Influence of seed priming and storage time on germination and enzymatic activity of selected Berberis species Tarun Belwal, Aarti Bisht, Indra D. Bhatt & Ranbeer S. Rawal
Plant Growth Regulation An International Journal on Plant Growth and Development ISSN 0167-6903 Plant Growth Regul DOI 10.1007/s10725-015-0051-0
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Author's personal copy Plant Growth Regul DOI 10.1007/s10725-015-0051-0
ORIGINAL PAPER
Influence of seed priming and storage time on germination and enzymatic activity of selected Berberis species Tarun Belwal1 • Aarti Bisht1 • Indra D. Bhatt1 • Ranbeer S. Rawal1
Received: 1 July 2014 / Accepted: 9 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Effect of various priming treatments, storage conditions and seed collection sites on seed germination of selected species of Berberis i.e. B. aristata and B. jaeschkeana was studied. Pre-soaking the seeds in different treatments followed by incubation at 25/10 °C (light: dark; 16:8 h) revealed variation in germination percentage, mean germination time (MGT), germination rate and seed vigour index. Seed treated with vermiwash (1:4 ratio) showed maximum germination percentage in B. aristata (92 %) and 1:10 ratio in B. jaeschkeana (50.6 %). Storage conditions (cold–dry for 1–4 months) and collection sites found to be effective for increasing germination percentage and germination rate in B. jaeschkeana at both the altitude (3200 and 3700 m asl) and B. aristata at higher altitude (2600 m asl) only. Further, germinated seedlings were analysed for enzymatic activity (Catalase and peroxidase) and found to be negatively correlated with catalase and positive with peroxidase activity for germination percentage in both species. Role of catalase and peroxidase in seed germination was associated with formation of reactive oxygen species which was found effective when treating with hydrogen peroxide. The potential for germination after various priming treatments especially vermiwash and thiourea is recommended and precursors such as hydrogen peroxide would be preferred in these species especially for
Electronic supplementary material The online version of this article (doi:10.1007/s10725-015-0051-0) contains supplementary material, which is available to authorized users. & Indra D. Bhatt
[email protected];
[email protected] 1
G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643, Uttarakhand, India
reducing the MGT. Cold–dry storage would be preferred for Berberis species of higher altitude population. Keywords Berberis Priming treatment Seed germination Enzyme activity Reactive oxygen species Hydrogen peroxide Abbreviations ROS Reactive oxygen species H2O2 Hydrogen peroxide CAT Catalase POD Peroxidase NADPH Nicotinamide adenine dinucleotide phosphate TU Thiourea VM Vermiwash GA3 Gibberellic acid CU Cow urine SA Salicylic acid MGT Mean germination time GP Germination percentage GR Germination rate PPP Pentose phosphate pathway SVI Seed vigour index IUCN International Union for Conservation of Nature
Introduction Seed priming is an effective way to overcome dormancy as it reduces the time between seed sowing and seedling emergence (Parera and Cantliffe 1994). The germination process starts with the rapid uptake of water (Phase I), followed by embryo expansion (Phase II), and emergence
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of the radicle (Phase III). Germination and dormancy has been reported to be influenced by a number of genetic and environmental factors that interact to maximize the long term chance of survival of the seeds in number of species (Koornneef et al. 2002), and Himalayan species are no exception. Among others, the genus Berberis is well known for its active compound ‘Berberine’, which has anti-diabetic, antimicrobial, anti-cancer, anti-lipidemic and anti-oxidant properties (Potdar et al. 2012). In India, the genus Berberis accounts for over 55 species, of which 24 species (44 %) are reported from Uttarakhand (Rao et al. 1998a, b; Tiwari et al. 2012). At present, Berberis species are being harvested from the wild for extraction of valuable drug ‘Berberine’, and also for removal as a host of dreaded wheat rust i.e. Puccinia graminis tritici (Pritchard 1911). This has imposed threat to the different species of Berberis in the region and a number of species of the genus have found place in the list of threatened category. In this context, it is imperative to develop reproducible propagation protocol so that the threatened status of different species of the genus can be minimized. As such, scanty information on seed germination of species of Berberis is available