STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions Laboratory of Cytogenetics and Plant Breeding | Page No.: 48 Results and discussions 4.1: Germplasm collections of ginger in India: Considering importance of germplasm conservation, total 12 cultivars of ginger were collected from India for the present study (Plate: III) and well maintained in botanical garden of Department of Botany, Shivaji University, Kolhapur. In India about 900 accessions of ginger are conserved in different places. Majority of ginger cultivars are conserved in Indian Institute of Spices Research, Calicut followed by Orissa University of Agriculture and Technology, Pottangi, Orissa. In India, ginger has rich cultivar diversity, and major growing cultivars that are specific to the area are mostly known by place names. Kerala has the more diversity followed by northeastern region of India. At present, more than 70 ginger cultivars possessing varying quality attributes and yield potential are being cultivated in India (Ravindran and Nirmal Babu, 2004). There are 15 agroclimatic zones in India, in present work attempts have been made to collect germplasm from maximum agroclimatic zones and total 12 cultivars from 10 agroclimatic zones were collected. Rio de Janeiro cultivar was introduced into India from Brazil and has become very popular in Kerala. 4.2: HPLC analysis of 6-gingerol from different cultivars of ginger: According to Leverington, (1975) and Connell and Sutherland, (1969) the main pungent principles extracted from the rhizomes were 6-gingerol, 8-gingerol, and 10-gingerol, and in terms of pungency 6-gingerol was the most pungent compounds, (Govindarajan, 1979 and 1982) Hence, 6-gingerol was chosen for the present study. Calibration Curve: The linearity of the proposed analytical method for determination of the 6- gingerol was evaluated by analyzing four concentration levels of standard solution. Each concentration was repeated three times. The calibration curve of the standard was constructed with the correlation coefficients (R 2 ) above 0.9975. The results of the regression equations were y = 3.65e+004 X +3.44e+004.
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STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 48
Results and discussions
4.1: Germplasm collections of ginger in India:
Considering importance of germplasm conservation, total 12 cultivars of
ginger were collected from India for the present study (Plate: III) and well
maintained in botanical garden of Department of Botany, Shivaji University,
Kolhapur. In India about 900 accessions of ginger are conserved in different
places. Majority of ginger cultivars are conserved in Indian Institute of Spices
Research, Calicut followed by Orissa University of Agriculture and Technology,
Pottangi, Orissa.
In India, ginger has rich cultivar diversity, and major growing cultivars that
are specific to the area are mostly known by place names. Kerala has the more
diversity followed by northeastern region of India. At present, more than 70
ginger cultivars possessing varying quality attributes and yield potential are being
cultivated in India (Ravindran and Nirmal Babu, 2004). There are 15 agroclimatic
zones in India, in present work attempts have been made to collect germplasm
from maximum agroclimatic zones and total 12 cultivars from 10 agroclimatic
zones were collected. Rio de Janeiro cultivar was introduced into India from
Brazil and has become very popular in Kerala.
4.2: HPLC analysis of 6-gingerol from different cultivars of ginger:
According to Leverington, (1975) and Connell and Sutherland, (1969) the
main pungent principles extracted from the rhizomes were 6-gingerol, 8-gingerol,
and 10-gingerol, and in terms of pungency 6-gingerol was the most pungent
compounds, (Govindarajan, 1979 and 1982) Hence, 6-gingerol was chosen for
the present study.
Calibration Curve:
The linearity of the proposed analytical method for determination of the 6-
gingerol was evaluated by analyzing four concentration levels of standard solution.
Each concentration was repeated three times. The calibration curve of the standard
was constructed with the correlation coefficients (R2) above 0.9975. The results of
the regression equations were y = 3.65e+004 X +3.44e+004.
STUDIES IN ZINGIBER OFFICINALE ROSCOE
Laboratory of Cytogenetics and Plant Breeding
The calibration graph for 6
result by linear regression analysis showed a very good linear relationship between
peak area and concentration.
Figure: 4.1 a - d: Chromatogramgingerol and e: Calibration graph of standard solution of 6
OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.:
The calibration graph for 6-gingerol, was shown in (Figure:
result by linear regression analysis showed a very good linear relationship between
peak area and concentration.
d: Chromatogram of different concentration of standard 6Calibration graph of standard solution of 6-gingerol
(a)
(e)
(c)
Results and Discussions
Page No.: 49
4.1 a - e). The
result by linear regression analysis showed a very good linear relationship between
standard 6- gingerol
(b)
(e)
(d)
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 50
6- Gingerol content by HPLC:
The 6-gingerol contents, calculated using the standard calibration curve
(R2 = 0.998), were varied from 0.1% to 0.2 % (Table 4.1). The 6-gingerol content
for the ginger cultivars ranged from 0.2% in Rajasthan to 0.1% in Udaipur which
was shown in fig. 4.2 a to l.
The results perfectly matched with the observations made by Nybe and
coworkers (1982), which determined gingerol content in different ginger cultivars
and found to be highest in Rio De Janero and lowest in Wynad. Xiang et al.
(2008) analysed 6-gingerol content from ginger by HPLC, which showed 6-
gingerol content was 0.39% in dried ginger, 0.10% in baked ginger and 0.19% in
fresh ginger. Using different drying methods, Hawlader et al. (2006) determined
6-gingerol content and it was 0.6% in normal air drying. Determination of
gingerol by LC-MS from raw herb and dried aqueous extract was done by
Samiuela et al. (2007). During determination of 6-gingerol, extraction by
sonication and methanol solvent showed maximum yield. 6-gingerol content in
dried aqueous extract was 0.18% while raw herb extract had 0.93%. Zachariah et
al. (1993) evaluated germplasm for oleoresin content and found range for 6-
gingerol content in between 0.3% to 0.7%. Ravindran and Nirmal Babu, (2004)
recorded many ginger cultivars with high oleoresin, such as Rio de Janeiro, Ernad
Chernad, Wynad, Kunnamangalam, and Meppayyur.
STUDIES IN ZINGIBER OFFICINALE ROSCOE
Laboratory of Cytogenetics and Plant Breeding
Figure: 4.2 Chromat
Himachal Pradesh, d:
Rio-de-Janero, i:
OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.:
4.2 Chromatograms of different varieties a: Satara, b:
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 60
Figure: 4.5 Dendrogram of ginger cultivars
1
2
3
4
5
I
II
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 61
4.5: Correlations between biochemical parameters:
Statistical analysis for all biochemical parameters showed positive
correlations (Figure: 4.6 a to j). DPPH activity and phenolic content were
significantly correlated (R2 = 0.449). Thus, the three cultivars Rajasthan, Rio De
Janero and Sagar (Table: 4.3), that ranked highest for antioxidant activity also
ranked within the top four for phenolic content. Because phenolic compounds are
some of the most important water soluble antioxidants and can be present at high
concentrations in plants, the correlation between these two traits was expected.
Antioxidant activity increased proportionally to the phenolic content and a linear
relationship between DPPH-radical scavenging activity and total phenolic was
established. In case of flavonoid and phenolic there was also strong correlation
(R2 = 0.880) but p value is not significant (Table: 4.5). All the ginger cultivars
which contained high phenolic compounds exhibited high antioxidant activity
when determined by DPPH and FRAP assays. It, thus, confirms that phenolic
compounds have an important role in antioxidant activities (Harborne, 1998).
Correlation between antioxidant activity and phenolic compounds were
significant in Bulgarian medicinal plants (Ivanova, et al. 2005), Chinese
medicinal plants (Zheng and Wang, 2001), some fruits, vegetables and grain
products (Velioglu et al. 1998). The phenolic hydroxyl groups present in plant
antioxidants have redox properties (Shahidi and Wanasundara, 1992 and Pietta,
2000) allowing them to act as a reducing agent and a hydrogen donator in the two
assays. Thus, phenolic compounds could be the major antioxidant in these ginger
cultivars.
