BIOCHEMICAL CHARACTERIZATION BASED ON SDS-PAGE … · 10 BR010 Dhola Chhota Gotia (DCG) U.P. Land race 11 BR275 NC-57923 NBPGR Selection 12 BR271 NC-57913 NBPGR Selection 13 BR286

___________________________

Corresponding author: S. Shukla, National Botanical Research Institute, Rana Pratap Marg,

Lucknow-226001, U.P., India Tel: 91-522-2297936; Fax: 91-522-2205836; e-mail:

[email protected]

UDC 575.630

DOI: 10.2298/GENSR1503029V Original scientific paper

BIOCHEMICAL CHARACTERIZATION BASED ON SDS-PAGE ANALYSIS AND

CORRELATION AMONG TRAITS IN OPIUM POPPY (Papaver somniferum L.)

GERMPLASM

Nidhi VERMA1, *Sudhir SHUKLA

1, Kusum YADAV

2, Brij K. MISHRA

1 and Anu RASTOGI

1

1Genetics and Plant Breeding, National Botanical Research Institute, Lucknow-226001, U.P., India

2Department of Biochemistry, University of Lucknow, Lucknow-226020, U.P., India

Verma N., S. Shukla, K. Yadav, B. K. Mishra

and A. Rastogi (2015):

Biochemical characterization based on SDS-PAGE analysis and correlation among traits

in opium poppy (Papaver somniferum L.) germplasm.- Genetika, Vol 47, No. 3,1029 -

1050.

This research aimed to investigate the genetic diversity based on the pattern of

seed storage protein and to establish correlation between alkaloid and seed protein

content among 124 germplasm lines of opium poppy (Papaver somniferum L.). Twenty

seven polypeptide bands in range of 6 to 205 kDa were recorded. Similarity index was

calculated by using Jaccard’s Similarity index and cluster analysis was performed using

UPGMA model. Polymorphism was observed in three variable regions i.e., high, medium

and low molecular weight, among which bands of 10.4, 20, 22, 24, 30, 32, 33, 49 and 205

kDa’s were common in all the germplasms but other bands showed variation. All the 124

germplasms were broadly grouped into 13 clusters based on average linkage method.

None of the trait showed significant correlation with seed protein content. The differential

protein profile of the germplasms revealed wide variability and diversity among the

studied germplasms which could be further used in hybridization programme to obtain

maximum gain.

Key words: Alkaloids; Correlation; Opium Poppy; Path analysis; Seed storage

protein

.

INTRODUCTION

Opium poppy (Papaver somniferum L.) is a medicinal plant of immense importance and

one of the cheapest sources for raw opium having more than 80 alkaloids of medicinal value,

among which five alkaloids i.e. morphine, codeine, thebaine, narcotine and papaverine are the

1030 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

major ones (FRICK et al., 2005; SHUKLA et al., 2006, 2010). However, raw opium is used as

sedative, antispasmodic, hypnotic and sudorific is also used in diarrhea and dysentery (MISHRA et

al., 2010; CHATERJEE et al., 2010). Apart from being used mainly for pharmaceutical purposes,

poppy seeds are invaluable source of plant based protein supplement for human consumption

(AZCAN et al., 2004; OZCAN and ATALAY, 2006). Poppy seeds contain protein upto 24% and are

used whole or ground as an ingredient in many foods (SINGH et al., 1995, 1998). India has a rich

diversity of opium poppy germplasms (SINGH et al., 1997). The efficient management and

utilization of germplasms requires detailed knowledge of genetic diversity of economic traits for

proper characterization of populations to facilitate designing of efficient breeding strategies to

accomplish specific objectives (SHUKLA et al., 2010). Morphological characterization is the first

step in description and classification of germplasm (BHARGAVA et al., 2007, 2008). Several

attempts have been made to study the diversity in opium poppy based on morphological traits (LAL

et al., 1996; BHANDARI et al., 1997; SAINI and KAICKER, 1987; SINGH et al., 1998, 2003, 2004;

TIWARI et al., 2001). However the efforts to assess the diversity based on chemotypic traits were

also done (SHUKLA et al., 2006, 2010; YADAV et al., 2006). But, it is quite difficult to identify

potentially distinct germplasm lines based only on morphological traits as these are easily

influenced by the environment and thus less reliable. This has led to the development of new stable

parameters such as use of their genetic material (nucleic acids and proteins) as a tool for

varietal/germplasm identification (CRUZ et al., 1994; TAMKOC and ARSLAN, 2010). Many tools are

now available for studying variability and relationships among germplasms/accessions that include

seed protein electrophoresis, isozymes and various types of molecular markers (DOYLE et al.,

1992). Seed proteins are used as biomarkers because they are physiologically stable, easy to

handle and direct gene products where environment has little influence. Seed protein analysis

through sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used

to resolve inter or intra specific genetic diversity studies (OMONHINMIN and OGUNBODEDE, 2013)

identification and characterization of accessions, and phylogenetic relationship studies of the

accessions (TAMKOC and ARSLAN, 2010). Seed protein variations also provide information on the

relationship among the seeds collected from various geographical regions (SHOTWELL and

LARKINS, 1989; MURPHY et al., 1990; ANWAR et al., 2003; JAVAID et al., 2004). Since so much

efforts have been done to characterize the available germplasms of opium poppy, but till to date

the characterization based on molecular marker is not done. So the present study was undertaken

with the objectives (i) to gather the reliable information of genetic diversity in available

germplasms of opium poppy (ii) to study the association of seed protein with opium and seed yield

and its alkaloids.