and systematic investigation is yet to be carried out. Thakur et al. (2005) demonstrated the improvement in germination of B. aristata using acid scarification treatment as compared to control. The effect of storage conditions on germination of B. aristata was also studied (Thakur et al. 2006). Completion of germination is the critical step as it requires the activation of a complex regulatory system, which is controlled by intrinsic and extrinsic factors. Seed germination occurs when enzyme activity is restored after hydration of the protein (Mayer and Poljakoff 1989). Various treatments such as plant growth hormones (gibberellic acid, indole butyric acid) and chemicals (salicylic acid, thiourea, sulphuric acid, polyethylene glycol, etc.) have been reported to be used as priming agent. Besides, organic treatments such as vermiwash and cow urine are also reported to be effective for seed germination (Hatti et al. 2010). Imbibed seeds generate reactive oxygen species (ROS) during water uptake (Wojtyla et al. 2006) and show a positive effect in releasing seed dormancy (Oracz et al. 2007). Antioxidant enzymes such as catalase, peroxidase, superoxidase, etc. have been known to scavenge ROS, and thus help in seed germination. Various pre-treatments have shown increased germination and are well correlated with antioxidant enzyme activity (Hendricks and Taylorson 1975). Berberis species are known for physiologically nondeep dormancy. Various pretreatments such as scarification (Pelton 1956), stratification (dry–hot, dry–cold, wet–cold) (Pelton 1956; Cavieres and Arroyo 2000) are reported to
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break seed dormancy (Schwienbacher et al. 2011). Among others, the dry–cold stratification is considered more relevant to those species, which experiences low temperature during winter. Similar reports on other Himalayan species are available where chilling pretreatment found to be effective [Myrica esculenta (Bhatt et al. 2000); Cornus capitata (Airi et al. 2005)]. Considering the above, the present study was initiated to determine the effect of different priming treatments, storage conditions, and role of catalase and peroxidase enzymes on the germination of two selected species of Berberis i.e. B. aristata and B. jaeschkeana. Also, effect of H2O2 was tested to study its role in germination percentage and mean germination time.
Materials and methods Plant material Two species of Berberis i.e. B. aristata and B. jaeschkeana were selected in the present study based on their medicinal and conservation importance. Also, both the species are source of ‘Berberine’ which is highly valued in preparation of different medicines. Brief botanical and ecological notes of both the species are given below: Berberis aristata DC (Family—Berberidaceae) is a shrub, 1–3 m tall, with spiny yellow stem, subsessile, obovate or elliptic leaves, inflorescence a drooping raceme, 4–6 cm long, flowers dense, bright yellow. B. aristata is distributed from Himachal Pradesh to Nepal between altitudes of 1800–2700 m (Gaur 1999). The species is medicinally important and reported to be useful in eye diseases, stomach infection, piles, remittent fevers, jaundice, boils, ulcer, skin diseases, wound, painful urination, etc. Besides, the species contain an active ingredient ‘berberine’ which is being used in preparation of different medicines. The natural regeneration of the species is low; however, extraction from wild is very high. This has resulted its depletion in wild is very fast. Therefore, there is a need to develop seed germination protocol so as to large number of seedling for plantation could be achieved. Berberis jaeschkeana Schneider (Family—Berberidaceae) is a shrub, up to 1.5 m tall with angular stem, trifid 10–15 mm long spines, sessile, subsessile leaves, umbellate or subumbellate inflorescence, up to 8-flowered, and oblong, ovoid berries. B. jaeschkeana is distributed in entire Himalayan region between 3000–5200 m and considered endemic to Himalayan region (Andola et al. 2010). The species is medicinally important and source of ‘berberine’. As such no medicinal properties of the species are solely reported, however, it is used as a substitute of the B. aristata in the Himalayan region. Generally, roots are
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extracted for the various purposes as for other species of Berberis (Andola et al. 2010). Natural regeneration of the species low and propagation through any means is not reported. The species is also threatened in its natural habitats, therefore, development of seed propagation protocol is essentially required.
