- Email: [email protected]
Genetic variability and diversity in Indian germplasm of opium poppy (Papaver somniferum L.)
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap
Genetic variability and diversity in Indian germplasm of opium poppy (Papaver somniferum L.) ⁎
Rashmi Lahiria, , R.K. Lala, Nupur Srivastavab, Karuna Shankerb a b
Department of Genetics and Plant Breeding, CSIR-CIMAP, Lucknow, India Department of Analytical Chemistry, CSIR-CIMAP, Lucknow, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Canonical analysis D2-statistics Genetic diversity Opium poppy Variability
Prevailing diversity among germplasm in a crop assists invaluably in crop improvement program. Genetic divergence in 60 germplasm lines of opium poppy was studied based on 13 morphometric characters by using Mahalonobis D2 statistics. The germplasm lines belong to the different eco-geographical origin and are being maintained at CSIR-CIMAP, Lucknow. The ANOVA revealed significant amount of differentiation among germplasm. The genotypes were grouped in eight diverse clusters. Cluster I is the largest group comprises 40 genotypes followed by Cluster II, consisting 12 genotypes and cluster III and IV has 2 genotypes while rest 4 clusters have only a single individual. The maximum intra-cluster distance was observed in cluster IV (93153.35) and minimum for Cluster I (33360.9). Inter-cluster D2 values range from 115261.4 to 4717153. The minimum was between Cluster I and II (115261.4) followed by IV and V (128735.4) and maximum between Cluster I and VII (4717153) followed by Cluster VI and VII (4655531). Based on the mean performance genotypes, G-1 and G53 can be further selected for exploitation in hybrid breeding program for poppy crop improvement.
1. Introduction Opium poppy is a well known medicinal plant which serves as a source of pharmaceutically important alkaloids in medicinal history. Opium poppy (Papaver somniferum L.) belongs to the family ‘Papaveraceae’ which produces more than 100 different benzylisoquinoline alkaloids, including morphine and codeine as analgesic, papaverine as muscle relaxant, noscapine as anti-tumorigenic drug, and sanguinarine as antimicrobial agent (Facchini and Park, 2003). Because of its rich alkaloid content, opium poppy is an important medicinal crop for many countries growing poppy such as Turkey, India, and Bulgaria (Celik et al., 2016; Prajapati et al., 2002; Shukla et al., 2010; Gumuscu and Arslan 1999; Gumuscu and Arslan 2008). Apart from pharmaceutical use, poppy seeds also serve as an invaluable source of plant based protein supplement for human consumption (Ozcan and Atalay 2006; Azcan et al., 2004). The majority of commercially used poppy cultivars contain low genetic variation (Acharya and Sharma, 2009), thus knowledge of genetic diversity of economic traits is needed for proper characterization of population and for designing efficient breeding strategies to accomplish breeding objectives (Shukla et al., 2010). Natural genetic variability has been exploited from the very beginning of agriculture, to fulfill the requirement of a growing
population. Genetic diversity provides the base for biodiversity and diversity between species, within species, and that of ecosystems, which provides information about genetic divergence (Saini and Kaicker, 1987). Genetic diversity is associated with prevention of narrowing the genetic base and provides opportunity to develop new and improved cultivars with desirable characteristics. Genetic diversity explores the existing variations present in crop genotypes which can be identified through specific statistical method. With the increasing global demand there is a need to develop poppy with high opium and seed yielding varieties integrated with high specific alkaloids. Now-a-days research on poppy is heading in three directions, first is producing cultivars with high alkaloid content in the capsule husk, and the second is producing cultivars with low alkaloids or zero morphine. Third is cultivar with high seed yield. Thus, it becomes necessary to develop varieties for dual purposes having high alkaloids coupled with high seed yield. The present investigation was carried out to study, the degree of genetic divergence and variability present in the 60 germplasm/Indian landraces of opium poppy based on thirteen traits. Genetic divergence was quantified by multivariate analysis by assessing the proximity of accessions with each other, and classifying them in different groups/ clusters for further utilization of identified diverse genotype(s) for further hybrid/heterosis breeding program of opium poppy.
⁎
Corresponding author. E-mail addresses: [email protected] (R. Lahiri), [email protected] (R.K. Lal), [email protected] (N. Srivastava), [email protected] (K. Shanker). http://dx.doi.org/10.1016/j.jarmap.2017.10.001 Received 15 February 2017; Received in revised form 5 October 2017; Accepted 12 October 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Lahiri, R., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.10.001
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
2. Materials and methods
following- (Singh and Chaudhary, 1979). Analysis of variance was carried out following the procedure given by Panse and Sukhatme (1976). The data on 13 morpho-metric traits were analyzed using Mahanonobis’s generalized distance D2-statistics (Mahalanobis, 1936) and canonical analysis. The clustering was done on the basis of Tocher’s methods (Rao, 1952).
2.1. Materials The experimental material for the present study comprises of 60 accessions/Indian landraces of opium poppy (Papaver somniferum L.) which were collected from different eco-geographical regions of India were evaluated for two consecutive years (2013–2014 and 2014–2015) at CSIR- Central Institute of Medicinal and Aromatic Plants, Lucknow.
3. Results The analysis of variance showed significant differences among the genotypes for all the thirteen characters studied, which indicated the presence of high genetic variability among the present set of materials. On the basis of D2 values for all possible n (n-1)/2 = 1770 pairs of accessions, where ‘n’ is number of genotype, all the 60 experimental materials were grouped into eight diverse clusters (Figs. 1 and 2). Cluster I was the largest group that comprised 40 genotypes followed by Cluster II, consisted of 12 genotypes and clusters III and IV had two genotypes in each. Rest 4 clusters, Cluster V, VI, VII, and VIII have single genotype in each. Maximum intra-cluster distance was observed
2.2. Experimental site The present experiment was conducted at the experimental field of Genetics and Plant Breeding, CSIR-CIMAP, Lucknow, located at 26.5° N latitude and 80.50° E longitude and 120 m above sea level. 2.3. Experimental design All 60 genotypes were grown for two consecutive years (2013–14 and 2014–15) with three replications in a randomized block design (RBD). In each replication, one single row of each entry was grown with spacing of 10 cm within rows and 40 cm between rows. All required standard cultural practices were followed throughout the crop season which include pre-sowing, the addition of farmyard manure at the rate of 10t/ha and 80, 40, 40 kg/ha of nitrogen (N2), phosphorus (P2O5) and potassium (K2O), respectively as basal covering of land. An additional dose of nitrogen of 40 kg/ha was top dressed in two equal splits at 40 days and 60 days after sowing and sprayed with the fungicide redomil 0.2% at 45 and 60 days after sowing. The experimental field was irrigated as and when required. Morpho-metric data were recorded on five competitive randomly selected plants in each line for following thirteen traits- days to 50% flowering, plant height (cm), capsules/ plant, capsule size, seed yield/plant (gm), capsule husk yield/plant (gm); alkaloid yield/plant (%) – different includes five alkaloid i.e. morphine, codeine, thebaine, papaverine and narcotine, obtained from each selected plant in percentage. 2.4. Chemical analysis 2.4.1. 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 & 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. 2.4.2. 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. 2.5. Statistical analysis The pooled mean data of two years were statistically analyzed using the CSIR-CIMAP, Lucknow developed Statistical Software 4.0 version which is available in the Genetics and Plant Breeding Division of the institute based on Singh and Chaudhary (1979) and Panse and Sukhatme (1976). The mean, standard error, ranges were determined
Fig. 1. Cluster diagram with their distances of 60 genotypes in opium poppy germplasm.
