaverine. Mostly the clusters comprised of accessions with different possible combinations of alka‐ loids comprising high in one alkaloid with high or low of another. Generally the percentage of morphine content was higher than the sum of four other alkaloids except in one cluster where narcotine content was slightly higher than morphine. Based on their study they con‐ cluded that successful breeding for specific alkaloids or a combination of alkaloids could be achieved by using these accessions in hybridization programme.

#### **7. Creation of variability through hybridization**

A breeding programme focused to develop improved varieties requires knowledge about the genetic variability that exists for the concern trait. It is documented that sufficient varia‐ tion for composition and content of secondary metabolites occurs in a number of medicinal plant. Several studies have been carried out in opium poppy to study the existing variability in different set of materials which showed varying results for composition of secondary me‐ tabolites and other chemical compounds along with morphological variations. Singh et al. [49] found that F8 genotypes obtained through interspecific cross between *Papaver somnife‐ rum* and *Papaver setigerum* had higher oil (>40%) and fatty acid concentration than respective parental species. They also obtained varying results for linoleic (68%-74.4%) and oleic acid (13.6%-20.3%) content in F8 genotypes. High oleic desaturation ratio and C18 polyunsaturat‐ ed fatty acid with very low linolenic (18:3) acid (0.37%) indicated the possibility of using poppy oil for edible purposes. However, oleic (18:1) acid was not correlated with other fatty acids, except for significant negative correlation with linoleic (C18:2) acid. Ozturk and Gun‐ lu [50] conducted correlation and path coefficient analysis for qualitative and quantitative traits in four poppy cultivars in Central Anatolia. They found statistically significant differ‐ ences for all the studied traits among all the four genotypes. Positive and significant correla‐ tion of morphine yield with morphine content, seed yield, capsule yield, oil yield; capsule yield with oil yield; seed yield with capsule yield, oil yield were noticed. Through path anal‐ ysis, it was noticed that morphine content, capsule yield, seed yield and oil yield had posi‐ tive direct effect on morphine yield. Yadav et al. [51] analyzed F1 and F2 generations of a twenty parents fractional diallel cross in opium poppy (*P. somniferum* L.) to estimate the combining ability of the crosses based on ten quantitative and five qualitative (alkaloids) traits. The results indicated that significant differences exists among the parents for all the traits and GCA (General Combining Ability) and SCA (Specific Combining Ability) compo‐ nents of variances were also significant for all the traits. However, SCA component of var‐ iance (σ<sup>2</sup> s) was predominant which indicated the preponderance of non-additive gene effect for all the traits except for leaves/plant and papaverine in F1 hybrids. The average degree of dominance (σ <sup>2</sup> s/ σ <sup>2</sup> g) was more than unity indicated over dominance and also confirmed the non-additive mode of gene action. They suggested that the inclusion of good general combiners in a multiple crossing program or an intermating among the population involv‐ ing all possible crosses subjected to biparental mating can be expected to offer maximum promise in breeding for higher opium and seed yield and alkaloid content. In an another study, Yadav et al. [52] examined combining ability for yield and its component traits along with morphine content to elucidate the inheritance pattern governing these traits and also to identify potential genotypes which could be further exploited in breeding programmes. They noticed that most of the traits were governed by non-additive gene action while addi‐ tive gene action was also important for some other traits. They found three best parents viz., BR-232, BR-245 and BR-234 as good general combiners which could be used in hybridization programme aiming at maximum gain. Similarly, Kumar and Patra [53] also studied inheri‐ tance pattern for quantitative traits in four single crosses in opium poppy. They found that simple additive, dominance and epistatic genetic components were significant for inheri‐ tance of the traits under study. They also noticed differential gene actions with differential magnitude for different traits and concluded that following biparental mating followed by recurrent selection for desired recombinants may be utilized for genetic upgradation of opi‐ um poppy crop. Mishra et al. [54] evaluated progenies of randomly selected individuals from 14 promising hybrids over F2 to F6 generations for opium and seed yield and their con‐ tributing traits for the formulation of effective selection strategy in opium poppy (*P. somnife‐ rum* L.). They observed that in general heritability and genetic gain declined from generation to generation. They obtained a cross MOP541 x BR241 which showed similar pattern for ge‐ netic gain in all the traits. The values of broad sense heritability decreased from F2 to F6 gen‐ eration for most of the traits. Matyasova et al. [55] evaluated 57 cultivars of opium poppy comparing the groups of values representing the indicators of production-significant mor‐

et al. [44] studied genetic divergence using 101 germplasm lines of different ecogeographical origin for seed and opium yield per plant and its 8 component traits following multivariate and canonical analysis. They grouped the germplasm into 13 clusters on the basis of multi‐ variate analysis which was also confirmed by canonical analysis. 68% genotypes were found genetically close to each other and grouped in 6 clusters while apparent diversity was no‐ ticed for 32 percent of the genotypes who diverged into rest 7 clusters. They concluded that the genotypes in clusters IX, X, XI and XII had greater potential as breeding stock by virtue of high mean values of one or more component characters and high statistical distances among them. Yadav et al. [45] made an effort to study the genetic divergence in a genetically distinct new stock of opium poppy using cluster and principle component analysis. They found that a large amount of variability exists among the accessions and formed 8 clusters from which some accessions were recommended which can be used in hybridization pro‐ gramme to get desirable transgressive segregants. Similarly, Yadav et al. [46] assessed genet‐ ic divergence in 110 population (20 parents and 90 F1 hybrids) using multivariate analysis. All the entries were grouped into 14 clusters which indicated substantial diversity among parental genotypes which had potential to release considerable variation in their crosses. Similarly, Brezinova et al. [47] evaluated 404 genotypes of poppy from world collection to assess genetic diversity over the selected traits based on their morphological characteristic to create a digitalized visual documentation. On the basis of morphometric analysis, the im‐ portant diversity in observed traits were recognized in agro-climatic conditions of Slovakia, documented by statistical characteristics and by digitalized documentation of accessions. Di‐ versity based on alkaloid spectrum in 122 accessions of indigenous opium poppy was un‐ dertaken by Shukla et al. [48]. They obtained 11 clusters based on extent of correlation between five major alkaloids i.e. morphine, codeine, thebaine, narcotine and papaverine. Mostly the clusters comprised of accessions with different possible combinations of alka‐ loids comprising high in one alkaloid with high or low of another. Generally the percentage of morphine content was higher than the sum of four other alkaloids except in one cluster where narcotine content was slightly higher than morphine. Based on their study they con‐ cluded that successful breeding for specific alkaloids or a combination of alkaloids could be

220 Plant Breeding from Laboratories to Fields

achieved by using these accessions in hybridization programme.

A breeding programme focused to develop improved varieties requires knowledge about the genetic variability that exists for the concern trait. It is documented that sufficient varia‐ tion for composition and content of secondary metabolites occurs in a number of medicinal plant. Several studies have been carried out in opium poppy to study the existing variability in different set of materials which showed varying results for composition of secondary me‐ tabolites and other chemical compounds along with morphological variations. Singh et al. [49] found that F8 genotypes obtained through interspecific cross between *Papaver somnife‐ rum* and *Papaver setigerum* had higher oil (>40%) and fatty acid concentration than respective parental species. They also obtained varying results for linoleic (68%-74.4%) and oleic acid

**7. Creation of variability through hybridization**

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 traits.