MATERIALS AND METHODS

Plant Material

The experimental material for the study comprised of dry seeds of 124 distinct germplasm

lines of opium poppy (Papaver somniferum L.) maintained at Dept. of Genetics and Plant

Breeding, National Botanical Research Institute, Lucknow, India. The description and origin of

each germplasm is presented in Table 1. The germplasm lines were evaluated in randomized block

design with three replications. The rows of each germplasm per replication were grown with row

to row distance 30cm and plant to plant 10cm. Each row was three meter in length.

N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1031

HPLC Analysis

The pooled latex (opium) of four lancing of five plants from each germplasm per

replication was collected. Similarly the seeds of five plants per replication of each germplasm were

collected and weighed. The mean data of of opium yield/plant (mg) and seed yield/plant (g) were

used for statistical analysis. Five major alkaloids viz. morphine, codeine, thebaine, narcotine and

papaverine in opium latex were quantified through HPLC, Waters (Milford, USA) following the

method suggested by KHANNA and SHUKLA (1986).

Table1. Source and origin of 124 germplasm lines of opium poppy (P. somniferum L.)

S. No. Germplasm Collection Origin Source

1 BR001 Ghazipur (GZ) U.P. Land race

2 BR002 Bhakua (BK) U.P. Land race

3 BR003 Kalidandi(KD) U.P. Cultivar

4 BR004 MOP- 47 Raj. Land race

5 BR005 NBRI 2 (NB-2) NBRI Selection

6 BR006 Sugapankhi (SP) U.P. Land race

7 BR007 Kali Dandi Baunia (KDB) U.P. Land race

8 BR009 M -11 U.P. Land race

9 BR008 Telia (TE) U.P. Land race

10 BR010 Dhola Chhota Gotia (DCG) U.P. Land race

11 BR275 NC-57923 NBPGR Selection



14 BR306 MOP-217 M.P.(Mandsaur) Selection



17 BR311 ND -7 NDAUT Selection









26 BR329 Ran Jhatak Raj. Landrace

27 BR027 Te x M-11 U.P. Sel. Intra-specific

28 BR321 Dholia Raj. Landrace




32 BR320 NBPGR-11 NBPGR Selection

33 BR292 UO-1185 Raj.(Udaipur) Selection

1032 GENETIKA, Vol. 47, No.3, 1029-1050, 2015








41 BR304 IC-140 NBPGR Selection









50 BR331 GP-74 NBRI Sel. Inter-specific



53 BR314 ND-1186 NDAUT Selection




57 BR315 NDHS-101 NDAUT Selection

58 BR316 NDHS-201 NDAUT Selection



61 BR322 Shayama CIMAP Selection

62 BR323 Shweta CIMAP Selection

63 BR324 Sanchita CIMAP Selection

64 BR325 Vivek CIMAP Selection

65 BR334 Big Cap CIMAP Selection

66 BR222 Papline NBRI. Selection

67 BR220 NBRI-1 NBRI Sel. Intra-specific



70 BR328 BROP-1 NBRI Sel. Intra-specific






76 BR046 IS-1 NBRI Sel. Inter-specific

77 BR047 IS-2-1 NBRI Sel. Inter-specific

78 BR059 IS-2-12 NBRI Sel. Inter-specific










87 BR056A IS-11A NBRI Sel. Inter-specific

88 BR056B IS-11B NBRI Sel. Inter-specific




92 BR061 IS16 NBRI Sel. Inter-specific








100 BR113 Rajasthan Raj. Selection


102 BR223 Aphuri U.P. Landrace

103 BR247 Jhalawar (Raj.) Raj. Selection

104 BR-248 Jhalawar (Raj.) Raj. Selection




108 BR-253 Garoth (M.P.) M.P. Selection

109 BR-254 Pratapgarh (Raj.) Raj. Selection




113 BR-258 Kota (Raj.) Raj. Selection

114 BR-259 Kota (Raj.) Raj. Selection

115 BR-260 Neemuch II (M.P.) M.P. Selection

116 BR-261 Mandsaur III (M.P.) M.P. Selection

117 BR-262 Mandsaur III (M.P.) M.P. Selection

118 BR064 Chittor III (Raj.) Raj. Selection

119 BR-265 Chittor III (Raj.) Raj. Selection

120 BR-266 Jaora I (M.P.) M.P. Selection

121 BR-267 Jaora I (M.P.) M.P. Selection

122 BR-268 Mandsaur I (M.P.) M.P. Selection

1034 GENETIKA, Vol. 47, No.3, 1029-1050, 2015



CIMAP Central Institute of Medicinal and Aromatic Plants, Lucknow, U.P.

NBPGR National Bureau of Plant Genetic Resources, New Delhi

NBRI National Botanical Research Institute, Lucknow, U.P.

NDAUT Narendra Deva University of Agriculture and Technology, Faizabad, U.P.

M.P. Madhya Pradesh, Raj. Rajasthan

Protein Extraction

For the extraction of protein, 50mg defatted seeds of each opium poppy germplasm were

crushed and grounded into fine powder in liquid nitrogen using mortar and pestle. Grounded

samples were taken in a 1.5 ml eppendorf and 1ml of ‘Modified Laemmli SDS extraction Buffer’

(LAEMMLI, U.K. 1970) was added in each sample, sonicated for 15 minutes and centrifuged at

15,000 rpm for 10 minutes at 40C temperature. The supernatant was taken and pellet was

discarded. 10µL of Protease Inhibitor (PI) was added in each 500µL of sample supernatant, kept it

for overnight and centrifuged again at 15,000 rpm for 10 minutes at 40C. The final clear

supernatant of treated sample was taken in another eppendorf for further quantification.