was recorded for different time period (0, 2, 4 up to 120 h). Weight gain value near constant was considered as completely imbibed and recorded as imbibition time. An imbibition curve was plotted between weight gain (g) and time period (h). Seed germination test
Seed collection Ripened fruits were collected from Kedarnath area (West Himalaya), Uttarakhand (2200–3700 m asl) from ten different individuals of target species i.e. B. aristata and B. jaeschkeana during the months of September to October 2012. Collection details of site with altitude, latitude and longitude is given (Table 1). Herbarium specimens of each species were prepared and identified from Botanical Survey of India (BSI, Dehradun, Uttarakhand) and deposited at G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora (Acc. No.–BSI– 114119–B. aristata; 114108–B. jaeschkeana). Collected fruits were brought to the laboratory and mixed properly and then seeds were separated from the fruits. Damaged seeds were removed from the bulk. To study the effect of different pre-treatments, fifty seeds from each species in triplicates were randomly selected and subjected to each priming solution concentration. To study the effect of cold storage on seed germination, seeds were stored at 4 ± 2 °C in dark for 1, 2 and 4 months prior to germination test. A set of fifty seeds in triplicates (as control) were sown immediately for comparing the results. Seed viability To study the seed viability, fifty seeds in triplicates were soaked in a solution of 0.1 % of 2,3,5-Triphenyl Tetrazolium Chloride (TTC) for 24 h at 30 ± 1 °C in an oven (Moore 1973). Staining intensity was examined under microscope, and seeds with completely red stained embryo were considered as viable. Imbibition curve To determine the imbibition time, twenty-five seeds in triplicate were soaked in distilled water and the weight gain
Seeds were surface sterilized with Tween 20 solution (1–2 drops) followed by 0.1 % HgCl2 for 5 min, and then washed thoroughly under tap water followed by five times with distilled water. Six treatments i.e. gibberellic acid (GA3), salicylic acid (SA), thiourea (TU) each in 5–20 lM, vermiwash (VW), cow urine (CU), each in a 1:4 and 1:10 ratio and distilled water (DW) were used along with control. In each case, fifty seeds in triplicates were soaked in these solutions for 72 h. Treatment like GA3, SA, and TU were first dissolved in small amount of ethyl alcohol and then made a volume of the solution with distilled water. While vermiwash and cow urine was directly used with 1:4 and 1:10 ratio (1 volume of cow urine/vermiwash and 4/10 volume of distilled water). Selection of the treatment was based on the results of preliminary trials as well as the published report on other Himalayan plant species (Bhatt et al. 2000; Airi et al. 2005, 2009). The pre-soaked seeds were then transferred on moist filter paper in a Petri dish (9 cm diameter) and incubated for 40 days at a daily temperature regime of 25/10 °C (light:dark; 16:8 h) and relative humidity of 65 ± 2 % in seed germinator (UTS Sales, Delhi). Filter papers changed within a week or before, if there is any sign of contamination. Seeds kept for dry storage (at 4° ± 2 °C) for 1, 2 and 4 months following incubation as above mentioned method. Seeds were exposed to cool white fluorescent light (25–30 lmol m-2 s-1) for 16 h/day and were moistened at every 2 days interval with distilled water. To study the effect of Hydrogen Peroxide (H2O2) on seed germination, seeds of each species were treated with different H2O2 concentrations (5, 10, 20 mM) and incubated in above mentioned conditions. For control, sterilized seeds without any treatment were used and transferred to germination condition same as described earlier. Seed germination was monitored regularly and
