Micropropagation of Swertia chirayita -...

Micropropagation

TERI University-Ph.D. Thesis, 2006

Micropropagation of Swertia chirayita

4.1 Introduction

In vitro culture is an efficient method for ex situ conservation of plant

biodiversity and multiplication of the endangered species. It enables the

propagation of the endangered species from minimum plant material that is

available for propagation. S. chirayita, though a widely used medicinal plant, is

now a rare and endangered species, a consequence of its ruthless and destructive

harvesting. Further, low germination percentage and poor viability of the seeds,

long gestation periods and delicate field handling are some of the factors that

discourage commercial cultivation and agro-technology development of S.

chirayita.

Considering the problems beset with conventional propagation of the species

and its endangered status, efforts have been made to propagate the plant by in

vitro techniques. Wawrosch et al. (1999) described adventitious shoot formation

from root explants of in vitro germinated seedlings of S. chirata. The

regenerated shoots were merely 3 mm in length, making handling of cultures a

difficult task. Hence a two-step regeneration protocol was optimised in order to

reduce the problems of callus formation, hyperhydricity and small shoot size.

Ahuja et al. (2003) filed patent for medium composition for faster propagation

of S. chirayita from mature explants. The patent report cited a shoot

multiplication fold that ranged from 11.4 to 26.2, while the rooting frequency

reportedly ranged between 50-80% in time duration of about 8 weeks, and a

survival of 70% was reported.

The present study describes axillary multiplication of S. chirayita from nodal

explants derived from four-week-old aseptic seedlings and successful

establishment of the plantlets in field. In this study, clusters obtained from the

seedling-derived nodal explants gave a multiplication fold of 4.5 every four

weeks. Hundred percent rooting was obtained within four weeks of inoculation

on rooting medium. We also describe here a regeneration protocol from leaf

explants procured from in vitro cultures.

4

Micropropagation

TERI University - Ph.D. Thesis, 2006

4.2 Results

Micropropagation from seedling derived nodal explants

4.2.1. Seed germination and culture establishment

A protocol for the axillary multiplication of S. chirayita was established in this

study. In absence of mature explants, S. chirayita seedlings were used for

procuring initial nodal explants for culture initiation. The surface sterilised, GA3

pre-treated seeds showed 80% germination. In absence of pre-treatment with

GA3, the seeds failed to germinate. These seeds were allowed to grow in half

strength (MS major inorganic salts and iron reduced to half the original MS

strength) semi-solid MS medium supplemented with 3% sucrose, and later,

four-week-old seedlings were used for further in vitro studies.

4.2.2 Culture establishment

4.2.2.1 Effect of type and concentration of cytokinins on shoot bud

induction

Preliminary experiments were conducted to test the effect of equimolar

concentration of BAP, Kn and 2iP with MS basal as control on shoot bud

induction response of nodal explants (Figure 4.1). Sugar concentration was kept

constant at 3% and the medium was gelled with 0.8% agar. Data was recorded

after four weeks of culture period.

The nodal explants cultured on MS basal and on MS medium supplemented

with 3 µM each of BAP, Kn or 2iP separately, showed a significant variation in

terms of number of shoot buds induced per explant (Figure 4.2). A maximum

number of 5.9 and 5.8 shoot buds were induced on MS medium supplemented

with 3 M each of 2iP and BAP, respectively. On the other hand, only 4.9 shoot

buds per explant were induced on medium supplemented with an equimolar

concentration of Kn. The response from nodal explants was stabilised after the

fifth subculture and, by that time, the nodal cultures started forming shoot

clusters (Figure 4.1 c, d). The shoot bud induction response on basal medium

remained almost negligible with only 1.65 shoot buds being formed per explant.

Our results thus conclusively showed that cytokinins were essential for shoot

bud induction and multiplication. The effect of cytokinins on cluster elongation

was however not significant and remained more or less similar to that observed

on the basal medium used as control in the study (Figure 4.2). The nodal

explants initially inoculated on medium supplemented with BAP, Kn and 2iP

were further transferred to similar medium for multiplication. The shoot

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clusters obtained on BAP and Kn enriched medium were found to retain their

vigour and health. However, shoots multiplying on 2iP-enriched medium started

showing signs of hyper-hydricity by the end of the fifth passage. These shoots

were subsequently removed and excluded from further experimental studies.

Further experiments during the culture establishment phase were carried out

with MS medium supplemented with 3 M BAP as constant.

4.2.2.2 Effect of sugar concentration on the multiplication rate

The sugar concentration was varied from 1 to 5% to test its effect on

morphogenic response (Table 4.1). A gradual increase was observed in the

number of shoot buds induced per nodal explant as the sucrose concentration

was increased from 1 to 3%. However, an increase in sucrose concentration

beyond 3% resulted in decline in the number of shoot buds per explant. Hence,

maximum number of 4.27 shoots with an average height of 2.43 cm was

obtained from each nodal explant inoculated on medium with 3% sucrose.

Table 4.1 Effect of the varying sugar concentration on shoot multiplication

Sugar concentration (%) Shoot Buds Shoot length (cm)

1 1.5± 0.35c* 0.85±0.11b

2 2.8±0.46b 1.11±0.34b

3 4.27±0.22a 2.43±0.20a

4 2.96±0.23b 1.23±0.22b

5 2.64±0.26b 1.25±0.09b

LSD (0.05)** 0.58 0.38

On the other hand, multiplication fold reduced to a minimum of 1.5 when the

explants were cultured on medium supplemented with 1% sucrose and the

average cluster height also declined to 0.85 cm. Also, no significant variation in

shoot bud induction per explant was observed on medium supplemented with 2,

4 and 5% sucrose concentrations. Despite the quantitative variation in number

of shoot buds induced, the shoots remained healthy with dark green leaves

irrespective of sucrose concentrations.

* Means followed by the same letter within the column are not significantly different and the

values represent mean±standard deviation ** Least Significant Difference (LSD) helps in determining directly if two means differ

significantly. If the sample means differ more than the LSD, it is considered that the two are

significantly different.

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On the whole, varying sucrose concentration did have a significant effect on

the multiplication fold and shoot elongation as was evident from the highly

significant P values, both for cluster multiplication and elongation.

4.2.2.3 Effect of different gelling agents

The effect of different gelling agents viz. agar, agar gel and gelrite was

ascertained on explant multiplication and elongation (Table 4.2). Maximum

number of shoot buds (5.9/explant) were induced in medium gelled with 0.8%

agar, followed by 0.4% agar-gel and 0.2% gelrite, which resulted in 5 and 4.3

multiplication fold respectively.

However, completely contrasting results were observed for cluster elongation

in this experiment.

Table 4.2 Effect of the different gelling agents on multiplication fold and shoot elongation

Gelling agent Shoot buds Shoot length (cm)

Agar 5.9±0.69a 2.1±0.14b

Agar gel 5±0.10b 2.5±0.28b

Gelrite 4.3±0.06b 3.29±0.17a

LSD (0.05) 0.81 0.42

Shoot elongation obtained on gelrite medium was significantly higher as

compared to that obtained on medium gelled with agar-gel and agar (Figure

4.3). However, vitrification of shoots was observed on gelrite medium. The

variations observed for explant multiplication and shoot elongation on agar and

agar gel medium, were however, found to be statistically non significant, as

evaluated by Duncan’s Multiple Range Test (DMRT). Considering the higher

multiplication obtained on agar gelled medium, agar was employed as the

gelling agent in all subsequent studies.