Protein Quantification

The concentration of the extracted protein samples were determined by Bradford assay

(BRADFORD, 1976) using different concentrations of the samples against control in microtitre plate

(ELISA plate). Relative concentrations of all the samples were calculated using the following

formula at 595nm.

Protein in Sample (µg/µL) = (O.D. obs – O.D. blank) * Standard Factor

µL of sample taken

Where, Standard factor = 18.3 calculated as per the calculations based on standard.

Protein Profiling

Protein profiling of extracted samples were done through SDS-PAGE using 15%

separating gel and 5% stacking gel. Fixed amount of protein samples were loaded into the stacking

gel wells with prior heating of samples at 90-950C for 15 minutes. Electrophoresis was carried out

at 28mA and 150mV. The gels were stained with 0.25% (w/v) Coomassie brilliant blue R250

solution overnight and destained with methanol and acetic acid for about three hours and were

scanned for binary analysis of bands.


Protein Imaging and Data analysis

RF value and molecular weight of each protein band was determined using standard

molecular marker ranged from 3-205 kDa. Electropherogram for each germplasm was scored and

the presence [1] and absence [0] of each band was noted. Presence and absence of bands were

entered in the binary data matrix. Based on the results of electrophoretic band spectra, similarity

index was calculated by using Jaccard’s Similarity index through SPSS software. Further a

dendrogram was constructed using NTSYS software based on the UPGMA model (1973).

I n the present study attempt was made to characterize 124 opium poppy germplasm using SDS-

PAGE protein profiling of the seed storage proteins, simultaneously association of seed protein

content with opium, seed yield and alkaloid content were also established. The results showed that

amount of protein in different germplasm lines ranged from 0.610 to 2.530µg/µl. The highest

protein content was observed in germplasm BR56A followed by BR269, BR270 and BR266

(Table 2). The seed yield and opium yield per plant ranged from 0.55 to 5.13 g/plant and 222.67 to

2573.33 mg/plant respectively. The amount of morphine ranged from 8.82 to 17.59%, codeine

from 0.89 to 4.00%, thebaine from 0.92 to 8.06%, narcotine from 1.24 to 11.40% and papaverine

from 0.00 to 5.16 %.

The seed proteins of all the germplams were separated by SDS-PAGE electrophoresis and

the gel pictures of electrophoresis were used for binary analysis (Figure 1).

Table 2. Total alkaloids, seed and opium yield per plant and seed protein content in different

germplasm lines of opium poppy (P.somniferum L.)

S.S. No. Germplasm M% C% T% N% P%

SY

(g/plt)

OY

(mg/plt)

SP (µg/µl)