2
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Fig. 2. Spatial distribution of 60 genetic stocks/accessions in opium poppy accessions.
Table 1 Distribution of opium poppy accessions in different cluster and their geographical locations. S. No.
Cluster
Number of accessions
Accessions
Origin of accessions
1
Ist
40
UP, Rajasthan, New Delhi, MP
2
IInd
12
G-1, G-2, G-4, G-6, G-7, G-8, G-10, G-12, G-13, G-14, G-16, G-20,G-22, G-24, G-29, G-30, G-40, G-42, G-47, G-48, G-54, G-55, G-59, G-60, G-61,G-62, G-68, G-69,G-70, G-75, G-76, G-77, G-82, G-85, G-86, G-92, G93, G-95, G-100 G-9, G-18, G-19, G-32, G-35, G-65, G-66, G-87, G-89, G-97, G-99, G-104
3 4 5 6 7 8
IIIrd IVth Vth VIth VIIth VIIIth
2 2 1 1 1 1
G-53, G-56 G-57, G-58 G-49 G-50 G-51 G-90
MP, Rajasthan, New Delhi, UP, UP, New Delhi UP Slovak Republic UP UP UP
content (0.0673%) along with low mean value for number of capsules, capsule index, codeine, thebaine, and narcotine content. Cluster VII has high mean values for capsule husk yield (7.303 g), codeine (0.305%) and thebaine (0.0315%) content (Prajapati et al., 2002). Cluster VIII includes genotypes having high peduncle length and high narcotine content while these have low mean values for days to maturity, seed yield, and capsule husk yield. It is also reported that genotypes within the cluster with a high degree of divergence would produce more desirable breeding materials for achieving maximum genetic gain (Kumar Bose and Pradhan, 2006). From the obtained results it could be concluded that accession with high seed yield and low morphine and papaverine content present in cluster I, accessions with low codeine, thebaine, and narcotine content from cluster V could be selected as parents for the hybridization program to fulfill the objective of poppy
in cluster IV (93153.35) and the minimum for Cluster I (33360.9). Inter cluster distance ranges from 115261.4–4717153. The minimum was between Cluster I and II (115261.4) followed by IV and V (128735.4) and maximum was between Cluster I and VII (4717153) followed by Cluster VI and VII (4655531) (Tables 1 and 2). Based on the cluster means (Table 3) the important cluster for high morphine content was Cluster III that possesses highest morphine content (0.249%) and low or negligible papaverine along with low seed (6.997 g) and capsule husk yield (5.334 g). Cluster IV had the highest mean for plant height (105.76 g), capsule index (0.904) and low mean for no. of capsules (1.917) and negligible papaverine. Cluster V had higher mean for days to flowering (140.94), number of capsule (2.667) with lowest plant height (91.37 cm). Cluster VI had highest mean for days to 50% flowering (104.17), seed yield (8.517 g) and papaverine 3
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Table 2 Estimates of inter- and intra-cluster distances among eight cluster of poppy. Cluster
I
II
III
IV
V
VI
VII
VIII
Mean
I
33360.9 (182.649)
115261.4 (339.501) 48885 (221.099)
2499505 (1580.982) 2208807 (1486.205) 33530.89 (183.114)
1149814 (1072.293) 990012.1 (994.993) 301928.1 (549.479) 93153.35 (305.210)
1590573 (1261.179) 1259500 (1122.274) 195216.9 (441.833) 128735.4 (358.797) 0 0
361002.1 (1900.000) 138511.1 (372.170) 2592753 (1610.202) 1339208 (1157.241) 1453019 (1205.412) 0 0
4717153 (2171.900) 4259130 (2063.765) 366371.6 (605.286) 1270438 (1127.137) 936428.8 (967.692) 4655531 (2157.667) 0 0
406832 (637.833) 340718.5 (583.710) 1363013 (1167.481) 513103.4 (716.312) 818596.3 (904.763) 601239.5 (775.396) 2976122 (1725.144) 0 0
1359188
II III IV V VI VII VIII
1170103 1195141 783494.9 797758.7 1392658 2397647 877453.1
Table 3 Cluster mean of the thirteen economic traits in opium poppy.
I II III IV V VI VII VIII
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
101.89 102.46 101.86 101.46 101.33 104.17 100.73 102.08
99.64 97.96 102.46 105.76 91.37 98.46 102.65 99.23
21.81 20.63 21.60 21.35 20.45 20.78 18.38 23.88
132.34 131.75 135.42 134.78 140.94 136.11 134.00 122.72
2.113 2.005 2.417 1.917 2.667 1.667 2.333 2.333
0.899 0.884 0.890 0.904 0.881 0.820 0.869 0.886
7.102 7.139 6.997 7.328 7.794 8.517 7.344 6.220
6.094 6.106 5.334 6.530 6.588 6.548 7.303 4.408
0.085 0.102 0.249 0.124 0.169 0.102 0.192 0.145
0.008 0.017 0.226 0.154 0.171 0 0.305 0.068
0.007 0.008 0.021 0.017 0.008 0.005 0.031 0.060
0.004 0.031 0 0 0.026 0.067 0.005 0.014
0.013 0.029 0.019 0.019 0.036 0 0.006 0.044
Where X1 = Days to flowering, X2 = plant height (cm), X3 = peduncle length (cm), X4 = days to maturity, X5 = number of capsule/plant, X6 = capsule index, X7 = seed yield/plant (g), X8 = capsule husk yield/plant, X9 = morphine content (%), X10 = codeine content (%), X11 = thebaine content (%), X12 = papaverine content (%), X13 = narcotine content (%).
