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RESULTS
The results of two years investigation from 2009-2011 have been described under
the following headings like Micro propagation , estimation of primary metabolites,
chlorophyll content and presence of secondary metabolites in Withania somnifera and
Adhatoda vasica . The in vitro propagation of both the plants was done under the
influence of different growth regulators BAP, NAA and kinetin. The concentrations of
growth hormone used was 0.10, 0.25, 0.50, 0.75 and1.00mgl-1. The effect of different
concentration of growth hormone was estimated by studying the parameters like shoots
per explants, average shoot length, nodes per shoots, leaves per shoots and bud break. All
the investigations were made in three replicates.
(I). Micropropagation in Withania somnifera.
(1.0) Effects of plant growth regulators (BAP) in micro propagation of Withania
somnifera.
(1.1) Effects of BAP (mgl-1) in shoots per explant of Withania somnifera in the year
2009-2010.
The effect of BAP on shoots per explants was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1concentration. Shoots/explant was ranging from 1.3-3.1 in all the
concentrations of BAP supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1of BAP, while minimum value was noted in 0.10mgl-
1concentration. The nodal segments of Withania somnifera showed shoots per explant
ranging from 3.0 to 3.1 on MS medium supplemented with 1.0 mgl-1concentration of
BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 3.0, 3.1 and 3.0 respectively,
with an average value of 3.03. An increasing trend was observed in shoots/explant value
with the increase in BAP concentration except the concentration 0.50 mgl-1concentration
where value remained unchanged.
In statistical analysis, F- value was calculated 176.3784 at the 0 probability. The
co-efficient of variation was determined 3.60% for different concentrations of BAP in
shoot per explants raised from nodal segment of Withania somnifera. The standard error
of mean and standard error of differences was calculated 0.0279 and 0.00282 respectively
(Table1 & Plate 1A).
(1.2) Effects of BAP (mgl-1) in shoots per explant of Withania somnifera in 2010-
2011.
The effect of BAP on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoots/explant was ranging from 1.2-3.2 in all the
concentrations of BAP supplemented with media. Maximum mean value of shoots/
explant was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed shoots per
explant ranging from 3.0 to 3.2 on MS medium supplemented with 1.0 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 3.2, 3.0 and 3.1
respectively, with an average value of 3.10. An increasing trend was observed in
shoots/explant value with the increase in BAP concentration.
In statistical analysis the F- value was calculated 125.8750 at the 0 probability.
The co-efficient of variation was found to be 4.67%, for different concentrations of BAP
in shoots per explant raised from Withania somnifera. The standard error of mean and
standard error of differences obtained was 0.0356 and 0.0051 respectively (Table 2&
Plate 1B).
(1.3) Effects of BAP (mgl-1) in average shoot length of Withania somnifera in 2009-
2010.
The effect of BAP on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 1.1cm-6.8cm in all
the concentrations of BAP supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1 of BAP while minimum value was noted in
0.50mgl-1concentration. The nodal segments of Withania somnifera showed average
shoot length ranging from 6.7cm to 6.8cm on MS medium supplemented with 1.0 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 6.8cm, 6.7cm
and 6.8cm respectively, with an average value of 6.76cm. An increasing trend was
observed in average shoot length value with the increase in BAP concentration except the
concentration 0.50 mgl-1concentration where minimum value obtained.
In statistical analysis, F- value was calculated 2398.7125 at the 0 probability. The
co-efficient of variation was determined 2.43% for different concentrations of BAP in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0295 and 0.0032
respectively (Table 3).
(1.4) Effects of BAP (mgl-1) in average shoot length of Withania somnifera in 2010-
2011.
The effect of BAP on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 0.6cm-6.8cm in all
the concentrations of BAP supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1of BAP while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed average
shoot length ranging from 6.7cm to 6.8cm on MS medium supplemented with 1.0 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 6.7cm, 6.8cm
and 6.8cm respectively, with an average value of 6.76cm. An increasing trend was
observed in average shoot length value with the increase in BAP concentration.
In statistical analysis, F- value was calculated 1457.3842 at the 0 probability. The
co-efficient of variation was determined 3.43% for different concentrations of BAP in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0356 and 0.0051
respectively (Table 4).
(1.5) Effects of BAP (mgl-1) in nodes per shoot of Withania somnifera in 2009-2010.
The effect of BAP on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 0.0-6.2 in all the
concentrations of BAP supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1of BAP while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed nodes per
shoot ranging from 6.0 to 6.2 on MS medium supplemented with 1.0 mgl-1 concentration
of BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 6.0, 6.2 and 6.2 respectively,
with an average value of 6.13. An increasing trend was observed in nodes per shoot value
with the increase in BAP concentration except the concentration 0.10mgl-1concentration
where no value obtained.
In statistical analysis, F- value was calculated 3303.5911 at the 0 probability. The
co-efficient of variation was determined 2.75% for different concentrations of BAP in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0263 and 0.0023 respectively
(Table 5).
(1.6) Effects of BAP (mgl-1) in nodes per shoot of Withania somnifera in 2010-2011.
The effect of BAP on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 0.0-6.2 in all the
concentrations of BAP supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1of BAP while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed nodes per
shoot ranging from 6.1 to 6.2 on MS medium supplemented with 1.0 mgl-1 concentration
of BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 6.2, 6.1 and 6.1 respectively,
with an average value of 6.13. An increasing trend was observed in nodes per shoot value
with the increase in BAP concentration except the concentration 0.10 mgl-1concentration
where no value obtained.
In statistical analysis, F- value was calculated 5019.6879 at the 0 probability. The
co-efficient of variation was determined 2.17% for different concentrations of BAP in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0229 and 0.0014 respectively
(Table 6).
(1.7) Effects of BAP (mgl-1) in leaves per shoots of Withania somnifera in 2009-2010.
The effect of BAP on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 0.0-22.4 in all the
concentrations of BAP supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of BAP while no value was noted in 0.10 mgl-1
concentration. The nodal segments of Withania somnifera showed leaves per shoot
ranging from 22.3 to 22.4 on MS medium supplemented with 1.0 mgl-1 concentration of
BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 22.4, 22.3 and 22.3 respectively,
with an average value of 22.33. An increasing trend was observed in leaves per shoots
value with the increase in BAP concentration except the concentration
0.10mgl-1 concentration where no value obtained.
In statistical analysis, F- value was calculated 100092.55 at the 0 probability. The
co-efficient of variation was determined 0.45% for different concentrations of BAP in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0211 and 0.0009 respectively
(Table 7).
(1.8) Effects of BAP (mgl-1) in leaves per shoot of Withania somnifera in 2010-2011.
The effect of BAP on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 0.0-22.4 in all the
concentrations of BAP supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1 of BAP while no value was noted in 0.10 mgl-1
concentration. The nodal segments of Withania somnifera showed leaves per shoot
ranging from 22.2 to 22.4 on MS medium supplemented with 1.0 mgl-1 concentration of
BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 22.2, 22.4 and 22.3 respectively,
with an average value of 22.30. An increasing trend was observed in leaves per shoot
value with the increase in BAP concentration except the concentration
0.10mgl-1concentration where no value obtained.
In statistical analysis, F- value was calculated 32394.037 at the 0 probability. The
co-efficient of variation was determined 0.78% for different concentrations of BAP in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0295 and 0.0032 respectively
(Table 8).
(1.9) Effects of BAP (mgl-1) in bud break of Withania somnifera in 2009-2010.
The nodal segment of Withania somnifera showed bud break response ranging
from 40% to 100% on MS medium with all the five concentration of BAP. In all the
concentrations maximum value obtained was 100% and minimum value was 40%. The
average value was noted to be 88.0% for all the concentrations from 0.10 to 1.00mgl-1.
From the concentration of 0.25mgl-1 to 1.00mgl-1 of BAP, the values of experimental
replicates were 100%, and for these concentrations the average values were determined
100% and at the concentration of 0.10 mgl-1 of BAP the average value was noted to be
40%.
In statistical analysis, F- value was calculated 0.00 at the 0 probability. The co-
efficient of variation was determined 0.00 for different concentrations of BAP in bud
break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was also calculated 0.00 (Table 9).
(1.10) Effects of BAP (mgl-1) in bud break of Withania somnifera in 2010-2011.
The nodal segment of Withania somnifera showed bud break response ranging
from 30% to 100% on MS medium with all the five concentration of BAP. In all the
concentrations maximum value obtained was 100% and minimum value was 30%. The
average value was noted to be 84.64% for all the concentrations from 0.10 to 1.00mgl-1.
From the concentration of 0.50mgl-1 to 1.00mgl-1 of BAP, the values of experimental
replicates were 100%, and for these concentrations the average value was 100%, at the
concentration of 0.10 mgl-1 of BAP the average value was noted to be 36.6% and at the
concentration of 0.25 mgl-1 of BAP the average value was found to be 86.6%.
In statistical analysis, F- value was calculated 75.3333 at the 0 probability. The
co-efficient of variation was determined 6.47% for different concentrations of BAP in
bud break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was calculated 1.8257 and 14.1421 respectively (Table 10).
(2.0) Effects of plant growth regulators (NAA) in micro propagation of Withania
somnifera.
(2.1) Effects of NAA (mgl-1) in shoots per explant of Withania somnifera in 2009-
2010.
The effect of NAA on shoots per explants was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants was ranging from 1.0-1.5 in all the
concentrations of NAA supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1of NAA, while minimum value was noted in
0.25mgl-1concentration. The nodal segments of Withania somnifera showed shoots per
explants ranging from 1.4 to 1.5 on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 1.4, 1.5 and 1.4
respectively, with an average value of 1.43. An increasing trend was observed in
shoots/explant value with the increase in NAA concentration except the concentration
0.25 mgl-1concentration where minimum value obtained.
In statistical analysis, F- value was calculated 83.7143 at the 0 probability. The
co-efficient of variation was determined 2.96% for different concentrations of NAA in
leaves per shoots raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0194 and 0.00047 respectively
(Table 11).
(2.2) Effects of NAA (mgl-1) in shoots per explant of Withania somnifera in 2010-
2011.
The effect of NAA on shoots per explants was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoots/explant was ranging from 1.0-1.6 in all the
concentrations of NAA supplemented with media. Maximum mean value of shoots/
explant was obtained in 1.0 mgl-1of NAA, while minimum value was noted in 0.25 mgl-1
and 0.50 mgl-1 concentrations. The nodal segments of Withania somnifera showed shoots
per explant ranging from 1.4 to 1.6 on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 1.6, 1.4 and 1.4
respectively, with an average value of 1.46. An increasing trend was observed in
shoots/explant value with the increase in NAA concentration except the concentration
0.25 mgl-1and 0.50 mgl-1 concentration where minimum value obtained.
In statistical analysis the F- value was calculated 11.1020 at the 0 probability. The
co-efficient of variation was determined 7.57%, for different concentrations of NAA in
shoots per explant raised from Withania somnifera. The standard error of mean and
standard error of differences obtained was 0.0311 and 0.0037 respectively (Table 12).
(2.3) Effects of NAA (mgl-1) in average shoot length of Withania somnifera in 2009-
2010.
The effects of NAA on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 0.5cm-4.2cm in all
the concentrations of NAA supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1 of NAA while minimum value was noted in 0.10
mgl-1 concentration. The nodal segments of Withania somnifera showed average shoot
length ranging from 4.0cm to 4.2cm on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 4.0cm, 4.2cm
and 4.1cm respectively, with an average value of 4.10cm. An increasing trend was
observed in average shoot length value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 928.790 at the 0 probability. The
co-efficient of variation was determined 4.40% for different concentrations of NAA in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0279 and 0.0028
respectively (Table 13).
(2.4) Effects of NAA (mgl-1) in average shoot length of Withania somnifera in 2010-
2011.
The effects of NAA on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 0.5cm-4.2cm in all
the concentrations of NAA supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1of NAA, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed average
shoot length ranging from 4.0cm to 4.2cm on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 4.0cm, 4.2cm
and 4.1cm respectively, with an average value of 4.10cm. An increasing trend was
observed in average shoot length value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 726.936 at the 0 probability. The
co-efficient of variation was determined 4.83% for different concentrations of NAA in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0311 and 0.0037
respectively (Table 14).
(2.5) Effects of NAA (mgl-1) in nodes per shoot of Withania somnifera in 2009-2010.
The effects of NAA on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 1.0-4.8 in all the
concentrations of NAA supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed nodes per
shoot ranging from 4.6 to 4.8 on MS medium supplemented with 1.0 mgl-1 concentration
of NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In
this concentration the values of experimental replicates were 4.8, 4.7 and 4.6
respectively, with an average value of 4.70. An increasing trend was observed in nodes
per shoot value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 1397.248 at the 0 probability. The
co-efficient of variation was determined 2.37% for different concentrations of NAA in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0246 and 0.0018 respectively
(Table 15 & Plate 1C).
(2.6) Effects of NAA (mgl-1) in nodes per shoot of Withania somnifera in 2010-2011.
The effects of NAA on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 1.0-4.8 in all the
concentrations of NAA supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed nodes per
shoot ranging from 4.6 to 4.8 on MS medium supplemented with 1.0 mgl-1 concentration
of NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In
this concentration the values of experimental replicates were 4.6, 4.8 and 4.6
respectively, with an average value of 4.66. An increasing trend was observed in nodes
per shoot value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 597.332 at the 0 probability. The
co-efficient of variation was determined 3.50% for different concentrations of NAA in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0326 and 0.0042 respectively
(Table 16 & Plate 1D).
(2.7) Effects of NAA (mgl-1) in leaves per shoot of Withania somnifera in 2009-2010.