1 BR001 12.46 4.23 2.00 11.36 0.00 2.61 187.00 1.590

2 BR002 15.96 3.72 1.50 10.54 0.00 3.64 216.00 1.790

3 BR003 17.33 3.47 1.75 10.31 0.60 3.45 169.33 1.530

4 BR004 15.08 2.26 1.92 10.96 0.00 3.42 192.67 1.590

5 BR005 20.79 1.82 1.52 9.76 0.00 3.16 173.53 1.730

6 BR006 15.33 2.95 1.93 9.59 0.00 2.78 162.00 1.720

7 BR007 14.35 2.73 2.06 8.38 0.00 2.54 243.33 1.360

8 BR009 15.96 3.19 2.12 10.56 0.00 3.10 226.40 1.709

9 BR008 12.37 2.80 1.61 9.79 0.00 3.70 156.40 1.550

10 BR010 12.72 3.87 1.93 8.93 0.00 5.13 171.33 1.750

11 BR275 11.68 4.51 2.32 10.34 0.00 4.29 187.23 1.570

12 BR271 13.06 3.37 2.47 9.99 0.00 2.86 218.33 1.820

13 BR286 15.68 3.43 1.58 7.67 0.00 3.73 180.00 1.360

14 BR306 14.26 2.81 1.54 9.94 0.63 1.20 82.00 1.400

15 BR285 14.87 3.48 1.38 10.80 0.80 2.80 150.13 0.732

16 BR284 18.45 3.04 5.30 17.92 4.08 3.77 197.50 1.270

1036 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

17 BR311 14.49 4.44 1.69 13.12 1.67 2.50 162.90 1.610

18 BR283 13.99 4.69 2.90 15.34 5.27 2.97 171.20 1.430

19 BR282 11.36 6.76 4.85 15.09 5.78 2.99 197.40 1.990

20 BR281 14.48 4.38 7.03 12.70 6.04 2.25 224.00 2.450

21 BR280 16.79 5.19 2.13 13.40 2.30 1.66 189.40 2.210

22 BR279 18.65 3.23 3.97 10.10 3.49 2.83 137.30 2.404

23 BR278 16.62 2.59 3.30 3.41 0.00 3.88 242.60 2.050

24 BR277 13.51 4.27 2.17 6.92 0.14 2.69 243.80 1.940

25 BR276 14.35 4.89 1.57 8.11 2.60 2.69 215.50 2.220

26 BR329 18.31 3.62 1.69 8.28 0.33 2.73 393.20 1.680

27 BR027 14.62 5.04 2.91 9.23 0.00 3.30 259.50 1.850

28 BR321 14.33 5.84 2.46 8.95 0.00 2.76 235.70 1.850

29 BR272 20.40 2.56 1.86 9.69 0.30 2.70 213.50 1.840

30 BR273 17.60 3.03 2.05 8.68 0.00 2.13 162.80 1.830

31 BR274 18.96 4.20 1.95 10.67 0.00 2.21 160.80 2.203

32 BR320 17.83 2.94 1.83 10.08 0.00 2.60 119.50 2.020

33 BR292 18.51 3.20 1.44 11.37 0.00 1.89 112.40 2.002

34 BR294 15.86 2.37 2.44 8.37 2.79 2.53 157.40 2.310

35 BR293 12.62 4.96 3.12 8.21 0.36 3.07 200.40 2.130

36 BR295 18.76 3.48 3.66 6.11 0.00 3.07 229.00 2.130

37 BR296 16.87 3.28 2.03 11.04 3.89 3.62 175.80 1.440

38 BR297 17.73 3.10 1.31 9.66 1.39 3.65 136.50 1.570

39 BR298 19.25 2.89 1.52 8.98 0.23 2.89 150.70 1.550

40 BR299 16.33 3.16 2.50 9.58 0.26 3.65 227.33 1.760

41 BR304 16.45 4.68 4.52 5.90 0.59 3.05 126.33 1.250

42 BR301 12.98 3.48 3.52 9.16 0.59 2.51 248.00 2.220

43 BR290 14.70 3.43 3.18 7.97 2.22 3.16 211.33 1.650

44 BR289 18.70 2.48 2.66 10.30 3.97 2.94 205.67 1.380

45 BR288 18.77 1.85 1.34 7.99 1.56 2.69 184.50 1.630

46 BR287 14.27 2.01 2.57 7.89 2.18 4.07 154.80 1.870

47 BR303 13.39 2.10 1.84 8.40 1.86 2.17 164.40 1.620

48 BR300 18.43 2.75 1.31 13.94 0.50 3.20 195.50 1.840

49 BR302 15.24 2.97 2.52 13.88 0.25 2.49 266.00 1.360

50 BR331 15.41 3.48 3.16 11.50 0.57 3.33 191.30 1.770

51 BR293 14.82 3.68 3.01 6.67 1.19 2.11 247.10 1.810

52 BR310 12.79 2.28 2.28 5.51 5.06 2.58 186.00 1.660


53 BR314 14.42 3.21 3.16 8.21 4.06 2.16 194.50 1.600

54 BR317 15.00 2.77 2.23 7.89 0.59 4.34 193.40 2.000

55 BR305 12.12 3.27 2.33 10.31 3.19 2.75 270.37 2.220

56 BR309 15.18 3.47 2.45 6.06 0.27 3.25 213.07 2.260

57 BR315 13.69 3.50 8.37 8.08 0.00 2.39 215.37 1.290

58 BR316 14.10 3.89 3.49 9.26 0.00 3.31 230.50 1.800

59 BR319 20.86 3.66 1.80 8.21 0.00 2.48 257.40 1.860

60 BR318 17.15 3.44 2.67 8.95 0.00 2.29 159.80 1.710

61 BR322 17.66 3.10 1.60 8.22 0.00 2.12 232.00 1.750

62 BR323 20.69 3.24 2.03 7.93 0.00 2.39 157.70 1.790

63 BR324 17.35 3.39 2.37 10.81 0.00 3.02 163.50 1.920

64 BR325 17.28 3.42 1.16 8.00 0.28 2.75 179.10 1.480

65 BR334 11.66 3.04 1.94 8.32 5.98 4.01 128.70 0.890

66 BR222 14.25 3.14 1.22 6.50 0.00 3.92 260.50 1.520

67 BR220 17.73 3.40 1.71 9.11 1.42 2.66 288.40 0.810

68 BR326 14.03 3.00 1.12 5.06 1.53 3.67 303.00 1.660

69 BR327 12.52 4.06 1.12 3.74 0.00 3.27 203.13 0.620

70 BR328 15.39 3.48 2.04 4.88 3.72 2.53 207.50 0.680

71 BR307 10.78 2.99 1.00 4.68 0.00 2.14 245.50 1.620

72 BR308 9.20 3.72 0.70 5.31 0.00 2.49 257.33 0.610

73 BR312 13.78 3.11 1.27 6.28 0.00 3.26 211.50 1.470

74 BR313 15.93 3.03 1.76 4.33 3.86 3.02 162.10 1.990

75 BR291 14.82 3.07 1.47 8.39 2.03 2.36 218.50 1.540

76 BR046 14.52 4.57 2.37 7.82 0.14 0.63 223.97 1.480

77 BR047 10.49 2.63 4.03 3.29 2.30 0.58 185.00 1.520

78 BR059 11.47 3.64 2.80 8.11 0.00 2.18 125.00 1.520

79 BR048 11.98 3.71 1.55 5.54 2.68 3.22 231.00 2.060

80 BR049 12.92 4.92 2.19 7.84 0.61 3.52 208.00 2.360

81 BR050 11.02 3.39 2.67 8.75 4.20 3.42 215.70 1.570

82 BR051 9.35 4.79 2.22 7.61 0.95 4.91 183.33 1.700