recombination breeding programmes. The clustering pattern reflects the closeness between the genotypes. The clusters containing solitary genotype might be due to the parentage of the genotype and geographical origin. Majority of the results suggest no good correspondence between genetic divergence and geographical origin of genotypes. The maximum divergence was observed in cluster IV (Table 1). It is reported that genotypes within the clusters with high degree of divergence would produce more desirable breeding materials for achieving maximum genetic advance. Therefore due emphasis should be given on the members of cluster IV for selection of parents for hybridization programme. The other clusters showing high intra cluster values were cluster II, III and I and the minimum or nil intra cluster values were exhibited by cluster V, VI, VII and VIII this indicated negligible genetic diversity among the genotype for each character. The maximum inter cluster divergence was observed between cluster I and VII (Table 2). Highly divergent genotypes would produce a broad spectrum of variability in the subsequent generation for morphological and quality traits enabling further selection and improvement. Studies reported that selection of parents for hybridization should be from two clusters having wider inter clusters distance to get maximum variability in the segregating generations (Pradhan and Ray 1990; Rahman et al., 1997). Hybridizaion between genotypes of different cluster is required for the development of desirable genotypes. Studies by Singh et al. (1996) suggested utility of recombination between genotypes of different clusters In nutshell in the present investigation, it is suggested that hybridization programme within the divergent cluster are expected to give promising and desirable recombinants in the segregating generations. The two accessions namely, G-1 and G-53 were found promising for dual purpose i.e. for high seed yield and high morphine content (Table 5). These two accessions recommended for further commercial cultivation on large scale.
with high seed yield and low amount of alkaloids. Accessions among the cluster separated by high D2 values could be further used in the hybridization program for obtaining a wide range of variations among the segregates. The grouping obtained through D2 statistics based on two dimensional Z1 and Z2 chart, the contribution of characters towards divergence of genotypes revealed that in vector (Z1) contribution toward genetic divergence in the primary axis of differentiation were due to seed yield, codeine, papaverine, thebaine content. In vector II (Z2) which was the secondary axis of differentiation, papaverine, narcotine, days to 50% flowering were important. It was also revealed that vectors (I and II) were positive for days to 50% flowering and, papaverine. Such results indicate that the character contribute the maximum towards divergence (Table 4). Among the important economical characters in poppy, seed yield occupies an important place, beside high nutritive value it is a good source of healthy edible oil. For high seed yield the selected genotypes are G-8 (9.553gm), G-18 (9.019gm), G-24 (8.933gm), G-32 (8.792gm) and G-9 (8.616gm). Capsule husk is another important character of poppy as capsule straw is a source of alkaloids which was obtained by processing of unlanced capsule. The accessions G-18 (8.823gm), G-19 (8.566gm), G-29 (8.236gm), G-59 (8.275gm) and G- 60 (8.033gm) were found promising for poppy husk yield (Table 5). Similarly accession, G-1 (0.262), G-53 (0.307), G-61 (0.233) and G-65 (0.237) for morphine, G-49 (0.171), G-51 (0.305), G-53 (0.221) and G-56 (0.230) for codeine, G-10 (0.0246), G-51 (0.0243), G-51 (0.0315) and G-53 (0.0322) and G-57 (0.0262) for thebaine content were also found promising. 4. Discussion The analysis of variance showed high variances for most of the characters, which favour selection and utilization in future 4
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Table 4 Character contributions (%), rank, mean and other allied genetic parameters of the thirteen traits in opium poppy. Parameter X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
CC (%) Ranks Z1 Vector CTPA (%) CCPAR Z2 Vector CTSA(%) CCSAR Range
3.53 7 0.0004 0.0336 5 −0.0012 −0.1805 9 88.04–108.36
3.897 6 −0.0007 −0.0542 12 −0.0012 −0.1716 8 18.38–26.39
1.733 1 0.0004 0.0279 7 0.0002 0.0319 4 122.44–141.94
10.711 2 0.0004 0.0317 6 −0.0012 −0.1813 10 1–3.167
4.517 5 −0.0002 −0.0174 11 −0.0017 −0.2454 11 0.78–1.016
6.913 4 0.00007 0.0053 9 −0.0003 −0.0476 5 4.34–9.55
7.973 3 0.0002 0.0218 8 −0.0009 −0.1400 7 3.66–8.82
53.568 1 0.0012 0.0930 4 −0.0003 −0.0431 5 0–0.66
0.392 1 0.9609 72.6645 1 −0.2486 −35.277 13 0–7.06
1.099 1 0.1060 8.0219 3 −0.0678 −9.6259 12 0–0.060
0.791 1 0.2554 19.3154 2 0.9640 136.77 1 0–0.076
2.549 8 −0.0017 −0.1355 13 0.0638 9.0542 2 0–0.055
2.319 9th −0.0001 −0.0083 10 0.0003 0.0520 3 91.05–107.05
Where X1 = Days to flowering, X2 = plant height (cm), X3 = peduncle length (cm), X4 = days to maturity, X5 = number of capsule/plant, X6 = capsule index, X7 = seed yield/plant (g), X8 = capsule husk yield/plant, X9 = morphine content (%), X10 = codeine content (%), X11 = thebaine content (%), X12 = papaverine content (%), X13 = narcotine content (%).