4.2.3 Culture multiplication

4.2.3.1 Effect of different cytokinins on multiplication

The effect of BAP, 2iP and Kn at concentrations varying between 0.5 to 10.0 M

was studied on multiplication and growth response of shoot clusters (Figure 4.4)

Since, good cluster growth was obtained after the first five to six sub-cultures,

data for multiplication fold was, henceforth, recorded as the number of clusters,

instead of shoot buds (also referred as explants or propagules).

Among the eighteen combinations tested, a maximum of 3.6 multiplication

fold was obtained for the clusters inoculated on medium supplemented with 4

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M BAP (Figure 4.4). It was observed that the type and concentration of

cytokinin had a significant effect on the multiplication fold obtained on each

tested media combination. The lowest multiplication rate of 1.2 was obtained on

MS basal medium preceded by 2-fold multiplication on medium supplemented

with 0.5 M 2iP. For each type of cytokinin tested, the multiplication fold

showed a consistent increase upto a certain level (Figure 4.4), beyond which the

multiplication rate declined.

Also, the type of cytokinin and their concentration had a definite effect on

the cluster height. Among the three cytokinins tested, 2iP showed the most

optimal effect on cluster elongation. The elongation obtained for six

concentrations of 2iP ranged from a minimal of 2.13±0.09 cm to a maximum of

2.73±0.57. In case of BAP, these values ranged between 0.9±0.11 and 2.6±0.44

cm. Thus, the six different concentrations of BAP showed the highest variance in

terms of shoot elongation. At the highest concentration of 10 M BAP, the shoot

clusters showed stunted shoot growth, though, an average multiplication fold of

2.5 was obtained from each cluster. In case of explants inoculated in media with

different concentrations of Kn no variation on shoot elongation was observed.

A prolonged incubation of six weeks in culture medium, instead of the usual

4-week culture period, resulted in significant increase in cluster elongation.

However, this increase in cluster height was attributed to increase in internodal

distance. Hence, even after six weeks of incubation period, there was no increase

in the number of explants or the multiplication fold. Since, 4 M BAP produced

the most desirable results, both in terms of multiplication fold (3.6) and

cluster elongation (2.6 cm), further experiments were conducted with MS

medium gelled with 0.8% agar and supplemented with 3% sucrose

and 4 M BAP as constant. The media composition was employed as control

media while conducting further experiments for fine-tuning the media

composition.

4.2.3.2. Synergistic effect of BAP along with 2iP and Kn on cluster

multiplication and elongation

Synergistic effect of 4 M BAP with varying concentration of 2iP and Kn was

studied for cluster multiplication and elongation. The average multiplication

fold varied significantly (P= 0.0071) with the type and concentration of

cytokinin that was added to the control medium (MS + 4 M BAP + 3% sucrose).

For instance, maximum multiplication fold of 4.5 was obtained on MS + 4 M

BAP + 1.5 M 2iP medium, which was followed by 3.9-fold multiplication on

medium MS + 4 M BAP + 0.5 M 2iP (Table 4.3). A further increase in

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concentration of 2iP to 3 and 4 M, did not increase the multiplication fold,

which remained static at 3.7 for both the concentrations tested. It was observed

that the addition of Kn to control medium (MS + 4 M BAP) did not have any

significant effect on the regeneration response (Table 4.3). At 0.5 and 1.5 M

Kn, multiplication fold of 3.6 and 3.5 were obtained, which decreased to 3 and

2.9 folds when the concentration of kinetin was further increased to 3 and 4 M,

respectively.

The increase in shoot length for the above-discussed medium combinations,

was found to be statistically non- significant with a P value of 0.524.

Table 4.3 Synergistic effects of BAP (benzyl amino purine) with varying concentration of 2iP (6-[ , di-methylallylamino] purine) and Kn (kinetin), on multiplication fold and cluster height after 4 weeks of culture period

Cytokinins ( M) Number of propagules Cluster height (cm)

BAP 2iP Kn

4 0.5 - 3.9±0.43ab 2.2±0.10d

4 1.5 - 4.5±0.40a 2.8±0.3a

4 3 - 3.7±0.30bc 2.7±0.05abc

4 4 - 3.7±0.20bc 2.5±0.05abcd

4 - 0.5 3.0±0.23cd 2.2±0.16cd

4 - 1.5 3.6±0.49bcd 2.7±0.32ab

4 - 3 3.5±0.41bcd 2.4±0.32abcd

4 - 4 2.9±0.2d 2.4±0.32abcd

4 (control) - - 3.4±0.72bcd 2.3±0.21bcd

LSD 0.704 0.40

Maximum elongation of 2.8 and 2.7 cm was obtained for shoot clusters cultured

on control medium supplemented with 1.5 iP and 1.5 M Kn as well as 3 M

2iP separately.

4.2.3.3. Synergistic effect of 4 M BAP along with auxins on growth

of Swertia chirayita

Auxins, namely, IBA, IAA and NAA were tested along with 4 M of BAP to study

their effect on cluster multiplication and elongation. ANOVA for data recorded

for cluster multiplication and elongation showed highly significant P values for

the two variables. Nevertheless, addition of auxins to the control medium did

not have a positive effect on multiplication fold and cluster elongation (Table

4.4).

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Table 4.4 Synergistic effects of BAP and auxins on multiplication. All the concentrations are in M

BAP IBA IAA NAA Number of

propagules#

Cluster height

(cm)#

4 0.5 - - 2.43±0.15 bcd 2.35±0.36 ab

4 1 - - 2.5±0.2 bc 2.59±0.24 a

4 - 0.5 - 2.79±0.13 ab 2.53±0.28 ab

4 - 1 - 2.6±0.28 b 2.15±0.18b

4 - - 0.5 1.84±0.15 cde 1.41±0.17c

4 - - 1 1.75± 0.3 de 1.12±0.07 c

4 (control) - - - 3.32±0.9a 2.6±0.17 a

Basal medium - - - 1.65±0.21e 1.28±0.02 c

LSD (0.05) 0.65 0.38

Whereas 3.3 multiplication fold was obtained on the control medium (MS+ 4

M BAP), 2.79 fold multiplication was obtained on medium combination MS + 4

M BAP + 0.5 M IAA, followed by 2.6 multiplication fold on MS + 4 M + 1 M

IAA. The cluster height recorded for the two concentrations of IAA were 2.53

and 2.15 cm, respectively.

IBA when tested at 0.5 and 1 M concentration, along with 4 M BAP gave a

multiplication fold of 2.43 and 2.5, respectively. The cluster height recorded at

these two concentrations averaged to 2.35 and 2.59, respectively.

The effect of supplementing the control medium (MS + 4 M BAP) with 0.5

and 1.0 M NAA, were inhibitory both in terms of multiplication fold and cluster

elongation. Multiplication fold of 1.84 and 1.75 were obtained at 0.5 and 1.0 M

NAA and cluster height of 1.41 and 1.12 were recorded at these two

concentration, respectively. The cultures on NAA supplemented medium also

exhibited basal callusing.