83 BR052 15.91 2.40 2.44 6.57 0.00 4.64 178.60 1.890

84 BR053 16.28 2.37 1.62 9.47 0.00 1.92 216.00 1.420

85 BR054 13.00 2.73 2.67 7.19 4.77 2.64 264.30 1.800

86 BR055 14.90 2.61 1.37 9.95 0.00 2.66 149.50 1.780

87 BR056A 17.16 2.76 1.08 8.04 0.00 2.25 130.33 2.520

88 BR056B 16.03 2.71 2.83 8.43 3.10 2.31 192.33 2.370

1038 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

89 BR057 14.27 2.29 0.77 11.36 2.61 2.64 141.30 1.640

90 BR058 11.99 3.98 3.05 12.99 4.07 2.79 171.60 1.080

91 BR060 15.13 3.40 0.72 10.70 0.32 3.79 153.50 1.590

92 BR061 16.14 2.88 1.96 7.84 0.67 3.23 237.00 1.402

93 BR062 11.47 1.95 0.81 7.34 0.66 2.77 263.60 2.020

94 BR063 12.64 1.74 0.61 6.31 0.00 3.21 218.60 1.705

95 BR064 11.03 2.16 1.74 5.72 0.00 3.03 173.30 1.910

96 BR065 11.64 1.68 2.10 8.75 0.00 4.14 120.00 2.030

97 BR066 10.50 2.45 2.79 6.34 0.00 2.87 126.20 1.970

98 BR067 16.19 1.96 1.55 3.78 0.00 2.28 146.00 2.180

99 BR068 16.64 3.54 1.64 5.84 1.29 2.42 159.67 2.170

100 BR113 18.48 3.03 1.68 5.66 1.35 1.86 137.60 1.600

101 BR330 18.68 2.09 1.08 5.41 0.25 3.54 134.33 1.500

102 BR223 20.26 1.68 0.82 5.40 0.51 4.71 112.53 2.200

103 BR247 16.11 2.00 0.78 6.82 3.30 3.12 158.67 1.900

104 BR-248 15.62 3.69 1.26 5.31 1.53 3.79 134.73 1.900

105 BR-249 16.01 3.44 1.32 4.55 2.21 3.05 142.67 1.800

106 BR-250 16.44 2.07 1.23 6.51 1.17 4.00 131.33 1.300

107 BR-251 17.30 2.21 0.85 4.76 2.81 3.78 138.13 1.800

108 BR-253 16.35 2.65 1.16 5.45 2.37 1.74 152.13 1.600

109 BR-254 15.09 3.17 1.07 5.44 2.58 2.80 159.87 1.400

110 BR-255 17.80 3.02 0.91 7.18 0.09 3.12 164.80 1.800

111 BR-256 19.38 1.57 1.04 6.37 0.06 2.33 192.73 1.400

112 BR-257 18.49 3.16 1.11 6.48 1.50 3.38 151.53 2.000

113 BR-258 15.09 3.26 1.35 5.53 1.40 1.82 152.47 1.600

114 BR-259 17.90 3.58 1.75 4.42 2.43 2.74 200.83 1.400

115 BR-260 16.82 3.60 1.55 5.80 0.88 3.13 151.33 1.300

116 BR-261 18.21 3.61 1.57 5.58 1.47 2.74 143.60 1.600

117 BR-262 17.72 2.95 0.89 5.17 0.00 2.72 151.17 1.600

118 BR064 14.55 2.49 0.61 6.11 2.19 4.96 191.90 1.400

119 BR-265 15.87 2.52 0.80 5.78 0.36 3.59 149.67 2.000

120 BR-266 17.93 2.74 1.12 6.35 0.94 3.11 125.33 2.500

121 BR-267 18.13 3.01 1.26 3.70 1.50 2.93 220.00 1.900

122 BR-268 15.15 1.66 0.86 7.04 2.27 3.04 200.00 1.800

123 BR-269 17.63 3.06 1.09 3.47 1.37 2.92 392.67 2.500

124 BR-270 15.08 1.62 0.90 7.14 2.31 2.95 396.20 2.500


Mean+

SE

15.43+

0.23

3.20+

0.08

2.03+

0.10

8.13+

0.24

1.26+

0.14

2.95+

0.07

191.66+

4.80

1.73+

0.03

M, morphine; C, codeine; T, thebaine; N, narcotine; P, papaverine; SY, seed yield (g) per plant; OY, opium yield (mg)

per plant; SP, seed protein (µg) per µl

a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Contd.

1040 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

(j) (k) (l)

(m) (n) (o)

Figure 1. Seed protein pattern in 124 germplasm lines of opium poppy (P. somniferum L.) (a-o)

Genetic Diversity Evaluation

The protein profiling of 124 germplasms gave a total of 27 bands. Polymorphism was

observed in three variable regions i.e., high, medium and low molecular weight, among which

bands of 10.4, 20, 22, 24, 30, 32, 33, 49 and 205 kDa’s were common in all the germplasms while

other bands showed variation. The protein profile of the germplasm also showed that seeds of most

of the germplasm lines contained majorly low molecular weight proteins (<75kDa). Some protein

bands showed high expressions and their expression pattern also varied in different germplasm

lines. It was noticed that germplasm lines BR-321, BR-320, BR-287, BR-302, BR-331, BR-247,

BR-249, BR-268 and BR-269 do not have higher molecular weight proteins. The germplasms

namely BR-001, BR-002, BR-006, BR-007, BR-009, BR-008, BR-276, BR-029, BR-027, BR-273,

BR-320, BR-292, BR-293 and BR-049 possessed majorly expressing seed protein bands of 49kDa,

43 kDa, 33 kDa, 32 kDa and 30 kDa. The protein bands of size 26 kDa, 25kDa and 27kDa were

unique bands which were present in few germplasm lines viz., BR-283, BR-282, BR-272, BR-273,

BR-320, BR-298, BR-299, BR-304, BR-301, BR-290, BR-289, BR-309, BR-315, BR-308, BR-

249, BR-250, BR-251, BR-253, BR-254, BR-256, BR-257, BR-258, BR-259, BR-260, BR-261,


BR-262, BR-064, BR-265, BR-266, BR-267, BR-268 and BR-270. The 14.3kDa band was a

unique band with low molecular weight present in germplasms BR-319, BR-318, BR-322, BR-

323, BR-324, BR-325, BR-312, BR-313, BR-291, BR-046, BR-047, BR-059, BR-048, BR-049,

BR-050, BR-051, BR-056B, BR-057, BR-068 and BR-248 only.