Table 5 Mean performances of five trait specific promising accessions of opium poppy. Characters
Accession
Seed Yield
G-8 G-9 G-18 G-24 G-32 G-18 G-19 G-29 G-59 G-60 G-1 G-53 G-61 G-65 G-49 G-51 G-53 G-56 G-57 G-10 G-11 G-51 G-53 G-57 G-1 G-53
Capsule husk yield
Morphine content
Codeine content
Thebaine content
Dual purpose
DF
PH
PL
DM
NC
CI
SY
CHY
M
C
T
98.413 103.013 103.086 103.506 104.536 103.086 106.153 102.843 102.43 103.143 98.413 101.12 103.16 101.623 101.326 100.726 101.12 102.603 100.8 102.58 107.05 100.726 101.12 100.8 98.413 101.12
102.416 99.613 99.606 100.446 97.26 99.606 102.886 98.71 101.166 92.876 89.633 97.769 100.98 104.16 91.369 102.65 97.769 107.156 107.216 103.943 102.95 102.65 97.769 107.216 89.633 97.769
21.53 20.433 20.843 21.343 19.503 20.843 20.073 19.88 26.096 23.393 22.883 22.82 21.463 21.826 20.453 18.383 22.82 20.376 20.233 23.566 21.173 18.383 22.82 20.233 22.883 22.82
130.053 135.556 132.776 131.5 135.223 132.776 129.166 137.39 133.89 141.943 139.333 134.666 125.666 127 140.943 134 134.666 136.166 137.556 136.39 136.336 134 134.666 137.556 139.333 134.666
1.777 1.555 1.666 1.666 2.111 1.666 1.888 2.444 3.167 2.166 2.444 2.9444 1.611 2.166 2.666 2.333 2.9444 1.888 1.778 1.666 1.389 2.333 2.9444 1.778 2.444 2.9444
0.938 0.795 0.933 0.936 0.892 0.933 0.932 0.898 0.844 0.932 0.906 0.894 0.899 0.907 0.881 0.868 0.894 0.885 0.914 0.912 0.882 0.868 0.894 0.914 0.906 0.894
9.553 8.616 9.019 8.933 8.792 9.019 8.402 7.789 7.669 8.402 8.234 7.289 6.044 5.535 7.794 7.344 7.289 6.704 7.01 7.346 7.924 7.344 7.289 7.01 8.234 7.289
7.466 6.008 8.823 6.586 6.255 8.823 8.566 8.236 8.275 8.033 6.091 6.668 4.532 5.329 6.588 7.303 6.668 5.633 6.396 5.765 6.793 7.303 6.668 6.396 6.091 6.668
0.0413 0.0623 0.0382 0.0053 0.0087 0.0382 0.07066 0.03476 0.0453 0.0661 0.262 0.307 0.233 0.237 0.169 0.192 0.307 0.191 0.142 0.05723 0.0263 0.192 0.307 0.142 0.262 0.307
0.066 0.007 0.025 0 0.0021 0.025 0.026 0.0065 0 0 0 0.2215 0.0224 0.0302 0.1714 0.3056 0.2215 0.2307 0.1734 0.021 0.006 0.3056 0.2215 0.1734 0 0.2215
0.0133 0.0228 0.0052 0.0058 0 0.0052 0.0063 0 0 0 0.0052 0.0322 0.0325 0.0163 0.0077 0.0315 0.0322 0.0113 0.0262 0.0246 0.0243 0.0315 0.0322 0.0262 0.0052 0.0322
P 0.0011 0.0312 0.0274 0.0028 0.029 0.0274 0.0312 0 0.0135 0 0.0221 0 0.0184 0.0387 0.0762 0.0045 0 0 0 0.0032 0.0015 0.0045 0 0 0.0221 0
N 0.0315 0.0425 0.0124 0.0024 0.0157 0.0124 0.0113 0.0214 0.0122 0 0.0163 0.0227 0.0227 0.0096 0.0367 0.0064 0.0227 0.0167 0.0195 0.0234 0.0165 0.0064 0.0227 0.0195 0.0163 0.0227
Conflict of interest
5. Conclusions
The authors declare that they have no conflict of interest.
Genetic divergence among 60 genetic stock of opium poppy to represent a wide range of variation with respect to yield has been estimated using D2 statistics. Selection of promising accessions based on genetic divergence along with better seed yield. Selected five promising accessions on the basis of genetic divergence with better seed yield G-8 (9.553gm), G-18 (9.019gm), G-24 (8.933gm), G-32 (8.792gm) and G-9 (8.616gm). For Capsule husk yield accession G-18 (8.823gm), G-19 (8.566gm), G-29 (8.236gm), G-59 (8.275gm) and G-60 (8.033gm) were found promising. For dual purpose i.e. for high seed and high morphine content accession G-1 (seed yield-8.234 gm and morphine content7.289) and G-53 (seed yield 7.289 gm and morphine content 0.307) was found promising and can be utilized for further improvement. On the basis of observation recorded in divergent clusters of poppy it can be concluded that hybridization between diverse parents among different clusters may be useful and help in coping with limitation pertaining with the improvement of poppy.
Funding Author Rashmi Lahiri thanks CSIR, New Delhi for providing Senior Research Fellowship. The funding agency plays no role in study design, collection, analysis and in paper writing. Acknowledgements Authors thanks to the Director, CSIR-CIMAP, Lucknow, India for the encouragement and facilities provided during the investigation. References Acharya, H.S., Sharma, V., 2009. Molecular characterization of opium poppy (Papaver somniferum) germplasm. American Journal of Infectious Diseases 5, 148–153. Azcan, N., Kalender, B.O., Kara, M., 2004. Investigation of Turkish poppy seeds and seed
5
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
(Papaver somniferum L.). Varieties Grasas Y Aceites 57, 169–174. Panse, V.G., Sukhatme, P.V., 1976. Statistical Methods for Agricultural Workers. ICAR, New Delhi, pp. 361. Pradhan, K., Ray, R., 1990. Genetic divergence in rice. Oryza 27 (4), 415–418. Prajapati, S., Bajpai, S., Singh, D., Luthra, R., Gupta, M.M., Kumar, S., 2002. Alkaloid profiles of the Indian land races of the opium poppy Papaver somniferum L. Genetic Resources and Crop Evolution 49 (2), 183–188. Rahman, M., Acharya, B., Sukla Pande, S.N.K., 1997. Genetic divergence in low land rice genotypes. Oryza 34 (3), 209–212. Rao, C.R., 1952. Advanced Statistical Methods in Biometric Research. John Wiley and Sons, New York, pp. 1–104. Saini, H.C., Kaicker, U.S., 1987. Genetic diversity in opium poppy. The Indian Journal of Genetics and Plant Breeding 47 (3), 291–296. Shukla, S., Yadav, H.K., Rastogi, A., Mishra, B.K., Singh, S.P., 2010. Diversity in relation to breeding for specific alkaloids in opium poppy (Papaver somniferum L.). Czech Journal of Genetics and Plant Breeding 46, 164–169. Singh, R.K., Chaudhary, B.D., 1979. Variance and Covariance Analysis. Biometrical Methods in Quantitative Genetic Analysis. Kalyani Publisher, New Delhi (India), pp. 57. Singh, A.K., Singh, S.B., Singh, S.M., 1996. Genetic divergence in scented and fine genotype of rice. Annals of Agriculture Research 17, 163–166.