4.2.3.4 Effect of casein hydrolysate and adenine on cluster


The addition of the two supplements, namely, adenine and casein hydrolysate

did not have a promotory effect on the overall regeneration response. Adenine

was added to the multiplication medium at 125 mgl-1 and 250 mgl-1

concentrations. Similarly, casein hydrolysate was added to the control medium

at 250 and 500 mgl-1 concentrations.

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There was a general decline in multiplication rate when any of the two

additives were added in either of the two concentrations. In fact, the analysis of

variance revealed no significant effect of additives on cluster multiplication

(P=0.047). In case of cluster elongation, the effect of additives was again not

stimulatory, though, the variation obtained on different medium combinations

varied significantly from each other (Table 4.5). A reduction in cluster height

was observed when adenine was added to multiplication medium at either of the

two concentrations. In case of casein hydrolysate, at 250 mgl-1 concentration the

cluster height remained similar to that obtained on the multiplication medium

without additives (2.6); though, a further increase in concentration culminated

in decline of cluster height to 1.6 cm.

Table 4.5 Effect of casein hydrolysate and adenine on shoot multiplication

Adenine (mgl-1) CH (mgl-1) Number of

propagules

Shoot length (cm)

125 - 2.15±0.2b 1.38±0.1c

250 - 2.3±0.2b 1.68±0.6b

- 250 2.7±0.5ab 2.6±0.1a

- 500 2.3±0.2b 1.6±0.2bc

Control medium - 3.32±0.9a 2.6±0.17a

LSD (0.05) 0.79 0.26

Finally, it was concluded that in the present study, of the tested media, MS

medium supplemented with 4 M BAP and 1.5 M 2iP was the most optimal

multiplication medium, producing an average of 4.5 clusters per explant with

an average height of 2.8 cm (Table 4.3). Shoots obtained from the above

experiments were subsequently used for standardising rooting in S. chirayita.

4.2.4 Rooting

4.2.4.1 Effect of different auxins and activated charcoal on root

induction

A two-factor experiment was designed to study the effect of auxins as well as

activated charcoal on root induction in S. chirayita plantlets. MS ½ (major

inorganic salt and iron reduced to half the original MS strength) with 3% sucrose

was initially used for optimising the rooting medium and root inducing growth

regulators (IAA, NAA, IBA, Figure 4.5) were tested at an equimolar

concentration of 1 M. It was observed that on MS ½ basal medium only a few

root initials were formed which gained a length of 0.3 cm only even after four

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weeks of incubation. On the contrary, 100% rooting was observed on MS ½

medium supplemented with 1 M of IAA, IBA or NAA separately.

In the present experiment MS ½ + 1 M NAA was found most optimal for

root induction and an average of 6.5 roots per shoot were induced, which gained

a length of 0.69 cm, after 4 weeks of incubation (Table 4.6). In case of medium

supplemented with auxins IBA and IAA, the root length remained more or less

consistent for the two media (0.49 and 0.50 respectively), however the number

of roots induced on IBA supplemented medium (11.5) far exceeded that obtained

on IAA or NAA supplemented media (7.8 and 6.5 respectively).

Table 4.6 Duncan’s multiple range tests for the effect of different auxins on root induction

Growth Regulators (1 M)

Shoot length (cm) Root length (cm) Root Number

Basal 2.33± 0.10 ab 0.30±0.19 c 2.83±1.9c

IBA 2.53±0.36a 0.49± 0.12b 11.5±2.4a

IAA 2.56± 0.32a 0.50±0.14b 7.8±0.75b

NAA 2.26± 0.18b 0.69± 0.23a 6.5±1.04b

LSD 0.05 0.22 0.14 1.73

The analysis of variance of the recorded experimental data revealed that the

influence of auxins was highly significant both for the number of roots induced

(P= 0.000) and the root length (P=0.0004) recorded on each media

combination.

The influence of auxins on shoot elongation was however not highly

significant as is evident from ANOVA which gave a P value of 0.029. The shoots

inoculated on IBA and IAA supplemented medium grew to a length of 2.5 cm

each, while those inoculated on NAA supplemented medium averaged to 2.2 cm,

which was approximately the same as recorded for shoots cultured on MS ½

basal medium (2.3 cm).

This experiment also highlighted the influence of activated charcoal on the

quality of roots. The shoots inoculated on medium without activated charcoal

developed basal callusing and the roots remained thick, small and tuberous

(Figure 4.6a). Addition of activated charcoal improved the quality of roots. The

roots formed were longer and thinner (Figure 4.6b). The basal callusing

observed in shoots inoculated on medium devoid of activated charcoal was

conspicuously absent in media combinations supplemented with activated

charcoal. Hence, in the subsequent rooting experiments activated charcoal

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supplemented medium was chosen for further experimentation to avoid basal

callusing.

Table 4.7 Duncan’s multiple range test for the effect of activated charcoal on root formation in Swertia chirayita

Medium Shoot length# (cm)

Root length# (cm) Root Number#

Without activated

charcoal

2.58±0.28a 0.37±0.17b 7.75±4.35a

With activated charcoal 2.26±0.15b 0.61±0.19a 6.5±2.53a

LSD 0.05 0.158 0.10 1.22

Analyses of variance for the recorded data showed that activated charcoal had a

highly significant effect on shoot length (P= 0.0005) and root length (P=0.0001,

Table 4.7). The average root length on medium containing activated charcoal

was 0.61 cm as compared to 0.37 cm for roots formed on medium without

activated charcoal. Similarly, shoots inoculated on medium without activated

charcoal gained an average length of 2.58 cm as compared to 2.26 cm of average

length of shoots inoculated on medium with activated charcoal. However, the

number of roots inoculated on medium with and without activated charcoal (7.7

and 6.5, respectively) remained statistically non-significant as is also deduced

from the ANOVA results. The interaction of growth regulators and activated

charcoal was found to be non significant for all the three variables (Table 4.8).

Further experiments were conducted with MS ½ medium supplemented with 1

M NAA, and 500 mgl-1 activated charcoal.

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Table 4.8 Analysis of variance (ANOVA) for the effect of auxins (growth regulators) and activated charcoal on the theree variables namely, shoot length, root length and root number

Source Shoot length Root length Root numbers

Degree

of

freedom

Mean

square

Probability Degree

of

freedom

Mean

square

Probability Degree

of

freedom

Mean

square

Probability

Individual

factor

Growth

regulator

(GR)

3 0.13 0.029* 3 0.14 0.0004*** 3 76.8 0.000***

Activated

charcoal

(AC)

1 0.63 0.0005*** 1 0.34 0.0001*** 1 8.1 0.604ns

Interaction

(GR X AC) 3 0.075 0.12ns

3 0.029 0.132ns

3 5.5 0.076ns

*** Very highly significant P<0.0001; ** highly significant P < 0.001, * less significant P<0.01; ns non significant

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4.2.4.2 Effect of different medium strengths on rooting

With the above mentioned factors (500 mgl-1 activated charcoal + 1 M NAA)

as constant, MS strengths were varied to study the effect of salt strengths on

root induction. The salt strengths were varied as shown in Table 4.9. The

roots induced on MS full strength medium were found to be small, tuberous

and were formed as dense clusters when compared to those induced on

medium with reduced MS salt strength. It was observed that shoot length

remained more or less same as the salt strengths were reduced from full to

half and quarter strengths (2.9, 2.4 and 2.7 cm, respectively); however, a

further decrease in salt strengths to one-eighth and one-tenth of the original

had a bolting effect on shoot length which shot to 4.76 and 4.38 cm,

respectively. This induced lankiness in the shoots, thus, adversely affecting

the plant vigour. These shoots were not suitable for further hardening due to

their lankiness.