Table 1 Site characteristics of selected Berberis species Species
Fruit ripening period
Collection site
Altitude (m asl)
Latitude (N)
Longitude (E)
Berberis aristata
Aug to Sep
1
2200
30°380 11.800
078°580 35.100
2600
0
00
079°020 50.000
0
00
2 Berberis jaeschkeana
Oct to Nov
30°40 59.0
1
3200
30°42 37.0
079°030 59.200
2
3700
30°440 26.600
079°030 53.300
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Author's personal copy Plant Growth Regul Table 2 Effect of different treatments on germination percentage, rate, mean germination time, seed vigour index and enzymatic activities in seedlings of Berberis aristata Treatments
Concentration
GP
Control
–
48.00 ± 4.71e
DW SA
GA3
TU
1.67 ± 0.33
54.67 ± 2.72
5 lM
10.67 ± 2.65
f
10 lM
0.00
20 lM
0.00
5 lM
80.00 ± 8.15abc
1.67 ± 0.17
10 lM 20 lM
89.33 ± 4.90a 72.00 ± 7.06bc
5 lM
66.67 ± 3.59
–
cd
72.00 ± 4.71
bc
20 lM
84.00 ± 4.08
ab
01:04
92.00 ± 2.35a
01:10 CU
MGT
de
10 lM VW
GR
01:04 01:10
78.67 ± 5.92
abc
86.67 ± 3.59
a
78.67 ± 4.90
abc
cd
SVI
10.33 ± 0.88d
1.33 ± 0.33
d
abc
14.67 ± 0.67
0.53 ± 0.03
e
e
Catalase
1433.73 ± 27.22
g
f
2108.67 ± 39.40
h
Peroxidase
20.00 ± 1.56ab
14.00 ± 2.12d
bc
21.00 ± 2.12bc
a
23.00 ± 2.12
5.00 ± 1.02e 0.00
18.00 ± 2.70
7.67 ± 0.67
487.70 ± 37.63
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
16.67 ± 1.45a
2576.80 ± 37.49d
10.00 ± 1.02ef
24.00 ± 2.04b
2.07 ± 0.23bc 2.23 ± 0.23bc
15.00 ± 1.16ab 11.33 ± 0.88d
3591.90 ± 56.88a 2603.50 ± 57.21d
10.00 ± 1.56ef 9.00 ± 1.18f
22.00 ± 2.12bc 15.00 ± 1.56d
1.83 ± 0.17bcd
15.67 ± 0.88a
2245.17 ± 36.30e
17.00 ± 2.12bc
22.00 ± 2.12bc
d
d
cd
2.17 ± 0.44
bc
2.40 ± 0.10
ab
12.33 ± 0.33
d
2657.47 ± 79.01
c
bcde
23.00 ± 1.18bc
15.00 ± 1.76
ef
24.00 ± 1.56b
12.00 ± 1.00
2864.47 ± 39.61
14.00 ± 1.56 cd
2.40 ± 0.12ab
12.67 ± 0.67bcd
3612.87 ± 49.02a
10.00 ± 1.02ef
2.40 ± 0.23
ab
bcd
b
bc
18.00 ± 1.56cd
2.40 ± 0.12
ab
def
23.00 ± 2.56bc
3.03 ± 0.17
a
bcd
19.00 ± 1.18bcd
12.67 ± 0.88
bcd
12.67 ± 0.67
d
10.67 ± 0.33
3164.40 ± 31.22
a
3578.13 ± 57.13
c
2846.73 ± 71.56
33.00 ± 2.04a
17.00 ± 2.12 11.00 ± 2.12 16.00 ± 1.56
Mean values followed by the same letter(s) in a column are not significantly different (p \ 0.05) based on DMRT GP germination percentage, GR germination rate, MGT mean germination time (days), SVI seed vigour index, DW distilled water, SA salicilic acid, GA3 gibberellic acid, TU thiourea, VW vermiwash, CU cow urine
Table 3 Effect of different treatments on germination percentage, rate, mean germination time, seed vigour index and enzymatic activities in seedlings of Berberis jaeschkeana Treatment
Concentration
GP
Control
–
10.67 ± 1.36f
DW
–
24.00 ± 4.71
e
SA
5 lM
13.33 ± 1.36f
0.53 ± 0.03f
10.67 ± 0.88de
363.87 ± 26.21
10 lM
0.00
0.00
0.00
20 lM
0.00
0.00
0.00
5 lM
29.33 ± 1.36de
0.83 ± 0.15e
10 lM 20 lM
32.00 ± 2.35 cd 36.00 ± 2.35bcd
5 lM
50.67 ± 2.72a
GA3
TU
46.67 ± 1.36
a
20 lM
46.67 ± 3.59
a
01:04
38.67 ± 4.90bc
10 lM VW
01:10 CU
GR
01:04 01:10
50.67 ± 1.36
a
42.67 ± 3.59
ab
33.33 ± 1.36
cd
MGT
0.43 ± 0.03f 0.93 ± 0.12
SVI
12.00 ± 0.58cde
de
9.33 ± 1.45
e
Catalase h
166.77 ± 15.95 632.83 ± 30.63
f
52 ± 2.04a 19 ± 1.56
d
6 ± 1.02
fg
ef
8 ± 1.02
31 ± 1.56b
8 ± 1.18ef
0.00
0.00
0.00
0.00
0.00
0.00