oils. Chemistry of Natural Compounds 40, 370–375. Celik, I., Camci, H., Kose, A., Kosar, F.C., Doganlar, S., Frary, A., 2016. Molecular genetic diversity and association mapping of morphine content and agronomic traits in Turkish opium poppy (Papaver somniferum) germplasm. Molecular Breeding 36 (4), 1–3. Facchini, P.J., Park, S.U., 2003. Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64 (1), 177–186. Gumuscu, A., Arslan, N., 1999. Comparing yield and yield components of some selected poppy (Papaver somniferum L.) lines. Turkish Journal of Agriculture & Forestry 4, 991–997. Gumuscu, A., Arslan, N., 2008. Research on heterosis on yield and yield components of some poppy hybrid lines. Journal of Agricultural Science 14, 365–373. Gupta, M.M., Verma, R.K., 1996. Combined thin-layer chromatography-densitometry method for the quantitative estimation of major alkaloids in poppy straw samples. Indian Journal of Pharmaceutical Sciences 58 (4), 161. Kumar Bose, L., Pradhan, S.K., 2006. Genetic divergence in deepwater rice genotypes. Journal of Central European Agriculture 6 (4), 635–640. Mahalanobis, P.C., 1936. On the generalized distance in statistics. Proceedings of the National Institute of Sciences of India 2, 49–55. Ozcan, M.M., Atalay, C., 2006. Determination of seed and oil properties of some poppy
6
Contents lists available at ScienceDirect
Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap
Genetic variability and diversity in Indian germplasm of opium poppy (Papaver somniferum L.) ⁎
Rashmi Lahiria, , R.K. Lala, Nupur Srivastavab, Karuna Shankerb a b
Department of Genetics and Plant Breeding, CSIR-CIMAP, Lucknow, India Department of Analytical Chemistry, CSIR-CIMAP, Lucknow, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Canonical analysis D2-statistics Genetic diversity Opium poppy Variability
Prevailing diversity among germplasm in a crop assists invaluably in crop improvement program. Genetic divergence in 60 germplasm lines of opium poppy was studied based on 13 morphometric characters by using Mahalonobis D2 statistics. The germplasm lines belong to the different eco-geographical origin and are being maintained at CSIR-CIMAP, Lucknow. The ANOVA revealed significant amount of differentiation among germplasm. The genotypes were grouped in eight diverse clusters. Cluster I is the largest group comprises 40 genotypes followed by Cluster II, consisting 12 genotypes and cluster III and IV has 2 genotypes while rest 4 clusters have only a single individual. The maximum intra-cluster distance was observed in cluster IV (93153.35) and minimum for Cluster I (33360.9). Inter-cluster D2 values range from 115261.4 to 4717153. The minimum was between Cluster I and II (115261.4) followed by IV and V (128735.4) and maximum between Cluster I and VII (4717153) followed by Cluster VI and VII (4655531). Based on the mean performance genotypes, G-1 and G53 can be further selected for exploitation in hybrid breeding program for poppy crop improvement.
1. Introduction Opium poppy is a well known medicinal plant which serves as a source of pharmaceutically important alkaloids in medicinal history. Opium poppy (Papaver somniferum L.) belongs to the family ‘Papaveraceae’ which produces more than 100 different benzylisoquinoline alkaloids, including morphine and codeine as analgesic, papaverine as muscle relaxant, noscapine as anti-tumorigenic drug, and sanguinarine as antimicrobial agent (Facchini and Park, 2003). Because of its rich alkaloid content, opium poppy is an important medicinal crop for many countries growing poppy such as Turkey, India, and Bulgaria (Celik et al., 2016; Prajapati et al., 2002; Shukla et al., 2010; Gumuscu and Arslan 1999; Gumuscu and Arslan 2008). Apart from pharmaceutical use, poppy seeds also serve as an invaluable source of plant based protein supplement for human consumption (Ozcan and Atalay 2006; Azcan et al., 2004). The majority of commercially used poppy cultivars contain low genetic variation (Acharya and Sharma, 2009), thus knowledge of genetic diversity of economic traits is needed for proper characterization of population and for designing efficient breeding strategies to accomplish breeding objectives (Shukla et al., 2010). Natural genetic variability has been exploited from the very beginning of agriculture, to fulfill the requirement of a growing
population. Genetic diversity provides the base for biodiversity and diversity between species, within species, and that of ecosystems, which provides information about genetic divergence (Saini and Kaicker, 1987). Genetic diversity is associated with prevention of narrowing the genetic base and provides opportunity to develop new and improved cultivars with desirable characteristics. Genetic diversity explores the existing variations present in crop genotypes which can be identified through specific statistical method. With the increasing global demand there is a need to develop poppy with high opium and seed yielding varieties integrated with high specific alkaloids. Now-a-days research on poppy is heading in three directions, first is producing cultivars with high alkaloid content in the capsule husk, and the second is producing cultivars with low alkaloids or zero morphine. Third is cultivar with high seed yield. Thus, it becomes necessary to develop varieties for dual purposes having high alkaloids coupled with high seed yield. The present investigation was carried out to study, the degree of genetic divergence and variability present in the 60 germplasm/Indian landraces of opium poppy based on thirteen traits. Genetic divergence was quantified by multivariate analysis by assessing the proximity of accessions with each other, and classifying them in different groups/ clusters for further utilization of identified diverse genotype(s) for further hybrid/heterosis breeding program of opium poppy.
⁎
Corresponding author. E-mail addresses: [email protected] (R. Lahiri), [email protected] (R.K. Lal), [email protected] (N. Srivastava), [email protected] (K. Shanker). http://dx.doi.org/10.1016/j.jarmap.2017.10.001 Received 15 February 2017; Received in revised form 5 October 2017; Accepted 12 October 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Lahiri, R., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.10.001
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
2. Materials and methods
following- (Singh and Chaudhary, 1979). Analysis of variance was carried out following the procedure given by Panse and Sukhatme (1976). The data on 13 morpho-metric traits were analyzed using Mahanonobis’s generalized distance D2-statistics (Mahalanobis, 1936) and canonical analysis. The clustering was done on the basis of Tocher’s methods (Rao, 1952).
2.1. Materials The experimental material for the present study comprises of 60 accessions/Indian landraces of opium poppy (Papaver somniferum L.) which were collected from different eco-geographical regions of India were evaluated for two consecutive years (2013–2014 and 2014–2015) at CSIR- Central Institute of Medicinal and Aromatic Plants, Lucknow.
3. Results The analysis of variance showed significant differences among the genotypes for all the thirteen characters studied, which indicated the presence of high genetic variability among the present set of materials. On the basis of D2 values for all possible n (n-1)/2 = 1770 pairs of accessions, where ‘n’ is number of genotype, all the 60 experimental materials were grouped into eight diverse clusters (Figs. 1 and 2). Cluster I was the largest group that comprised 40 genotypes followed by Cluster II, consisted of 12 genotypes and clusters III and IV had two genotypes in each. Rest 4 clusters, Cluster V, VI, VII, and VIII have single genotype in each. Maximum intra-cluster distance was observed
2.2. Experimental site The present experiment was conducted at the experimental field of Genetics and Plant Breeding, CSIR-CIMAP, Lucknow, located at 26.5° N latitude and 80.50° E longitude and 120 m above sea level. 2.3. Experimental design All 60 genotypes were grown for two consecutive years (2013–14 and 2014–15) with three replications in a randomized block design (RBD). In each replication, one single row of each entry was grown with spacing of 10 cm within rows and 40 cm between rows. All required standard cultural practices were followed throughout the crop season which include pre-sowing, the addition of farmyard manure at the rate of 10t/ha and 80, 40, 40 kg/ha of nitrogen (N2), phosphorus (P2O5) and potassium (K2O), respectively as basal covering of land. An additional dose of nitrogen of 40 kg/ha was top dressed in two equal splits at 40 days and 60 days after sowing and sprayed with the fungicide redomil 0.2% at 45 and 60 days after sowing. The experimental field was irrigated as and when required. Morpho-metric data were recorded on five competitive randomly selected plants in each line for following thirteen traits- days to 50% flowering, plant height (cm), capsules/ plant, capsule size, seed yield/plant (gm), capsule husk yield/plant (gm); alkaloid yield/plant (%) – different includes five alkaloid i.e. morphine, codeine, thebaine, papaverine and narcotine, obtained from each selected plant in percentage. 2.4. Chemical analysis 2.4.1. 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 & 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. 2.4.2. 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. 2.5. Statistical analysis The pooled mean data of two years were statistically analyzed using the CSIR-CIMAP, Lucknow developed Statistical Software 4.0 version which is available in the Genetics and Plant Breeding Division of the institute based on Singh and Chaudhary (1979) and Panse and Sukhatme (1976). The mean, standard error, ranges were determined
Fig. 1. Cluster diagram with their distances of 60 genotypes in opium poppy germplasm.