Table 4.9 Effect of the different MS strengths on root induction and plant growth

Variation in MS

major and Iron

Medium

code

Shoot length

(cm)

Root length

(cm)

Root

number

Full SSR1 2.93±0.36b 0.59±0.07b 9.16±1.5a

Half SSR2 2.49±0.13b 1.18±0.32a 9.6±0.89a

Quarter SSR3 2.74±0.47b 1.10±0.35a 11.16±1.24a

One-eighth SSR4 4.76±0.73a 0.84±0.52ab 10.2±2.5a

One-tenth SSR5 4.38±0.55a 0.53±0.27b 10.93±2.7a

MS organics only SSR6 2.78±0.39b 0c 0b

No salt (water agar) SSR7 1.76±0.07c 0c 0b

LSD (0.05) - 0.51 0.476 1.92

The number of roots induced on these media combinations was however more

or less the same, with a lowest of 9 and a highest of 11 roots. However, the

variations in the salt strengths did induce a variable response in terms of root

length. The length of roots increased on MS ½ and ¼ medium (1.18 and 1.1,

respectively) when compared to root length on MS full (0.59). A further

reduction in salt strength did not result in further increase in root length,

instead the root length steadily declined from 0.84 to 0.53 cm as salt strength

was decreased from MS 1/8 to MS 1/10. In medium combination coded SSR6,

MS inorganic salts were completely eliminated and only MS organic salts were

added. On the other hand in medium coded SSR7 both, MS inorganic and

organic salts were completely eliminated (water agar medium). However,

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sucrose + 1 M NAA was supplemented in all the medium combinations tested.

In the later two medium combinations (lacking MS salts) a conspicuous absence

of root induction was observed. On the basis of these observations, MS ½ was

considered most optimal for root induction in S. chirayita.

4.2.4.3 Effect of sugar concentration on root induction

The effect of sucrose concentration (1-3%) was tested. It was observed that on

increasing the sugar concentration from 1 to 3% in the rooting medium, a

distinct increase in shoot length from 1.59 to 2.4 was observed. The number (5,

5.9 and 6.9) of induced roots, however, remained statistically similar at 1, 1.5

and 2% sugar concentration, respectively (Table 4.10). Only at 3% sucrose

concentration a statistically significant increase was observed in the number of

roots formed. However, root length was significantly affected by sucrose

concentration as compared to the root numbers. It was observed that root length

increased from 0.34 to 1 and 1.5 on medium supplemented with 1, 1.5 and 2%

sucrose, respectively. A further increase to 3% did not enhance the root length

and an average of 0.96 cm long roots were recorded. 2% sucrose concentration

was thus considered to be the most optimal for root induction with respect to

root number and root length.

Table 4.10 Effect of the varying sugar concentration on root induction and plant growth

Sucrose

concentration (%)

Root length (cm) Root number Shoot length (cm)

1 0.34±0.2 b 5.28±1.23b 1.59±0.31b

1.5 1.04± 0.4 ab 5.94±1.08b 1.97±0.41ab

2 1.49± 0.6a 6.96±0.6b 2.43±0.6a

3 0.96± 0.61 ab 9.10±1.71a 2.44±0.1a

LSD (0.05) 0.751 1.85 0.62

4.2.4.4 Effect of the medium content in culture vessel on root

induction

An attempt was made to evaluate the effect of the medium volume in the culture

vessels on root induction as well as on plant vigour. The volume of medium in

the culture vessel was varied from 20 ml to 60 ml and 15 shoots were inoculated

in each culture vessel. It was observed that the amount of medium in the culture

vessel does not affect root induction in S. chirayita (Table 4.11). The number of

roots induced and the length gained by them remained statistically non

significant for 20, 30 and 40 ml of medium pouring. However, 20 and 30 ml of

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the medium in 400 ml culture jars dried very soon due to exhaustion of media

and this in turn adversely affected the growth of the plant. The leaves dried and

the plants wilted within the culture period of 4 weeks itself. In case of 40, 50 and

60 ml of medium, the plant remained healthy. In fact, a significant increase in

length was observed in shoots (3.1 and 3.05 respectively) inoculated in culture

vessels with 50 and 60 ml of medium as compared to an average of 2.7 cm long

plantlets in 40 ml medium. However, this increase in shoot length resulted in

lanky plants, which adversely affects the hardening process. Therefore, it was

concluded that 40 ml of medium volume was optimal for 15 shoots in each

culture vessel.

Table 4.11 Effect of the different volumes of medium poured in the culture vessel on percentage rooting

Medium Volume

(ml)

Shoot length

(cm)

Root length (cm) Root number

20 2±0.63b 1.21±0.6a 4.6±0.02b

30 2.23±0.12b 1.45±0.32a 6.48±0.85b

40 2.75±0.39b 0.954±0.23a 6.84±2.1b

50 3.10±0.29a 1.23±0.2a 10.27±1.79a

60 3.05±0.3a 1.18±0.26a 12.05±1.2S

LSD (0.05) 0.51 0.476 2.20

4.2.5 Acclimatisation and field transplantations

4.2.5.1 Ex vitro hardening and effect of different months on the

survival rate

At a commercial scale hardening is carried out in the green house so as to

harden plants in large numbers. The green house experiments were conducted at

Gual Pahari (Gurgaon, Haryana), where the temperatures vary throughout the

year. The ambient temperature extremes recorded for the region varied from a

maximum of 40 0C to a minimum of 3 0C round the year. The temperature

conditions were maintained within a range of 11 to 27 0C throughout the year in

the green house. The rooted plants were, therefore, taken out during different

months of the year to record percentage survival of the plants. A minimal

survival rate of 20-35% was recorded during the months of May, June, July and

August (Table 4.12). However, the plants taken out after September showed a

substantial increase in survival percentage.