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 62
Table 4.5: ANOVA of the correlations between different traits
Correlations P value Remark R2 value Flavonoid vs Phenolics >0.05 Not significant 0.880 Flavonoid vs FRAP <0.001 Extremely significant 0.301 Flavonoid vs DPPH <0.001 Extremely significant 0.589 Phenolics vs DPPH <0.001 Extremely significant 0.449 DPPH vs FRAP <0.001 Extremely significant 0.330 Gingerol vs Flavonoid >0.05 Not significant 0.898 Gingerol vs FRAP <0.001 Extremely significant 0.731 Gingerol vs Phenolics >0.05 Not significant 0.872 Gingerol vs DPPH <0.001 Extremely significant 0.850 Phenolics vs FRAP <0.001 Extremely significant 0.433
Repeated measures analysis of variance
Tukey – Kramer multiple comparisons tests
Figure: 4.6 Correlations between biochemical parameters
R² = 0.880
0.5
0.7
0.9
1.1
1.3
1.5
1.7
0.1 0.2 0.3 0.4
g/10
0g T
anni
c ac
id e
q.
Flavonoid g/100g Quercetin eq.
(a) Correlation between Flavonoid and Phenolics
R² = 0.301
350
1350
2350
3350
4350
5350
0.1 0.15 0.2 0.25 0.3 0.35 0.4
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(b) Correlation between Flavonoid and Antioxidant (FRAP)
R² = 0.589
500700900
110013001500170019002100
0.1 0.15 0.2 0.25 0.3 0.35 0.4
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(c) Correlation between Flavonoid and Antioxidant (DPPH)
R² = 0.449
500700900
110013001500170019002100
0.6 0.8 1 1.2 1.4 1.6 1.8
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(d) Correlation between Phenolics and Antioxidant (DPPH)
R² = 0.330
500700900
110013001500170019002100
500 1500 2500 3500 4500 5500
µM A
scor
bic
eq.
FRAP µM Ascorbic eq.
(e) Correlation between Antioxidant tests: FRAP and DPPH
R² = 0.898
0.000.050.100.150.200.250.300.350.400.45
0.1 0.12 0.14 0.16 0.18 0.2 0.22
g/10
0g Q
uerc
etin
eq.
% Gingerol
(f) Correlation between Gingerol and Flavonoid content
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 63
4.6: Morphological mutations induced by EMS and Gamma rays in Ginger
cv. Satara:
VM1 Generation:
The parameters selected for morphological mutants in VM1 and VM2
generation comprised:
• Germination percentage
• Chlorophyll mutant percentage
• Height of shoot/tiller per rhizome
• Number of shoots/tillers per rhizome
• Number of leaves per shoot/tiller of rhizome
• Weight of rhizome
4.6a: Germination studies:
Rhizome germination was studied in EMS treated and gamma irradiated
rhizomes of ginger and results are depicted in Table.4.6.
Germination percentage:
Various physical and chemical mutagens are known to affect germination
of seed. Seed germination in M1 is one of the parameters and used as an index in
determining the effects of mutagens on plants (Konzak et al., 1972).
R² = 0.731
0
1000
2000
3000
4000
5000
6000
0.1 0.12 0.14 0.16 0.18 0.2 0.22
µM A
scor
bic
acid
eq.
% Gingerol
(g) Correlation between Gingerol and Antioxidant activity (FRAP)
R² = 0.872
0.50
0.70
0.90
1.10
1.30
1.50
1.70
0.1 0.12 0.14 0.16 0.18 0.2 0.22
g/10
0g T
anni
c ac
id e
q.
% Gingerol
(h) Correlation between Gingerol and Phenolic content
R² = 0.850
750
950
1150
1350
1550
1750
1950
0.1 0.12 0.14 0.16 0.18 0.2 0.22
µM A
scor
bic
acid
eq.
% Gingerol
(i) Correlation between Gingerol and Antioxidant activity (DPPH)
R² = 0.433
350
1350
2350
3350
4350
5350
6350
0.6 0.8 1 1.2 1.4 1.6 1.8
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(j) Correlation between Phenolics and Antioxidant (FRAP)
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 64
The effects of mutagens on seed germination are depicted in Table 4.6. It
has been observed that germination percentage in control is 100% (Table 4.6).
Both the mutagens used in present study had inhibitory effects on seed
germination. The reduction in germination of seed was also expressed as percent
lethality. The germination percentage ranged from 80% to 96% in case of EMS
(0.10% to 0.25%) 8 hrs treatments (Figure: 4.7), while for EMS (0.30% t0
0.60%) 4 hrs treatment germination was in the range of 64% to 96% (Figure:
4.8). The germination percentage ranged from 4% to 88% for gamma rays
treatments (Figure: 4.9). In general germination was decreased with increase in
gamma rays dose over control. Jayachandran (1989) treated the cv. Rio de
Janeiro with gamma rays at 0.5 to 1.5 KRAD and Ethylmethane sulfonate (EMS)
at 2.0 to 10.0 mM. In his study the LD50 i for sprouting and survival was
between 0.5 and 1.0 KR of gamma rays. However, in present study the LD 50
was 0.500KRAD.
The impact and the tolerance level of the biological material to a mutagen
are manifested in M1 generation itself in terms of germination and lethality
(Gaul, 1957). In the present investigation germination and survival percentage
decreased with increasing dose/ concentration. Similar results were observed by
Ghanavat (2000) in Psophocarpus, Gaikwad (2002) in Lentil, Dhanavel et al.
(2008) in Vigna and Kavithamani et al. (2008) in Glycine. The decreased
percentage of germination might be due to drastic injury implicated upon the
cellular system at the molecular level which resulted in either lowering down or
completely inhibiting the physiology of germination (Gustaffson, 1944; Bilquiz
and Martin, 1961; Raghuvanshi and Singh, 1976; Tarar and Dnyansagar, 1983
and Chandra and Tarar, 1987). The decrease in germination percentage has been
attributed mainly to the interference by the mutagen with metabolic activities of
the seed (Micke, 1958 and 1961, Gottschalk and Schieb, 1960 and Sjodin 1962).
The general inhibition of germination and increased lethality could be due to the
lowering of the rate of mitotic proliferation and the consequent delay in cell
division and repair of damaged DNA (Hutterman et al., 1978).
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Table: 4.6: -Effects of different mutagens on germination percentage of
Ginger in VM1
Treatment Concentration Germination % Lethality% Control Control 100 --
0.30% 21.30 08.30 0.389 1.248 * * 31.25 5.66 0.181 0.534 n s 0.40% 23.95 09.60 0.400 1.104 ns 32.05 5.64 0.175 0.461 n s 0.50% 23.54 10.31 0.437 1.349 ns 31.04 4.73 0.152 0.473 n s 0.60% 18.76 08.41 0.448 1.684 * * 30.24 4.24 0.140 0.526 n s
Gamma Rays (KR)
0.125 22.29 10.43 0.467 1.279 * * 32.11 3.83 0.119 0.381 n s 0.250 21.73 07.91 0.364 0.938 * * 32.05 4.53 0.141 0.401 n s 0.375 25.10 09.22 0.367 1.198 ns 25.5 7.57 0.296 1.094 * * 0.500 23.50 08.41 0.357 1.344 ns 32.87 4.40 0.133 0.697 n s 0.750 22.42 13.04 0.581 4.932 ns 31.33 4.27 0.136 1.103 n s 1.000 25.66 07.76 0.302 4.485 ns 32.27 4.60 0.142 1.913 n s
COV: Coefficient of Variation; SEM: Standard Error of Mean *** extremely significant (P<0.001), * * Good significant (P< 0.01), * fairly significant (P<0.05) and ns - Not
significant - Dunnett compares all vs control
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
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Jayachandran (1989) analyzed the VM2 generation of ginger and found a
significant reduction in plant height as the dose increased. Pelc and Howard
(1955) and Gorden (1957) have suggested that the possible interference of
irradiation with synthesis of new DNA may lead to inhibition of growth. Evans
(1965), while studying the effects of radiation on meristematic cells considered
growth reduction was to be due to cumulative expression of mitotic cycle delay.
