Genetic Variability, Correlation and Path Analysis among Quantitative and Qualitative Traits of Opium Poppy (Papaver somniferum L.)

The Opium poppy is one of the ancient therapeutic plants in the history of mankind’s. It belongs to the family ‘Papaveraceae’ and mainly cultivated in Southeast Asian and some European countries for instance Turkey, India, China, Bulgaria and Tasmania on commercial basis (Celik et al., 2016). This magical medicinal plant produces over 100 different kinds of benzylisoquinoline alkaloids uses in pharmaceutical as well as nutraceutical industries for remedial purpose. Morphine, codeine, papaverine, thebaine, narcotine and nosacapine are six most economically significant opium alkaloids, extracted naturally in huge amount and directly uses in drug industries. The opium extracts/alkaloids are uses as sedative, analgesic, antitussive, muscle relaxant anti-tumorigenic and antimicrobial agent in many treatments (Facchini and Park, 2003). The source of the opium latex is quite matured green capsule of this plant. Opium poppy is a self-pollinated crop with varied degree of out crossing (2 -70%) depending upon the types of varieties, rapidity of wind, bees visits, insects and climatic conditions etc. (Khanna and Shukla, 1983) and (Bhandari, 1990). The global trend shows that the consumption of opium alkaloids and its derivatives are growing day by day in recent years. According to (Khatik and Dodiya, 2019) Morphine, which is the main narcotic component of opium, showed tenfold demand in last two decades. To meets the National and International demand, India plays an important role as it produces half of the opium consumed by the world’s pharmaceutical industries that’s why its position is number one on running board as largest opium licit producing and exporting country in the world. Globally, the opium poppy licit is under the firm control of Central Bureau of Narcotics and it’s headquarter at Vienna, Austria. But at various locations its illegitimate cultivation is also being practiced such as Golden Crescent (Iran, Afghanistan and Pakistan) and Golden Triangle (Thailand, Burma and Myanmar) (Mishra et al., 2013). To avoid drug abuses and illegal practicing of opium poppy extracts it is necessary to move on towards CPS (Concentrated poppy straw) system in which the opium alkaloids were extracted from dried capsules. (Acharya and Sharma, 2009) reported that majority of opium poppy cultivars used for commercial purpose having low genetic variation. Accessibility of genetic variability is very essential for purpose of selection, genetic improvement and development of high yielding cultivars. Therefore, it is important to carry out the effectual breeding strategies for proper designing and characterization of population of economic traits for genetic variability (Shukla et al., 2010).
       
Keeping in mind, the current study was carried out for possible genetic improvement in the existing germplasm/accessions of opium poppy. Genetic variability, correlation and path coefficients analysis was undertaken to understand the interrelationship among the thirteen economically viable traits for developing a suitable selection strategy in thirty germplasm/indian landraces of Papaver somniferum L. collected from different geographical origins.

Experimental material
 
The experimental material for the present study consist of of 30 accessions/Indian landraces of opium poppy (Papaver somniferum L.) which were collected from different eco-geographical regions of India (Table 1).
 

 
Experimental site and design
 
The 30 genotypes of Opium poppy including different breeding lines and landraces were grown in year (2017-18) in randomized complete-block designs (RCBD) with three replications at the research farm of CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, located at 26.5° N latitude and 80.50° E longitude and 120 m above sea level. Plants were planted in rows of 4m long and 50 cm apart. The plants received normal intercultural operations, irrigation and fertilizer applications (120 kg N, 80 kg P₂O₅ and 60 kg K₂O.ha^-1). Morpho-matric data were recorded on five competitive randomly selected plants in each line for following thirteen traits- days to 50% flowering, plant height (cm), Pedicel length (cm), capsules/plant, leaves/plant, capsule index, seed yield(g)/plant, capsule husk yield (g)/plant, alkaloid content (%) in poppy straw includes five major alkaloid i.e. morphine, codeine, thebaine, papaverine and narcotine (Fig 1).
 

 
Chemical analysis
 
Sample preparation and analysis
 
The capsule husk (1 g) was grinded to fine powder and extracted (3 ×10 ml) in water bath with methanol by boiling for 30 min. The solution was pooled and concentrated and then re-dissolved in 1.0 ml methanol. The samples were centrifuged at 10,000 rpm and then HPTLC analysis is performed. The chemical, i.e. morphine, codeine, thebaine, papaverine and narcotine were provided by Government Opium and Alkaloid Works, Neemuch, India. The purity of each referred opiate was found 98% by HPLC peak and normalization method. The stock solution was prepared separately for each estimated compound. The quantification was done using linear regression curve.
 
