(i). micropropagation in withania somnifera. ) in shoots...

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


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.



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



shoot length was obtained in 1.0 mgl-1of BAP while minimum value was noted in






observed in average shoot length value with the increase in BAP concentration.






(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


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.



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



shoot was obtained in 1.0 mgl-1of BAP while minimum value was noted in



of BAP. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this



with the increase in BAP concentration except the concentration 0.10 mgl-1concentration

where no value obtained.





(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



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.



leaves per shoot raised from nodal segment of Withania somnifera. The standard error of


(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


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





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.





(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.


from 30% to 100% on MS medium with all the five concentration of BAP. In all the




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%.



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


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


shoots/explant value with the increase in NAA concentration except the concentration

0.25 mgl-1concentration where minimum value obtained.


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


(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


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





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


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


of NAA. In this concentration the values of experimental replicates were 4.0cm, 4.2cm


observed in average shoot length value with the increase in NAA concentration.






(2.4) Effects of NAA (mgl-1) in average shoot length of Withania somnifera in 2010-

2011.




shoot length was obtained in 1.0 mgl-1of NAA, while minimum value was noted in












(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


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



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.





(Table 15 & Plate 1C).

(2.6) Effects of NAA (mgl-1) in nodes per shoot of Withania somnifera in 2010-2011.




shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in



of NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In



per shoot value with the increase in NAA concentration.





(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


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



NAA. The maximum value was noted at the concentration of 1.00mgl-1 of BAP. In this



value with the increase in NAA concentration.





(Table 17).

(2.8) Effects of NAA (mgl-1) in leaves per shoot of Withania somnifera in 2010-2011.




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


NAA. The maximum value was noted at the concentration of 1.00mgl-1 of NAA. In this








(Table 18).

(2.9) Effects of NAA (mgl-1) in bud break of Withania somnifera in 2009-2010.


from 80% to 100% on MS medium with all the five concentration of NAA. In all 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



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.


from 90% to 100% on MS medium with all the five concentration of NAA. In all the


average value was calculated 98.66% for all the concentrations from 0.10 to 1.00mgl-1.




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



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



(3.2) Effects of kinetin (mgl-1) in shoots per explant of Withania somnifera in 2010-

2011.


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






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


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




of kinetin. In this concentration the values of experimental replicates were 3.2cm, 3.0cm


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.


co-efficient of variation was determined 3.03% for different concentrations of kinetin in




(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.




error of mean and standard error of differences was calculated 0.0356and 0.0051


(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


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


kinetin. The maximum value was noted at the concentration of 0.75mgl-1 of kinetin. In



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.




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.




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





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.




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


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




respectively, with an average value of 28.50. An increasing trend was observed in leaves

per shoot value with the increase in kinetin concentration.





(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


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





respectively, with an average value of 28.53. An increasing trend was observed in leaves

per shoot value with the increase in kinetin concentration.





(Table 28 & Plate 1F).

(3.9) Effects of kinetin (mgl-1) in bud break of Withania somnifera in 2009-2010.


from 40% to 100% on MS medium with all the five concentration of kinetin. In all the




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%.


efficient of variation was determined 0.00 for different concentrations of kinetin in bud



(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



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



F value : 125.8750 at probability = 0.00

Coefficient of Variation : 4.67% Standard error of Mean : 0.0356


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



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



S.N. Concentration

(mgl-1)


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






Table-5

Effect of different level of BAP in MS medium on nodes/shoot raised from nodal


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





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


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






Table-7

Effect of different level of BAP in MS medium on leaves/shoot raised from nodal


S.N. Concentration

(mgl-1)

Leaves per shoots

R1 R2 R3 Mean


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




Coefficient of Variation : 0.45% Standard error of Mean : 0.0211 Standard error of Difference : 0.0009

Table-8



S.N. Concentration

(mgl-1)