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Table 4.12 Acclimatisation success rate of the micropropagated S. chirayita plantlets in green house at Gual pahari, Haryana, India, during different months of the year

Month Temperature (oC) Relative humidity (%)

In Green House

Survival

percentage

Ambient Green house Minimum Maximum

Minimum Maximum Minimum Maximum

Dec 5.2 26 11.25 16.1 77.25 80 93.96

Jan 3 22.3 11.75 18.5 63.75 78.5 95.4

Feb 8.3 28.2 14.8 19.8 63 78.8 96.4

March 16.4 32.3 17.25 20 68.5 80.5 97

April 21.2 38.4 18.4 21.6 65.2 80.4 90

May 24.7 39.3 24.3 27.0 80.0 81.3 22

June 25.5 37 25 27 76.75 80.3 20

July 26 34.6 24.75 26.8 77.75 80 26

August 25.3 35.8 24.25 27.2 80 81.5 35

Sept 22 38.1 21.25 24.8 78 80.5 80

Oct-Nov 13.8 17.9 20 23 77.25 80 86

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In fact, a marked increase in percent survival from 80% (September) to 97%

(March) was observed. Hence, based on these observations it was concluded that

months of December to April are most conducive for successful hardening of S.

chirayita plantlets in green house conditions. Figure 4.7a shows tissue-cultured

plantlets freshly transferred to polybags and kept in green house for

acclimatisation. Figure 4.7b represents the fully hardened plants in nursery

ready for transplantation to the field. A total of 3596 plants were subjected to

hardening during the months of December to March, and high survival

percentage of 94.5 was recorded. Finally, a total of 3400 S. chirayita plantlets

were transferred to the shade area. These plants were taken to Mukteshwar

(Uttaranchal state) at an altitude of 2286 m above sea level for final

transplantation in March and April.

4.2.5.2 In vitro hardening

During the hardening trials in green house lowest survival percentage was

observed from May to August, when temperatures ranged between maximum of

27 0C to a minimum of 24 0C. Hence, in vitro hardening trials were conducted in

growth room with controlled conditions of temperature (22 2 0C) during the

months of May and August. A successful hardening response of 95.5% and

92.7% was obtained in May and August, respectively. These plants were

transplanted at Mandal (1568 m above sea level) and Mukteshwar (2286 m

above sea level) in the months of June and September. Figure 4.8 shows the

plantlets during in vitro hardening and Figure 4.9 represents the different stages

from rooting to successful hardening of S. chirayita plantlets.

4.2.5.3 Effect of different potting mixes on initial survival and plant

growth

Different combination of coir-peat and soil were tested during transplantation

trials. However, no significant variation was found with respect to percent

survival in the different potting mixes tested and a high survival percentage of

97-99 was recorded. However, the growth of the plant was found to vary

depending on the potting mixes. Highest plant elongation (1.9 cm) was achieved

in potting mixes of coir peat and soil in varying ratios of 1:2, 1:4 and 1:5. When

soil and coir peat were used solely as potting mixes, the plant height did not

increase beyond 1.5 cm. In case of potting mix containing soil and FYM in the

ratio of 4:1, there was a substantial increase in the number of leaves produced

per plant (12) as compared to an average of 8 leaves per plant in other potting

mixes (Table 4.13). Since the potting mix containing coir peat and soil in the

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ratio of 1:2 recorded a survival of 99% plants which gained an average height 1.9

cm, it was chosen for large scale hardening of S. chirayita plantlets. Based on

the results of the previous experiment (4.2.5.1), this particular experiment was

carried out in the month of March (green house temperature ranged between 17

to 20 oC), during which the highest hardening survival of 97% was recorded.

Table 4.13 Effect of different potting mixes on the survival rate and other growth statistics.

The values represent mean±standard deviation of 50 plantlets in three repeated experiments

Potting mixes

Coir peat: soil

Survival rate

Plant height (cm) Pair of leaves per plant

Leaf length (cm)

1:0 97% 1.5±0.79b 4.3±0.49b 2.6±0.67c

1:1 99% 1.75±0.36ab 4.25±0.57b 3.85±1.17ab

1:2 99% 1.93±0.33a 4.5±0.52b 4.5±0.67a

1:4 98% 1.93±0.33a 4.16±0.38b 4.14±0.76ab

1:5 99% 1.9±0.5a 4.0±0.0b 3.9±0.57ab

0:1 99% 1.47±0.3b 4.3±0.48b 3.94±0.57ab

Soil 4: FYM1 97% 1.5±0.36b 6.08±0.79a 2.6±0.58c

LSD (0.05) - 0.31 0.43 0.63

4.2.5.4 Transplantation to field

S. chirayita plants, acclimatised in greenhouse (at Micropropagation

Technology Park, Gurgaon, Haryana) were taken to Mandal (Uttaranchal, India)

for transplantation. S. chirayita is a plant of high altitude and therefore,

Mandal, located at an altitude of 1568 m above sea level in the Himalayas

(Figure 1.1) served as an ideal field location.

Table 4.14 Growth parameters of S. chirayita recorded six months after transplantation to

the field at Mandal. The values represent the mean ± SD for 50 plants per plot

Plot No. Biomass (gm) Area (sq. inch) Plant height (cm) Root length (cm) Survival (%)

Plot I 179±14.2 235±14.5 18.4± 0.86 15.8±0.35 86

Plot II 106± 9.1 241±16.3 15.3±0.50 12.3±0.35 80

Plot III 83.3±1.9 259±45.2 12.25±1.1 11.3±0.73 16

Figure 4.10a shows the well-hardened plants freshly transferred to the field at

Mandal. An average survival of 65.3% was recorded after six months of growth

(Table 4.14). The plots were made within the poly-houses and in one particular

plot a very low survival of 16% was recorded whereas in the remaining two 80

and 86% survival was recorded. The third plot was towards the lower side of the

hill, which remained exposed to the sun throughout the day. This may have

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likely resulted in wilting of the plantlets at an early stage, immediately after

transplantation, thus resulting in the high mortality in the initial stages itself.

The results emphasise the significance of initial care required while handling the

tissue culture raised plants.

Besides recording the shoot length and area occupied by the plant,

destructive harvesting was done to determine the root length and the average

biomass of the plants. The root length averaged from 11 to 15 cm, whereas,

biomass per plant (fresh weight) averaged to 122 gm. Figure 4.10b show the

transplanted S. chirayita plantlets after six months of growth. Figure 4.11 gives a

comparative picture of the micropropagated plants at three months, six months

and after one year of growth. After the first year of growth the plant gained an

average of two-feet height as is evident in the figure. After bolting, the plant

enters into flowering phase (Figure 4.12 a). The pictures represent the flowering

twig (Figure 4.12 a, c) and a closer view of the flower (Figure 4.12 d). Figure

4.12b represents the root (the most potent part of the plant) after the completion

of flowering season. These illustrations represent the success of transplantation

trial carried out at Mandal.

Micropropagation


A schematic presentation for mass propagation of Swertia chirayita

**94.5% hardening represents the average of acclimatisation success obtained

for the months of December to April, which were found most conducive for

hardening in greenhouse conditions

Seeds

Transfer of four week-old seedlings to MS + 3µM BAP + 3%sugar + 0.8% agar

Germination on MS ½ medium + 3%sugar + 0.8% agar

(80% germination)

Subculture on MS with 4 µM BAP + 1.5 µM 2iP+ 3%sucrose + 0.8% agar (every 4 wk @ 4.5 multiplication fold)

Transfer of rootable shoots to MS ½ + 2% sucrose + 1µM NAA + 500 mgl-1 activated charcoal + 0.8% agar (4 wk)

(100% rooting)

Hardening of rooted shoots in green house in poly-bags containing

potting mix of coir-peat and soil (1:2) (15 days, 94.5% hardening**)

success)

Maintenance of hardened plants in nursery before final field trials at higher altitudes (temperate conditions)

Micropropagation


Production plan for 100,000 plants of Swertia chirayita - a schematic presentation

A surplus of 7000 plants was produced, keeping margin for contamination and

mortality during hardening.