The effects of NAA on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 6.0-22.4 in all the
concentrations of NAA supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Withania somnifera showed leaves per shoot
ranging from 22.2 to 22.4 on MS medium supplemented with 1.0 mgl-1 concentration of
NAA. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 22.4, 22.2 and 22.3 respectively,
with an average value of 22.33. An increasing trend was observed in leaves per shoot
value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 13297.563 at the 0 probability. The
co-efficient of variation was determined 0.73% for different concentrations of NAA in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0341 and 0.0047 respectively
(Table 17).
(2.8) Effects of NAA (mgl-1) in leaves per shoot of Withania somnifera in 2010-2011.
The effects of NAA on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 6.2-22.4 in all the
concentrations of NAA supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of NAA while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Withania somnifera showed leaves per shoots
ranging from 22.2 to 22.4 on MS medium supplemented with 1.0 mgl-1 concentration of
NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In this
concentration the values of experimental replicates were 22.4, 22.3 and 22.2 respectively,
with an average value of 22.36. An increasing trend was observed in leaves per shoot
value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 13864.613 at the 0 probability. The
co-efficient of variation was determined 0.70% for different concentrations of BAP in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0341 and 0.0047 respectively
(Table 18).
(2.9) Effects of NAA (mgl-1) in bud break of Withania somnifera in 2009-2010.
The nodal segment of Withania somnifera showed bud break response ranging
from 80% to 100% on MS medium with all the five concentration of NAA. In all the
concentrations maximum value obtained was 100% and minimum value was 80%. The
average value was found to be 97.33% for all the concentrations from 0.10 to 1.00mgl-1.
In statistical analysis, F- value was calculated 4.000 at the 0.045 probability. The
co-efficient of variation was determined 5.31% for different concentrations of NAA in
bud break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was 1.721 and 12.570 respectively (Table 19).
(2.10) Effects of NAA (mgl-1) in bud break of Withania somnifera in 2010-2011.
The nodal segment of Withania somnifera showed bud break response ranging
from 90% to 100% on MS medium with all the five concentration of NAA. In all the
concentrations maximum value obtained was 100% and minimum value was 90%. The
average value was calculated 98.66% for all the concentrations from 0.10 to 1.00mgl-1.
In statistical analysis, F- value was calculated 4.000 at the 0.045 probability. The
co-efficient of variation was determined 2.62% for different concentrations of NAA in
bud break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was 0.0860 and 3.142 respectively (Table 20).
(3.0) Effects of plant growth regulators (kinetin) in micro propagation of Withania
somnifera.
(3.1) Effects of kinetin (mgl-1) in shoot per explant of Withania somnifera in 2009-
2010.
The effects of kinetin on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants were ranging from 1.0-1.3 in all the
concentrations of kinetin supplemented with media. Maximum mean value of shoots/
explant was obtained in 1.0 mgl-1of kinetin while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Withania somnifera showed shoots per
explant ranging from 1.0 to 1.3 on MS medium supplemented with 1.0 mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 1.00mgl-1
of kinetin. In this concentration the values of experimental replicates were 1.0, 1.3 and
1.2 respectively, with an average value of 1.16. An increasing trend was observed in
shoots/explant value with the increase in kinetin concentration except the concentration
0.50 mgl-1concentration where decreases value was observed.
In statistical analysis, F- value was calculated 2.3636 at the 0.1397 probability.
The co-efficient of variation was determined 3.82% for different concentrations of kinetin
in shoot per explants raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0211 and 0.0009
respectively (Table 21).
(3.2) Effects of kinetin (mgl-1) in shoots per explant of Withania somnifera in 2010-
2011.
The effects of kinetin on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1concentration. Shoots/explant were ranging from 1.0-1.2 in all the
concentrations of kinetin supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.25
mgl-1 concentration. The nodal segments of Withania somnifera showed shoots per
explants ranging from 1.1 to 1.2 on MS medium supplemented with 1.00mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 1.00mgl-1
of kinetin. In this concentration the values of experimental replicates were 1.1, 1.2 and
1.1 respectively, with an average value of 1.13. An increasing trend was observed in
shoots/explant value with the increase in kinetin concentration except the concentration
0.25 mgl-1concentration where decreases value was observed.
In statistical analysis the F- value was calculated 1.4286 at the 0.3088 probability.
The co-efficient of variation was found to be 5.38%, for different concentrations of
kinetin in shoots per explant raised from Withania somnifera. The standard error of mean
and standard error of differences obtained was 0.0246 and 0.0018 respectively (Table
22).
(3.3) Effects of kinetin (mgl-1) in average shoot length of Withania somnifera in 2009-
2010.
The effect of kinetin on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 1.5cm-3.2cm in all
the concentrations of kinetin supplemented with media. Maximum mean value of average
shoot length was obtained in 0.75 mgl-1of kinetin while minimum value was noted in
1.00mgl-1concentration. The nodal segments of Withania somnifera showed average
shoot length ranging from 3.0cm to 3.2cm on MS medium supplemented with 0.75 mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 0.75mgl-1
of kinetin. In this concentration the values of experimental replicates were 3.2cm, 3.0cm
and 3.1cm respectively, with an average value of 3.10cm. An increasing trend was
observed in average shoot length value with the increase in kinetin concentration except
the concentration 1.00mgl-1 where higher concentrations of kinetin in MS medium
reduced the value of average shoot length.
In statistical analysis, F- value was calculated 169.562 at the 0 probability. The
co-efficient of variation was determined 3.03% for different concentrations of kinetin in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0263 and 0.0023
respectively (Table 23).
(3.4) Effects of kinetin (mgl-1) in average shoot length of Withania somnifera in 2010-
2011.
The effects of kinetin on average shoot length was studied in 0.10, 0.25, 0.50,
0.75 and1.00mgl-1 concentration. Average shoot length were ranging from 1.4cm-3.2cm
in all the concentrations of kinetin supplemented with media. Maximum mean value of
average shoot length was obtained in 0.75 mgl-1of kinetin while minimum value was
noted in 1.00mgl-1concentration. The nodal segments of Withania somnifera showed
average shoot length ranging from 3.0cm to 3.2cm on MS medium supplemented with
0.75 mgl-1 concentration of kinetin. The maximum value was noted at the concentration
of 0.75mgl-1 of kinetin. In this concentration the values of experimental replicates were
3.0cm, 3.2cm and 3.1cm respectively, with an average value of 3.10. An increasing trend
was observed in average shoot length value with the increase in kinetin concentration
except the concentration 1.00mgl-1 where higher concentrations of kinetin in MS medium
reduced the value of average shoot length.
In statistical analysis, F- value was calculated 97.0321 at the 0 probability. The
co-efficient of variation was determined 4.32% for different concentrations of kinetin in
average shoot length raised from nodal segment of Withania somnifera. The standard
error of mean and standard error of differences was calculated 0.0356and 0.0051
respectively (Table 24).
(3.5) Effects of kinetin (mgl-1) in nodes per shoot of Withania somnifera in 2009-
2010.
The effects of kinetin on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.0-6.1 in all the
concentrations of kinetin supplemented with media. Maximum mean value of nodes per
shoot was obtained in 0.75mgl-1of kinetin, while minimum value was noted in 1.00 mgl-1
concentration. The nodal segments of Withania somnifera showed nodes per shoot
ranging from 6.0 to 6.1 on MS medium supplemented with 0.75 mgl-1 concentration of
kinetin. The maximum value was noted at the concentration of 0.75mgl-1 of kinetin. In
this concentration the values of experimental replicates were 6.0, 6.0 and 6.1
respectively, with an average value of 6.10. An increasing trend was observed in nodes
per shoot value with the increase in kinetin concentration except the concentration
1.00mgl-1 where higher concentrations of kinetin in MS medium reduced the value of
average shoot length.
In statistical analysis, F- value was calculated 407.4717 at the 0 probability. The
co-efficient of variation was determined 1.90% for different concentrations of kinetin in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0326 and 0.0042respectively
(Table 25).
(3.6) Effects of kinetin (mgl-1) in nodes per shoot of Withania somnifera in 2010-
2011.
The effects of kinetin on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.2-6.2 in all the
concentrations of kinetin supplemented with media. Maximum mean value of nodes per
shoot was obtained in 0.75mgl-1of kinetin, while minimum value was noted in 1.00 mgl-1
concentration. The nodal segments of Withania somnifera showed nodes per shoot
ranging from 6.1 to 6.2 on MS medium supplemented with 0.75 mgl-1 concentration of
kinetin. The maximum value was noted at the concentration of 0.75mgl-1 of kinetin. In
this concentration the values of experimental replicates were 6.2, 6.1 and 6.2
respectively, with an average value of 6.16. An increasing trend was observed in nodes
per shoot value with the increase in kinetin concentration except the concentration
1.00mgl-1 where higher concentrations of kinetin in MS medium reduced the value of
average shoot length.
In statistical analysis, F- value was calculated 111.3439 at the 0 probability. The
co-efficient of variation was determined 1.86% for different concentrations of kinetin in
nodes per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0356and 0.0051respectively
(Table 26).
(3.7) Effects of kinetin (mgl-1) in leaves per shoot of Withania somnifera in 2009-
2010.
The effects of kinetin on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 26.0-28.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.10
mgl-1 concentration. The nodal segments of Withania somnifera showed leaves per shoot
ranging from 28.4 to 28.6 on MS medium supplemented with 1.0 mgl-1 concentration of
kinetin. The maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In
this concentration the values of experimental replicates were 28.6, 28.4 and 28.5
respectively, with an average value of 28.50. An increasing trend was observed in leaves
per shoot value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 1001.998 at the 0 probability. The
co-efficient of variation was determined 0.20% for different concentrations of kinetin in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0229and 0.0014respectively
(Table 27 & Plate 1E).
(3.8) Effects of kinetin (mgl-1) in leaves per shoot of Withania somnifera in 2010-
2011.
The effects of kinetin on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 26.0-28.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.25 mgl-1
concentration. The nodal segments of Withania somnifera showed leaves per shoot
ranging from 28.4 to 28.6 on MS medium supplemented with 1.0 mgl-1 concentration of
kinetin. The maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In
this concentration the values of experimental replicates were 28.4, 28.6 and 28.6
respectively, with an average value of 28.53. An increasing trend was observed in leaves
per shoot value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 819.8063 at the 0 probability. The
co-efficient of variation was determined 0.27% for different concentrations of kinetin in
leaves per shoot raised from nodal segment of Withania somnifera. The standard error of
mean and standard error of differences was calculated 0.0263and 0.0023respectively
(Table 28 & Plate 1F).
(3.9) Effects of kinetin (mgl-1) in bud break of Withania somnifera in 2009-2010.
The nodal segment of Withania somnifera showed bud break response ranging
from 40% to 100% on MS medium with all the five concentration of kinetin. In all the
concentrations maximum value obtained was 100% and minimum value was 40%. The
average value was noted to be 88.0% for all the concentrations from 0.10 to 1.00mgl-1.
From the concentration of 0.25mgl-1 to 1.00mgl-1 of BAP, the values of experimental
replicates were 100%, and for these concentrations the average values were determined
100% and at the concentration of 0.10 mgl-1 of BAP the average value was noted to be
40%.
In statistical analysis, F- value was calculated 0.00 at the 0 probability. The co-
efficient of variation was determined 0.00 for different concentrations of kinetin in bud
break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was also calculated 0.00 (Table 9).
(3.10) Effects of kinetin (mgl-1) in bud break of Withania somnifera in 2010-2011.
The nodal segment of Withania somnifera showed bud break response reported
100% on MS medium with all the five concentration of kinetin so the average value was
found to be 100% for all the concentrations from 0.10 to 1.00mgl-1.
According to the statistical database, in the calculated value of ANOVA the F-
value, co-efficient of variation was calculated 0.00 for different concentrations of kinetin
in bud break from Withania somnifera. The standard error of mean and standard error of
differences obtained was calculated 0.00 (Table 30).
II. Estimation of primary metabolites in Withania somnifera
Quantitative estimation of primary metabolites like carbohydrates, protein and
chlorophyll content was done in different parts of normal and regenerated plants.
(i) Estimation of reducing sugar and total sugar in normal and regenerated plants:-
Withania somnifera the amount of reducing sugar determined in normal root was
128.30.4 µg/g, while it was 151.70.1 µg/g in regenerated root. The amount of reducing
sugar present in normal stem was 140.10.2 µg/g while, 289.20.2 µg/g in regenerated
stem. The amount of reducing sugar present in regenerated leaf was 151.40.3 µg/g as
compared to 121.3±0.3 µg/g in normal leaf. The amount of reducing sugar in callus was
240.10.9 µg/g (Fig.-2).The total sugar value was ranged from 148 to 440.2µg/g while
reducing sugar in 121.3- 289.2 µg/g. Maximum total sugar was obtained 440.2µg/g in
callus, while minimum in 148µg/g in normal stem (Fig.-3). Reducing sugar was obtained
maximum 289 µg/g in regenerated stem while, minimum 121.3 in normal leaf.
Comparative maximum value of total sugar was determined in callus, while reducing
sugar in regenerated stem (Table-31).
(ii) Estimation of total soluble proteins and TCA precipitated protein in normal and
regenerated plants:-
Withania somnifera the amount of total soluble proteins determined in normal
root was 210.10.2 µg/g while it was 280.1µg/g in regenerated root. The amount of
total soluble proteins present in normal stem was 261.0µg/g while, 302.6µg/g in
regenerated stem. The amount of total soluble proteins present in regenerated leaf was
502.1 µg/g as compared to 401.3 µg/g in normal leaf. The amount of total
soluble proteins in callus was 510.3µg/g (Fig.-4). The TCA precipitated protein
value was ranged from 101.1 to 210.1µg/g while total soluble proteins in 210.1-
510.3µg/g. Maximum TCA precipitated protein was obtained 210.1µg/g in callus, while
minimum in 101.1µg/g in normal root (Fig.-5). Total soluble proteins was obtained
maximum 510.3 µg/g in callus while, minimum 210.1in normal root. Maximum value of
TCA precipitated protein and total soluble proteins were determined in callus (Table-32).