14.33 ± 0.88abc
840.27 ± 36.06e
27 ± 1.76bc
3 ± 1.02gh
0.93 ± 0.09de 1.13 ± 0.09 cd
16.33 ± 1.33a 12.33 ± 1.33bcd
812.53 ± 33.12e 681.47 ± 34.61f
47 ± 2.04a 48 ± 2.04a
8 ± 0.59ef 11 ± 1.02de
1.20 ± 0.06cd
15.00 ± 0.58ab
1393.67 ± 46.74b
19 ± 1.56d
39 ± 2.12a
a
25 ± 2.12
c
25 ± 1.56b
1171.57 ± 29.80
30 ± 1.56
bc
22 ± 1.18b
1066.60 ± 27.85d
50 ± 1.76a
1.33 ± 0.07
abc
1.53 ± 0.15
a
1.23 ± 0.09bc 1.50 ± 0.06
ab
1.50 ± 0.12
ab
1.17 ± 0.12
cd
13.67 ± 1.20
abc
12.33 ± 0.88
bcd
9.33 ± 0.33e
g
Peroxidase
1583.27 ± 35.51
c
12.33 ± 0.88
bcd
a
10.67 ± 0.33
de
1349.37 ± 37.96
10.33 ± 0.88
de
e
1653.67 ± 68.56
b
874.87 ± 24.26
12 ± 1.02cd
30 ± 2.04
bc
14 ± 1.18cd
32 ± 1.56
b
15 ± 1.18c
47 ± 2.12
a
14 ± 1.18cd
Mean values followed by the same letter(s) in a column are not significantly different (p \ 0.05) based on DMRT GP germination percentage, GR germination rate, MGT mean germination time (days), SVI seed vigour index, DW distilled water, SA salicilic acid, GA3 gibberellic acid, TU thiourea, VW vermiwash, CU cow urine
seed considered to be germinated, if the radicle was visible through naked eye. Data on germination percentage (GP), germination rate (GR), mean germination time (MGT), and seed vigor index (SVI) were calculated using the following formulae:
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Germination Percentage ðGPÞ ¼ GN=SN 100; Germination Rate ðGRÞ ¼ R Sn =N; Mean Germination Time ðMGTÞ ¼ ðN1 T1 þ N2 T2 . . .. . .Nn Tn Þ=GN; Seed Vigor Index ðSVIÞ ¼ GP SL
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where, GN—total number of seed germinated; SN—total number of seed tested; Sn—number of seed germinating each day; N—the days to germination; N1T1—number of seed germinated at time T1; N2T2—number of seed germinated at time T2; and SL—the mean seedling length. Enzyme extraction and assay Seedlings were collected after 40 days of germination for estimation of catalase and peroxidase enzyme activity. The extraction of enzymes was performed following the method described by Kar and Mishra (1976). The seedlings (1 g) were ground with chilled mortar and pestle with 10 ml of cold phosphate buffer (0.1 M, pH 7.0). The homogenate was mixed and spin down in a refrigerated centrifuge (Hitachi; Model: Himac CR 22G) at 30,710 g for 15 min at 4 °C. Supernatant was collected and stored at -20 °C for enzyme assays. Enzyme assay Catalase (CAT) activity Total catalase activity was determined spectrophotometrically following the method of Beers and Sizer (1952). 2.9 ml of 0.036 % H2O2 was mixed with 0.1 ml of extract and changes in absorbance was recorded at 240 nm using spectrophotometer (U- 2001, Hitachi, Japan). The enzyme activity was expressed in U/mg of protein. Peroxidase (POD) activity Total peroxidase activity was determined spectrophotometrically following the method described by Putter (1974). 0.05 ml of Guaiacol solution (20 mM) was added with 2.9 ml of 0.036 % H2O2 and mixed immediately with inversion. Finally, 0.1 ml of enzyme extract (100 mg/ml) was added to the mixture and change in absorbance was recorded at 436 nm using spectrophotometer (U- 2001, Hitachi, Japan). The enzyme activity was expressed in U/mg of protein.
SPSS version 16. Correlation coefficient (r) and coefficient of determination (r2) were determined to find out the relationship between germination percentage and enzymatic activity using Microsoft Excel 2007.