Validation
 
Thin layer chromatography (TLC)-densitometry was used to quantify the five major opium alkaloids morphine, codeine, thebaine, papaverine and narcotine (Gupta and Verma, 1996). Toluene-acetonemethanol-ammonia (40:40:6:2) v/v was used as a mobile phase. Silica gel Plates 60 F254 (Merck, Darmstadt, Germany) after derivatization using Dragendorff reagent were scanned at 540 nm.
 
Statistical analysis
 
The pooled mean data were statistically analysed by using Windostat statistical software 4.0 versions available at Department of Genetics and Plant Breeding, CSIR-CIMAP, Lucknow based on (Singh and Chaudhary, 1979) and (Panse and Sukhatme, 1976). The mean values of all the traits were subjected to correlation and path coefficient analyses (Dewey and Lu, 1959). Genetic parameters, including broad sense heritability (h² _bs), genetic advance (GA) and genotypic and environmental coefficient of variation (GCV and ECV) were estimated from the components of variance by using the following formulas.
   
h² _(bs) = Heritability in broad sense.
σ_p = Phenotypic standard deviation of the trait.
k = Standard selection differential which is 2.06 at 5 per cent selection intensity.

Accessions variability
 
The variability existing among the 30 accessions was assessed through analysis of variance (ANOVA), which showed significant differences among the accessions for different morphological traits and alkaloids (Table 2). A large variation between minimum and maximum mean values of accessions for each trait was also visible (Table 3). The plant height was varied from 85.0 cm to 135.6 cm and pedicel length was varied from 14.33 cm to 28.66 cm. Days to 50% flowering was varied from 26.66 days to 38.33days, seed yield ranged from 0.43 (g)/plant to 3.06 (g)/plant and Husk yield was varied from 0.38 (g)/plant to 1.64 (g)/plant. Among the alkaloids, morphine yield was ranged from 0.026% to 0.089% and papavarine was ranged from 0.017% to 0.450%. Genetic parameters for the heritable and non-heritable components of variation were computed. Among the agro-morphological characters, the maximum amount of genotypic coefficient of variation (GCV) was observed for seed yield (g)/plant (35.52 %) followed by capsules/plant (32.77%) and husk yield (g)/plant (24.03%) suggesting effective selection for these traits for genetic improvement of opium (Table 4).
 

 

 

       
Highest phenotypic coefficient of variation (PCV) was noticed for capsule/plant (39.70%) followed by seed yield (g)/plant (36.35%) and lowest PCV was observed for capsule/plant (9.69%) (Table 4). Among alkaloids, highest GCV was observed for Thebaine (136.79%) followed by Codeine (103.30%) and Nosacapine (44.50%). Highest PCV was observed for Codeine (137.23%) followed by Thebaine (137.04%) and Nosacapine (44.95%). Phenotypic coefficient of variation was higher than the genotypic coefficient of variation for all the traits studied. This clearly indicates that observed variation was not only due to genotype alone but also due to environment.
       
A high heritability estimate (h² _bs) with corresponding high genetic advance (GA) is more reliable for selection than that with low genetic advance (GA). This indicated that simple selection will help in genetic improvement of these traits. Among the morph-metric traits, highest heritability was noticed for thebaine (99.64%) followed by nosacapine (98.02%), papervine (97.74%) and seed yield (g)/plant (95.51%). Something, alike findings were also reported for variability on genotypic and phenotypic level and high heritability to the characters viz., plant height, diameter of main capsule, days of 50% flowering, number of leaves per plant, husk yield per plant and seed yield per plant by (Jain et al., 2005) & (Singh and Singh, 2008). Thus the genetic advance has an added edge over heritability as a guiding factor to breeders in a selection programme (Singh and Singh, 1981). The GA as percent mean varied from minimum (2.43%) for husk yield (g)/plant to maximum (885.71%) for thebaine followed by (789.17%) for nosacapine, morphine (328.07%), papervine (77.93%), no. of capsule/plant (46.14%) and seed yield (g)/plant (37.61%). Which suggests that the maximum improvement may be achieved for thebaine followed by nosacapine since it had high heritability coupled with GA. The GAM for thebaine and nosacapine were high because of extreme variation present in the materials. High heritability coupled with high GAM and GCV for the traits thebaine and nosacapine suggests that the traits are primarily controlled by additive gene action. In the same way, related outcome were also reported for Genotypic and genotypic variability, broad sense heritability, genetic advance under selection and interrelationship of traits in 74 and 122 accessions of opium poppy by (Yadav et al., 2005 and 2006).
 