(iii) Estimation of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in
normal and regenerated plants:-
The amount of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in
normal leaf of Withania somnifera was 0.0210.01mg/g, 0.0120.05 mg/g, 0.0250.01
mg/g and 0.0040.03 mg/g comparatively as compared to 0.0120.02 mg/g, 0.0220.01
mg/g, 0.0350.03 mg/g and 0.0070.01 mg/g in regenerated leaf (Table-33, Fig.-6 and
Plate 6A).
III. Estimation of Secondary Cell Metabolites in Withania somnifera
Root, stem and leaf of W. somnifera exhibited differences in the presence of
secondary cell metabolites. In qualitative analysis of plant parts alkaloid, glycosides and
phenols were present in leaf, while steroid was present in both root and stem when
extracted with ethanol (Table-34). When extraction was done with methanol, alkaloids,
steroids, glycosides and phenols were present in leaf, while steroid was present in roots
and glycosides and phenols in stem (Table-35 and Plate 7A).
PLANT-1
(Withania somnifera)
Table-1
Effect of different level of BAP in MS medium on shoots/explant raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.4 1.3 1.4 1.36
2. 0.25 2.0 2.2 2.0 2.06
3. 0.50 2.1 2.0 2.1 2.06
4. 0.75 2.4 2.4 2.3 2.36
5. 1.00 3.0 3.1 3.0 3.03
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 176.3784 at probability = 0.00 Coefficient of Variation : 3.60% Standard error of Mean : 0.0279
Standard error of Difference : 0.0028
Table-2
Effect of different level of BAP in MS medium on shoots/explant raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explants
R1 R2 R3 Mean
1. 0.10 1.4 1.2 1.2 1.26
2. 0.25 2.0 2.2 2.1 2.10
3. 0.50 2.1 2.0 2.2 2.10
4. 0.75 2.5 2.4 2.6 2.50
5. 1.00 3.2 3.0 3.1 3.10
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 125.8750 at probability = 0.00
Coefficient of Variation : 4.67% Standard error of Mean : 0.0356
Standard error of Difference : 0.0051
Table-3
Effect of different level of BAP in MS medium on average shoot length raised from
nodal explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 2.0 2.2 2.0 2.06
2. 0.25 2.2 2.2 2.1 2.16
3. 0.50 1.2 1.2 1.1 1.16
4. 0.75 5.2 5.0 5.0 5.06
5. 1.00 6.8 6.7 6.8 6.76
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 2398.7125 at probability = 0.00 Coefficient of Variation : 2.43%
Standard error of Mean : 0.0295 Standard error of Difference : 0.0032
Table-4
Effect of different level of BAP in MS medium on average shoot length raised from
nodal explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 0.6 0.7 0.6 0.63
2. 0.25 2.2 2.4 2.0 2.20
3. 0.50 2.3 2.2 2.3 2.26
4. 0.75 3.2 3.4 3.3 3.30
5. 1.00 6.7 6.8 6.8 6.76
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1457.3842 at probability = 0.00
Coefficient of Variation : 3.43% Standard error of Mean : 0.0356
Standard error of Difference : 0.0051
Table-5
Effect of different level of BAP in MS medium on nodes/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 1.2 1.2 1.1 1.16
3. 0.50 2.6 2.6 2.6 2.60
4. 0.75 2.8 2.7 2.7 2.73
5. 1.00 6.0 6.2 6.2 6.13
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 3303.5911 at probability = 0.00 Coefficient of Variation : 2.75%
Standard error of Mean : 0.0263 Standard error of Difference : 0.0023
Table-6
Effect of different level of BAP in MS medium on nodes/shoot from nodal explants
of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 1.2 1.3 1.2 1.23
3. 0.50 2.7 2.8 2.8 2.76
4. 0.75 2.9 2.8 2.8 2.83
5. 1.00 6.2 6.1 6.1 6.13
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 5019.6879 at probability = 0.00
Coefficient of Variation : 2.17% Standard error of Mean : 0.0229
Standard error of Difference : 0.0014
Table-7
Effect of different level of BAP in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoots
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 5.2 5.0 5.0 5.06
3. 0.50 10.6 10.5 10.5 10.53
4. 0.75 16.2 16.1 16.2 16.16
5. 1.00 22.4 22.3 22.3 22.33
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 100092.5555 at probability = 0.00
Coefficient of Variation : 0.45% Standard error of Mean : 0.0211 Standard error of Difference : 0.0009
Table-8
Effect of different level of BAP in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 5.1 5.3 5.2 5.20
3. 0.50 10.4 10.5 10.4 10.43
4. 0.75 16.3 16.1 16.2 16.20
5. 1.00 22.2 22.4 22.3 22.30
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 32394.0373 at probability = 0.00 Coefficient of Variation : 0.78% Standard error of Mean : 0.0295
Standard error of Difference : 0.0032
Table-9
Effect of different level of BAP in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 40 40 40 40
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 0.00 at probability = 0.00
Coefficient of Variation : 0.00% Standard error of Mean : 0.00
Standard error of Difference : 0.00
Table-10
Effect of different level of BAP in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 30 40 40 36.6
2. 0.25 80 80 100 86.6
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 75.3333 at probability = 0.00 Coefficient of Variation : 6.47%
Standard error of Mean : 1.8257 Standard error of Difference : 14.142
Table-11
Effect of different level of NAA in MS medium on shoots/explant raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.0 1.1 1.0 1.03
2. 0.25 1.0 1.0 1.0 1.00
3. 0.50 1.0 1.1 1.1 1.06
4. 0.75 1.2 1.3 1.2 1.23
5. 1.00 1.4 1.5 1.4 1.43
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 83.7143 at probability = 0.00
Coefficient of Variation : 2.96% Standard error of Mean : 0.0194
Standard error of Difference : 0.0004
Table-12
Effect of different level of NAA in MS medium on shoots/explant raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.2 1.1 1.0 1.10
2. 0.25 1.0 1.1 1.1 1.06
3. 0.50 1.0 1.2 1.0 1.06
4. 0.75 1.3 1.3 1.2 1.26
5. 1.00 1.6 1.4 1.4 1.46
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 11.1020 at probability = 0.0024 Coefficient of Variation : 7.57%
Standard error of Mean : 0.0311 Standard error of Difference : 0.0037
Table-13
Effect of different level of NAA in MS medium on average shoot length raised from
nodal explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 0.6 0.5 0.5 0.53
2. 0.25 1.0 1.1 1.1 1.06
3. 0.50 1.3 1.3 1.2 1.26
4. 0.75 2.0 2.2 2.1 2.10
5. 1.00 4.0 4.2 4.1 4.10
R1, R2, R3 - Experimental Replicates Statistical analysis
F value : 928.7900 at probability = 0.00 Coefficient of Variation : 4.40%
Standard error of Mean : 0.0279 Standard error of Difference : 0.0028
Table-14
Effect of different level of NAA in MS medium on average shoot length raised from
nodal explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 0.5 0.6 0.6 0.56
2. 0.25 1.2 1.2 1.0 1.13
3. 0.50 1.2 1.3 1.3 1.26
4. 0.75 2.0 2.1 2.0 2.03
5. 1.00 4.2 4.0 4.1 4.10
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 726.9367 at probability = 0.00 Coefficient of Variation : 4.86%
Standard error of Mean : 0.0311 Standard error of Difference : 0.0037
Table-15
Effect of different level of NAA in MS medium on nodes/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 1.0 1.0 1.0 1.00
2. 0.25 2.0 2.1 2.0 2.03
3. 0.50 2.7 2.6 2.5 2.60
4. 0.75 3.0 3.1 3.0 3.03
5. 1.00 4.8 4.7 4.6 4.70
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1397.2481 at probability = 0.00
Coefficient of Variation : 2.37% Standard error of Mean : 0.02462 Standard error of Difference : 0.00188
Table-16
Effect of different level of NAA in MS medium on nodes/shoot raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 1.1 1.0 1.1 1.06
2. 0.25 2.0 2.1 2.0 2.03
3. 0.50 2.6 2.7 2.7 2.66
4. 0.75 3.2 3.0 3.2 3.13
5. 1.00 4.6 4.8 4.6 4.66
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 597.3326 at probability = 0.00
Coefficient of Variation : 3.50% Standard error of Mean : 0.0326
Standard error of Difference : 0.0042
Table-17
Effect of different level of NAA in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 6.0 6.1 6.0 6.03
2. 0.25 8.0 8.2 8.1 8.10
3. 0.50 16.2 16.4 16.3 16.3
4. 0.75 16.8 16.7 16.9 16.8
5. 1.00 22.4 22.2 22.3 22.33
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 13297.191 at probability = 0.00 Coefficient of Variation : 0.73%
Standard error of Mean : 0.0341 Standard error of Difference : 0.0047
Table-18
Effect of different level of NAA in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 6.3 6.2 6.4 6.30
2. 0.25 8.2 8.0 8.1 8.10
3. 0.50 16.0 16.2 16.0 16.06
4. 0.75 16.7 16.6 16.8 16.70
5. 1.00 22.4 22.3 22.4 22.36
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 13864.6133 at probability = 0.00
Coefficient of Variation : 0.70% Standard error of Mean : 0.0341 Standard error of Difference : 0.0047
Table-19
Effect of different level of NAA in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N
.
Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 80 80 100 86.66
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 4.000 at probability = 0.0453
Coefficient of Variation : 5.31% Standard error of Mean : 1.7213
Standard error of Difference : 12.570
Table-20
Effect of different level of NAA in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 90 90 100 93.33
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 4.0000 at probability = 0.0453 Coefficient of Variation : 2.62% Standard error of Mean : 0.0860
Standard error of Difference : 3.1428
Table-21
Effect of different level of kinetin in MS medium on shoots/explant raised from
nodal explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explants
R1 R2 R3 Mean
1. 0.10 1.0 1.1 1.1 1.06
2. 0.25 1.0 1.2 1.2 1.13
3. 0.50 1.0 1.2 1.1 1.10
4. 0.75 1.0 1.2 1.2 1.13
5. 1.00 1.0 1.3 1.2 1.16
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 2.3636 at probability = 0.1397
Coefficient of Variation : 3.82% Standard error of Mean : 0.0211
Standard error of Difference : 0.0009
Table-22
Effect of different level of kinetin in MS medium on shoot/explant raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.0 1.2 1.1 1.10
2. 0.25 1.0 1.1 1.0 1.03
3. 0.50 1.0 1.2 1.2 1.13
4. 0.75 1.1 1.2 1.0 1.10
5. 1.00 1.1 1.2 1.1 1.13
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1.4286 at probability = 0.3088
Coefficient of Variation : 5.38% Standard error of Mean : 0.0246 Standard error of Difference : 0.0018
Table-23
Effect of different level of kinetin in MS medium on average shoot length raised
from nodal explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 2.5 2.4 2.3 2.40
2. 0.25 2.5 2.5 2.4 2.46
3. 0.50 2.6 2.4 2.6 2.53
4. 0.75 3.2 3.0 3.1 3.10
5. 1.00 1.6 1.5 1.6 1.56
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 169.5626 at probability = 0.00 Coefficient of Variation : 3.03% Standard error of Mean : 0.02630
Standard error of Difference : 0.002357
Table-24
Effect of different level of kinetin in MS medium on average shoot length raised
from nodal explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 2.4 2.3 2.5 2.40
2. 0.25 2.5 2.4 2.4 2.43
3. 0.50 2.4 2.5 2.6 2.50
4. 0.75 3.0 3.2 3.1 3.10
5. 1.00 1.4 1.6 1.4 1.46
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 97.0321 at probability = 0.00 Coefficient of Variation : 4.32%
Standard error of Mean : 0.0356 Standard error of Difference : 0.0051
Table-25
Effect of different level of kinetin in MS medium on nodes/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 5.2 5.1 5.2 5.16
2. 0.25 5.0 5.2 5.2 5.13
3. 0.50 5.2 5.4 5.3 5.30
4. 0.75 6.0 6.0 6.1 6.10
5. 1.00 3.2 3.0 3.1 3.10
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 407.4717 at probability = 0.00
Coefficient of Variation : 1.90% Standard error of Mean : 0.03265
Standard error of Difference : 0.00424
Table-26
Effect of different level of kinetin in MS medium on nodes/shoot raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 5.0 5.2 5.2 5.13
2. 0.25 5.1 5.0 5.1 5.06
3. 0.50 5.2 5.0 5.0 5.06
4. 0.75 6.2 6.1 6.2 6.16
5. 1.00 3.3 3.4 3.2 3.30
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 111.3439 at probability = 0.00
Coefficient of Variation : 1.86% Standard error of Mean : 0.0356 Standard error of Difference : 0.0051
Table-27
Effect of different level of kinetin in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 26.2 26.0 26.1 26.10
2. 0.25 28.2 28.1 28.3 28.20
3. 0.50 28.3 28.2 28.4 28.30
4. 0.75 28.3 28.2 28.4 28.30
5. 1.00 28.6 28.4 28.5 28.50
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1001.9984 at probability = 0.00 Coefficient of Variation : 0.20%
Standard error of Mean : 0.0229 Standard error of Difference : 0.0014
Table-28
Effect of different level of kinetin in MS medium on leaves/shoot raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 26.1 26.0 26.2 26.10
2. 0.25 26.0 26.1 26.1 26.06
3. 0.50 28.1 28.2 28.2 28.16
4. 0.75 28.1 28.0 28.2 28.10
5. 1.00 28.4 28.6 28.6 28.53
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 819.8063 at probability = 0.00 Coefficient of Variation : 0.27%
Standard error of Mean : 0.0263 Standard error of Difference : 0.0023
Table-29
Effect of different level of kinetin in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 100 100 100 100
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 0.00 at probability = 0.00
Coefficient of Variation : 0.00% Standard error of Mean : 0.00
Standard error of Difference : 0.00
Table-30
Effect of different level of kinetin in MS medium on bud break raised from nodal
explants of W. somnifera in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 100 100 100 100
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 0.0000 at probability = 0.00 Coefficient of Variation : 0.00%
Standard error of Mean : 0.00 Standard error of Difference : 0.00
Table-31
Estimation of total sugar and reducing sugar in normal and regenerated plant of W.
somnifera.