Multiplication cycle (4 culture passages of 4 wk each) with 4.5 fold multiplication

21,200 shoot clusters

Hardening in green house

Final passage before rooting

12 nodal cultures

Establishment phase (5 passages of 4 wk each)

52 shoot clusters

Transfer of single shoots to rooting medium (4 wk)

(21,200*5=1,06,000 shoots)

12 nodal explants derived from four-week-old in vitro germinated

seedlings

(Week 1)

(4 wk)

(24 wk)

(40 wk)

(44 wk)

(48 wk)

(52 wk)

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4.2.6 Cost Calculations

The cost of producing micropropagated plants at the Micropropagation

Technology Park (MTP) of The Energy and Resources Institute (TERI) have

been divided into two parts, namely, the direct cost and the indirect cost.

4.2.6.1 Direct Cost

Direct cost includes media cost, inoculation cost as well as the hardening cost,

at full occupancy and the costs incurred towards manpower and electricity

consumption. The parameters that decide the direct cost of the plant are

multiplication fold, rooting percentage and the hardening survival. The

parameters taken into consideration during production of 100,000 plants of S.

chirayita have been summarised below:

Parameters

Multiplication fold 4.5 in four weeks

Rooting percentage 100%

Hardening survival percentage 95%

Number of clusters in multiplication 5 shoot-clusters per culture

vessel

Number of shoots in rooting 15 shoots per culture vessel

Considering a 5% loss during the hardening stage and keeping a margin of 1%

for contamination, production of 1,07,00o plants was targeted in order to

produce a total of 1,00,000 successfully hardened plants of chiretta. It was

estimated from back calculations that a minimum of thirteen explants would be

required to eventually produce 1,07,000 rooted plants in eleven culture passages

(5 culture passages during establishment phase + 5 multiplication cycles + 1

rooting passage). Thus, during the establishment phase 13 nodal explants were

initially taken from 13 seedlings. The nodal explants established themselves into

clusters at the end of the fifth culture. In case of nodal explants 6 explants per

culture vessels were inoculated during the culture establishment stage. In case of

shoot clusters, five clusters were inoculated per culture vessel and a total of

fifteen rootable shoots were inoculated per culture vessel. Media pouring in each

jar was maintained at 40 ml throughout the production period. Table 4.15 gives

Inoculation cost includes the cost of consumables such as tissue paper, blades, rectified

spirit, sterile water, cling films, papers, sterilisation beads, savlon, garbage bag etc.

Culture vessel here refers to 400 ml jars used for commercial production of the chiretta

plants

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details regarding the media cosumption and number of culture vessels used

during production of 100,000 plants of S. chirayita.

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Table 4.15 Details of medium consumption and number of culture vessels used during production of 100,000 plants of Swertia chirayita

Stage Culture

passage

Number of

shoots/clusters

No. of culture vessels

(400 ml jars)

Medium consumption

(in litres, @ 40 ml/

culture vessel)

Establishment

phase

5 culture

passages

13 nodal explants 2*5 (6 nodal

explants/culture vessel)

0.4

Multiplication

phase

6th 58 clusters 12 (5 clusters/culture

vessel)

0.5

7th 261 clusters 52 2.08

8th 1174 clusters 235 9.4

9th 5284 clusters 1057 42.28

10th 23,778 clusters 4756 190.24

Rooting 11th 23,777*5=1,07,000

shoots

7133 (15 shoots/culture

vessel)

285.32

Grand total 13,255.04 530.2 litres

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A. Manpower cost Work area (working

efficiency)

Man days (= work load/

working efficiency)

Rate (Rs/day) Cost incurred (Rs)

Media lab (media preparation

@ 135 l/ day)

3.92 (=530.2/135) 615 2,415.40

Washing area (jar washing @

438 jars/day)

30.19 (=13225.04/438) 138.46 4,180.70

Inoculation room (35 jars /

day)

377.85 (=13225.04/35) 615 232,382.8

Supervisor clean area 58 1201 69,658.00

Transfer room labour (1500

plantlets/day)

71.33 (=107000/1500) 138.46 9,876.80

Maintenance + look after 30 138.46 4,153.80

Supervisor in green house 30 615 18,450.00

Subtotal of A 341,117.50

Mandays for clean area supervisor were allocated on the basis of the proportion of plant growth room occupied by the chiretta plants for the 11 in vitro culture

passages during the production of 100,000 plants. The capacity of plant growth room at MTP is 70,000 jars. Thus, 13,255 jars of chiretta occupied 18.89%

(=13255/70000) of the plant growth room. Hence the mandays were calculated as a product of the proportion of area occupied and the total number of days

during which the growth room was actually used.

The manpower used in the green house area was calculated as the product of the proportion of greenhouse facility used for hardening 100,000 plants for a

period of 15 days. Since the 100,000 plants can be hardened in two lots (average capacity of green house at MTP is 50,000 plants) over a period of 15 days

each, 30 man-days each of supervisor and casual labour (for maintenance and look after) would be required.

Micropropagation


B. Electricity Electricity consumption

during

Cost (Rs) Cost incurred (Rs)

Autoclaving

Dry cycle

Media cycle

Unit cost (Rs)

9

9

Units consumed per

cycle

22.5

45

Total number of cycles

20

32

4,050 (=9*22.5*20)

12,960 (=9*45*32)

Laminar flow (two

seater)

Electricity cost / flow /

day

Flow required for - -

184.93 189 days - 34,951.77 (=184.93*189)

Plant growth room 0.17 / jar/ day 13225.04 jars - 2,248.25

(=0.17*13225.04)

Green house 1.5 / plant 107000 plants 160,500 (=1.5*107000)

Subtotal of B 214,710.02

C. Media cost

Break up Cost per litre (Rs) Total media (l) Cost incurred (Rs)

LPG cost towards media melting 0.6 530.2 318.12

Material cost of media 28.1 530.2 14,898.62

Subtotal of C 15,216.74

Calculated on the basis of the number of sterile petri plates required during inoculations of 13225.04 jars

Calculated on the basis of the total number of media jars to be autoclaved and the capacity of the autoclave

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D. Inoculation cost Cost / jar/ day (Rs) Jars/day Cost incurred (Rs)

37.85 377.85 (= total number

of jars / jars inoculated per day)

14301.93

Subtotal of D 14301.93

Direct cost of 100,000 chiretta plant = grand total of A + B + C + D

= Rs 283949 + Rs 214710.02 + Rs 15216.74 + Rs 341117.5

= Rs 585346.2

Direct cost of one chiretta plant = (585346.2/100000) = Rs 5.85

4.2.6.2 Indirect Cost

The indirect cost towards production of micropropagated plants at a commercial

scale is an important parameter to be considered. It includes the administrative

cost, depreciation of the infrastructure and equipment, maintenance cost,

professional cost associated with supervision as well as transport of the plants to

the transplantation site. The cost of following was taken into account while

calculating the indirect cost of the micropropagated chiretta plants

A. Annual administrative cost

B. Annual maintenance cost

C. Depreciation of building

D. Depreciation of equipment

E. Transport

F. Professional cost

A. Annual administrative cost

The annual administrative and maintenance cost on a yearly basis were

calculated separately from the expenses incurred round the year, during which

an average of 140, 000 plants (70% of the production capacity) were produced.