4.6d: Number of shoots/tillers per rhizome:
The effects of different mutagens on number of tillers per plant are
depicted in Table: 4.9. The average tiller number in control was 2.2 in VM1 and
5.04 in VM2 generation (Table: 4.9). The treatments of EMS 8 hrs viz. 0.10%,
0.15% and 0.20%, 0.40% in EMS 4 hrs treatment and 0.250 KR and 0.500 KR of
gamma rays treatment showed average shoot number more than control in VM2.
P values significant only at 0.375 KR gamma rays treatment otherwise it was not
significant for all other remaining treatments in VM2. The correlation between
VM1 and VM2 shows R2 = 0.279 (Figure: 4.12).
It is revealed from the present studies that the mean tiller number in the
VM2 showed shifts in both the directions. These results has also are in agreement
with the observations reported by Jaychandran (1989) in ginger.
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 72
Table: 4.9 Effects of different mutagens concentration on number of shoot per plant in VM1 and VM2 generations of Ginger
0.10% 1.61 0.920 0.571 0.200 n s 5.38 1.716 0.319 0.374 n s 0.15% 1.91 0.928 0.485 0.189 n s 5.29 1.573 0.297 0.321 n s 0.20% 1.86 0.833 0.447 0.177 n s 5.35 1.531 0.286 0.342 n s 0.25% 1.80 0.894 0.496 0.200 n s 5.00 1.511 0.302 0.322 n s
EMS 4hrs
0.30% 2.22 0.812 0.365 0.173 n s 5.04 1.132 0.224 0.241 n s 0.40% 2.83 1.20 0.424 0.245 n s 5.79 1.503 0.259 0.306 n s 0.50% 2.21 0.998 0.451 0.208 n s 4.34 1.300 0.299 0.271 n s 0.60% 1.68 0.793 0.472 0.198 n s 4.12 1.310 0.317 0.327 n s
Gamma Rays (KR)
0.125 2.57 0.870 0.338 0.189 n s 4.80 1.120 0.233 0.263 n s 0.250 3.09 0.889 0.287 0.194 * 5.61 1.160 0.206 0.253 n s 0.375 3.00 1.095 0.365 0.273 n s 3.00 1.095 0.365 0.273 * * 0.500 3.71 1.603 0.432 0.606 * * 5.85 1.573 0.268 0.594 n s 0.750 1.75 0.957 0.546 0.478 n s 3.75 0.957 0.255 0.478 n s 1.000 3.00 0.000 0.000 0.000 n s 5.00 0.000 0.000 0.000 n s
COV: Coefficient of Variation; SEM: Standard Error of Mean *** Extremely significant (P<0.001), * * Good significant (P< 0.01), * fairly significant (P<0.05) and ns -
Not significant - Dunnett compares all vs control
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 73
Figure: 4.12 Correlation of number of shoots in VM1 and VM2
4.6e: Number of leaves per shoot/tiller of rhizome:
The correlation between VM1 and VM2 was R2 = 0.021 (Figure: 4.13), it
was very low as compared to other traits. The average number of leaves in
control was 10.52 in VM1and 11.79 in VM2 generation (Table: 4.10). The
treatments which showed mean number of leaves more than that of control were
the 0.40% of EMS 4 hrs treatment and 0.250 KR, 500 KR and 1KR of gamma
rays. The p values are significant for all EMS 8 hrs treatments and gamma rays
0.375KR treatment in VM2.
R² = 0.279
2
3
4
5
6
7
1 1.5 2 2.5 3 3.5 4
VM
2
VM 1
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 74
Table: 4.10: Effects of different mutagens concentration on number of leaves per plant in VM1and VM2 generations of Ginger
0.30% 6.53 4.89 0.748 0.688 ** 11.15 2.60 0.233 0.248 n s 0.40% 6.55 4.62 0.705 0.562 ** 11.97 2.74 0.228 0.230 n s 0.50% 7.24 4.71 0.650 0.668 ** 11.52 2.52 0.218 0.252 n s 0.60% 6.76 3.59 0.539 0.719 ** 10.67 2.59 0.242 0.322 n s
Gamma Rays (KR)
0.125 6.72 4.72 0.702 0.607 ** 11.68 2.45 0.209 0.245 n s 0.250 7.21 4.64 0.643 0.510 ** 12.04 2.91 0.241 0.260 n s 0.375 7.89 3.44 0.435 0.541 * 08.27 3.68 0.444 0.531 ** 0.500 7.84 3.59 0.457 0.774 n s 12.25 2.93 0.239 0.463 n s 0.750 8.42 2.99 0.355 1.131 n s 11.20 2.11 0.188 0.545 n s 1.000 8.33 5.04 0.605 2.906 n s 12.00 3.39 0.282 1.517 n s
COV: Coefficient of Variation; SEM: Standard Error of Mean * * Good significant (P< 0.01), * fairly significant (P<0.05) and ns - Not significant
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
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Figure: 4.13 Correlation of leaf number in VM1 and VM2
4.6f: Weight of rhizome:
The effects of different mutagens on rhizome weight are reported in Table
4.11. The average weight of rhizome of control was 235.54 gm in VM2. The
concentration of (0.20%) EMS 4 hrs treatment, (0.40%) EMS 8 hrs treatment and
gamma rays 0.250 KR and 0.500 KR had shown average weight than the control
(Table: 4.11). The correlation between VM1 and VM2 was R2 = 0.275 (Figure:
4.14). The highest average weight of rhizome was found in 0.40% EMS 8 hrs
treatment (284.41 gm) followed by Gamma rays 0.500 KR treatment (275.14
gm). The weight of rhizome was increased about ten times in VM2 as compared
to VM1. Findings in the present studies are in agreement with the observations
reported by Jaychandran (1989) in ginger.
R² = 0.021
7
8
9
10
11
12
13
5 6 7 8 9 10 11 12
VM
2
VM1
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
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Table: 4.11 Effects of different mutagens concentration on weight of rhizome per plant in VM1 and VM2 generations of Ginger
VM1 VM2
Treatment Concentration Mean ± S.D. COV SEM P value Mean ± S.D. COV SEM P values
Control -- 29.72 10.22 0.343 2.04 ** 235.54 61.61 0.261 12.57 n s
EMS 8
hrs.
0.10% 26.00 11.43 0.439 2.55 n s 219.95 99.30 0.451 21.67 n s
0.15% 32.18 13.07 0.406 2.78 n s 261.45 89.42 0.342 18.25 n s
0.20% 27.27 10.22 0.374 2.17 n s 240.95 40.19 0.166 19.27 n s
0.25% 23.95 9.50 0.396 2.12 n s 225.40 77.19 0.342 16.50 n s
EMS 4hrs
0.30% 23.63 7.66 0.324 1.63 n s 236.68 37.97 0.160 14.49 n s
0.40% 37.54 18.40 0.490 3.92 n s 284.41 103.27 0.363 21.08 n s
0.50% 36.22 16.24 0.448 3.46 n s 198.00 66.95 0.338 13.96 n s
0.60% 18.30 5.73 0.313 1.59 * 196.87 69.57 0.353 17.39 n s
Gamma
Rays (KR)
0.125 25.31 8.51 0.336 1.95 n s 226.90 66.27 0.292 14.46 n s
0.250 23.76 11.01 0.463 2.40 n s 273.00 84.53 0.309 18.44 n s
0.375 21.93 8.24 0.375 2.06 n s 108.25 41.27 0.381 10.31 **
0.500 36.28 11.20 0.308 4.23 n s 275.14 97.05 0.352 36.68 n s
0.750 23.25 4.99 0.214 2.49 n s 179.75 69.00 0.383 34.50 n s
1.000 30.00 0.00 0.000 0.00 n s 231.00 00.00 0.000 00.00 n s
COV: Coefficient of Variation; SEM: Standard Error of Mean * * Good significant (P< 0.01), * fairly significant (P<0.05) and ns - Not significant
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 77
Figure: 4.14 Correlation of Rhizome weight in VM1 and VM2
The results obtained in the present investigation showed a shieft of mean
on positive and negative direction in almost all the characters studied compared
to the control. The data on quantitative characters revealed that mutagens not
only altered the mean values but also created genetic variability for polygenic
traits. The increase in induced variations and decreased mean values were
observed by Brock (1965), Goud et al., (1969 and 1971), Perssons and Hagberg
(1969), Singh et al. (1977), Reddy (1991) and Bale (1999). It should be
concluded that mutagenic treatments are capable of inducing polygenic
variability and this feature can be exploited by the plant breeders for the genetic
improvement of desirable traits through proper selection.