2
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Fig. 2. Spatial distribution of 60 genetic stocks/accessions in opium poppy accessions.
Table 1 Distribution of opium poppy accessions in different cluster and their geographical locations. S. No.
Cluster
Number of accessions
Accessions
Origin of accessions
1
Ist
40
UP, Rajasthan, New Delhi, MP
2
IInd
12
G-1, G-2, G-4, G-6, G-7, G-8, G-10, G-12, G-13, G-14, G-16, G-20,G-22, G-24, G-29, G-30, G-40, G-42, G-47, G-48, G-54, G-55, G-59, G-60, G-61,G-62, G-68, G-69,G-70, G-75, G-76, G-77, G-82, G-85, G-86, G-92, G93, G-95, G-100 G-9, G-18, G-19, G-32, G-35, G-65, G-66, G-87, G-89, G-97, G-99, G-104
3 4 5 6 7 8
IIIrd IVth Vth VIth VIIth VIIIth
2 2 1 1 1 1
G-53, G-56 G-57, G-58 G-49 G-50 G-51 G-90
MP, Rajasthan, New Delhi, UP, UP, New Delhi UP Slovak Republic UP UP UP
content (0.0673%) along with low mean value for number of capsules, capsule index, codeine, thebaine, and narcotine content. Cluster VII has high mean values for capsule husk yield (7.303 g), codeine (0.305%) and thebaine (0.0315%) content (Prajapati et al., 2002). Cluster VIII includes genotypes having high peduncle length and high narcotine content while these have low mean values for days to maturity, seed yield, and capsule husk yield. It is also reported that genotypes within the cluster with a high degree of divergence would produce more desirable breeding materials for achieving maximum genetic gain (Kumar Bose and Pradhan, 2006). From the obtained results it could be concluded that accession with high seed yield and low morphine and papaverine content present in cluster I, accessions with low codeine, thebaine, and narcotine content from cluster V could be selected as parents for the hybridization program to fulfill the objective of poppy
in cluster IV (93153.35) and the minimum for Cluster I (33360.9). Inter cluster distance ranges from 115261.4–4717153. The minimum was between Cluster I and II (115261.4) followed by IV and V (128735.4) and maximum was between Cluster I and VII (4717153) followed by Cluster VI and VII (4655531) (Tables 1 and 2). Based on the cluster means (Table 3) the important cluster for high morphine content was Cluster III that possesses highest morphine content (0.249%) and low or negligible papaverine along with low seed (6.997 g) and capsule husk yield (5.334 g). Cluster IV had the highest mean for plant height (105.76 g), capsule index (0.904) and low mean for no. of capsules (1.917) and negligible papaverine. Cluster V had higher mean for days to flowering (140.94), number of capsule (2.667) with lowest plant height (91.37 cm). Cluster VI had highest mean for days to 50% flowering (104.17), seed yield (8.517 g) and papaverine 3
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Table 2 Estimates of inter- and intra-cluster distances among eight cluster of poppy. Cluster
I
II
III
IV
V
VI
VII
VIII
Mean
I
33360.9 (182.649)
115261.4 (339.501) 48885 (221.099)
2499505 (1580.982) 2208807 (1486.205) 33530.89 (183.114)
1149814 (1072.293) 990012.1 (994.993) 301928.1 (549.479) 93153.35 (305.210)
1590573 (1261.179) 1259500 (1122.274) 195216.9 (441.833) 128735.4 (358.797) 0 0
361002.1 (1900.000) 138511.1 (372.170) 2592753 (1610.202) 1339208 (1157.241) 1453019 (1205.412) 0 0
4717153 (2171.900) 4259130 (2063.765) 366371.6 (605.286) 1270438 (1127.137) 936428.8 (967.692) 4655531 (2157.667) 0 0
406832 (637.833) 340718.5 (583.710) 1363013 (1167.481) 513103.4 (716.312) 818596.3 (904.763) 601239.5 (775.396) 2976122 (1725.144) 0 0
1359188
II III IV V VI VII VIII
1170103 1195141 783494.9 797758.7 1392658 2397647 877453.1
Table 3 Cluster mean of the thirteen economic traits in opium poppy.
I II III IV V VI VII VIII
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
101.89 102.46 101.86 101.46 101.33 104.17 100.73 102.08
99.64 97.96 102.46 105.76 91.37 98.46 102.65 99.23
21.81 20.63 21.60 21.35 20.45 20.78 18.38 23.88
132.34 131.75 135.42 134.78 140.94 136.11 134.00 122.72
2.113 2.005 2.417 1.917 2.667 1.667 2.333 2.333
0.899 0.884 0.890 0.904 0.881 0.820 0.869 0.886
7.102 7.139 6.997 7.328 7.794 8.517 7.344 6.220
6.094 6.106 5.334 6.530 6.588 6.548 7.303 4.408
0.085 0.102 0.249 0.124 0.169 0.102 0.192 0.145
0.008 0.017 0.226 0.154 0.171 0 0.305 0.068
0.007 0.008 0.021 0.017 0.008 0.005 0.031 0.060
0.004 0.031 0 0 0.026 0.067 0.005 0.014
0.013 0.029 0.019 0.019 0.036 0 0.006 0.044
Where X1 = Days to flowering, X2 = plant height (cm), X3 = peduncle length (cm), X4 = days to maturity, X5 = number of capsule/plant, X6 = capsule index, X7 = seed yield/plant (g), X8 = capsule husk yield/plant, X9 = morphine content (%), X10 = codeine content (%), X11 = thebaine content (%), X12 = papaverine content (%), X13 = narcotine content (%).