Plant part Total sugars (g/g) Reducing sugars (g/g)
Normal stem 148.0 140.1
Regenerated stem 384.2 289.2
Normal Leaf 185.3 121.3
Regenerated Leaf 415.2 151.4
Normal root 151.6 128.3
Regenerated root 321.7 151.7
Callus 440.2 240.1
Table-32
Estimation of total soluble protein and TCA precipitated protein in normal and
regenerated plants of W. somnifera.
Plant part Soluble Protein (g/g) TCA pptd. Protein
(g/g)
Normal stem 261.0 130.0
Regenerated stem 302.6 180.1
Normal Leaf 401.3 101.2
Regenerated Leaf 502.1 142.3
Normal root 210.1 101.1
Regenerated root 280.1 141.2
Callus 510.3 210.1
Table- 33
Chlorophyll content in normal and regenerated plants of W. somnifera.
Pigment
(mg/g)
Normal Plant Regenerated plant
Chlorophyll a 0.011 0.012
Chlorophyll b 0.020 0.022
Total Chlorophyll 0.032 0.035
Caratenoids 0.006 0.007
Table-34
Presence of secondary metabolites in root, stem and leaf of W. somnifera when
extracted with ethanol.
Table-35
Presence of secondary metabolites in root, stem and leaf of W. somnifera when
extracted with methanol.
Chemical
compounds
Root Stem Leaf
Alkaloids + - +
Steroids + - +
Glycosides - + +
Terpenoids + - -
Phenols - + +
Chemical
compounds
Root Stem Leaf
Alkaloids + - +
Steroids + + -
Glycosides - + +
Terpenoids + - -
Phenols + - +
Micro propagation of Adhatoda vasica.
(1.0) Effects of plant growth regulators (BAP) in Adhatoda vasica.
(1.1) Effects of BAP (mgl-1) in shoots per explant of Adhatoda vasica in the year
2009-2010.
The effects of BAP on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants were ranging from 1.2-2.4 in all the
concentrations of BAP supplemented with media. Maximum mean value of shoot/
explants was obtained in 0.75mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed shoots per
explant ranging from 2.2 to 2.4 on MS medium supplemented with 0.75 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 0.75mgl-1
of BAP. In this concentration the values of experimental replicates were 2.2, 2.3 and 2.4
respectively, with an average value of 2.30. An increasing trend was observed in
shoots/explant value with the increase in BAP concentration where higher concentrations
of BAP in MS medium reduced the value of shoots per explant.
In statistical analysis, F- value was calculated 51.7857 at the 0 probability. The
co-efficient of variation was determined 5.00% for different concentrations of BAP in
shoot per explant raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0326 and 0.0042 respectively
(Table 36 & Plate 2C).
(1.2) Effects of BAP (mgl-1) in shoot per explant of Adhatoda vasica in 2010-2011.
The effect of BAP on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants were ranging from 1.2-2.3 in all the
concentrations of BAP supplemented with media. Maximum mean value of shoot/
explant was obtained in 1.00mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed shoots per
explant ranging from 2.1 to 2.3 on MS medium supplemented with 1.00 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 2.2, 2.1 and 2.3
respectively, with an average value of 2.20. An increasing trend was observed in
shoots/explant value with the increase in BAP concentration where in the 0.75 mgl-1
concentrations of BAP the value of shoots per explant was reduced.
In statistical analysis the F- value was calculated 186.6250 at the 0 probability.
The co-efficient of variation was found to be 2.95%, for different concentrations of BAP
in shoot per explant raised from Adhatoda vasica. The standard error of mean and
standard error of differences obtained was 0.0229 and 0.0014 respectively (Table 37&
Plate 2D).
(1.3) Effects of BAP (mgl-1) in average shoot length of Adhatoda vasica in 2009-2010.
The effects of BAP on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 0.8cm-4.6cm in all
the concentrations of BAP supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed average shoot
length ranging from 4.4cm to 4.6cm on MS medium supplemented with 1.0 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 4.4cm, 4.5cm
and 4.6cm respectively, with an average value of 4.50cm. An increasing trend was
observed in average shoot length value with the increase in BAP concentration.
In statistical analysis, F- value was calculated 760.0007 at the 0 probability. The
co-efficient of variation was determined 4.77% for different concentrations of BAP in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0311 and 0.0037 respectively
(Table 38).
(1.4) Effects of BAP (mgl-1) in average shoot length of Adhatoda vasica in 2010-2011.
The effects of BAP on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 0.7cm-4.5cm in all
the concentrations of BAP supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed average shoot
length ranging from 4.3cm to 4.5cm on MS medium supplemented with 1.0 mgl-1
concentration of BAP. The maximum value was noted at the concentration of 1.00mgl-1
of BAP. In this concentration the values of experimental replicates were 4.5cm, 4.3cm
and 4.4cm respectively, with an average value of 4.40cm. An increasing trend was
observed in average shoot length value with the increase in BAP concentration.
In statistical analysis, F- value was calculated 1022.667 at the 0 probability. The
co-efficient of variation was determined 3.90% for different concentrations of BAP in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0279 and 0.0028 respectively
(Table 39).
(1.5) Effects of BAP (mgl-1) in nodes per shoot of Adhatoda vasica in 2009-2010.
The effects of BAP on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.4-7.4 in all the
concentrations of BAP supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1of BAP, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed nodes per shoot
ranging from 7.2 to 7.4 on MS medium supplemented with 1.0 mgl-1 concentration of
BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 7.2, 7.3 and 7.4 respectively,
with an average value of 7.30. An increasing trend was observed in nodes per shoot value
with the increase in BAP concentration.
In statistical analysis, F- value was calculated 362.0734 at the 0 probability. The
co-efficient of variation was determined 2.43% for different concentrations of BAP in
nodes per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0448and 0.0084 respectively
(Table 40).
(1.6) Effects of BAP (mgl-1) in nodes per shoot of Adhatoda vasica in 2010-2011.
The effects of BAP on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.4-7.2 in all the
concentrations of BAP supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in
0.10 mgl-1concentration. The nodal segments of Adhatoda vasica showed nodes per shoot
ranging from 7.1 to 7.2 on MS medium supplemented with 1.0 mgl-1 concentration of
BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 7.1, 7.2 and 7.2 respectively,
with an average value of 7.16. An increasing trend was observed in nodes per shoot value
with the increase in BAP concentration.
In statistical analysis, F- value was calculated 833.6241 at the 0 probability. The
co-efficient of variation was determined 1.62% for different concentrations of BAP in
nodes per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0311 and 0.0037 respectively
(Table 41).
(1.7) Effects of BAP (mgl-1) in leaves per shoot of Adhatoda vasica in 2009-2010.
The effects of BAP on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 3.6-7.7 in all the
concentrations of BAP supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 7.5 to 7.7 on MS medium supplemented with 1.0 mgl-1 concentration of BAP. The
maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 7.7, 7.5 and 7.6 respectively,
with an average value of 7.60. An increasing trend was observed in leaves per shoots
value with the increase in BAP concentration.
In statistical analysis, F- value was calculated 856.5356 at the 0 probability. The
co-efficient of variation was determined 1.77% for different concentrations of BAP in
leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0326 and 0.0042 respectively
(Table 42).
(1.8) Effects of BAP (mgl-1) in leaves per shoot of Adhatoda vasica in 2010-2011.
The effects of BAP on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 3.6-7.6 in all the
concentrations of BAP supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 7.5 to 7.6 on MS medium supplemented with 1.0 mgl-1 concentration of BAP. The
maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 7.5, 7.6 and 7.6 respectively,
with an average value of 7.56. An increasing trend was observed in leaves per shoot
value with the increase in BAP concentration.
In statistical analysis, F- value was calculated 1752.0006 at the 0 probability. The
co-efficient of variation was determined 1.24% for different concentrations of BAP in
leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0246 and 0.0018 respectively
(Table 43).
(1.9) Effects of BAP (mgl-1) in bud break of Adhatoda vasica in 2009-2010.
The nodal segment of Adhatoda vasica showed bud break response reported
100% on MS medium with all the five concentration of BAP so the average value was
calculated 100% for all the concentrations from 0.10 to 1.00mgl-1.
In statistical analysis, F- value was calculated 0.00 at the 0 probability. The co-
efficient of variation was determined 0.00 for different concentrations of BAP in bud
break from nodal segment of Withania somnifera. The standard error of mean and
standard error of differences was also calculated 0.00 (Table 44).
(1.10) Effects of BAP (mgl-1) in bud break of Adhatoda vasica in 2010-2011.
The nodal segment of Adhatoda vasica showed bud break response reported
100% on MS medium with all the five concentration of BAP so the average value was
calculated 100% for all the concentrations from 0.10 to 1.00mgl-1.
According to the statistical database, in the calculated value of ANOVA the F-
value, co-efficient of variation was observed 0.00 for different concentrations of BAP in
bud break from Adhatoda vasica. The standard error of mean and standard error of
differences obtained was calculated 0.00 (Table 45).
(2.0) Effects of plant growth regulators (NAA) in Adhatoda vasica.
(2.1) Effects of NAA (mgl-1) in shoots per explant of Adhatoda vasica in 2009-2010.
The effects of NAA on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoots/explant was ranging from 1.0 -1.5 in all the
concentrations of NAA supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1of NAA, while minimum value was noted in
0.25mgl-1concentration. The nodal segments of Adhatoda vasica showed shoots per
explant ranging from 1.4 to 1.5 on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 1.4, 1.5 and 1.4
respectively, with an average value of 1.43.An increasing trend was observed in
shoots/explant value with the increase in NAA concentration except the concentration
0.25 mgl-1concentration where minimum value obtained.
In statistical analysis, F- value was calculated 24.842 at the 0 probability. The co-
efficient of variation was determined 6.15% for different concentrations of NAA in
leaves per shoots raised from nodal segment of Adhatoda vasica The standard error of
mean and standard error of differences was calculated 0.0279 and 0.00282 respectively
(Table 46).
(2.2) Effects of NAA (mgl-1) in shoots per explant of Adhatoda vasica in 2010-2011.
The effects of NAA on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants were ranging from 1.0-1.8 in all the
concentrations of NAA supplemented with media. Maximum mean value of shoot/
explants was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in 0.10mgl-1
and 0.25 mgl-1 concentration. The nodal segments of Adhatoda vasica showed shoots per
explant ranging from 1.7 to 1.8 on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 1.7, 1.8 and 1.7
respectively, with an average value of 1.73. An increasing trend was observed in
shoots/explant value with the increase in NAA concentration except the concentration
0.10 mgl-1and 0.25 mgl-1 concentration where same value obtained.
In statistical analysis the F- value was calculated 23.200 at the 0 probability. The
co-efficient of variation was calculated 7.48%, for different concentrations of NAA in
shoots per explant raised from Adhatoda vasica. The standard error of mean and standard
error of differences obtained was 0.0326 and 0.0042 respectively (Table 47).
(2.3) Effects of NAA (mgl-1) in average shoot length of Adhatoda vasica in 2009-2010.
The effects of NAA on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 1.5cm-3.2cm in all
the concentrations of NAA supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in 0.10
mgl-1 concentration. The nodal segments of Adhatoda vasica showed average shoot
length ranging from 3.1cm to 3.2cm on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 3.1cm, 3.2cm
and 3.1cm respectively, with an average value of 3.13cm. An increasing trend was
observed in average shoot length value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 173.349 at the 0 probability. The
co-efficient of variation was determined 3.84% for different concentrations of NAA in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0295 and 0.00329 respectively
(Table 48).
(2.4) Effects of NAA (mgl-1) in average shoot length of Adhatoda vasica in 2010-2011.
The effects of NAA on average shoot length was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Average shoot length were ranging from 1.4cm-3.4cm in all
the concentrations of NAA supplemented with media. Maximum mean value of average
shoot length was obtained in 1.0 mgl-1of NAA, while minimum value was noted in 0.10
mgl-1 concentration. The nodal segments of Adhatoda vasica showed average shoot
length ranging from 3.3cm to 3.4cm on MS medium supplemented with 1.0 mgl-1
concentration of NAA. The maximum value was noted at the concentration of 1.00mgl-1
of NAA. In this concentration the values of experimental replicates were 3.3cm, 3.4cm
and 3.3cm respectively, with an average value of 3.33cm. An increasing trend was
observed in average shoot length value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 133.9730 at the 0 probability. The
co-efficient of variation was determined 5.19% for different concentrations of NAA in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0370 and 0.0056 respectively
(Table 49).
(2.5) Effects of NAA (mgl-1) in nodes per shoot of Adhatoda vasica in 2009-2010.
The effects of NAA on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.6-8.4 in all the
concentrations of NAA supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1of BAP, while minimum value was noted in
0.10 mgl-1concentration. The nodal segments of Adhatoda vasica showed nodes per shoot
ranging from 8.2 to 8.4 on MS medium supplemented with 1.0 mgl-1 concentration of
NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In this
concentration the values of experimental replicates were 8.4, 8.3 and 8.2 respectively,
with an average value of 8.30. An increasing trend was observed in nodes per shoot value
with the increase in NAA concentration.
In statistical analysis, F- value was calculated 1396.733 at the 0 probability. The
co-efficient of variation was determined 1.54% for different concentrations of NAA in
nodes per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0311and 0.0037 respectively
(Table 50).