R² = 0.275
100
150
200
250
300
15 20 25 30 35 40
VM
2
VM1
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Laboratory of Cytogenetics and Plant Breeding
4.7: Biochemical studies in EMS 4 hrs treatment:
Analysis of 6-gingerol:
The 6 – gingerol content of the control and irradiated material
were determined by using HPLC
0.16% of 6-gingerol in control
it was 0.18%. As the dose increased the 6
Lowest 6- gingerol content
plants.
Figure: 4.15: Chromatograms0.40%, d: EMS 4hrs 0.50% and e
OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.:
studies in EMS 4 hrs treatment:
gingerol content of the control and irradiated material
were determined by using HPLC (Figure: 4.15 a - e). The HPLC result shows
gingerol in control (Table 4.12) while for 0.30% EMS 4 hrs treatment
it was 0.18%. As the dose increased the 6-gingerol content was decreased.
gingerol content (0.09%) was observed in 60%EMS 4hrs treat
gingerol content of the control and irradiated materials of ginger
The HPLC result shows
EMS 4 hrs treatment
gingerol content was decreased.
EMS 4hrs treated
c: EMS 4hrs EMS 4hrs 0.60%.
(b)
(d)
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 79
Total Phenolic content:
Phenolic content of the ginger treated with different concentrations of
EMS 4 hrs treatments were determined using Folin Ciocalteu method and the
results were expressed in Tannic acid equivalent. Phenolic content found to be
decreased as concentration increased. Highest phenolic content (1.266 gm/100gm
dry weight of ginger) was recorded for 0.30% EMS 4 hrs treatments, which was
twofold more than 0.60% EMS, 4 hrs treatment.
Total Flavonoid content:
Flavonoid content was expressed in quercetin equivalent which was
highest in 0.30% EMS 4 hrs treatment. The flavonoid content also decreased with
increasing EMS 4 hrs concentrations. Lowest amount of flavonoid (0.197
gm/100gm dry weight of ginger) was recorded in 0.60% EMS 4 hrs treated
plants.
Antioxidant activity:
Antioxidant activities were measured by DPPH and FRAP method and
expressed in terms of Ascorbic acid equivalent. The highest activity was recorded
in 0.30 % EMS 4 hrs treated plants for both DPPH and FRAP (Table 4.12). The
trend of decrease in activity as increase in dose of mutagen was observed for both
DPPH and FRAP (Figure: 4.16).
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 80
Table: 4.12: Effects of various concentrations of EMS 4 hrs treatments on 6 - gingerol, Flavonoid and Phenolic contents and DPPH and FRAP activity in ginger
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 81
Figure: 4.16 Effects of various concentrations of EMS 4 hrs treatments on 6 - gingerol, Flavonoid and Phenolic contents in gm/100gm of dry wt eq. and
DPPH and FRAP activity in mM
4.8: Correlations between biochemical parameters in various concentrations
of EMS 4 hrs treatmets:
All the correlations were of positive R values (Figure 4.17: a to j), which
are depicted in Table 4.13. Strong correlations were found between Gingerol and
DPPH (R2 = 0.911) followed by Phenolics and DPPH (R2 = 0.910). The lowest
correlation was recorded between flavonoid and FRAP (R2 = 0.301).
Table: 4.13 Correlations between different biochemical traits in various
concentrations of EMS 4 hrs treated plants
Correlations P value Remark R2 value Flavonoid vs Phenolics >0.05 Not significant 0.880 Flavonoid vs FRAP <0.001 Extremely significant 0.301 Flavonoid vs DPPH <0.001 Extremely significant 0.589 Phenolics vs DPPH <0.001 Extremely significant 0.910 DPPH vs FRAP <0.01 Significant 0.859 6-gingerol vs Flavonoid >0.05 Not significant 0.773 6-gingerol vs FRAP <0.001 Extremely significant 0.856 6-gingerol vs Phenolics >0.05 Not significant 0.911 6-gingerol vs DPPH <0.001 Extremely significant 0.985 Phenolics vs FRAP <0.001 Extremely significant 0.433
0
0.5
1
1.5
2
2.5
3
3.5
Control 0.3 0.4 0.5 0.6
EMS 4 hrs treatments
Gingerol % Flavonoid Phenolic FRAP DPPH
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 82
Figure: 4.17 Correlations between different biochemical traits in various concentrations of EMS 4 hrs treated plants
R² = 0.880
0.60.70.80.9
11.11.21.31.4
0.15 0.2 0.25 0.3 0.35
g/10
0g T
anni
c ac
id e
q.
Flavonoid g/100g Quercetin eq.
(a) Correlation between Flavonoid and Phenolics
R² = 0.301
500
1000
1500
2000
2500
3000
3500
0.15 0.2 0.25 0.3 0.35
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(b) Correlation between Flavonoid and Antioxidant (FRAP)
R² = 0.433
500
1000
1500
2000
2500
3000
3500
0.4 0.6 0.8 1 1.2 1.4
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(c) Correlation between Phenolics and Antioxidant (FRAP)
R² = 0.589
500
700
900
1100
1300
1500
1700
0.15 0.2 0.25 0.3 0.35
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(d) Correlation between Flavonoid and Antioxidant (DPPH)
R² = 0.910
500
700
900
1100
1300
1500
1700
0.4 0.6 0.8 1 1.2 1.4
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(e) Correlation between Phenolics and Antioxidant (DPPH)
R² = 0.891
500
700
900
1100
1300
1500
1700
500 1000 1500 2000 2500 3000 3500
µM A
scor
bic
eq.
FRAP µM Ascorbic eq.
(f) Correlation between Antioxidant tests: FRAP and DPPH
R² = 0.773
0.000.050.100.150.200.250.300.350.40
0.08 0.1 0.12 0.14 0.16 0.18 0.2
g/10
0g Q
uerc
etin
eq.
% Gingerol
(g) Correlation between 6- gingerol and Flavonoid content
R² = 0.911
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0.08 0.1 0.12 0.14 0.16 0.18 0.2
g/10
0g T
anni
c ac
id e
q.
% Gingerol
(h) Correlation between 6-gingerol and Phenolic content
R² = 0.856
0500
100015002000250030003500
0.08 0.1 0.12 0.14 0.16 0.18 0.2
µM A
scor
bic
acid
eq.
% Gingerol
(i) Correlation between 6-gingerol and Antioxidant activity (FRAP)
R² = 0.985
0200400600800
10001200140016001800
0.08 0.1 0.12 0.14 0.16 0.18 0.2
µM A
scor
bic
acid
eq.
% Gingerol
(j) Correlation between 6-gingerol and Antioxidant activity (DPPH)
STUDIES IN ZINGIBER OFFICINALE
Laboratory of Cytogenetics and Plant Breeding
4.9: Biochemical studies in EMS 8 hrs treatment:
Analysis of 6-gingerol:
The HPLC analysis was done for the quantification of
content of the control and irradiated material
highest 6-gingerol content (0.175%)
while lowest (0.16%) for
4.14). There was no definite trend observed
increase or decrease the concentrations.