The administrative cost totalled to Rs. 500,000.

A. Annual administrative cost Yearly

Administartive

cost (Rs)

Cost per plant in Rs

(=total yearly cost / total

number of plants

produced round the year)

Total

plants

produced

Cost incurred in Rs

(= cost / plant *

total number of

plants produced)

500,000 0.357 (=500000/140000) 107,000 38,214.28

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B. Annual repair and maintenance cost Yearly

Maintenance

cost (Rs)

Cost per plant in Rs

(=total yearly cost

/ total number of

plants produced

round the year)

Total

plants

produced

Cost incurred

in Rs (= cost

/ plant *

total number

of plants

produced)

673,162 0.48

(=673162/140000)

107,000 38,246.88

C. Depreciation of building

The original cost of MTP is estimated at Rs. 1,50,51,000. A depreciation of 5%

flat was levied on this cost and was added accordingly towards production cost

of the plants.

Original

cost (Rs)

Yearly

depreciation

(@ 5% flat)

Cost per plant

(Rs)

Total Cost incurred (Rs)

1,50,51,000 752,550 0.537

(=722550/140000)

57,516.32

(=[722550/140000]/107000)

D. Depreciation of equipment

Total cost of equipments

Equipment Cost (Rs)

Hot air oven 36,575

Refrigerator 36,519

Refrigerator 38,000

Glass bead steriliser 19,855

pH meter 10,044

Laminar flow 248,240

Autoclave 500,590

Autoclave 280,732

Grand total 1,170,555

The depreciation cost on equipments at a rate of 10% flat comes out as Original

cost

(Rs)

Yearly

depreciation

(@ 10 %

flat)

Cost per plant

(Rs)

Total Cost incurred (Rs)

1,170,555 1,17,055.5 0.0836

(=117055.5/140000)

8,946.378

(=[117055.5/140000]/107000)

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E. Transport

The transport cost included the cost incurred towards loading of 100,000 plants

and transportation. This has been calculated as follows:

Loading cost Loading

efficiency

Man days

(=Total

plants/

working

efficiency)

Rate (Rs) Cost incurred (Rs)

1000

plants/man-

hour

13.3 man days 138.46 1846.1

The transport cost of 100,000 plants was calculated at Rs. 0.40 per plant.

Vehicle cost Cost per (Rs) Total

plants

transported

Cost incurred

(= cost /

plant * total

number of

plants

transported)

0.40 100,000 40,000

Total transport cost = loading cost + vehicle cost = Rs 1846.1 + Rs 40000 = Rs

41846.1

F. Professional cost Man days Rate per day (Rs) Cost incurred (Rs)

26 8457.20 219,887.20

Indirect cost of 100,000 chiretta plant = grand total of A + B + C + D + E + F

= Rs 38214.28 + Rs 11464.28 + Rs 57516.32 + Rs 8946.37 + Rs 41846.1 + Rs

219,887.20

= Indirect cost of one chiretta plant = (417909.70 / 100000) = Rs 4.17

4.2.6.3 Total Cost

Per plant cost of chiretta thus turns out to be = direct cost + indirect cost

= Rs 5.85 + Rs 4.17

Taken as 10% of the 52 weeks (260 man days) required for producing 100,000 chiretta

plants

Micropropagation


= Rs 10.02

Based on these calculations it can be inferred that the production of chiretta is

not a commercially feasible proposition. However, the present protocol can be

used for initial bulking up of the chiretta plantlets, which can later be

reintroduced in their natural habitat and established in herbal gardens as a

viable conservation measure. Once populations of elite chiretta germplasm has

been established, further propagation can be undertaken through seeds.

Micropropagation


Regeneration in Swertia chirayita from leaf explants

4.2.7.1 Response of different explants for regeneration

Leaf, internode and root explants of S. chirayita from in vitro grown plants were

tested for callus induction and regeneration response. The root explants and

internodal segments were cut into ca. 1 cm segments. The leaf explants were cut

into ca. 1 cm X 1 cm sections and were inoculated on medium slants. The MS

medium was modified for the regeneration experiments; with major and iron

salts reduced to half of the original MS salt strength. The rest of the components

were kept as the original MS medium. In a preliminary experiment, effect of

2,4,5-T (0.5 to 4 mg l-1) along with 0.5 mgl-1 BAP was tested for callus and shoot

bud induction response. The explants showing viable response were transferred

to fresh medium of similar composition after six weeks. Higher concentration of

2,4,5-T, ranging from 1-4 mgl-1 resulted in water uptake (visible as swelling of

explants) as well as cell proliferation (expansion of explants). The explants

remained green for 12 weeks (2 culture passages) and subsequently turned

brown and died, without showing any evidence of callus induction or

organogenesis. The root explants and internodal segments showed no response

at 0.5 mg l-1 2,4,5-T, though in case of leaf explants, organogenesis and callus

induction was observed.

Different combinations of auxins and cytokinins were also tested for the

regeneration response. On the medium combination supplemented with NAA

and IBA, each at 0.75 mgl-1 concentrations, shoot buds were induced in root

explants only (Figure 4.13). However, on repeating the experiments, it was

observed that this response was sporadic and not reproducible. These

preliminary experiments enabled selection of leaf explants for further

experimentation with MS half supplemented with BAP and 2,4,5-T (0.5 mgl-1

each).

4.2.6.2 Effect of varying concentration of BAP on callus induction

and shoot regeneration from leaf explants

The effect of varying concentrations of BAP

The BAP concentration was also varied to study its effect on shoot regeneration.

The regeneration response increased with increase in concentration of BAP from

0.25 to 2 mgl-1 (Figure 4.14). However, an increase in BAP concentration beyond

2 mgl-1 resulted in a decline in percentage regeneration. Callus formation was

conspicuous in most of the media combinations tested. On the other hand, in

absence of any growth regulator, the leaf explants exhibited minor curling and

Micropropagation


retained their green colour; however, no callus formation was noticed. The leaf

explants inoculated on medium supplemented with BAP at concentrations

ranging from 0.25 to 2.0 mgl-1 showed yellowish nodular growth, callus

induction and formation of root like outgrowths. Figure 4.15a illustrates the leaf

explant exhibiting initial swelling on regeneration medium whereas Figure 4.15b

show callus formed on leaf explant inoculated on MS 1/2 medium supplemented

with 2 mgl-1 BAP and 0.5 ml-1 2,4,5-T.