Cluster Analysis

All the 124 germplasms were broadly grouped into 13 clusters based on average linkage

method (Figure 2). The mean values of germplasms falling in each cluster are presented in Table

2. Cluster I had 17 different germplasm lines and Cluster II had largest number of germplasms

(44). Cluster III, IV and V comprised of 32, 5 and 2 germplasm lines respectively. Cluster VI had

six germplasms and cluster VII had two germplasms while cluster VIII and IX had one germplasm

in each. Cluster X had five germplasms and Clusters XI, XII and XIII had three germplasms in

each cluster.

Figure 2. Dendrogram showing 13 major and various other sub clusters in opium poppy (P. somniferum L.).

1042 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

Similarity Index

The similarity index between germplasm ranged from 0-1%. Based on the similarity

index, the similarities between germplasms is classified as exact similar (1.00), very similar (0.9

and 0.8), moderate similar (0.7 and 0.6) and less similar (<0.3).

Correlation Studies

Table 3. Genotypic and phenotypic (in parenthesis) correlation among various traits in opium poppy (P.

somniferum L.) Characters Codeine Thebaine Narcotine Papaverine Seed Yield

(mg)

Opium Yield

(mg)

Seed Protein (µg/µl)

Morphine -0.3422**

(-0.2679)

0.0183

(0.0529)

0.1396

(0.1852)

-0.3673**

(-0.3150)

0.0908

(0.0770)

-0.4022**

(-0.3677)

-0.1196

(-0.1097)

Codeine 0.0716

(0.0823)

-0.0509

(-0.0207)

-0.1761

(-0.1557)

-0.1830

(-0.1517)

0.0763

(0.0695)

-0.1177

(-0.1078)

Thebaine 0.1526

(0.2099)

0.1001

(0.1099)

-0.2004

(-0.1651)

0.0127

(0.0112)

-0.0224

(-0.0205)

Narcotine 0.1608

(0.1681)

-0.0222

(0.0016)

0.1284

(0.1154)

0.0049

(0.0053)

Papaverine -0.0002

(0.0086)

0.2131*

(0.2055)

0.1214

(0.1188)

Seed Yield (mg)

0.1381 (0.1191)

0.0224 (0.0202)

Opium

Yield (mg)

0.1849

(0.1840)

* & ** Significance at 5% and 1% respectively

The genotypic correlation coefficient between different pair was similar in sign and

nature to the corresponding phenotypic correlation coefficient (Table 3). However, genotypic

correlation coefficient was generally higher in magnitude for all the traits than the corresponding

phenotypic correlation coefficient. In the present study, none of the trait showed significant

correlation with seed protein content. Seed protein was negatively correlated with morphine (-

0.1196), codeine (-0.1177) and thebaine (-0.0224) while positively correlated with narcotine

(0.0049), papaverine (0.1214), seed yield (0.0224) and opium yield (0.1849). Seed yield was

positively correlated with morphine (0.0908), opium yield (0.1381) and seed protein (0.0224)

while negatively correlated with codeine (-0.1830), thebaine (-0.2004), narcotine (-0.0222) and

papaverine (-0.0002). Opium yield had positive significant correlation with papaverine (0.2131)

and negative significant correlation with morphine (-0.4022) while positive correlation with all the

traits. Morphine had negative significant correlation with codeine (-0.3422), papavarine (-0.3673)

and opium yield (-0.4022) while had positive but non-significant correlation with thebaine

(0.0183), narcotine (0.1396) and seed yield (0.0908). Codeine had positive correlation with

thebaine (0.0716) and opium yield (0.0763). Thebaine had positive correlation with narcotine


(0.1526), papaverine (0.1001) and opium yield (0.0127) while narcotine had positive correlation

with papaverine (0.1608), opium yield (0.1284) and seed protein (0.0049). Papaverine had

significant negative correlation with morphine (-0.3673) but positive correlation with opium yield

(0.2131).

Path coefficient analysis

Figure 3. Path diagrams showing path coefficients and correlation of Seed protein, Seed yield and Opium

yield with different traits in Opium Poppy (P.somniferum L.)

1044 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

Correlation coefficient measure the mutual association between two variables but doesn’t

permit the cause and effect relationship of traits contributing directly or indirectly towards the

economic yield, whereas path coefficient is partially standardized regression coefficient and as

such measure the direct influence of one variable upon another and specifies the causes and

measure their relative importance (SINGH et al., 2004). Path coefficient analysis was carried out as

direct and indirect effect using genotypic correlation coefficient among various characters to

estimate the direct and indirect effect of seven characters of manifestation on seed protein content,

seed yield and opium yield separately (Figure 3). Path analysis based on seed protein content

exhibited that morphine, codeine, thebaine, narcotine and seed yield had negative direct effect

towards seed protein while morphine and seed yield indirectly affected via codeine but codeine,

thebaine and narcotine indirectly affected via seed yield, however seed protein exhibited negative

genotypic correlation. Papaverine and opium yield had positive direct effect towards seed protein

while indirectly affected via narcotine, however seed protein exhibited positive genotypic

correlation. The path analysis based on genotypic correlation of seed yield showed morphine,

narcotine and opium yield had positive direct effect towards seed yield while morphine and opium

yield indirectly affected via narcotine, however seed yield exhibited positive genotypic correlation.