recombination breeding programmes. The clustering pattern reflects the closeness between the genotypes. The clusters containing solitary genotype might be due to the parentage of the genotype and geographical origin. Majority of the results suggest no good correspondence between genetic divergence and geographical origin of genotypes. The maximum divergence was observed in cluster IV (Table 1). It is reported that genotypes within the clusters with high degree of divergence would produce more desirable breeding materials for achieving maximum genetic advance. Therefore due emphasis should be given on the members of cluster IV for selection of parents for hybridization programme. The other clusters showing high intra cluster values were cluster II, III and I and the minimum or nil intra cluster values were exhibited by cluster V, VI, VII and VIII this indicated negligible genetic diversity among the genotype for each character. The maximum inter cluster divergence was observed between cluster I and VII (Table 2). Highly divergent genotypes would produce a broad spectrum of variability in the subsequent generation for morphological and quality traits enabling further selection and improvement. Studies reported that selection of parents for hybridization should be from two clusters having wider inter clusters distance to get maximum variability in the segregating generations (Pradhan and Ray 1990; Rahman et al., 1997). Hybridizaion between genotypes of different cluster is required for the development of desirable genotypes. Studies by Singh et al. (1996) suggested utility of recombination between genotypes of different clusters In nutshell in the present investigation, it is suggested that hybridization programme within the divergent cluster are expected to give promising and desirable recombinants in the segregating generations. The two accessions namely, G-1 and G-53 were found promising for dual purpose i.e. for high seed yield and high morphine content (Table 5). These two accessions recommended for further commercial cultivation on large scale.
with high seed yield and low amount of alkaloids. Accessions among the cluster separated by high D2 values could be further used in the hybridization program for obtaining a wide range of variations among the segregates. The grouping obtained through D2 statistics based on two dimensional Z1 and Z2 chart, the contribution of characters towards divergence of genotypes revealed that in vector (Z1) contribution toward genetic divergence in the primary axis of differentiation were due to seed yield, codeine, papaverine, thebaine content. In vector II (Z2) which was the secondary axis of differentiation, papaverine, narcotine, days to 50% flowering were important. It was also revealed that vectors (I and II) were positive for days to 50% flowering and, papaverine. Such results indicate that the character contribute the maximum towards divergence (Table 4). Among the important economical characters in poppy, seed yield occupies an important place, beside high nutritive value it is a good source of healthy edible oil. For high seed yield the selected genotypes are G-8 (9.553gm), G-18 (9.019gm), G-24 (8.933gm), G-32 (8.792gm) and G-9 (8.616gm). Capsule husk is another important character of poppy as capsule straw is a source of alkaloids which was obtained by processing of unlanced capsule. The accessions G-18 (8.823gm), G-19 (8.566gm), G-29 (8.236gm), G-59 (8.275gm) and G- 60 (8.033gm) were found promising for poppy husk yield (Table 5). Similarly accession, G-1 (0.262), G-53 (0.307), G-61 (0.233) and G-65 (0.237) for morphine, G-49 (0.171), G-51 (0.305), G-53 (0.221) and G-56 (0.230) for codeine, G-10 (0.0246), G-51 (0.0243), G-51 (0.0315) and G-53 (0.0322) and G-57 (0.0262) for thebaine content were also found promising. 4. Discussion The analysis of variance showed high variances for most of the characters, which favour selection and utilization in future 4
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
Table 4 Character contributions (%), rank, mean and other allied genetic parameters of the thirteen traits in opium poppy. Parameter X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
CC (%) Ranks Z1 Vector CTPA (%) CCPAR Z2 Vector CTSA(%) CCSAR Range
3.53 7 0.0004 0.0336 5 −0.0012 −0.1805 9 88.04–108.36
3.897 6 −0.0007 −0.0542 12 −0.0012 −0.1716 8 18.38–26.39
1.733 1 0.0004 0.0279 7 0.0002 0.0319 4 122.44–141.94
10.711 2 0.0004 0.0317 6 −0.0012 −0.1813 10 1–3.167
4.517 5 −0.0002 −0.0174 11 −0.0017 −0.2454 11 0.78–1.016
6.913 4 0.00007 0.0053 9 −0.0003 −0.0476 5 4.34–9.55
7.973 3 0.0002 0.0218 8 −0.0009 −0.1400 7 3.66–8.82
53.568 1 0.0012 0.0930 4 −0.0003 −0.0431 5 0–0.66
0.392 1 0.9609 72.6645 1 −0.2486 −35.277 13 0–7.06
1.099 1 0.1060 8.0219 3 −0.0678 −9.6259 12 0–0.060
0.791 1 0.2554 19.3154 2 0.9640 136.77 1 0–0.076
2.549 8 −0.0017 −0.1355 13 0.0638 9.0542 2 0–0.055
2.319 9th −0.0001 −0.0083 10 0.0003 0.0520 3 91.05–107.05
Where X1 = Days to flowering, X2 = plant height (cm), X3 = peduncle length (cm), X4 = days to maturity, X5 = number of capsule/plant, X6 = capsule index, X7 = seed yield/plant (g), X8 = capsule husk yield/plant, X9 = morphine content (%), X10 = codeine content (%), X11 = thebaine content (%), X12 = papaverine content (%), X13 = narcotine content (%).
Table 5 Mean performances of five trait specific promising accessions of opium poppy. Characters
Accession
Seed Yield
G-8 G-9 G-18 G-24 G-32 G-18 G-19 G-29 G-59 G-60 G-1 G-53 G-61 G-65 G-49 G-51 G-53 G-56 G-57 G-10 G-11 G-51 G-53 G-57 G-1 G-53
Capsule husk yield
Morphine content
Codeine content
Thebaine content
Dual purpose
DF
PH
PL
DM
NC
CI
SY
CHY
M
C
T
98.413 103.013 103.086 103.506 104.536 103.086 106.153 102.843 102.43 103.143 98.413 101.12 103.16 101.623 101.326 100.726 101.12 102.603 100.8 102.58 107.05 100.726 101.12 100.8 98.413 101.12
102.416 99.613 99.606 100.446 97.26 99.606 102.886 98.71 101.166 92.876 89.633 97.769 100.98 104.16 91.369 102.65 97.769 107.156 107.216 103.943 102.95 102.65 97.769 107.216 89.633 97.769
21.53 20.433 20.843 21.343 19.503 20.843 20.073 19.88 26.096 23.393 22.883 22.82 21.463 21.826 20.453 18.383 22.82 20.376 20.233 23.566 21.173 18.383 22.82 20.233 22.883 22.82