Figure: 4.18: Chromatogram
0.15%, d:
ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.:
Biochemical studies in EMS 8 hrs treatment:
HPLC analysis was done for the quantification of 6
content of the control and irradiated materials of ginger (Figure: 4.1
gingerol content (0.175%) was detected in 0.10% EMS 8 hrs
for 0.15% and 0.25% EMS 8 hrs treated plants (Table
ere was no definite trend observed in content of 6-gingerol
increase or decrease the concentrations.
: Chromatogram, a: Control, b: EMS 8hrs 0.10%, c
EMS 8hrs 0.20% and e: EMS 8hrs 0.25%
(a)
(c)
(e)
Results and Discussions
Page No.: 83
6 – gingerol
18 a - e). The
EMS 8 hrs treatment
ed plants (Table
gingerol during
: EMS 8hrs
(b)
(d)
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 84
Total Flavonoid content:
Flavonoid content was expressed in quercetin equivalent which was
The range of flavonoid content varies from 0.255 gm/100gm dry weight of ginger
(0.25% EMS 8 hrs treatment) to 0.440 gm/100gm dry weight of ginger (0.10%
EMS 8 hrs treatment).
Total Phenolic content:
Phenolic content of the ginger treated with different concentrations of
EMS 8 hrs treatments were determined using Folin Ciocalteu method and the
results were expressed in tannic acid equivalent. Highest phenolic content (1.407
gm/100gm dry weight of ginger) was recorded for 0.10% EMS 8 hrs treatment
and lowest content (1.106 gm/100gm dry weight of ginger) was recorded in
0.25% EMS 8 hrs treatment (Figure: 4.19).
Antioxidant activity:
DPPH and FRAP method was used to measure antioxidant activity. The
highest activity was recorded in 0.10 % EMS 8 hrs treatment for both DPPH and
FRAPS (Table 4.14) while lowest in 0.25 % EMS 8 hrs treatment.
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 85
Table: 4.14 Effects of various concentrations of EMS 8 hrs treatments on 6 - gingerol, Flavonoid and Phenolic contents and DPPH and FRAP activity in ginger
EMS 8 hrs in % 6 - gingerol %
Flavonoida ±sd Phenolicb ±sd FRAPc ±sd DPPHd ±sd
Control 0.1657 0.323 0.000394 1.240 0.002 2.731 0.00377 1.493 0.00302
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 86
Figure: 4.19 Effects of various concentrations of EMS 8 hrs treatments on 6 - gingerol, Flavonoid and Phenolic contents in gm/100 gm of dry weight and
DPPH and FRAP activity in Mm.
4.10: Correlations between biochemical parameters in various
concentrations of EMS 8 hrs treatments:
All the correlations were of positive R values (Figure 4.20 a to j), which
are depicted in Table 4.15. Strong correlations were found between Gingerol and
Flavonoid (R2 = 0.982) followed by FRAP and DPPH (R2 = 0.901). The lowest
activity was recorded between flavonoid and FRAP (R2 = 0.301).
Table: 4.15 Correlations between different biochemical traits in various
concentrations of EMS 8 hrs treated plants
Correlations P value Remark R2 value Flavonoid vs Phenolics >0.05 Not significant 0.880 Flavonoid vs FRAP <0.001 Extremely significant 0.301 Flavonoid vs DPPH <0.001 Extremely significant 0.589 Phenolics vs DPPH <0.001 Extremely significant 0.599 DPPH vs FRAP <0.01 Significant 0.901 6 -gingerol vs Flavonoid >0.05 Not significant 0.982 6- gingerol vs FRAP <0.001 Extremely significant 0.731 6 -gingerol vs Phenolics >0.05 Not significant 0.755 6- gingerol vs DPPH <0.001 Extremely significant 0.457 Phenolics vs FRAP <0.001 Extremely significant 0.689
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Control 0.1 0.15 0.2 0.25
EMS 8 hrs treatments
Gingerol % Flavonoid Phenolic FRAP DPPH
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 87
Figure: 4.20 Correlations between biochemical parameters in various
concentrations of EMS 8 hrs treatments
R² = 0.880
0.60.70.80.9
11.11.21.31.41.5
0.2 0.25 0.3 0.35 0.4 0.45 0.5
g/10
0g T
anni
c ac
id e
q.
Flavonoid g/100g Quercetin eq.
(a) Correlation between Flavonoid and Phenolics
R² = 0.301
500100015002000250030003500400045005000
0.2 0.25 0.3 0.35 0.4 0.45 0.5
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(b) Correlation between Flavonoid and Antioxidant (FRAP)
R² = 0.433
500100015002000250030003500400045005000
1 1.1 1.2 1.3 1.4 1.5
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(c) Correlation between Phenolics and Antioxidant (FRAP)
R² = 0.589
500700900
1100130015001700190021002300
0.2 0.25 0.3 0.35 0.4 0.45 0.5
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(d) Correlation between Flavonoid and Antioxidant (DPPH)
R² = 0.599
500700900
1100130015001700190021002300
1 1.1 1.2 1.3 1.4 1.5
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(e) Correlation between Phenolics and Antioxidant (DPPH)
R² = 0.901
500700900
1100130015001700190021002300
750 1750 2750 3750 4750
µM A
scor
bic
eq.
FRAP µM Ascorbic eq.
(f) Correlation between Antioxidant tests: FRAP and DPPH
R² = 0.982
0.00
0.10
0.20
0.30
0.40
0.50
0.15 0.155 0.16 0.165 0.17 0.175 0.18
g/10
0g Q
uerc
etin
eq.
% Gingerol
(g) Correlation between 6-gingerol and Flavonoid content
R² = 0.755
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
0.15 0.155 0.16 0.165 0.17 0.175 0.18
g/10
0g T
anni
c ac
id e
q.
% Gingerol
(h) Correlation between 6 -ingerol and Phenolic content
R² = 0.689
0
1000
2000
3000
4000
5000
0.15 0.155 0.16 0.165 0.17 0.175 0.18
µM A
scor
bic
acid
eq.
% Gingerol
(i) Correlation between 6 -gingerol and Antioxidant activity (FRAP)
R² = 0.457
1000120014001600180020002200
0.15 0.155 0.16 0.165 0.17 0.175 0.18
µM A
scor
bic
acid
eq.
% Gingerol
(j) Correlation between 6 -gingerol and Antioxidant activity (DPPH)
STUDIES IN ZINGIBER OFFICINALE ROSCOE
Laboratory of Cytogenetics and Plant Breeding
4.11: Biochemical studies in Gamma rays
Analysis of 6-gingerol:
Evaluation of 6
materials of ginger was done by HPLC.
gingerol content (pungent principle) of ginger
(Figure: 4.21 a-g). The concentration of 6
doses increased. The highest 6
KR Gamma rays treatment
treated plants (Table 4.16)
Figure: 4.21 Chromatograms: a
Gamma rays 0.250 KR d
f: Gamma rays 0.750
OFFICINALE ROSCOE Chapter IV: Results and Discussions
Cytogenetics and Plant Breeding | Page No.:
Biochemical studies in Gamma rays induced mutations:
Evaluation of 6 – gingerol content of the control and gamma
of ginger was done by HPLC. The effect of gamma radiation on the 6
gingerol content (pungent principle) of ginger was shown in Chromatograms
. The concentration of 6-gingerol continued to decrease as the
doses increased. The highest 6-gingerol content (0.19%) was detected in
treatment while lowest (0.12%) was in 1 KR gamma rays
(Table 4.16).