Results Seed viability Variations in viability among the species and altitude were recorded. For instance, viability of B. jaeschkeana seeds collected from two different altitudes was 93 % (3200 m asl) and 75 % (3700 m asl), while the viability of B. aristata seeds were 70 % (2200 m asl) and 85 % (2600 m asl). Photographs of both viable and non viable seeds of B. jaeschkeana are presented (Suppl. 1). Imbibitions time The imbibitions time was found to be 72 h for both the species. The weight gain was increased till 72 h of soaking but reached to a constant value as the soaking time further increased (Fig. 1). Priming effects on seed germination Berberis aristata Variation in germination percentage among different pretreatment was observed. Highest germination (92 %) was found in vermiwash treatment at 1:4 dilution followed by GA3 at 10 lM concentration (Table 2). Lowest germination percentage (10.66 %) was recorded in case of salicylic acid at a concentration of 5 lM. The germination rate (3 seeds/day) achieved after cow urine treatment at 1:10 dilution which was reduced to 0.5 seeds/day in salicylic acid treatment at 5 lM concentration. MGT was found to be reduced up to 8 days in salicylic acid treatment and 17 days in gibberellic acid at 5 lM concentration. Different 0.35
Statistical analysis Weight gain (g)
A total of 23 treatments in all the experiments were conducted. This includes, priming treatments—15 including Control; Storage conditions—4; and H2O2 treatment—4. All the experiments were conducted in a completely randomized block design. Duncan’s Multiple Range Test (DMRT) was estimated separately for comparison of means of germination percentage, mean germination time, germination rate, seed vigour index, and enzymatic activity among different treatments and storage conditions by using
B. aristata B. jaeschkeana
0.3 0.25 0.2 0.15 0.1 0.05 0 0
2
4
6
12
24
48
72
96
120
Time (h)
Fig. 1 Imbibitions curve of selected Berberis species
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concentrations/dilutions of treatments did not significantly affect germination percentage (Table 2). Seed vigor index (SVI) was found higher in vermiwash treatment (1:4) and lower in case of salicylic acid treatment. Overall vermiwash was found to be the best treatment amongst others. When compared with control, the germination percentage increased two-folds in vermiwash (1:4 dilutions) treatment. Berberis jaeschkeana Thiourea (5 lM) and vermiwash at 1:10 dilution showed highest germination (50.6 %) as compared to control (10.6 %) (Table 3). Germination percentage in GA3 and vermiwash treatment was increased with increasing concentrations; however, thiourea and cow urine inhibited germination percentage with increasing concentrations. The mean germination time was reduced to 9 days after vermiwash at 1:4 dilution. Germination rate was also found maximum as 1.5 seeds germinated/day in case of TU (20 lM), CU (1:4) and vermiwash (1:10) treatment. Seed vigour index (SVI) was found to be maximum in case of VW (1:10) treatment and lowest in case of control. As compared to others, vermiwash and thiourea was found to be best treatment for seed germination of B. jaeschkeana. Catalase (CAT) and peroxidase (POD) activity Berberis aristata Higher POD activity and lower CAT activity was recorded in vermiwash treatment at 1:4 dilution, however, this was opposite in case of salicylic acid treatment (5 lM) (Table 2). A significant (p \ 0.05) positive correlation was found between germination percentage and peroxidase activity; however, with catalase activity a