Codeine, thebaine, papaverine and seed protein had negative direct effect towards seed yield while

codeine and thebaine indirectly affected via seed protein, however seed yield exhibited negative

genotypic correlation except for seed protein. Path analysis of opium yield revealed that morphine

and codeine had negative direct effect towards opium yield while morphine indirectly affected via

codeine and papaverine, however opium yield exhibited negative genotypic correlation with

morphine and positive with codeine. Thebaine, narcotine, papaverine, seed yield and seed protein

had positive direct effect towards opium yield while narcotine and seed yield indirectly affected

via morphine, however opium yield exhibited positive genotypic correlation.

DISSCUSSION

The frequent occurrences of insufficient discrimination of germplasms by grow out test which

consequently results inability to confirm distinctness. This fact encouraged us to investigate

complementary method of characterizing germplasms based on the comparison of genetic diversity

following conventional method. Seed protein electrophoresis was performed by total soluble seed

proteins separated by SDS-PAGE electrophoresis. The knowledge of genetic diversity is a useful

tool in gene-bank management and breeding experiments like tagging of germplasm, identification

and/or elimination of duplicates in the gene stock, establishment of core collections and sorting of

populations for genome mapping experiments (KAGA et al., 1996). Since many cultivars are

closely and genetically related, but it is difficult to distinguish the germplasms on the basis of

morphological characters alone due to interaction of environment (TAMKOC and ARSLAN, 2010).

So the hereditary materials are used as genetic markers for the studies on genetic diversity. The

hereditary information (DNA) is expressed in distinguishable traits as RNA and proteins

(STOYANOVA et al., 2011). Proteins are generally polymorphic and heritable and are direct

products of active genes due to which they are the source of good genetic markers and thus

polymorphism in protein profiles reflects the changes in the active part of the genome (GARIMA et

al., 2013). These are independent of environment and display variations through polymorphisms.

Hence, seed protein electrophoresis are being used successfully as a biochemical marker in the

assessment of diversity and confirming cultivar identity (STOYANOVA et al., 2011). The use of

seed storage proteins as biochemical markers to screen the germplasms is cost, time and labour


effective in assessing the genetic variations of wild species germplasms and even though have

been used as genetic markers to study the genetic diversity, identifying variations among species,

screening of cultivars and establishing genome relationships (RADWAN et al., 2013). Various

techniques are available to analyze the protein polymorphism but polyacrylamide gel

electrophoresis (PAGE) is preferred due to rapid analysis and simplicity for unfolding genetic

variations among different plant species (CHITTORA and PUROHIT, 2012). In the present study seed

protein electrophoresis was performed by total soluble seed proteins separated by SDS-PAGE. The

differential protein profile pattern for most of the germplasms revealed diversity among the studied

germplasms. Similar findings were also observed by SINGH et al. (2004) and YADAV et al. (2007a,

b) based on their studies on morphological traits.

Polymorphism was observed in three variable regions i.e. high, medium and low

molecular weight which suggested that the origin of these germplasms is from the same genetic

background. Earlier, similar findings based on chemotypic traits were also observed in the same

set of germplasm lines (SHUKLA et al., 2010). The similarity in seed storage protein banding

pattern for some germplasm lines confirmed that the investigated germplasms were collected in

narrow restricted area which is also reflected by significant and higher genotypic correlation than

phenotypic correlation (SINGH et al., 2003, 2004; SHUKLA et al., 2010). Major expressing protein

bands of different molecular weights found in most of the germplasm also had higher amount of

protein content which could be attributed to higher metabolic activity in the germplasm lines. Only

four protein bands of molecular weights 14.3, 25, 26 and 27 from total protein bands were

uniquely present in ample number of germplasm which could ease in identification of these

distinct germplasm.

The clustering grouped all the 124 germplasms into 13 distinct clusters depending upon

the similarity of seed protein profile. The diversity analysis based on morphological similarity also

clustered the same germplasm lines into 13 clusters (SINGH et al., 2004). In the present study,

cluster size varied from 1 to 50 germplasm lines. All clusters contained accessions of different

origin except cluster VIII and IX having single germplasm each. The germplasms involved in

clustering are group of Indian landraces, improved varieties and selections obtained through

different breeding programmes and maintained at National Botanical Research Institute, Lucknow

for the last four decades (SINGH et al., 2004). The germplasm lines of common geographical origin

were distributed over several clusters which indicated partial role of geographical distribution in

the genetic diversity of opium poppy germplasms of Indian origin (SINGH, 1991; SINGH et al.,2003,

2004). Thus, it could be assumed that geographical diversity though important but may not be a

crucial factor in determining genetic divergence.

A strong correlation and higher heritability for economically-important characters are

highly desirable in breeding and selection programme. Correlation measures the mutual

association between two variables, which aids in determining the most effective procedures for

selection of desirable trait (UDENSI and IKPEME, 2012). The magnitude of genotypic and

phenotypic correlation coefficient observed similar trends for most of the characters and also

showed higher genotypic correlation coefficient than phenotypic correlation which could be

explained by low environmental effect on investigated agronomic traits (FALCONER, 1989; MEHTA

et al., 2006; AKRAM et al., 2008; YADAV et al., 2006). The higher values of genotypic correlation

coefficient might also be due the fact that the germplasm lines are superior but their expressions

are lessened under the influence of environment (MEHTA et al., 2006).

1046 GENETIKA, Vol. 47, No.3, 1029-1050, 2015

The present study also explored the probable reasons of increase or decrease of different

alkaloids based on their biosynthetic pathways. The morphine content in general was the major

constituent followed by narcotine, codeine, thebaine and papaverine (Table 2). The seed protein

content was positively correlated with seed and opium yield, while opium yield had positive

correlation with codeine, thebaine, narcotine and papaverine which is due to the fact that the

increase in quantitative characters can elevate the quality traits as also reported by CALIMAN et al.