(2.6) Effects of NAA (mgl-1) in nodes per shoot of Adhatoda vasica in 2010-2011.
The effects of NAA on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 3.2-8.4 in all the
concentrations of NAA supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in
0.10 mgl-1concentration. The nodal segments of Adhatoda vasica showed nodes per shoot
ranging from 8.2 to 8.4 on MS medium supplemented with 1.0 mgl-1 concentration of
NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In this
concentration the values of experimental replicates were 8.2, 8.4 and 8.3 respectively,
with an average value of 8.30. An increasing trend was observed in nodes per shoot value
with the increase in NAA concentration except the concentration of 0.75mgl-1 where the
value remained decreases.
In statistical analysis, F- value was calculated 454.657 at the 0 probability. The
co-efficient of variation was determined 2.85% for different concentrations of NAA in
nodes per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0516 and 0.0011 respectively
(Table 51).
(2.7) Effects of NAA (mgl-1) in leaves per shoot of Adhatoda vasica in 2009-2010.
The effects of NAA on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 3.2-7.4 in all the
concentrations of NAA supplemented with media. Maximum mean value of shoot
explants was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 7.2 to 7.4 on MS medium supplemented with 1.0 mgl-1 concentration of NAA. The
maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this
concentration the values of experimental replicates were 7.4, 7.2 and 7.3 respectively,
with an average value of 7.30. An increasing trend was observed in leaves per shoot
value with the increase in NAA concentration except the concentration of 0.75mgl-1
where the value remained decreases.
In statistical analysis, F- value was calculated 0.9346 at the 0 probability. The co-
efficient of variation was determined 31.48% for different concentrations of NAA in
leaves per shoots raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.542 and 1.2468 respectively
(Table 52 & Plate 2A).
(2.8) Effects of NAA (mgl-1) in leaves per shoot of Adhatoda vasica in 2010-2011.
The effects of NAA on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 3.1-8.6 in all the
concentrations of NAA supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 8.4 to 8.6 on MS medium supplemented with 1.0 mgl-1 concentration of NAA. The
maximum value was noted at the concentration of 1.00mgl-1 of NAA. In this
concentration the values of experimental replicates were 8.5, 8.6 and 8.4 respectively,
with an average value of 8.50. An increasing trend was observed in leaves per shoot
value with the increase in NAA concentration.
In statistical analysis, F- value was calculated 571.040 at the 0 probability. The
co-efficient of variation was determined 2.66% for different concentrations of NAA in
leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0527 and 0.0011 respectively
(Table 53 & Plate 2B).
(2.9) Effects of NAA (mgl-1) in bud break of Adhatoda vasica in 2009-2010.
The nodal segment of Adhatoda vasica showed bud break response ranging from
80% to 100% on MS medium with all the five concentration of NAA. In all the
concentrations maximum value obtained was 100% and minimum value was 80%. The
average value was calculated 89.33% for all the concentrations from 0.10 to 1.00mgl-1.
In statistical analysis, F- value was calculated 4.8571 at the 0.0277 probability.
The co-efficient of variation was determined 7.43% for different concentrations of NAA
in bud break raised from nodal segment of Adhatoda vasica. The standard error of mean
and standard error of differences was calculated 2.277 and 21.999 respectively (Table
54).
(2.10) Effects of NAA (mgl-1) in bud break of Adhatoda vasica in 2010-2011.
The nodal segment of Adhatoda vasica showed bud break response ranging from
80% to 100% on MS medium with all the five concentration of NAA. In all the
concentrations maximum value obtained was 100% and minimum value was 80%. The
average value was found to be 89.33% for all the concentrations from 0.10 to 1.00mgl-1.
In statistical analysis, F- value was calculated 11.500 at the 0.0021 probability.
The co-efficient of variation was determined 5.78% for different concentrations of NAA
in bud break raised from nodal segment of Adhatoda vasica. The standard error of mean
and standard error of differences was calculated 1.721 and 12.570 respectively (Table
55).
(3.0) Effects of plant growth regulators (kinetin) in Adhatoda vasica.
(3.1) Effects of kinetin (mgl-1) in shoot per explant of Adhatoda vasica in 2009-2010.
The effects of kinetin on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoot/explants were ranging from 0.0-1.4 in all the
concentrations of kinetin supplemented with media. Maximum mean value of shoots/
explant was obtained in 1.0 mgl-1of kinetin, while minimum value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed shoots per
explant ranging from 1.3 to 1.4 on MS medium supplemented with 1.0 mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 1.00mgl-1
of kinetin. In this concentration the values of experimental replicates were 1.3, 1.4 and
1.3 respectively, with an average value of 1.33. An increasing trend was observed in
shoots/explant value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 228.666 at the 0.00 probability. The
co-efficient of variation was determined 9.07% for different concentrations of kinetin in
shoots per explant raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0263 and 0.00235 respectively
(Table 56).
(3.2) Effects of kinetin (mgl-1) in shoots per explant of Adhatoda vasica in 2010-2011.
The effects of kinetin on shoots per explant was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Shoots/explant was ranging from 0.0-1.3 in all the
concentrations of kinetin supplemented with media. Maximum mean value of shoots/
explant was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.10
mgl-1 concentration. The nodal segments of Adhatoda vasica showed shoots per explant
ranging from 1.2 to 1.3 on MS medium supplemented with 1.0 mgl-1 concentration of
kinetin. The maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In
this concentration the values of experimental replicates were 1.3, 1.2 and 1.3
respectively, with an average value of 1.26. An increasing trend was observed in
shoots/explant value with the increase in kinetin concentration except the concentration
0.75 mgl-1concentration where decreases value was observed.
In statistical analysis the F- value was calculated 221.241 at the 0.00 probability.
The co-efficient of variation was calculated 9.75%, for different concentrations of kinetin
in shoot per explant raised from Adhatoda vasica. The standard error of mean and
standard error of differences obtained was 0.0263 and 0.0023 respectively (Table 57).
(3.3) Effects of kinetin (mgl-1) in average shoot length of Adhatoda vasica in 2009-
2010.
The effects of kinetin on average shoot length was studied in 0.10, 0.25, 0.50,
0.75 and1.00mgl-1 concentration. Average shoot length were ranging from 0.0cm-1.4cm
in all the concentrations of kinetin supplemented with media. Maximum mean value of
average shoot length was obtained in 1.00 mgl-1of kinetin, while minimum value was
noted in 0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed average
shoot length ranging from 1.2cm to 1.4cm on MS medium supplemented with 1.00 mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 1.00mgl-1
of kinetin. In this concentration the values of experimental replicates were 1.4cm, 1.3cm
and 1.2cm respectively, with an average value of 1.30cm. An increasing trend was
observed in average shoot length value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 473.963 at the 0 probability. The
co-efficient of variation was determined 8.04% for different concentrations of kinetin in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0211 and 0.0009 respectively
(Table 58).
(3.4) Effects of kinetin (mgl-1) in average shoot length of Adhatoda vasica in 2010-
2011.
The effects of kinetin on average shoot length was studied in 0.10, 0.25, 0.50,
0.75 and1.00mgl-1 concentration. Average shoot length were ranging from 0.0-1.4cm in
all the concentrations of kinetin supplemented with media. Maximum mean value of
average shoot length was obtained in 1.00 mgl-1of kinetin, while no value was noted in
0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed average shoot
length ranging from 1.3cm to 1.4cm on MS medium supplemented with 1.00 mgl-1
concentration of kinetin. The maximum value was noted at the concentration of 1.00mgl-1
of kinetin. In this concentration the values of experimental replicates were 1.4cm, 1.3cm
and 1.4cm respectively, with an average value of 1.36cm. An increasing trend was
observed in average shoot length value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 118.777 at the 0 probability. The
co-efficient of variation was determined 15.64% for different concentrations of kinetin in
average shoot length raised from nodal segment of Adhatoda vasica. The standard error
of mean and standard error of differences was calculated 0.0326and 0.0042respectively
(Table 59).
(3.5) Effects of kinetin (mgl-1) in nodes per shoot of Adhatoda vasica in 2009-2010.
The effects of kinetin on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 0.0-4.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.00 mgl-1of kinetin, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed nodes per shoot ranging
from 4.3 to 4.6 on MS medium supplemented with 1.00 mgl-1 concentration of kinetin.
The maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In this
concentration the values of experimental replicates were 4.3, 4.6 and 4.4 respectively,
with an average value of 4.43. An increasing trend was observed in nodes per shoot value
with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 2693.503 at the 0 probability. The
co-efficient of variation was determined 2.96% for different concentrations of kinetin in
nodes per shoots raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0246 and 0.0018 respectively
(Table 60).
(3.6) Effects of kinetin (mgl-1) in nodes per shoot of Adhatoda vasica in 2010-2011.
The effects of kinetin on nodes per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Nodes per shoot were ranging from 0.0-2.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of nodes per
shoot was obtained in 1.00mgl-1of kinetin, while no value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed nodes per shoot ranging
from 2.5 to 2.6 on MS medium supplemented with 1.00 mgl-1 concentration of kinetin.
The maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In this
concentration the values of experimental replicates were 2.6, 2.5 and 2.5 respectively,
with an average value of 2.53. An increasing trend was observed in nodes per shoot value
with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 1462.706 at the 0 probability. The
co-efficient of variation was determined 3.60% for different concentrations of kinetin in
nodes per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0229and 0.0014respectively
(Table 61).
(3.7) Effects of kinetin (mgl-1) in leaves per shoot of Adhatoda vasica in 2009-2010.
The effect of kinetin on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 0.0-2.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 2.5 to 2.6 on MS medium supplemented with 1.0 mgl-1 concentration of kinetin. The
maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In this
concentration the values of experimental replicates were 2.6, 2.5 and 2.5 respectively,
with an average value of 2.53. An increasing trend was observed in leaves per shoot
value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 215.699 at the 0 probability. The
co-efficient of variation was determined 10.61% for different concentrations of kinetin in
leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0460and 0.0089respectively
(Table 62 & Plate 2E).
(3.8) Effects of kinetin (mgl-1) in leaves per shoot of Adhatoda vasica in 2010-2011.
The effect of kinetin on leaves per shoot was studied in 0.10, 0.25, 0.50, 0.75
and1.00mgl-1 concentration. Leaves per shoot were ranging from 0.0-4.6 in all the
concentrations of kinetin supplemented with media. Maximum mean value of leaves per
shoot was obtained in 1.0 mgl-1 of kinetin, while minimum value was noted in 0.10 mgl-1
concentration. The nodal segments of Adhatoda vasica showed leaves per shoot ranging
from 4.3 to 4.6 on MS medium supplemented with 1.0 mgl-1 concentration of kinetin. The
maximum value was noted at the concentration of 1.00mgl-1 of kinetin. In this
concentration the values of experimental replicates were 4.3, 4.6 and 4.6 respectively,
with an average value of 4.50. An increasing trend was observed in leaves per shoot
value with the increase in kinetin concentration.
In statistical analysis, F- value was calculated 1272.212 at the 0 probability. The
co-efficient of variation was determined 5.17% for different concentrations of kinetin in
leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of
mean and standard error of differences was calculated 0.0326and 0.0042respectively
(Table 63 & Plate 2F).
(3.9) Effects of kinetin (mgl-1) in bud break of Adhatoda vasica in 2009-2010.
The nodal segment of Adhatoda vasica showed bud break response ranging from
80% to 100% on MS medium with the concentration from 0.25mgl-1 to 1.00mgl-1 of
kinetin and at the concentration of 0.10mgl-1 no bud break was observed, so the average
value was calculated 68.00% for all the concentrations from 0.10 to 1.00mgl-1.
In the statistical analysis, F- value, co-efficient of variation was determined 0.00
for different concentrations of kinetin in bud break from Adhatoda vasica. The standard
error of mean and standard error of differences obtained was also calculated 0.00 (Table
64).
(3.10) Effects of kinetin (mgl-1) in bud break of Adhatoda vasica in 2010-2011.
The nodal segment of Adhatoda vasica showed bud break response ranging from
80% to 100% on MS medium with the concentration from 0.25mgl-1 to 1.00mgl-1 of
kinetin and at the concentration of 0.10mgl-1 no bud break was observed, so the average
value was calculated 68.00% for all the concentrations from 0.10 to 1.00mgl-1. The
maximum value was observed at the concentration of 1.00mgl-1 of kinetin, where the
values of experimental replicates were 100%, 80% and 100% respectively, and for this
concentration the average value was determined 93.3%.
In the statistical analysis, F- value was 160.000 at 0.00 probabilities; co-efficient
of variation was determined 7.75% for different concentrations of kinetin in bud break
from Adhatoda vasica. The standard error of mean and standard error of differences was
calculated 1.7213 and 12.5709 respectively (Table 65).
II. Estimation of Primary Metabolites in Adhatoda vasica
Quantitative estimation of primary metabolites like carbohydrates, protein and
chlorophyll content was done in different parts of normal and regenerated plants.
(i) Estimation of reducing sugar and total sugar in normal and regenerated
plants:-
Adhatoda vasica, the amount of reducing sugar determined in normal root was
208.1µg/g, while it was 259.2µg/g in regenerated root. The amount of reducing
sugar present in normal stem was 168.2µg/g while, 278.1µg/g in regenerated
stem. The amount of reducing sugar present in regenerated leaf was 165.2µg/g as
compared to 130.0µg/g in normal leaf. The amount of reducing sugar in callus was
273.3µg/g (Fig.-8). The total sugar value was ranged from 168.34 to 334.30µg/g
while reducing sugar in 130.0- 278.1µg/g. Maximum total sugar was obtained
334.30µg/g in callus, while minimum in 168.34µg/g in normal leaf (Fig.-9). Reducing
sugar was obtained maximum 278.1 µg/g in regenerated stem while, minimum 130.0 in
normal leaf. Comparative maximum value of total sugar was determined in callus, while
reducing sugar in regenerated stem (Table-66).