130.053 135.556 132.776 131.5 135.223 132.776 129.166 137.39 133.89 141.943 139.333 134.666 125.666 127 140.943 134 134.666 136.166 137.556 136.39 136.336 134 134.666 137.556 139.333 134.666
1.777 1.555 1.666 1.666 2.111 1.666 1.888 2.444 3.167 2.166 2.444 2.9444 1.611 2.166 2.666 2.333 2.9444 1.888 1.778 1.666 1.389 2.333 2.9444 1.778 2.444 2.9444
0.938 0.795 0.933 0.936 0.892 0.933 0.932 0.898 0.844 0.932 0.906 0.894 0.899 0.907 0.881 0.868 0.894 0.885 0.914 0.912 0.882 0.868 0.894 0.914 0.906 0.894
9.553 8.616 9.019 8.933 8.792 9.019 8.402 7.789 7.669 8.402 8.234 7.289 6.044 5.535 7.794 7.344 7.289 6.704 7.01 7.346 7.924 7.344 7.289 7.01 8.234 7.289
7.466 6.008 8.823 6.586 6.255 8.823 8.566 8.236 8.275 8.033 6.091 6.668 4.532 5.329 6.588 7.303 6.668 5.633 6.396 5.765 6.793 7.303 6.668 6.396 6.091 6.668
0.0413 0.0623 0.0382 0.0053 0.0087 0.0382 0.07066 0.03476 0.0453 0.0661 0.262 0.307 0.233 0.237 0.169 0.192 0.307 0.191 0.142 0.05723 0.0263 0.192 0.307 0.142 0.262 0.307
0.066 0.007 0.025 0 0.0021 0.025 0.026 0.0065 0 0 0 0.2215 0.0224 0.0302 0.1714 0.3056 0.2215 0.2307 0.1734 0.021 0.006 0.3056 0.2215 0.1734 0 0.2215
0.0133 0.0228 0.0052 0.0058 0 0.0052 0.0063 0 0 0 0.0052 0.0322 0.0325 0.0163 0.0077 0.0315 0.0322 0.0113 0.0262 0.0246 0.0243 0.0315 0.0322 0.0262 0.0052 0.0322
P 0.0011 0.0312 0.0274 0.0028 0.029 0.0274 0.0312 0 0.0135 0 0.0221 0 0.0184 0.0387 0.0762 0.0045 0 0 0 0.0032 0.0015 0.0045 0 0 0.0221 0
N 0.0315 0.0425 0.0124 0.0024 0.0157 0.0124 0.0113 0.0214 0.0122 0 0.0163 0.0227 0.0227 0.0096 0.0367 0.0064 0.0227 0.0167 0.0195 0.0234 0.0165 0.0064 0.0227 0.0195 0.0163 0.0227
Conflict of interest
5. Conclusions
The authors declare that they have no conflict of interest.
Genetic divergence among 60 genetic stock of opium poppy to represent a wide range of variation with respect to yield has been estimated using D2 statistics. Selection of promising accessions based on genetic divergence along with better seed yield. Selected five promising accessions on the basis of genetic divergence with better seed yield G-8 (9.553gm), G-18 (9.019gm), G-24 (8.933gm), G-32 (8.792gm) and G-9 (8.616gm). For Capsule husk yield accession G-18 (8.823gm), G-19 (8.566gm), G-29 (8.236gm), G-59 (8.275gm) and G-60 (8.033gm) were found promising. For dual purpose i.e. for high seed and high morphine content accession G-1 (seed yield-8.234 gm and morphine content7.289) and G-53 (seed yield 7.289 gm and morphine content 0.307) was found promising and can be utilized for further improvement. On the basis of observation recorded in divergent clusters of poppy it can be concluded that hybridization between diverse parents among different clusters may be useful and help in coping with limitation pertaining with the improvement of poppy.
Funding Author Rashmi Lahiri thanks CSIR, New Delhi for providing Senior Research Fellowship. The funding agency plays no role in study design, collection, analysis and in paper writing. Acknowledgements Authors thanks to the Director, CSIR-CIMAP, Lucknow, India for the encouragement and facilities provided during the investigation. References Acharya, H.S., Sharma, V., 2009. Molecular characterization of opium poppy (Papaver somniferum) germplasm. American Journal of Infectious Diseases 5, 148–153. Azcan, N., Kalender, B.O., Kara, M., 2004. Investigation of Turkish poppy seeds and seed
5
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
R. Lahiri et al.
(Papaver somniferum L.). Varieties Grasas Y Aceites 57, 169–174. Panse, V.G., Sukhatme, P.V., 1976. Statistical Methods for Agricultural Workers. ICAR, New Delhi, pp. 361. Pradhan, K., Ray, R., 1990. Genetic divergence in rice. Oryza 27 (4), 415–418. Prajapati, S., Bajpai, S., Singh, D., Luthra, R., Gupta, M.M., Kumar, S., 2002. Alkaloid profiles of the Indian land races of the opium poppy Papaver somniferum L. Genetic Resources and Crop Evolution 49 (2), 183–188. Rahman, M., Acharya, B., Sukla Pande, S.N.K., 1997. Genetic divergence in low land rice genotypes. Oryza 34 (3), 209–212. Rao, C.R., 1952. Advanced Statistical Methods in Biometric Research. John Wiley and Sons, New York, pp. 1–104. Saini, H.C., Kaicker, U.S., 1987. Genetic diversity in opium poppy. The Indian Journal of Genetics and Plant Breeding 47 (3), 291–296. Shukla, S., Yadav, H.K., Rastogi, A., Mishra, B.K., Singh, S.P., 2010. Diversity in relation to breeding for specific alkaloids in opium poppy (Papaver somniferum L.). Czech Journal of Genetics and Plant Breeding 46, 164–169. Singh, R.K., Chaudhary, B.D., 1979. Variance and Covariance Analysis. Biometrical Methods in Quantitative Genetic Analysis. Kalyani Publisher, New Delhi (India), pp. 57. Singh, A.K., Singh, S.B., Singh, S.M., 1996. Genetic divergence in scented and fine genotype of rice. Annals of Agriculture Research 17, 163–166.
oils. Chemistry of Natural Compounds 40, 370–375. Celik, I., Camci, H., Kose, A., Kosar, F.C., Doganlar, S., Frary, A., 2016. Molecular genetic diversity and association mapping of morphine content and agronomic traits in Turkish opium poppy (Papaver somniferum) germplasm. Molecular Breeding 36 (4), 1–3. Facchini, P.J., Park, S.U., 2003. Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64 (1), 177–186. Gumuscu, A., Arslan, N., 1999. Comparing yield and yield components of some selected poppy (Papaver somniferum L.) lines. Turkish Journal of Agriculture & Forestry 4, 991–997. Gumuscu, A., Arslan, N., 2008. Research on heterosis on yield and yield components of some poppy hybrid lines. Journal of Agricultural Science 14, 365–373. Gupta, M.M., Verma, R.K., 1996. Combined thin-layer chromatography-densitometry method for the quantitative estimation of major alkaloids in poppy straw samples. Indian Journal of Pharmaceutical Sciences 58 (4), 161. Kumar Bose, L., Pradhan, S.K., 2006. Genetic divergence in deepwater rice genotypes. Journal of Central European Agriculture 6 (4), 635–640. Mahalanobis, P.C., 1936. On the generalized distance in statistics. Proceedings of the National Institute of Sciences of India 2, 49–55. Ozcan, M.M., Atalay, C., 2006. Determination of seed and oil properties of some poppy
6