Chromatograms: a: Control, b: Gamma rays 0.125
KR d: Gamma rays 0.375KR, e: Gamma rays 0.500
amma rays 0.750 KR and g: Gamma rays 1 KR
(a)
(c)
Results and Discussions
Page No.: 88
gamma irradiated
of gamma radiation on the 6-
Chromatograms
gingerol continued to decrease as the
was detected in 0.250
while lowest (0.12%) was in 1 KR gamma rays
amma rays 0.125 KR c:
amma rays 0.500 KR,
(b)
(d)
STUDIES IN ZINGIBER OFFICINALE ROSCOE
Laboratory of Cytogenetics and Plant Breeding
Total Flavonoid content:
Flavonoid content was expressed in Quercetin equivalent
0.334gm/100gm dry weight equivalent in control. The range of flavonoid content
of gamma rays doses varies from 0.197 to 0.311 gm/100gm of dry weight of
ginger which was low as compare to control. The content of flavonoid decreases
as dose increases (Figure:
Total Phenolic content:
The total phenolic content of irradiated and non
ginger was determined using the Folin
expressed as mg equivalents of
Table – 4.16. The phenolic content of the control (non
found to be 1.240 gm/100gm of dry weight.
total phenolic content showed
the dose increases. The highest total phenolic content (1.303 gm/100gm of dry
weight) found in 0.250KR treatment while lowest (0.853 gm/100gm of dry
weight) was in 1KR treatment.
from either the synthesis of new aromatic compounds (Nagieb, 1979) and/or the
acceleration of the accumulative phenol from the neighboring cells (Tomiyama,
1963).
OFFICINALE ROSCOE Chapter IV: Results and Discussions
Cytogenetics and Plant Breeding | Page No.:
Total Flavonoid content:
Flavonoid content was expressed in Quercetin equivalent
0.334gm/100gm dry weight equivalent in control. The range of flavonoid content
of gamma rays doses varies from 0.197 to 0.311 gm/100gm of dry weight of
ginger which was low as compare to control. The content of flavonoid decreases
ure: 4.22).
henolic content:
The total phenolic content of irradiated and non-irradiated samples of
was determined using the Folin–Ciocalteu phenol reagent. The results are
expressed as mg equivalents of Tannic acid/g dry weight of extract and given in
The phenolic content of the control (non-irradiated) sample was
found to be 1.240 gm/100gm of dry weight. For radiation-processed samples, the
showed initially increased and significantly
the dose increases. The highest total phenolic content (1.303 gm/100gm of dry
weight) found in 0.250KR treatment while lowest (0.853 gm/100gm of dry
weight) was in 1KR treatment. This increase of phenolic compounds may result
m either the synthesis of new aromatic compounds (Nagieb, 1979) and/or the
acceleration of the accumulative phenol from the neighboring cells (Tomiyama,
(e)
(g)
Results and Discussions
Page No.: 89
Flavonoid content was expressed in Quercetin equivalent which was
0.334gm/100gm dry weight equivalent in control. The range of flavonoid content
of gamma rays doses varies from 0.197 to 0.311 gm/100gm of dry weight of
ginger which was low as compare to control. The content of flavonoid decreases
irradiated samples of
Ciocalteu phenol reagent. The results are
ic acid/g dry weight of extract and given in
irradiated) sample was
processed samples, the
decreases as
the dose increases. The highest total phenolic content (1.303 gm/100gm of dry
weight) found in 0.250KR treatment while lowest (0.853 gm/100gm of dry
This increase of phenolic compounds may result
m either the synthesis of new aromatic compounds (Nagieb, 1979) and/or the
acceleration of the accumulative phenol from the neighboring cells (Tomiyama,
(f)
STUDIES IN ZINGIBER OFFICINALE ROSCOE Chapter IV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 90
Table: 4.16 Effects of various doses of gamma rays treatments on 6 - gingerol, Flavonoid and Phenolic contents and DPPH and FRAP activity in ginger
STUDIES IN ZINGIBER OFFICINALE ROSCOE ChapterIV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 91
Antioxidant activity:
The radical-scavenging activity of the irradiated and control ginger
samples were analyzed in methanol, using 1, 1-diphenyl-2-picrylhydrazyl radical
(DPPH) and FRAP. The reduction in the DPPH concentration is a measure of
scavenging activity. The highest activity was recorded in 0.250 KR of gamma
rays dose (Table 4.16) for both DPPH and FRAP while lowest in 1 KR of gamma
rays dose.
Figure: 4. 22 Effects of various doses of gamma rays treatments on 6 - gingerol, Flavonoid and Phenolic contents in gm/100gm of dry weight eq.
and DPPH and FRAP activity in Mm of ginger
4.12: Correlations between biochemical parameters in gamma rays
treatments:
Correlations between all the traits were positive (Figure 4.23 a to j) which
was depicted in table 4.17. Strong correlations was found between Phenolic and
Flavonoid (R2 = 0.880) followed by phenolic and DPPH (R2 = 0.703). The lowest
activity was recorded between flavonoid and FRAP (R2 = 0.301).
0
0.5
1
1.5
2
2.5
3
3.5
4
Control 0.125 0.25 0.375 0.5 0.75 1
Gamma rays in KR
Gingerol % Flavonoid Phenolic FRAP DPPH
STUDIES IN ZINGIBER OFFICINALE ROSCOE ChapterIV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 92
Table: 4.17 Correlation between biochemical parameters of different gamma
rays treatments
Correlations P value Remark R2 value Flavonoid vs Phenolics >0.05 Not significant 0.880 Flavonoid vs FRAP <0.001 Extremely significant 0.301 Flavonoid vs DPPH <0.001 Extremely significant 0.589 Phenolics vs DPPH <0.001 Extremely significant 0.703 DPPH vs FRAP <0.001 Extremely significant 0.619 6-gingerol vs Flavonoid >0.05 Not significant 0.687 6-gingerol vs FRAP <0.001 Extremely significant 0.673 6-gingerol vs Phenolics >0.05 Not significant 0.684 6-gingerol vs DPPH <0.001 Extremely significant 0.545 Phenolics vs FRAP <0.001 Extremely significant 0.433
Figure: 4.23 Correlations between biochemical parameters in gamma rays
treatments:
R² = 0.880
0.600.700.800.901.001.101.201.301.40
0.15 0.2 0.25 0.3 0.35
g/10
0g T
anni
c ac
id e
q.
Flavonoid g/100g Quercetin eq.
(a) Correlation between Flavonoid and Phenolics
R² = 0.301
1100
1600
2100
2600
3100
3600
4100
0.15 0.2 0.25 0.3 0.35
µM A
scor
bic
eq.
Flavonoid g/100g Quercetin eq.
(b) Correlation between Flavonoid and Antioxidant (FRAP)
R² = 0.589
1100
1200
1300
1400
1500
1600
1700
0.15 0.2 0.25 0.3 0.35
µM A
scor
bic
eq.
Flavonoids g/100g Quercetin eq.
(c) Correlation between Flavonoid and Antioxidant (DPPH)
R² = 0.703
1100
1200
1300
1400
1500
1600
1700
0.700 0.900 1.100 1.300 1.500
µM A
scor
bic
eq.
Phenolic g/100g Tannic acid eq.
(d) Correlation between Phenolics and Antioxidant (DPPH)
STUDIES IN ZINGIBER OFFICINALE ROSCOE ChapterIV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 93
There is no information available in the literature on the effects of
ionizing radiations and chemical mutagens on the phenolic content of ginger.
However, for other plant materials, diverse effects of radiation on the phenolic
content have been reported. Variyar et al. (1998) found increased amounts of
phenolic acids in irradiated cloves and nutmeg. The difference in the effect of
radiation on total phenolic content may be due to plant type, geographical and
environmental conditions, state of the sample (solid or dry), phenolic content
composition, extraction solvent, extraction procedures, temperature, dose of
gamma irradiation, etc.
The results obtained in the present investigations are in agreement with
the results observed by Sadowska (1975) in Peppermint. The results are in
conformity with Hegnauer (1975) and Levy (1982). They reported induced
mutations have resulted in significant changes in secondary metabolite. There is
evidence that mutagens (radiations) stimulate the metabolic activity of plants
R² = 0.619
1100
1200
1300
1400
1500
1600
1700
2000 2500 3000 3500 4000
µM A
scor
bic
eq.