Genotypic and phenotypic correlation
 
Study of association among the traits gives an idea to improve a particular character. The estimates of correlation coefficients are represented in (Table 6). The results of associations studies revealed that the trait seed yield (g)/plant was positively and significantly correlated with no. of capsule/plant (rg=0.735**, r_p=0.566**) at both genotypic and phenotypic level and no. of capsule was also positively correlated with pedicel length at genotypic level (r_g=0.429* r_p=0.160). Nosacapine was also positively and significantly correlated with seed yields (g)/plant (r_g= 0.670**, r_p= 0.643**) and no. of capsule/plant (r_g= 0.619**, r_p= 0.507**) at both genotypic and phenotypic level. The husk yield was positively correlated with no. of capsule/plant (r_g= 0.413*, r_p =0.257) and negatively correlated with plant height (r_g= -0.418*, r_p= -0.229) at genotypic level only. The papervine content with no. of leaves/plant was also positively correlated with each other at both genotypic and phenotypic level (r_g= 0.553**, r_p= 0.379*) while negatively correlated with plant height at genotypic level (r_g= -3.981*, r_p= -0.0008). Morphine content with pedicel length (r_g= 0.568**, r_p= 0.344), with seed yield (r_g= 0.376*, r_p= 0.340), with husk yield (r_g= 0.370*, r_p= 0.242) was positively correlated at genotypic level while, negatively correlated with capsule index (r_g= -0.388**, r_p= -0.345) at genotypic level. Thus, plants with more no. of capsules /plant were relatively high seed yielder. Hence, during selection plants with more capsules were to be selected to enhance the productivity of seeds and alkaloids i.e. morphine and nosacapine which was also positively and significantly concurrent with seed yields simultaneously. Similar studied were also carried out and reported earlier the extant of genetic variability, heritability, correlation and path analysis for different traits in germplasm of opium poppy (Bhandari et al., 1997; Singh et al., 2000; Shukla et al., 2003; Dubey 2010; Kara and Baydar 2018 and Yadav et al., 2005 and 2006).
 

 
Path analysis
 
The path analysis was estimated to study the direct and indirect effects of different traits on seed yield/plant (Table 5). The path coefficient study compiled that the trait which was direct responsible for highest total seed yield was made by no. of capsules/plant (1.343) followed by codeine (0.660), thebaine (0.596), nosacapine (0.538), days to 50% flowering (0.455) and plant height (0.226) (Table 5). Direct contribution of traits number of leaves/plant (-0.949), pedicel length (-0.704) showed negative effect on seed yield/plant but their indirect contribution were relatively large. The residual effect of 0.600 revealed that 90% of seed yield (g)/plant was contributed by the characters studied and thus indicated the adequacy of character. Therefore, the choice of the characters to be considered for improvement of seed yields were no. of capsule, days to 50 percent flowering, plant height, codeine, thebaine and nosacapine. The genetic associations along with their mean performance could be utilized efficiently for the selection of genetic stocks / parents in future hybridization programme for the improvement of opium poppy crop. These findings are in concord to the results obtained by (Mirjana et al., 2012 and Solanki et al., 2014) reported the high positive and direct effect of husk yield/plant and latex yield/plant over seed yield per plant. (Shukla et al., 2003; Singh et al., 2004; and Ozturk and Gunlu, 2008) have also reported high positive direct effect of plant height and capsule size on seed yield per plant in germplasm of opium poppy.
 

The meticulous study of analysis of variance, means, standard errors of means revealed highly significant difference for all the thirteen Agro-economically viable traits studies indicating thereby existence of considerable genetic variability among 30 genotypes/accessions. Availability of genetic variability is very important for genetic improvement and development of high yielding varieties. We studied genetic variability for thirteen traits in 30 opium poppy accession/germplasm in order to understand genetic variability and path coefficient. Highest genotypic and phenotypic coefficient of variation along with high genetic advance was noticed for thebaine and nosacapine. The lead of genetic associations finding showed that the traits capsule/plant and nosacapine was positively and significantly correlated with seed yield (g)/plant. Highest direct contribution for seed yield was also lead by number of traits i.e. capsules/plant followed by Codeine, thebaine, nosacapine, days to 50% flowering and plant height.

The authors thanks to the Director, Central Institute of Medicinal and Aromatic Plants (CSIR - CIMAP), Lucknow, UP (India) for providing mandatory facilities during the research work.