Leaves per shoot

R1 R2 R3 Mean


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





Table-9

Effect of different level of BAP in MS medium on bud break raised from nodal


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






Table-10



S.N. Concentration

(mgl-1)


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





Table-11

Effect of different level of NAA in MS medium on shoots/explant raised from nodal


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






Table-12



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





Table-13

Effect of different level of NAA in MS medium on average shoot length raised from


S.N. Concentration

(mgl-1)


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



Table-14



S.N. Concentration

(mgl-1)


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





Table-15

Effect of different level of NAA in MS medium on nodes/shoot raised from nodal


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





Table-16



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






Table-17

Effect of different level of NAA in MS medium on leaves/shoot raised from nodal


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





Table-18



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





Table-19

Effect of different level of NAA in MS medium on bud break raised from nodal


S.N

.

Concentration

(mgl-1)


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






Table-20



S.N. Concentration

(mgl-1)


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





Table-21

Effect of different level of kinetin in MS medium on shoots/explant raised from


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






Table-22

Effect of different level of kinetin in MS medium on shoot/explant raised from nodal


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





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)


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





Table-24


from nodal explants of W. somnifera in the year of 2010-2011.

S.N. Concentration

(mgl-1)


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





Table-25

Effect of different level of kinetin in MS medium on nodes/shoot raised from nodal


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






Table-26



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





Table-27

Effect of different level of kinetin in MS medium on leaves/shoot raised from nodal


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





Table-28



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





Table-29

Effect of different level of kinetin in MS medium on bud break raised from nodal


S.N. Concentration

(mgl-1)


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






Table-30



S.N. Concentration

(mgl-1)


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





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)







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


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





shoots/explant value with the increase in BAP concentration where higher concentrations

of BAP in MS medium reduced the value of shoots per explant.



shoot per explant raised from nodal segment of Adhatoda vasica. The standard error of


(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


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






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



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








average shoot length raised from nodal segment of Adhatoda vasica. The standard error


(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



shoot length was obtained in 1.0 mgl-1of BAP, while minimum value was noted in











(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




0.10mgl-1concentration. The nodal segments of Adhatoda vasica showed nodes per shoot





with the increase in BAP concentration.



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



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





with the increase in BAP concentration.





(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




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


with an average value of 7.60. An increasing trend was observed in leaves per shoots

value with the increase in BAP concentration.



leaves per shoot raised from nodal segment of Adhatoda vasica. The standard error of


(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





from 7.5 to 7.6 on MS medium supplemented with 1.0 mgl-1 concentration of BAP. The




value with the increase in BAP concentration.





(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.


efficient of variation was determined 0.00 for different concentrations of BAP in bud



(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


explants was obtained in 1.0 mgl-1of NAA, while minimum value was noted in





respectively, with an average value of 1.43.An increasing trend was observed in


0.25 mgl-1concentration where minimum value obtained.


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


(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


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






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.




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










(Table 48).

(2.4) Effects of NAA (mgl-1) in average shoot length of Adhatoda vasica in 2010-2011.




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










(Table 49).

(2.5) Effects of NAA (mgl-1) in nodes per shoot of Adhatoda vasica in 2009-2010.










with the increase in NAA concentration.




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.




shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in






with the increase in NAA concentration except the concentration of 0.75mgl-1 where the

value remained decreases.





(Table 51).

(2.7) Effects of NAA (mgl-1) in leaves per shoot of Adhatoda vasica in 2009-2010.




explants was obtained in 1.0 mgl-1 of BAP, while minimum value was noted in 0.10 mgl-1


from 7.2 to 7.4 on MS medium supplemented with 1.0 mgl-1 concentration of NAA. The




value with the increase in NAA concentration except the concentration of 0.75mgl-1

where the value remained decreases.


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


(Table 52 & Plate 2A).

(2.8) Effects of NAA (mgl-1) in leaves per shoot of Adhatoda vasica in 2010-2011.




shoot was obtained in 1.0 mgl-1 of NAA, while minimum value was noted in 0.10 mgl-1


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








(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


average value was calculated 89.33% for all the concentrations from 0.10 to 1.00mgl-1.