FRAP µM Ascorbic eq.
(e) Correlation between Antioxidant tests: FRAP and DPPH
R² = 0.687
0.000.050.100.150.200.250.300.350.40
0.10 0.12 0.14 0.16 0.18 0.20
g/10
0g Q
uerc
etin
eq.
% Gingerol
(f) Correlation between 6-gingerol and Flavonoid content
R² = 0.673
0500
1000150020002500300035004000
0.10 0.12 0.14 0.16 0.18 0.20
µM A
scor
bic
acid
eq.
% Gingerol
(g) Correlation between 6-gingerol and Antioxidant activity (FRAP)
R² = 0.684
0.0000.2000.4000.6000.8001.0001.2001.400
0.10 0.12 0.14 0.16 0.18 0.20
Phe
nolic
g/1
00g
Tan
nic
acid
eq
.
% Gingerol
(h )Correlation between 6-gingerol and Phenolic content
R² = 0.545
0200400600800
10001200140016001800
0.10 0.12 0.14 0.16 0.18 0.20
µM A
scor
bic
acid
eq.
% Gingerol
(i) Correlation between 6-gingerol and Antioxidant activity (DPPH)
R² = 0.433
1100120013001400150016001700
0.700 0.900 1.100 1.300 1.500FR
AP
µM
Asc
orbi
c eq
.
Phenolic g/100g Tannic acid eq.
(j) Correlation between Phenolics and Antioxidant (FRAP)
STUDIES IN ZINGIBER OFFICINALE ROSCOE ChapterIV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 94
such as respiration (Romani, 1966), glycolysis and oxidative phosphorylation
(Mergen and Johnson, 1964) and cytochrome oxidase and catalase activity
(Goldon, 1957) which may ultimately influence and enhance synthesis of plant
products (Kaul et al., 1973).
4.13: Callus induction studies:
Callus induction was observed in the three explants viz. leaf, rhizome and
pseudostem (Plate: VI a to c respectively) on MS basal medium supplemented
with various concentrations and combinations of 2, 4-D and NAA. There was a
wide range of variation in percentage of callus induction (Table 4.18). 2, 4-D
alone in all the concentrations tried (1.5 – 3.5 mg/l), was effective in inducing
callus from rhizome explant and (2 - 3.5 mg/l) from pseudo stem and leaf. The
positive response for rhizome explant was recorded on MS medium
supplemented with 2, 4-D (2.5 mg/l) with 60 % callusing. For the leaf and pseudo
However, 2, 4-D had no effect on callus induction when used in concentration
above 3.5 mg/l (Table 4.18) NAA 2.5 mg/l had an average callusing 35% for
rhizome explant and 25% for leaf and pseudo stem explant. NAA had no effect
on callus induction when used in concentrations below 1.5 mg/l but induced
callus when used in concentrations above 2 mg/l. Of the different growth
regulator tested, 2, 4-D was found to give good callus growth in ginger. This is in
agreement with observations reported by Nirmal Babu et al. (1992) and Kacker et
al. (1993). Application of different media such as MS, ½ MS, 1/3 MS and ¼ MS
with different concentrations of IAA, IBA and NAA did not produce satisfactory
results (Jabbarzadeh and Khosh-Khui, 2005). Callus induction in ginger was
reported by various workers (Pillai and Kumar, 1982, Sakamura and Suga, 1989,
Choi, 1991, Malamug, et al. 1991, Kacker, et al. 1993, Ilahi and Jabeen, 1992
and Samsudeen, 1996). Nirmal Babu (1997) revealed that callus can be
successfully grown from vegetative bud, young leaf, ovary, and anther tissues.
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Laboratory of Cytogenetics and Plant Breeding | Page No.: 95
Table: 4.18 Effects of different concentrations of growth regulators on induction of callus in Ginger
Sr. No. Medium combination
Percentage of Callus for
rhizome (After 45
days)
Percentage of Callus for Leaf (After
45 days)
Percentage of Callus for pseudo-stem
(After 45 days)
1 M. S. + 2, 4- D 1 mg/l Nil Nil Nil 2 M. S. + 2, 4- D 1.5 mg/l 15% Nil Nil 3 M. S. + 2, 4- D 2 mg/l 45% 15% 15% 4 M. S. + 2, 4- D 2.5 mg/l 60% 30% 35% 5 M. S. + 2, 4- D 3 mg/l 40% 15% 25% 6 M. S. + 2, 4- D 3.5 mg/l 10% 5% 15% 7 M. S. + 2, 4- D 4 mg/l Nil Nil Nil 8 M. S. + 2, 4- D 4.5 mg/l Nil Nil Nil 9 M. S. + NAA 1 mg/l Nil Nil Nil
10 M. S. + NAA 1.5 mg/l Nil 5% Nil 11 M. S. + NAA 2 mg/l 25% 10% 15% 12 M. S. + NAA 2.5 mg/l 35% 25% 25% 13 M. S.+ NAA 3 mg/l 30% 20% 20% 14 M. S.+ NAA 3.5mg/l 15% Nil 10% 15 M. S.+ NAA 4 mg/l Nil Nil Nil
Figures in table indicate response of 20 replicates
4.14: Micropropagation studies :
One of the main objectives of micropropagation is the establishment of
plants or clones that are uniform and predictable of selected qualities.
Sterilization efficiency:
Since the explants were taken from underground rhizomes, establishment
of aseptic culture was a major task. High rate of contamination in cultures was
reported when rhizomes or vegetative buds are used for micropropagation.
Standard methods of aseptic culture of plant tissues and organs were
followed in the present studies. Mercuric chloride (0.1%) solution a commonly
used sterilizing agent was tried in present study. The efficiency of this sterilizing
agent was obtained for axillary buds of rhizome (9 minutes of exposed period);
for leaves (2 minutes of exposed period) and pseudo stem (4 minutes of exposed
period). It was observed that rainy season favors the increased rate of
contamination.
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Laboratory of Cytogenetics and Plant Breeding | Page No.: 96
Among the different cytokinins used, BA was found to be most effective
for shoot induction and subsequent shoot multiplication, as compared with
kinetin. BA at the concentrations used (0.5 mg/l to 3.5 mg/l) exhibited 65-90%
frequency of shoot induction (Table 4.19). The frequency of response increased
to 90% with an average of 6.35 shoots per shoot explant when BA (2 mg/l) was
used. As the concentration of BA increased, it was found that the average
number of shoots also increased. The optimum shoot number recorded was 6.35
in the concentration of MS + BA2.0 mg/l (Plate: VI- e), which was found to
decrease further, when still higher concentrations of BA were used. The mean
root number ranged from 1.61 to 2.94 per shoot explant. The highest average root
number (2.94) was recorded in the 2 mg/l of BA concentration. In case of Kn it
was maximum (2.72) for 2.5mg/l concentration.
The percent shoot induction range for different concentrations of Kn (1mg
to 3.5mg/l) was 70 to 85%. Average shoots number was highest (5.44) for Kn
2.5mg/l concentration (Plate: VI - f). With increase in concentrations of Kn but
below the optimum level i.e. 2.5 mg/l the number of shoot increased. Present
investigation indicated that increasing concentrations of BA from 0.5 to a
maximum level of 3.5mg/l was responsible for shoot multiplication (Figure;
4.24). Bhagyalakshmi and singh (1988) obtained 4.0 shoots/explant of ginger on
MS medium fortified with BA at 1 mg/l. Such type of simultaneous production of
shoot and roots were reported earlier for a few species of Zingiberaceae by
Kuruvinashetty et al. (1982); Balachandran et al. (1990) and Borthakur and
Bordoloi (1992). Establishment of aseptic cultures was difficult in ginger, but
once a healthy culture was established, there was no further contamination.
Similar findings were observed by earlier investigators with ginger (Hosoki and
Sagava, 1977 and Inden and Ashira, 1988).