(2008). However, negative correlation between yield and quality trait was also prominent as in

case of opium and morphine content which has been reported earlier also (SHUKLA et al., 2003;

YADAV et al., 2004; BHANDARI et al., 1997). The significant and negative correlation of morphine

with codeine has occurred in the present study was also reported in our earlier studies (YADAV et

al., 2006). Contrary to our findings, BAJPAI et al., (2000) reported positive correlation between

morphine and codeine. Morphine is synthesized from thebaine via codeine (PSENAK, 1998). A

valid reason for the negative association among these alkaloids has been reported earlier (YADAV

et al., 2006). However, codeine was positively and significantly correlated with thebaine and

narcotine which might be due to the fact that the content of reticuline, the key branch point

intermediate of opium alkaloid synthesis was significantly correlated to that of codeinone, but had

negative correlation with codeine content as also reported by PRAJAPATI et al. (2002). This

suggests that codeinone reductase, the key enzyme which converts codeinone to codeine was

probably rate limiting and controls the accumulation of morphine in germplasm lines. The increase

in codeine and thebaine content in some of the identified germplasm lines may be due to less

enzymatic activity involved at a particular conversion step causing partial blocking of morphinane

pathway (Thebaine Codeine Morphine, SHUKLA et al., 2006).

The study also confirmed the findings of PRAJAPATI et al. (2002) that the total alkaloids

remain low in the germplasm lines deficient in both papaverine and narcotine as narcotine is one

of the major constituent of opium poppy (HOSZTFI, 1998). Since in mature seeds, type and amount

of proteins are more constant than other plant tissues therefore, the SDS-PAGE pattern of seed

storage proteins of opium poppy showed polymorphism on the basis of difference in protein

intensity among germplasm lines. The enhancement in seed protein content can be achieved

through selection of high seed and opium yielding plant types rich in morphine and thebaine from

the germplasm lines as there exist positive genotypic correlation and direct effect of these traits on

seed protein content.

CONCLUSIONS

Based on the present study desirable germplasm lines for seed storage protein have been

isolated which can be used in future hybridization programmes aimed to develop varieties rich in

seed storage proteins for commercial cultivation in various opium growing locations of the states

of India. Beside this, it was also concluded that the electrophoresis (SDS-PAGE) of seed storage

proteins can be economically used to assess genetic variation and relation in germplasm and also

to differentiate mutants from their parent genotypes. It was viewed that the seed storage protein

profiles could be a useful marker in germplasm identification.

Contribution of each Author: Nidhi Verma, Anu Rastogi conducted the experimentation, Sudhir

Shukla provided the idea and checking of the manuscript, Brij Kishore Mishra and Nidhi Verma

wrote the manuscript and Kusum Yadav provided specific comments, intellectual inputs on the

manuscript and checking of the manuscript.


ACKNOWLEDGEMENTS

The authors are thankful to the Director for encouragement and facilities provided during the

investigation. Nidhi Verma, Brij Kishore Mishra and Anu Rastogi thank the Council of Scientific

and Industrial Research, New Delhi for providing Research Fellowships. Financial assistance

provided from Narcotics Dept., Ministry of Finance, Govt. of India is duly acknowledged.

Received February02nd, 2015

Accepted October 20th, 2015

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BIOHEMIJSKA KARAKTERIZACIJA BAZIRANA NA SDS–PAGE ANALIZI

KORELACIJE IZMEĐU OSOBINA GERMPLAZME MAKA

(Papaver somniferum L.)

Nidhi VERMA1, *Sudhir SHUKLA

1, Kusum YADAV

2, Brij K MISHRA

1 i Anu RASTOGI

1

1 Genetika i oplemenjivanje biljaka, nacionalni botanički istraživački Instiut, Lucknow-226001,

U.P., India 2 Odelenje za biohemiju, Univerzitet Lucknow, Lucknow-226020, U.P., India

Izvod

Cilj ovih istraživanja je spiivanje genetičkog diverziteta među 124 linije germplazme, zasnovanog

na elektroforegramu rezervnihb proteina zrna i uspostavljanje korelacije između sadržaja

alkaloida i rezervnih roteina zrna maka (Papaver somniferum L.). Dobijeno je 27 polipeptidnih

traka molekulske težine od 6 do 205 kD. Indeks sličnosti je izračunat korišćenjem Jaccard’s

indeksa sličnosti a analiza klastera preko UPGMA modela. Utvrđen je polimorfizam u tri različita

regiona; visoke, srednje i male molekulske težine čije trake težine 10.4, 20, 22, 24, 30, 32, 33, 49 i

205 kDa’s su bile zajedničke kod svih germplazmi ali su ostale trake pokazale varijabilnost. Sve su

široko grupisane u 13 klastera zasnovanih na meetodi prosečne ukopčanosti. Ni jedna od osobina

nije pkazala značajne korelacije sa sadržajem proteina.Razlčiti profili proteina germplazme

potvrđuju široku varijabilnost i diverzitet između ispitivanih germplazmi, što može dalje da se

koristi u programima hibridizacije da bi se ostvarila maksimalna dobit.

. Primljeno 02.II 2015.

Odobreno 20. X. 2015.

Documents

BIOCHEMICAL CHARACTERIZATION BASED ON SDS-PAGE … · 10 BR010 Dhola Chhota Gotia (DCG) U.P. Land race 11 BR275 NC-57923 NBPGR Selection 12 BR271 NC-57913 NBPGR Selection 13 BR286