significant (p \ 0.01) negative correlation was observed (Fig. 2a). All the treatments, except gibberellic acid, showed positive relationship between germination percentage and peroxidase activity and negative with catalase activity. A significant (p \ 0.05) difference in germination percentage, catalase and peroxidase activity was recorded among all the treatments, however, catalase activity showed significant (p \ 0.05) difference only in vermiwash treatment at different concentrations. In case of peroxidase activity, gibberellic acid and vermiwash treatment showed significant (p \ 0.05 and p \ 0.005, respectively) differences at different concentrations (Table 2). Berberis jaeschkeana Treatment with Thiourea (5 lM) showed higher POD and lower CAT activity while lowest POD activity was observed in gibberellic acid (5 lM) treatment (Table 3). A
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significant (p \ 0.01) positive correlation between germination percentage and peroxidase activity was observed. However, catalase activity showed non-significant negative correlation with germination percentage (Fig. 2b). All treatments except gibberellic acid showed positive relationship between germination percentage and peroxidase activity and a negative relationship with catalase activity. A significant (p \ 0.05) differences in germination percentage, catalase and peroxidase activity was recorded among all the treatments. Catalase activity showed difference within all the treatments at its different concentrations/dilutions however, in case of peroxidase activity only GA3 and TU treatments at its different concentrations showed significant (p \ 0.005 and p \ 0.001, respectively) differences (Table 3). Exogenous treatment with hydrogen peroxide (H2O2) Pretreatment with different H2O2 concentrations (5, 10, 20 mM) showed the variation in percentage germination (Fig. 3). Both, B. jaeschkeana and B. aristata showed higher germination percentage at 20 mM H2O2 concentration which was three to five-folds higher than control. MGT was also reduced to 7 days in B. jaeschkeana and 6 days in B. aristata seeds after H2O2 treatment (10 mM) as compared to control. Storage effect A significant (p \ 0.05) variation in germination percentage of B. jaeschkeana in different cold storage time period was observed (Table 4). The highest germination percentage after 1 month of seed storage was 77 % (3200 m asl) and 42 % (3700 m asl) (Table 4). As the storage duration increases up to 4 months the germination percentage was decreases up to 72 and 61 %, respectively in the seeds collected from 3200 and 3700 m asl populations. In case of B. aristata, a significant [2200 m (p \ 0.05); 2600 m (p \ 0.05)] variation in germination percentage among all durations of cold storage was observed (Table 4). The highest germination percentage (53.35 %) in the seeds collected from 2600 m asl population after 2 months of cold storage and 28 % for 2200 m asl population without any storage was observed (Table 4). As the cold storage duration increases from 0 to 4 months the germination percentage decreases more than 75 % in the seeds collected from both the altitude. Storage time significantly affect the germination rate of B. jaeschkeana seeds, but in case of B. aristata only seed collected from 2600 m asl population showed significant (p \ 0.05) variation in germination rate (Table 4). The
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(a)
y = 0.2466x + 2.9073 R2 = 0.685; p