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Laboratory of Cytogenetics and Plant Breeding | Page No.: 97
Table: 4.19 Effects of different individual concentrations of growth
regulators on induction of shoots and roots in Ginger
Sr. No.
Medium combination
Percent response
Mean No. of shoots
Mean No. of roots
1. M. S. + BA 0.5mg/l 65 3.07 ±0.64 1.61 ±0.65 2. M. S. + BA 1mg/l 80 4.33 ±0.61 2.20 ±0.67 3. M. S. + BA 1.5mg/l 85 4.75 ±0.93 2.87 ±0.88 4. M. S. + BA 2mg/l 90 6.35 ±0.78 2.94 ±0.74 5. M. S. + BA 2.5mg/l 80 4.93 ±0.77 2.43 ±0.72 6. M. S. + BA 3mg/l 75 4.73 ±0.88 2.26 ±0.70 7. M. S. + BA 3.5mg/l 65 3.92 ±0.73 2.07 ±0.61 8. M. S. + Kin. 1mg/l 75 2.46 ±0.96 1.76 ±0.72 9. M. S. + Kin. 1.5mg/l 80 3.12 ±0.61 2.12 ±0.88 10. M. S. + Kin. 2mg/l 85 3.82 ±0.80 2.41 ±0.87 11. M. S. + Kin. 2.5mg/l 80 5.44 ±0.98 2.72 ±0.66 12. M. S. + Kin. 3mg/l 75 4.68 ±0.79 2.31 ±0.94 13. M. S. + Kin. 3.5mg/l 70 3.73 ±0.70 1.60 ±0.73
Figures in table indicate response of 20 replicates
Figure: 4.24 Effects of different individual concentration of growth
regulators on induction of shoots and roots in Ginger
0102030405060708090100
012345678
1 2 3 4 5 6 7 8 9 10 11 12 13
Num
ber
Medium + PGR Sr. No. From Table 4.19Mean No. of shoots Mean No. of roots Percent response
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Laboratory of Cytogenetics and Plant Breeding | Page No.: 98
The shoot and root response of the vegetative buds of ginger to various
concentrations and combinations of cytokinin and auxin is presented in Table
4.20. Shoot and root response were recorded after 45 days of culture on MS
supplemented with BAP, Kn and NAA at different concentrations. The highest %
response was noticed on M. S. + BA 2 + NAA 2.5 mg/l (90%) (Plate: VI- d and
g) and lowest on M. S. + Kn 2 + NAA 1mg/l (65%). The highest mean number of
shoots per explant was observed on MS supplemented with BA 2 + IAA 2.5 mg/l
which was 6.62 (Figure: 4.25) while lowest mean number of shoot on BA 2 +
IAA 1mg/l (3.62). During micropropagation, induction of both roots and shoots
in the same medium minimize the time taken for cloning considerably, which
eliminates the step of in vitro rooting and reduces the overall cost of
micropropagation. The highest mean number of roots per explant was observed
on M. S. + BA 2 + IAA 2.5 mg/l (5.06) and lowest was on M. S. + Kn 2 + NAA
1mg/l (2.38).
Our observations made in present studies are in agreement with the results
obtained by Nirmal Babu (1997), who tried MS basal medium supplemented with
auxin (NAA 0–4 mg/l) and cytokinin (BA 0–4 mg/1). Sakamura et al. (1986);
Charlwood et al. (1988) and Sakamura and Suga (1989) reported that BAP and
NAA combinations were best for shoot multiplication in ginger. The presence of
NAA at low concentrations resulted in good growth of culture, root induction,
and shoot multiplication and addition of BA at 2.5mg/l increased the multiple
shoot induction. BA alone at higher concentration (2.5mg/l) induced only
multiple shoots and rarely roots.
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Laboratory of Cytogenetics and Plant Breeding | Page No.: 99
Table: 4.20 Effect of different combinations of growth regulators on
induction of shoots and roots in Ginger
Sr. No. Medium combination Percent
response Mean No. of
shoots Mean No. of roots
1 M. S. + BA 2 + IAA 1mg/l 70 3.64 ±0.92 2.85 ±0.66 2 M. S. + BA 2 + IAA 1.5 mg/l 75 4.86 ±0.74 3.80 ±0.67 3 M. S. + BA 2 + IAA 2 mg/l 85 5.70 ±0.98 4.64 ±0.86 4 M. S. + BA 2 + IAA 2.5 mg/l 80 6.62 ±0.80 5.06 ±0.77 5 M. S. + BA 2 + IAA 3 mg/l 70 5.07 ±0.73 3.28 ±0.72 6 M. S. + Kn 2 + IAA 1mg/l 75 4.00 ±0.75 2.53 ±0.63 7 M. S. + Kn 2 + IAA 1.5 mg/l 80 4.56 ±0.72 3.18 ±0.75 8 M. S. + Kn 2 + IAA 2 mg/l 80 5.06 ±0.77 4.06 ±0.68 9 M. S. + Kn 2 + IAA 2.5 mg/l 85 6.29 ±0.77 5.05 ±0.74
10 M. S. + Kn 2 + IAA 3 mg/l 80 5.00 ±0.73 3.43 ±0.72 11 M. S. + BA 2 + NAA 1mg/l 80 4.06 ±0.85 2.31 ±0.60 12 M. S. + BA 2 + NAA 1.5 mg/l 75 4.53 ±0.51 3.06 ±0.59 13 M. S. + BA 2 + NAA 2 mg/l 85 5.76 ±0.83 4.35 ±0.86 14 M. S. + BA 2 + NAA 2.5 mg/l 90 6.61 ±0.77 4.83 ±0.92 15 M. S. + BA 2 + NAA 3 mg/l 85 5.64 ±0.86 3.35 ±0.86 16 M. S. + BA 2 + NAA 3.5 mg/l 70 4.57 ±0.64 2.92 ±0.73 17 M. S. + Kn 2 + NAA 1mg/l 65 4.38 ±0.65 2.38 ±0.50 18 M. S. + Kn 2 + NAA 1.5 mg/l 80 4.68 ±0.60 3.06 ±0.68 19 M. S. + Kn 2 + NAA 2 mg/l 85 5.23 ±0.75 4.11 ±0.69 20 M. S. + Kn 2 + NAA 2.5 mg/l 85 6.47 ±0.87 4.41 ±0.87 21 M. S. + Kn 2 + NAA 3 mg/l 75 5.23 ±0.83 3.41 ±0.87
Figures in table indicate response of 20 replicates
STUDIES IN ZINGIBER OFFICINALE ROSCOE ChapterIV: Results and Discussions
Laboratory of Cytogenetics and Plant Breeding | Page No.: 100
Figure: 4.25 Effect of different combination of growth regulator on
induction of shoots and roots in Ginger
4.15: Hardening and Acclimatization in Ginger:
Hardening and acclimatization are the crucial stages of tissue culture. In
the process of hardening, three different kinds of potting mixtures were tested for
the survival of the plants. During acclimatization, high relative humidity (85 –
90%) was maintained for the initial 20 to 30 days, and a single spray of a
commercial fungicide Bavistin (0.2 % w/v) was found effective for plantlets.
However, the humidity was reduced gradually for hardening and establishment of
plantlets. The survival rate of transplanted plantlets ranged from 70 to 95 percent
and the time taken for hardening was about 25 days in direct regenerated plants.
The different potting mixtures such as autoclaved garden soil, compost and sand
were used in variable proportions. In the present study, it was observed that the
potting mixture composing soil: sand: compost; (1:2:1) showed good response to
survival rate of the in vitro derived plants. This may probably be due to high sand
content in the mixture which favors the adequate aeration and growth of roots.
Furthermore, increasing the composition of sand (i.e. 1:3:1, 1:4:1 and 1:5:1) in
the potting mixture did not work in a positive way in terms of enhancement of
survival rate. The low survival percentage (55%) was observed in the mixture of