(ii) Estimation of total soluble proteins and TCA precipitated protein in normal and
regenerated plants:-
Adhatoda vasica the amount of total soluble proteins determined in normal root
was 207.6µg/g while it was 272.3µg/g in regenerated root. The amount of total
soluble proteins present in normal stem was 316.2µg/g while, 445.3µg/g in
regenerated stem. The amount of total soluble proteins present in regenerated leaf was
495.2µg/g as compared to 334.5µg/g in normal leaf. The amount of total soluble
proteins in callus was 398.3µg/g (Fig.-10). The TCA precipitated protein value was
ranged from 102.3to 294.3µg/g while total soluble proteins in 207.6- 445.3µg/g.
Maximum TCA precipitated protein was obtained 294.3µg/g in callus, while minimum in
102.3µg/g in normal stem(Fig.-11). Total soluble proteins was obtained maximum
495.2µg/g in regenerated leaf while, minimum 207.6µg/g in normal root. Comparative
maximum value of TCA precipitated protein was determined in callus, while total soluble
protein in regenerated leaf (Table-67).
(iii) Estimation of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in
normal and regenerated plants:-
In Adhatoda vasica the amount of chlorophyll a, chlorophyll b, total chlorophyll
and carotenoids in normal leaf of Adhatoda vasica was 0.153mg/g, 0.082
mg/g, 0.220 mg/g and 0.211 mg/g comparatively as compared to 0.221
mg/g, 0.093 mg/g, 0.302 mg/g and 0.248 mg/g in regenerated leaf
(Table-68, Fig.-12 and Plate 6B).
III. Estimation of Secondary Cell Metabolites in Adhatoda vasica
Root, stem and leaf of Adhatoda vasica also exhibited differences in the presence
of secondary cell metabolites. Different plant parts were extracted with ethanol and
methanol. Alkaloid, steroids and phenols were present in leaf and stem while glycosides
were present in root when extracted with ethanol (Table-69). When extracted with
methanol alkaloids, steroids and terpenoids were present in leaf, glycosides were present
in roots and phenols were present in stem (Table-70 and Plate 7B).
PLANT-1I
(Adhatoda vasica)
Table-36
Effect of different level of BAP in MS medium on shoots/explant raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explants
R1 R2 R3 Mean
1. 0.10 1.2 1.4 1.5 1.36
2. 0.25 1.7 1.6 1.7 1.66
3. 0.50 2.0 2.2 2.1 2.10
4. 0.75 2.2 2.3 2.4 2.30
5. 1.00 2.3 2.2 2.2 2.23
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 51.7857 at probability = 0.00
Coefficient of Variation : 5.00% Standard error of Mean : 0.0326 Standard error of Difference : 0.0042
Table-37
Effect of different level of BAP in MS medium on shoots/explant raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explants
R1 R2 R3 Mean
1. 0.10 1.3 1.2 1.2 1.23
2. 0.25 1.6 1.5 1.6 1.56
3. 0.50 2.2 2.1 2.1 2.13
4. 0.75 1.7 1.6 1.6 1.63
5. 1.00 2.2 2.1 2.3 2.20
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 186.6250 at probability = 0.0000 Coefficient of Variation : 2.95%
Standard error of Mean : 0.0229 Standard error of Difference : 0.0014
Table-38
Effect of different level of BAP in MS medium on average shoot length raised from
nodal explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 0.9 0.8 0.8 0.83
2. 0.25 1.4 1.3 1.2 1.30
3. 0.50 1.3 1.4 1.3 1.33
4. 0.75 1.6 1.8 1.7 1.70
5. 1.00 4.4 4.5 4.6 4.50
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 760.000 at probability = 0.00 Coefficient of Variation : 4.77%
Standard error of Mean : 0.0311 Standard error of Difference : 0.0037
Table-39
Effect of different level of BAP in MS medium on average shoot length raised from
nodal explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 0.8 0.7 0.8 0.76
2. 0.25 1.3 1.2 1.4 1.30
3. 0.50 1.3 1.4 1.4 1.36
4. 0.75 2.0 2.1 2.2 2.10
5. 1.00 4.5 4.3 4.4 4.40
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 1022.6676 at probability = 0.0000
Coefficient of Variation : 3.90% Standard error of Mean : 0.0279 Standard error of Difference : 0.0028
Table-40
Effect of different level of BAP in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 3.8 3.4 3.6 3.60
2. 0.25 4.5 4.6 4.4 4.50
3. 0.50 6.1 6.3 6.2 6.20
4. 0.75 6.1 6.2 6.2 6.16
5. 1.00 7.2 7.3 7.4 7.30
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 362.0734 at probability = 0.00
Coefficient of Variation : 2.43% Standard error of Mean : 0.0448
Standard error of Difference : 0.0084
Table-41
Effect of different level of BAP in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 3.4 3.5 3.5 3.46
2. 0.25 4.4 4.6 4.5 4.50
3. 0.50 6.2 6.1 6.3 6.20
4. 0.75 6.3 6.1 6.2 6.20
5. 1.00 7.1 7.2 7.2 7.16
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 833.6241 at probability = 0.0000 Coefficient of Variation : 1.62%
Standard error of Mean : 0.0311 Standard error of Difference : 0.0037
Table-42
Effect of different level of BAP in MS medium on leaves/shoot raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 3.7 3.6 3.7 3.66
2. 0.25 4.1 4.3 4.2 4.20
3. 0.50 5.2 5.3 5.2 5.23
4. 0.75 6.5 6.7 6.5 6.56
5. 1.00 7.7 7.5 7.6 7.60
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 856.5356 at probability = 0.00
Coefficient of Variation : 1.77% Standard error of Mean : 0.0326
Standard error of Difference : 0.0042
Table-43
Effect of different level of BAP in MS medium on leaves/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 3.6 3.7 3.6 3.63
2. 0.25 4.2 4.2 4.3 4.23
3. 0.50 5.1 5.2 5.2 5.16
4. 0.75 6.4 6.7 6.5 6.53
5. 1.00 7.5 7.6 7.6 7.56
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1752.0006 at probability = 0.0000 Coefficient of Variation : 1.24% Standard error of Mean : 0.0246
Standard error of Difference : 0.0018
Table-44
Effect of different level of BAP in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 100 100 100 100
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 0.000 at probability = 0.00
Coefficient of Variation : 0.00% Standard error of Mean : 0.00
Standard error of Difference : 0.00
Table-45
Effect of different level of BAP in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 80 100 100 93.33
2. 0.25 100 100 100 100
3. 0.50 100 100 100 100
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 0.000 at probability = 0.00
Coefficient of Variation : 0.00% Standard error of Mean : 0.00 Standard error of Difference : 0.00
Table-46
Effect of different level of NAA in MS medium on shoots/explant raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.0 1.2 1.1 1.10
2. 0.25 1.1 1.0 1.1 1.06
3. 0.50 1.2 1.4 1.3 1.30
4. 0.75 1.4 1.3 1.4 1.36
5. 1.00 1.6 1.7 1.6 1.63
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 24.8421 at probability = 0.0001
Coefficient of Variation : 6.15% Standard error of Mean : 0.0279
Standard error of Difference : 0.002
Table-47
Effect of different level of NAA in MS medium on shoots/explant raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 1.2 1.0 1.1 1.10
2. 0.25 1.0 1.2 1.1 1.10
3. 0.50 1.2 1.2 1.1 1.16
4. 0.75 1.3 1.2 1.4 1.30
5. 1.00 1.7 1.8 1.7 1.73
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 23.2000 at probability = 0.0002 Coefficient of Variation : 7.48% Standard error of Mean : 0.0326
Standard error of Difference : 0.0042
Table-48
Effect of different level of NAA in MS medium on average shoot length raised from
nodal explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 1.5 1.6 1.6 1.56
2. 0.25 1.7 1.6 1.7 1.66
3. 0.50 2.0 2.2 2.1 2.10
4. 0.75 2.0 2.2 2.3 2.16
5. 1.00 3.1 3.2 3.1 3.13
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 173.3499 at probability = 0.0000
Coefficient of Variation : 3.84% Standard error of Mean : 0.0295
Standard error of Difference : 0.0032
Table-49
Effect of different level of NAA in MS medium on average shoot length raised from
nodal explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 1.4 1.6 1.5 1.50
2. 0.25 1.5 1.6 1.5 1.50
3. 0.50 2.0 2.3 2.2 2.16
4. 0.75 2.2 2.0 2.3 2.16
5. 1.00 3.3 3.4 3.3 3.33
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 133.9730 at probability = 0.00
Coefficient of Variation : 5.19% Standard error of Mean : 0.0370
Standard error of Difference : 0.0056
Table-50
Effect of different level of NAA in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 3.8 3.6 3.7 3.70
2. 0.25 4.2 4.0 4.1 4.10
3. 0.50 6.1 6.3 6.2 6.20
4. 0.75 7.1 7.0 7.0 7.03
5. 1.00 8.4 8.3 8.2 8.30
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 1396.7335 at probability = 0.0000
Coefficient of Variation : 1.54% Standard error of Mean : 0.0311
Standard error of Difference : 0.0037
Table-51
Effect of different level of NAA in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 3.6 3.2 3.3 3.36
2. 0.25 4.1 4.0 4.2 3.36
3. 0.50 6.3 6.1 6.0 6.13
4. 0.75 5.4 5.6 5.3 5.43
5. 1.00 8.2 8.4 8.3 8.30
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 454.6578 at probability = 0.00 Coefficient of Variation : 2.85%
Standard error of Mean : 0.0516 Standard error of Difference : 0.0011
Table-52
Effect of different level of NAA in MS medium on leaves/shoot raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 3.6 3.2 3.3 3.36
2. 0.25 4.1 4.2 4.1 4.10
3. 0.50 6.9 6.8 6.8 6.83
4. 0.75 5.3 5.2 5.2 5.23
5. 1.00 7.4 7.2 7.3 7.30
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 0.9346 at probability = 0.0000
Coefficient of Variation : 31.48% Standard error of Mean : 0.5421
Standard error of Difference : 1.2468
Table-53
Effect of different level of NAA in MS medium on leaves/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 3.2 3.3 3.1 3.20
2. 0.25 4.2 4.1 4.3 3.20
3. 0.50 6.8 6.3 6.5 6.53
4. 0.75 7.0 7.2 7.1 7.10
5. 1.00 8.5 8.6 8.4 8.50
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 571.040 at probability = 0.00 Coefficient of Variation : 2.66%
Standard error of Mean : 0.0527 Standard error of Difference : 0.0011
Table-54
Effect of different level of NAA in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 80 80 80 80
2. 0.25 80 80 80 80
3. 0.50 80 100 80 86.66
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 4.8571 at probability = 0.0277
Coefficient of Variation : 7.43% Standard error of Mean : 2.2771
Standard error of Difference : 21.999
Table-55
Effect of different level of NAA in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 80 80 80 80
2. 0.25 80 80 80 80
3. 0.50 100 80 80 86.66
4. 0.75 100 100 100 100
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 11.5000 at probability = 0.0021 Coefficient of Variation : 5.78%
Standard error of Mean : 1.721 Standard error of Difference : 12.57
Table-56
Effect of different level of kinetin in MS medium on shoots/explant raised from
nodal explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.3 0.2 0.2 0.23
3. 0.50 1.0 1.1 1.0 1.03
4. 0.75 1.2 1.0 1.1 1.10
5. 1.00 1.3 1.4 1.3 1.33
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 228.6666 at probability = 0.0000 Coefficient of Variation : 9.07%
Standard error of Mean : 0.0263 Standard error of Difference : 0.0023
Table-57
Effect of different level of kinetin in MS medium on shoots/explant raised from
nodal explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Shoots per explant
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.1 0.2 0.1 0.13
3. 0.50 1.2 1.0 1.1 1.10
4. 0.75 1.0 1.1 1.1 1.06
5. 1.00 1.3 1.2 1.3 1.26
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 221.2414 at probability = 0.00
Coefficient of Variation : 9.75% Standard error of Mean : 0.0263
Standard error of Difference : 0.0023
Table-58
Effect of different level of kinetin in MS medium on average shoot length raised
from nodal explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.1 0.09 0.1 0.09
3. 0.50 0.4 0.5 0.4 0.43
4. 0.75 1.1 1.2 1.2 1.16
5. 1.00 1.4 1.3 1.2 1.30
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 473.9633 at probability = 0.0000
Coefficient of Variation : 8.04% Standard error of Mean : 0.0211
Standard error of Difference : 0.0009
Table-59
Effect of different level of kinetin in MS medium on average shoot length raised
from nodal explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Average shoot length in cm
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.3 0.1 0.1 0.16
3. 0.50 0.3 0.4 0.5 0.40
4. 0.75 1.0 1.2 1.1 1.10
5. 1.00 1.4 1.3 1.4 1.36
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 118.7778 at probability = 0.00 Coefficient of Variation : 15.64% Standard error of Mean : 0.0326
Standard error of Difference : 0.0042
Table-60
Effect of different level of kinetin in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.5 0.6 0.5 0.53
3. 0.50 2.2 2.3 2.1 2.20
4. 0.75 3.5 3.6 3.5 3.53
5. 1.00 4.3 4.6 4.4 4.43
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 2693.5030 at probability = 0.0000 Coefficient of Variation : 2.96% Standard error of Mean : 0.0246
Standard error of Difference : 0.0018
Table-61
Effect of different level of kinetin in MS medium on nodes/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Nodes per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.4 0.4 0.5 0.43
3. 0.50 2.1 2.0 2.1 2.06
4. 0.75 2.3 2.4 2.4 2.36
5. 1.00 2.6 2.5 2.5 2.53
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1462.7061 at probability = 0.00 Coefficient of Variation : 3.60%
Standard error of Mean : 0.0229 Standard error of Difference : 0.0014
Table-62
Effect of different level of kinetin in MS medium on leaves/shoot raised from nodal
explants of A. vasica after 30 days of incubation in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.3 0.2 0.3 0.26
3. 0.50 1.3 1.4 1.2 1.30
4. 0.75 2.1 2.4 2.6 2.36
5. 1.00 2.6 2.5 2.5 2.53
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 215.6991 at probability = 0.0000
Coefficient of Variation : 10.61% Standard error of Mean : 0.0460
Standard error of Difference : 0.0089
Table-63
Effect of different level of kinetin in MS medium on leaves/shoot raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Leaves per shoot
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 0.2 0.3 0.1 0.20
3. 0.50 1.2 1.3 1.3 1.26
4. 0.75 3.5 3.4 3.5 3.46
5. 1.00 4.3 4.6 4.6 4.50
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 1272.2124 at probability = 0.00 Coefficient of Variation : 5.17% Standard error of Mean : 0.0326
Standard error of Difference : 0.0042
Table-64
Effect of different level of kinetin in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2009-2010.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 80 80 80 80
3. 0.50 80 80 80 80
4. 0.75 80 80 80 80
5. 1.00 100 100 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis F value : 0.000 at probability = 0.00
Coefficient of Variation : 0.00% Standard error of Mean : 0.00
Standard error of Difference : 0.00
Table-65
Effect of different level of kinetin in MS medium on bud break raised from nodal
explants of A. vasica in the year of 2010-2011.