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.


80% to 100% on MS medium with all the five concentration of NAA. In all the


average value was found to be 89.33% for all the concentrations from 0.10 to 1.00mgl-1.


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.




explant was obtained in 1.0 mgl-1of kinetin, while minimum value was noted in






shoots/explant value with the increase in kinetin concentration.



shoots per explant raised from nodal segment of Adhatoda vasica. The standard error of


(Table 56).

(3.2) Effects of kinetin (mgl-1) in shoots per explant of Adhatoda vasica in 2010-2011.




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







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.


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





observed in average shoot length value with the increase in kinetin concentration.





(Table 58).

(3.4) Effects of kinetin (mgl-1) in average shoot length of Adhatoda vasica in 2010-

2011.


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






observed in average shoot length value with the increase in kinetin concentration.




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.




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



with the increase in kinetin concentration.



nodes per shoots raised from nodal segment of Adhatoda vasica. The standard error of


(Table 60).

(3.6) Effects of kinetin (mgl-1) in nodes per shoot of Adhatoda vasica in 2010-2011.




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



with the increase in kinetin concentration.





(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





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



value with the increase in kinetin concentration.





(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





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



value with the increase in kinetin concentration.





(Table 63 & Plate 2F).

(3.9) Effects of kinetin (mgl-1) in bud break of Adhatoda vasica in 2009-2010.


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.


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


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


Statistical analysis F value : 51.7857 at probability = 0.00


Table-37



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


Statistical analysis F value : 186.6250 at probability = 0.0000 Coefficient of Variation : 2.95%


Table-38


nodal explants of A. vasica in the year of 2009-2010.

S.N. Concentration

(mgl-1)


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


Statistical analysis F value : 760.000 at probability = 0.00 Coefficient of Variation : 4.77%


Table-39



S.N. Concentration

(mgl-1)


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




Table-40



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





Table-41



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





Table-42



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





Table-43



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





Table-44



S.N. Concentration

(mgl-1)


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





Table-45



S.N. Concentration

(mgl-1)


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




Table-46



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





Table-47



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





Table-48



S.N. Concentration

(mgl-1)


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





Table-49



S.N. Concentration

(mgl-1)


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





Table-50



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





Table-51



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





Table-52



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





Table-53



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





Table-54



S.N. Concentration

(mgl-1)


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





Table-55



S.N. Concentration

(mgl-1)


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





Table-56



S.N. Concentration

(mgl-1)

Shoots per explant

R1 R2 R3 Mean


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





Table-57



S.N. Concentration

(mgl-1)

Shoots per explant

R1 R2 R3 Mean


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





Table-58


from nodal explants of A. vasica in the year of 2009-2010.

S.N. Concentration

(mgl-1)


R1 R2 R3 Mean


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





Table-59


from nodal explants of A. vasica in the year of 2010-2011.

S.N. Concentration

(mgl-1)


R1 R2 R3 Mean


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





Table-60



S.N. Concentration

(mgl-1)

Nodes per shoot

R1 R2 R3 Mean


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





Table-61



S.N. Concentration

(mgl-1)

Nodes per shoot

R1 R2 R3 Mean


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





Table-62


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


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





Table-63



S.N. Concentration

(mgl-1)

Leaves per shoot

R1 R2 R3 Mean


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





Table-64



S.N. Concentration

(mgl-1)


R1 R2 R3 Mean


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





Table-65



S.N. Concentration

(mgl-1)


R1 R2 R3 Mean


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





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)







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)







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


Normal Regenerated

0

100

200

300

400

500

600


Co

ncen

trati

on

(g

/g)

Fig.4: Estimation of soluble protein in normal plant, regenerated plant


Normal regenerated

0

50

100

150

200

250


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


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).


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.

(i). micropropagation in withania somnifera. ) in shoots...

Documents