S.N. Concentration
(mgl-1)
Bud break response (%)
R1 R2 R3 Mean
1. 0.10 Nil Nil Nil Nil
2. 0.25 80 80 80 80
3. 0.50 80 80 80 80
4. 0.75 80 80 80 80
5. 1.00 100 80 100 100
R1, R2, R3 - Experimental Replicates
Statistical analysis
F value : 160.0000 at probability = 0.00 Coefficient of Variation : 7.75% Standard error of Mean : 1.7213
Standard error of Difference : 12.570
Table-66
Estimation of total sugar and reducing sugar in normal and regenerated plant
of Adhatoda vasica.
Plant part Total sugars (g/g) Reducing sugars (g/g)
Normal stem 241.20 168.2
Regenerated stem 302.33 278.1
Normal Leaf 168.34 130.0
Regenerated Leaf 218.64 165.2
Normal root 258.20 208.1
Regenerated root 310.00 259.2
Callus 334.30 273.3
Table-67
Estimation of soluble protein and TCA precipitated protein in normal and
regenerated plants of Adhatoda vasica.
Table- 68
Chlorophyll content in normal and regenerated plant of Adhatoda vasica.
Pigments
(mg/g)
Normal Plant Regenerated plant
Chlorophyll a 0.153 0.221
Chlorophyll b 0.082 0.093
Total Chlorophyll 0.220 0.302
Caratenoids 0.211 0.248
Plant part Soluble Protein (g/g) TCA pptd. Protein(g/g)
Normal stem 316.2 102.3
Regenerated stem 445.3 146.2
Normal Leaf 334.5 143.1
Regenerated Leaf 495.2 230.2
Normal root 207.6 135.4
Regenerated root 272.3 189.7
Callus 398.3 294.3
Table-69
Presence of secondary metabolites in root, stem and leaf of Adhatoda vasica
when extracted with ethanol.
Table-70
Presence of secondary metabolites in root, stem and leaf of Adhatoda vasica when
extracted with ethanol.
Chemical
compounds
Root Stem Leaf
Alkaloids + + +
Steroids - + +
Glycosides + - -
Terpenoids - - -
Phenols - + +
Chemical
compounds
Root Stem Leaf
Alkaloids + + +
Steroids - - +
Glycosides + - -
Terpenoids - - +
Phenols - + -
0
10
20
30
40
50
60
70
80
90
100
Jan - Feb Mar-Apr May-Jun July-Aug Sep-Oct Nov-Dec
Perc
en
tag
e (
%)
Fig.-1 Effect on seasonal variation on contamination and bud break in
cultures derived from nodal explants of W.somnifera.
Contamination Bud break
0
50
100
150
200
250
N. stem N. Leaf N. root Callus
Co
ncen
trati
on
(µg
/g)
Fig.2:Estimation of reducing sugars in normal plant, regenerated plant
and callus of W. somnifera.
Normal Regenerated
0
50
100
150
200
250
stem Leaf root Callus
Co
ncen
trati
on
(µg
/g)
Fig.3: Estimation of total sugars in normal plant, regenerated plant
and callus of W. somnifera.
Normal Regenerated
0
100
200
300
400
500
600
N. stem N. Leaf N. root Callus
Co
ncen
trati
on
(g
/g)
Fig.4: Estimation of soluble protein in normal plant, regenerated plant
and callus of W. somnifera.
Normal regenerated
0
50
100
150
200
250
N. stem N. Leaf N. root Callus
Co
ncen
trati
on
(µg
/g)
Fig.5:Estimation of TCA precipitated protein in normal plant, regenerated
plant and callus of W. somnifera.
Normal Regenerated
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
Chl. a Chl. b Total Chl. Caratenoids
Co
ncen
trati
on
(mg
/g)
Fig.6: Chlorophyll content in normal and regenerated plant of W. somnifera.
Normal Plant (mg/g) Regenerated plant (mg/g)
0
10
20
30
40
50
60
70
80
90
100
jan-fab mar-apr may-jun july-aug sep-oct nov-dec
Pe
rce
nta
ge
(%)
Fig.-7:Effect on seasonal variation on contamination and bud break in cultures
derived from nodal explants of A. vasica.
contamination bud break
0
50
100
150
200
250
300
350
400
N. stem N.Leaf N. root Callus
Co
nce
ntr
atio
n(µ
g/g
)
Fig.9:Estimation of total sugars in normal plant, regenerated plant and
callus of A. vasica.
Normal Regenerated
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
Chl. a Chl. b Total Chl. Caratenoids
Co
ncen
trati
on
(mg
/g)
Fig.12:Chlorophyll content in normal and regenerated plant of A. vasica.
Normal Plant (mg/g) Regenerated plant (mg/g)
0
50
100
150
200
250
N. stem N. Leaf N. root Callus
Co
ncen
trati
on
(g
/g)
Fig.11: Estimation of TCA precipitated protein in normal plant, regenerated
plant and callus of A. vasica.
Normal Regenerated
(2009-2010) (2010-2011)
(A) 0.10 0.25 0.50 0.75 1.00 (B) 0.25 0.50 0.75 1.00
Effect of different concentrations of BAP (mgl-1) in nodal segments of Withania
somnifera
(C) 0.25 0.50 0.75 1.00 (D) 0.10 0.25 0.50 0.75 1.00
Effect of different concentrations of NAA (mgl-1) in nodal segments of Withania
somnifera.
(E) 0.50 0.75 1.00 (F) 0.25 0.50 0.75 1.0
Effect of different concentrations of kinetin (mgl-1) in nodal segments of Withania
somnifera.
Plate-1
(2009-2010) (2010-2011)
(A) 0.25 0.50 0.75 1.00 (B) 0.10 0.25 0.50 0.75 1.00
Effect of different concentrations of NAA (mgl-1) in nodal segments of Adhatoda
vasica.
(C) 0.10 0.25 0.50 0.75 1.00 (D) 0.25 0.50 0.74 1.00
Effect of different concentrations of BAP (mgl-1) in nodal segments of Adhatoda
vasica.
(E) 0.10 0.25 0.50 0.75 1.00 (F) 0.25 0.50 0.50 1.00
C. Effect of different concentrations of kinetin (mgl-1) in nodal segments of Adhatoda
vasica.
Plate-2
A.Callus Culture in Withania somnifera
B. Callus Culture in Adhatoda vasica
Plate-3
A. Hardening of Withania somnifera
B. Hardening of Adhatoda vasica
Plate 4
A. Hardening of Withania somnifera
B. Hardening of Adhatoda vasica
Plate 5
A.Chlorophyll content in regenerated plant of Withania somnifera. .
Chlorophyll a Chlorophyll b Total Chlorophyll Carotenoids
B. Chlorophyll content in regenerated plant of Adhatoda vasica.
Chlorophyll a Chlorophyll b Total Chlorophyll Carotenoids
Plate 6
A. Secondary metabolites present in Withania somnifera.
Alkaloids Glycoside Steroids Phenol Flavonoids
B. Secondary metabolites present in Adhatoda vasica
Alkaloids Glycoside Steroids Phenol Flavonoid
Plate 7
Discussion
The present investigation on micro propagation, estimation of primary
metabolites, chlorophyll content and presence of secondary metabolites in Withania
somnifera and Adhatoda vasica are being discussed below.
I. Micropropagation
A. Shoot Bud Culture
For the selection of suitable explants, and establishing successful plant tissue
culture system, it is essential to have knowledge on natural propagation system of plants.
The time of the year when explants are collected from stock plants may have an influence
on auxiliary shoot out growth. The best period of the year for initiating shoot bud culture
from nodal explants of Withania somnifera and Adhatoda vasica was noted to be May-
June. During this period both the plant exhibited maximum bud break percentage, while
contamination was noted to be very low or no contamination in March-April.
Seasonal changes in plants and response of plants to contamination:
During in vitro propagation of some plants certain types of slow growing
microbial contaminants persist even after initial surface sterilization of explants. Such
contaminants may persist for many generations without being noticed and cause
reduction in vigour or chlorosis in propagated plantlets (Knauss and Miller, 1978). The
time of the year that the explants are collected from stock plants may have an influence
on axillary shoot outgrowth. The culture initiated from nodal explants of Withania
somnifera exhibited maximum contamination (90%) during July-August. In
corresponding period, the bud break response of explants was very low, while the nodal
explants of Adhatoda vasica showed maximum contamination during September-
October. This indicates that the bud break response and contamination of the cultures
varied depending on the season of the explants isolation from the mother plant.
Fluctuation in the environmental factors in different seasons had a definite effect on shoot
bud differentiation from explanted nodal segments in Withania somnifera and Adhatoda
vasica as similarly observed in other medicinal herbs including Ocimum species (Ahuja
et al., 1982; Pattnaik and Chand, 1996), Actinidia deliciosa (Kumar et al., 1998), Tridex
procumbens (Sahoo and Chand, 1998) and Solanum surattense (Swamy et al., 2004).
Various workers investigated the problem of microbial contamination of culture
especially serious when the tissue was derived from field grown material.
In this respect, the behavior of explant in culture may be decided critically by the
season and growth stage of the parent plant. Stone (1963) found better survival of
carnation meristem isolated from donor plants during the active growing season of spring
and autumn than those taken in summer and winter. Hohtola (1988) suggested that during
winter months decreased contamination might be due to the better resistance of the tissue
against microbes during active period of growth and/or susceptibility of microbes to the
decontaminants. Anaz and Vijay Kumar (1997) attributed the high level of contamination
during rainy month to the amount of inoculums present in the environment due to
favorable condition during the rainy months. To ensure complete conditions of the
explants it is essential to remove dirt and debris from the plant tissue. To improve wetting
of the tissue surface, a detergent or alcohol wash often precedes treatment with steriliants.
Ethanol partially removes hydrophobic waxes and resins, which protect micro organisms
from contact with aqueous steriliants (Kunneman and Faaij-Groenen, 1988).
Effects of explants:
Shoot tips and nodal segments were used as explants at establishment stage. Of
these two, nodal segments responded better than shoot tips. Nodal bud cultures were
found to be an efficient means of clonal multiplication of plants by several workers too.
Plantlets were successfully produced by culturing shoot tips with a couple of primordial
of Lupinus and Tropaeolum (Ball, 1946).
Direct in vitro clonal propagation for nodal explants had been achieved in
Euphorbia lathyris and Euphorbia plepus (Tideman and Hawker, 1982), Euphorbia
fulgens (Zhang et al., 1987), Jatropha curcas (Sardana, 1998), Wedelia chinensis
(Emmanuel et al., 2000), Ocimum sanctum (Shahzad and Siddiqui, 2000), Hyptis
suaveolens (Britto et al., 2001), Jatropha curcas (Rajore et al., 2002), Tinospora
cordifolia (Kumar et al., 2003) Withania somnifera (Vadawale et al., 2004), Centella
asiatica (George et al., 2004; Shashikala et al., 2005), Tabebuia serratifolia (Nery et
al.,2008), Elaeagnus angustifolia (Zeng et al., 2009), and Ceropegia thwaitesii
(Muthukrishnan et al., 2012). There were also records of studies that was done on various
other plants which used different other plant parts to induce callus such as leaves, nodes
and buds (Mungole et al., 2009). In vitro clonal multiplication of Kaempferia galanga
through rhizome buds was reported (Vincent et al., 1992; Geetha et al., 1997; Lakshmi
and Mythili, 2003). A few reports available described the K.galanga micro propagation
using rhizome pieces (Shirin et al., 2000, Swapna et al., 2004).
There are a number of reports regarding in vitro regeneration of Withania
somnifera L. Dunal by using various explants such as shoot tips (Sen and Sharma, 1991),
nodal segments (Tiwari and Singh, 1991), axillary meristems (Roja et al., 1991), axillary
shoots and hypocotyls and root segments (Rani and Grover, 1999), nodal segments
(Kulkarni et al., 2000).
Effects of used media
The earliest nutrient media used for growing plant tissues in vitro were based on
the nutrient formulations for whole plants. First significant attempt of isolated plant cells
on artificial nutrient medium was done by Haberlandt (1902). Subsequently various
culture media were reported to obtain cultured cells in diverse plant explants of several
taxa by the work of Hildebrandt et al. (1946), Nitsch (1951), Reinert and White(1956),
Murashige and Skoog (1962), White (1963), Gamborg et al. (1968), Schenck and
Hidebrandit (1972).
Among several media employed in tissue culture studies, the most suited
formulation was Murashige and Skoog (MS medium) which was well balanced micro and
macro nutrients besides having a highest concentration of nitrogen compared to other
media. MS formulation allowed for a further increase in the number of plant species that
could be cultured, many of them using only a defined medium consisting of macro and
micro nutrients, a carbon source, reduced nitrogen, vitamin B and growth regulators
(Gamborg et al., 1976). The MS salt formulation is now the most widely used nutrient
medium in plant tissue culture. In Withania somnifera and Adhatoda vasica the
formulation of (Murashige and Skoog’s, 1962) basal medium was found more suitable
than B5 medium for establishment of nodal explants, whereas micro propagation of
woody species, in general, present better propagation and rooting rates when cultured in
WP than in more concentrated culture media as MS (Thorpe et al., 1991; Mantovani et
al., 2001; Ferreira and Pasqual, 2008; Dutra et al., 2009). There are also documentations
of different media being used for callus induction purpose. Manivannan et al. (2010)
used N6 media to induce callus from elite Indian maize inbreeds immature embryo.
Shoots per explant, average shoot length and nodes per shoot were found more on
MS medium as compared to B5 medium suggested that the MS medium could be used
for establishment of the nodal explants of Withania somnifera and Adhatoda vasica. The
MS medium was found more suitable than other media for establishment of explants of
several species such as Plantago ovate ( Barna and Wakhlu,1989), Tylophora indica
(Chattopadhyay et al.,1992), Phyllanthus caroliniensis (Catapan et al., 2000), Punica
granatum (Rudra and Juwarkar, 2002), Tricosanthes dioica (Awal et al., 2005), Eclipta
alba (Husain and Anis, 2006), Mollungo nudicaulis (Nagesh and Shanthamma, 2011) and
Hymenocallis littoralis (Sundarasekar et al., 2012). Studies with different species have
shown that concentrations of micro and macro nutrients added to the culture medium
directly interfere in the development of in vitro plantlet cultures.
Effects of growth regulators
Growth regulator concentration in the culture medium is critical to control the
growth and morphogenesis. Generally, a high concentration of auxin and a low
concentration of cytokinin in the medium promoted abundant cell proliferation with the
proliferation of callus. On the other hand, low auxin and high cytokinin concentrations in
the medium resulted in the induction of shoot morphogenesis. Auxin alone or with a very
low concentration of cytokinin was found important in the induction of root primordial.
The role of cytokinins in shoot organogenesis is well established (Evans et al., 1983).
Different plants were expected to respond differently to various cytokinins and auxins
and this may be partly because of their endogenous hormonal levels. Supply of hormones
in a proper sequence was important to achieve a particular response. It is well established
that proper ratio of cytokinin and auxin is necessary for morphogenesis leading the
formation of complete plantlets (George and Sherrington, 1984).
Auxins could regulate and influence diverse response on a whole plant level, such
as tropism, apical dominance and root initiation and responses on cellular level such as
cell enlargement, division and differentiation (Hagen and Guilfoyle, 2002). It was clearly
documented that generally, high concentration of auxins and low cytokinins in the
medium promote abundant cell proliferation with the formation of callus (Shah et al.,
2003). Cytokinins act at the cellular level by inducing the expression of some genes,
promotion mitosis and chloroplast development but also on the organ level by releasing
buds from apical dominance or by inhibiting root growth (Riefler et al., 2006).
Thus auxin and cytokinins interacts in the control of many central developmental
processes, particularly in apical dominance and root and shoot development. The
different concentrations of BAP in MS medium influenced the shoot number per explant,
shoot length, node number per shoot and leaves number per shoot. Callus showed a
different response according to the growth regulators used. Kinetin seems to be essential
for further multiplication and growth of shoots. When explants with shoots and shoot
buds were sub cultured on the same medium, shoot buds did not develop into shoots, but
when subculture on medium containing kinetin, shoot development was seen.
In cultures raised from nodal explants of Withania somnifera and Adhatoda
vasica maximum numbers of shoots were produced on MS medium supplemented with
1.0mgl-1 BAP. These explants developed greater than two shoots per node while, in all
other concentrations of NAA and kinetin developed either two or less than two shoots per
explant. BAP, NAA and kinetin at a concentration range 0.1-1.0mgl-1 were tested for
assessing the optimum concentrations of the cytokinins for early sprouting and maximum
proliferation of axillary shoots. BAP was found to be more effective than NAA and
kinetin on the proliferation and development of Withania somnifera and Adhatoda vasica
shoots. Superiority of BAP over other cytokinin in producing in vitro shoots had also
been confirmed in other plants. The explants show shoot initiation after 7-10 days.
The medium with growth regulator BAP produced greater number of shoots than
the basal medium as also been reported in Zingiber officinale (Balachandran et al.,
1990); Hoppea odorata (Scott et al.,1995); Murraya koenigii (Rajendra and D’Souza
1998); Celastraus paniculata (Nair and Seeni, 2001); Vitex negundo (Chandramu et al.,
2003); Adhatoda vasica (Rahman et al.,2004); Arbus precatorius (Biswas et al., 2007);
Kaempferia galangal (Jinu and Aravindan , 2008); Hymenocallis littoralis (Aftab et al.,
2008); Ricinus communis (Nahar and Borna, 2012).
The synergistic effect of higher concentration of cytokinin with lower
concentration of auxin induced better shoot formation was reported by various workers in
different plant species (Mishra et al., 2004 and Vidya et al., 2005). However, some other
hormones have also been added by some of the workers along with BAP for some other
plants like Solanum viarum (Tejavathi and Bhuvana ,1996); Alpinia galangal (Anand and
Hariharan, 1997); Passiflora caerulea (Jasrai et al., 1999); Acorus calamus (Rani et al.,
2000); Vitex negundo (Thiruvengadan and Jayabalan ,2000); Hypericum patulum,
(Baruah et al., 2001); Cajanus cajan (Vijayakumari et al., 2001); Vitex negundo (Sikdar
et al., 2003); Triticum aestivum (Turhan and Baser, 2004); Plumbagl zeylanica (Chaplot
et al., 2006); Withania somnifera (Bansal and Baghel , 2010); Ricinus Communis (Alam
et al., 2010); Cicer arietinum (Riazuddin et al., 2012).
In the present study, when explants were cultured in MS medium supplemented
with various concentrations of kinetin, single healthy shoots were produced in the media
composition. This is in accordance with the results as reported earlier by Neeti and
Kothari (2005) in Eclipta alba. In the present investigation, higher concentration of
cytokinin reduced the shoot number as well as shoot length. A similar response was
observed in Centella asiatica (Nath and Buragohain, 2003) and Terminalia chebula.
(Shyamkumar et al., 2004).
Of the two cytokinins used for multiple shoot induction in the present study, BAP
induced more number of multiple shoots compared to kinetin. The superiority of BAP
over kinetin in inducing large number of multiple shoots has been reported in several
plants by several workers; Gymnema sylvestre (Komalavalli and Rao, 2000); Cunila
galioides (Fracaro and Echeverigaray, 2001); Stevia rebaudiana (Mousmi, 2008) and
Acacia catechu (Jain et al., 2009). Rooting of in vitro derived shoots of Withania
somnifera and Adhatoda vasica was achieved on MS medium supplemented with varying
concentration of IBA after 4 weeks of culture. Media having a low concentration of salts
have proven satisfactory for rooting of shoots micro propagation. Roots are mostly
induced in the presence of a suitable auxin in the medium. Auxin (IBA) induced root
formation which was accompanied by shoot elongation. But it was reported that NAA
along with BAP promoted both root and shoot regeneration and finally complete plantlets
(Kumar et al., 2003).
Statistical analysis
For statistical analysis, the data were subjected to one-way analysis of variance
(ANOVA) to assess treatment difference and interaction using the SPSS 11.3 statistical
package for windows. Each experiment was performed in triplicates. The experiment was
carried out in a completely randomized factorial design. When the ANOVA indicated
significant treatment effects (5 or 1%) based on the F-test, the standard error of mean was
used as a method to determine which treatments were statistically different from other
treatments.
According to this analysis if calculated value of F is greater than tabulated value
of F at 5 % level of significance then it was showed as F* and treatment difference was
said to be significant. If calculated value of F is greater than tabulated value of F at 1 %
level of significance then it was showed as F** and treatment difference was said to be
highly significant. If calculated value of F is less than or equal to tabulated value of F at 5
% level of significance then it was showed as ns and treatment difference was said to be
non-significant.
Estimation of primary metabolites
The data obtained during present study showed that the regenerated plants had
more primary metabolites when compared to normal plants, suggested that regenerated
plants have possibility of gene amplification. The primary metabolites such as sugars,
protein, lipid, chlorophyll and nucleic acids, which are common to all plants and are
involved in the primary metabolic process of building and maintaining plant cells
(Kaufman et al.,1999; Wink 1999).
From the presently recorded data, it appears that the regenerated plants have
better synthesizing machinery than normal plants which is probably related with high rate
of photosynthesis in regenerated plants as supported by higher amount of total
chlorophyll in tissue cultured plants.
In present study, efficient plant regeneration and metabolites estimation protocol
was developed for Withania somnifera and Adhatoda vasica, important medicinal plants.
In regenerated Withania somnifera the maximum amount of reducing sugar and total
sugar was noted 289.20.2 µg/g and415.2g/g. In regenerated Adhatoda vasica the
maximum amount of reducing sugar and total sugar was noted 278.1µg/g and
310.00g/g. Similarly the presence of reducing sugars and resins were reported in
Aganosma dichotoma (Wahi et al., 1984), Amaranthus viridis and Spinacea oleracea
(Naseer Banu et al., 2003) estimated sugar contents in which, A.viridis showed higher
sugar than S.oleracea.
In regenerated Withania somnifera the maximum amount of protein was noted
502.10.2 µg/g and415.2g/g, while in Adhatoda vasica the maximum amount of
protein was noted 495.2 µg/g, similar result was determined in Solanum
xanthocarpum (Udayakumar et al., 2003).
Estimation of secondary metabolites
In the present study, the different plant parts like root stem and leaf of Withania
somnifera and Adhatoda vasica had exhibited differences in the presence of secondary
metabolites.
Preliminary phytochemical screening of plant extract was reported in several
medicinal plants (Amerjothy et al., 2007). In the present investigation, extracts of
different plant parts of Withania somnifera and Adhatoda vasica had exhibited presence
of alkaloids, steroids, glycosides, terpenoids and phenols. It is well known that the
qualitative and quantitative contents of secondary metabolites in a plant show marked
variation, that is regulated by intrinsic factors (ontogeny and phenology) and also by
abiotic factors ( light, moisture, nutrient availability) and biotic factors such as different
physiological and growth stages (Harborne, 1993; Brooks and Feeney, 2000; Calixto,
2000; Sosa et al., 2005). In the case of plants used for medicinal purposes, all these
factors must be considered, besides the post harvesting managements.
In the present study, root of Withania somnifera had exhibited more number of
chemical compounds than the leaf and stem, whereas in case of Adhatoda vasica, the
more number of chemical compounds were noted in leaf and stem, when extracted with
ethanol and methanol. It was reported that the methanolic extracts of this plant generally
possess alkaloids and phenolics, which was reported by different workers as
antimicrobial compounds (Rao and Satyanarayan, 1977; Ahmad et al., 1998;
Perumalsamy et al., 1998; Valarmathy et al., 2010).
Alkaloids are ubiquitous secondary products of plants and about 4,000
representatives are known (Harborne, 1977; Witzell et al., 2003). They are frequently
found in fruits and vegetables and therefore are part of the human diet. They are also
responsible for the pharmacological effects of several medicinal plants. As a consequence
of their chemical diversity and biological functions, there is an increasing interest in this
group of phytochemicals as chemotaxonomic markers, as well as in their ecological role
and beneficial health effects in chronic and degenerative diseases. They disclose a wide
pharmacological profile including antioxidant, free radical scavenger, lipid peroxidation
inhibition, anti-inflammatory, anti-allergic, anticarcinogenic, anti-arthrithic, anxiolytic
and antihypertensive activities (Robards and Antolovich, 1997; Di Carlo et al., 1999).
Recent advances in the knowledge on the neuro pharmacological and cardiac effects of
flavonoids point out to their potential for the management of various psychiatric
conditions and cardiac insufficiencies including the treatment of hypertension, arrhythmia
and tachycardia (Johnson and Beart, 2004). Due to the multifunctional characteristics of
phytochemicals, the antioxidant efficacy of a plant extract is best evaluated based on
results obtained by commonly accepted assays, taking into account different oxidative
conditions, system compositions, and antioxidant mechanisms (Frankel & Meyer, 2000;
Prior, Wu, & Schaich, 2005)
It may be concluded that normal plants in synthesizing primary and secondary
metabolites, which can be commercially exploited.
Advantages of extracting secondary metabolites using plant tissue cultures are:-
1. The source of secondary metabolites that is most of the high plant has specific
agro-climatic requirements. Hence, specific metabolites can be produced all
through the year even in places where crops are not grown.
2. The already limited supply of these raw materials cannot be exhausted
considering the future needs.
3. It has also been found that cells under culture tend to produce greater amount of
these metabolites than that is accumulated in nature.
The major advantages of a cell culture system over the conventional cultivation of whole
plants are:
1. Useful compounds can be produced under controlled conditions independent of
climatic changes or soil conditions.
2. Cultured cells would be free of microbes and insects.
3. The cells of any plants, tropical or alpine, could easily be multiplied to yield their
specific metabolites.
4. Automated control of cell growth and rational regulation of metabolite processes
would reduce of labor costs and improve productivity.
5. Organic substances are extractable from callus cultures.