Figure 4.14 shows the graphical representation of the effect of varying

concentration of BAP on callus formation and shoot bud induction. At a

concentration range of 0.25 to 2.0 mgl-1, an increase in the percent regeneration

response from 16 to 76% was revealed. At 0.5, 1.0 and 2.0 mgl-1 concentrations of

BAP the leaf explants showed a regeneration response of 30, 36 and 76%,

respectively. The maximum numbers of 10 shoot buds per explant were induced

on medium supplemented with 1 and 2 mgl-1 BAP. A further increase in BAP

concentration to 3 mgl-1 led to a decline in regeneration response and only 16%

explants exhibited callus and shoot bud induction, whereas, the rest eventually

turned brown. A further increase of BAP to 4 mgl-1 led to browning of all the

explants inoculated on the medium.

The leaf explants inoculated on medium supplemented with 0.5 mgl-1, 1.0

mgl-1 and 2.0 mgl-1 of BAP when transferred to MS 1/2 basal medium (Figure

4.16), showed a substantial increase in shoots buds and shoot length. The

numbers of shoot buds increased from 8.3 (MS ½ + 0.5 mgl-1 2,4,5-T + 0.5 mgl-

1 BAP), 10.3 (MS ½ + 0.5 mgl-1 2,4,5-T + 1.0 mgl-1 BAP) and 10.7 (MS ½ + 0.5

mgl-1 2,4,5-T + 2 mgl-1 BAP) to 24, 34.8 and 43, respectively when transferred

from regeneration medium, as detailed in parenthesis, to MS 1/2 basal medium.

Figure 4.17 gives a comparison of the shoot bud induction in explants inoculated

on regeneration medium and then transferred to MS 1/2 basal. The shoot buds

elongated to an average of 3 cm after transfer to MS ½ basal medium (Figure

4.17 and 4.18).

The effect of additives on callus growth and shoot bud induction

The effect of three additives, namely casein hydrolysate (CH), glutamine and

adenine was analysed to study their effect on shoot bud induction from leaf

explants. Varying CH concentrations (125, 250 and 500 mgl-1) were tested

whereas glutamine and adenine were tested at 125 mgl-1 concentrations. It was

observed that the latter two, at the tested concentration did not have any

stimulatory effect on shoot bud induction. Though an initial callus formation

was observed on media supplemented with adenine and glutamine, the explants

Micropropagation


eventually turned brown (Figure 4.19). On the other hand, in case of CH, at

concentrations of 125 and 250 mgl-1, leaf explants showed expansion and

formation of nodular structures, without any shoot bud induction. As the

concentration of CH was increased to 500 mgl-1, the leaf explants exhibited

shoot bud regeneration. However, the addition of CH at any of the

concentrations did not improve the number of shoot buds induced on the

regeneration medium (MS ½ + 0.5 mgl-1 2,4,5-T + 2 mgl-1 BAP) as discussed in

the above section.

4.2.6.3 Effect of different gelling agents on explant growth and

callus induction

In order to study the influence of gelling agents on regeneration response, the

leaf explants were inoculated on medium gelled with 0.8% agar, 0.4% agar gel

and 0.2% gelrite. The medium composition was otherwise kept constant (MS ½

+ 0.5 mgl-1 2,4,5-T + 2 mgl-1 BAP). The initial expansion of the leaf explants was

observed for all the three gelling agents. However, gelrite and agar gel did not

favour any further growth and the explants turned brown by the end of the six-

week incubation period. In case of agar, the explants showed callus and shoot

bud induction. Hence, agar at 0.8% was consistently used in all further

experiments.

4.2.6.4 Effect of MS salt strength on the regeneration response of

leaf explants

The data recorded in percent values was arcsine transformed (section 2.6.2.1)

and the transformed data was subjected to analysis of variance. The data

expressed as percentage or proportions generally has a binomial distribution.

Since one of the assumptions of ANOVA is that the data should have a normal

distribution, therefore all the data recorded in percentage was arcsine

transformed prior to ANOVA.

The regeneration response of leaf explants varied significantly with

variations in MS salt strengths. Whereas on MS full strength medium only 24.7%

leaf explants exhibited shoot induction with an average of six shoots buds

induced per explant, on MS ½ and ¼ strength medium, an average of 78% and

44.7% explants showed regeneration response, respectively (Table 4.16). The

number of shoot buds per explant however did not vary much for these salt

strengths and an average of 11 and 10 shoots per explant were produced on MS

½ and MS ¼ media combinations, respectively.

Micropropagation


Table 4.16 Effect of MS salt strength on shoot buds induction in leaf explants

MS strength % Response Shoot buds/explant

MS full 24.7c (29.9)

# 6.3

b

MS half 78.4b (62.3) 11.3

a

MS quarter 44.7a (42) 10.3

a

LSD 7.7 3.05

# Values in the bracket ( ) represent the arcsine-transformed values

4.2.6.5 Effect of different sugar concentration on regeneration

frequency

Sucrose concentrations were varied from 1 to 5% to study its effect on shoot bud

induction in leaf explants. The leaf explants showed significant differences in

response to the five different sucrose concentrations tested. At 1% sucrose

concentration the leaf explants turned brown, whereas at 4% and 5%

concentration, though the explants remained green and an outgrowth of white,

root-like structures was observed, no shoot bud induction was recorded. On the

other hand, at 3% sucrose concentration, 78% explants responded to shoot

regeneration with an average of 7.4 shoot buds per explant. On medium

supplemented with 2% sucrose, only 47% explants responded producing an

average of 5.5 shoots per explant. Thus, a significant difference was observed on

medium supplemented with 2 and 3% sucrose with respect to the percentage of

explants responding to regeneration. However, no significant differences were

observed in the number of shoot buds induced per explant at these two sucrose

concentrations (Table 4.17).

Table 4.17 Effect of sugar concentration on regeneration response

Sucrose concentration

(%)

Response (%) Shoot buds/explant

1 0c (0)

# 0

b

2 47b (43.3) 5.5

a

3 78a (62) 7.4

a

4 0c (0) 0

b

5 0c (0) 0

b

LSD 12.52 2.3

# Values in the bracket ( ) represent the arcsine-transformed values

Eventually it was observed that MS 1/2 supplemented with 3% sucrose +

0.5 mgl-1 2,4,5-T + 2.0 mgl-1 BAP and solidified with 0.8% agar produced

Micropropagation


the most optimal results for shoot bud induction. On transfer of the explants to

the MS ½ medium an increase in number of shoot buds was observed. The

shoots developed roots on the MS ½ basal medium itself and were successfully

hardened. The clonal fidelity analysis (detailed in Chapter 5) of the regenerants

proved that no variations were developed during the regeneration process.

Micropropagation


4.3 Discussions

In spite of the immense demand for Swertia chirayita and the reported

scarcity in its supply, very few reports dealing with large-scale propagation of S.

chirayita or even its allied species are available in the literature. This situation

has remained practically unchanged since the seventies when in vitro culture of

S. japonica, another species of the genus Swertia (Miura et al. 1978 a, b) was

reported for the first time. Ahuja et al. (2003) patented micropropagation

protocol of S. chirayita, reporting a shoot multiplication rate ranging between

11.4-26.2 using single nodal explants and a 50-80% rooting at the end of 8 wk

inoculation period. In the present study an attempt has been made to develop a

micropropagation protocol for S. chirayita from seedling derived nodal

explants.

4.3.1. Seed germination

In absence of mature explants, S. chirayita seedlings were used for culture

initiation. Very few studies on germination and viability of S. chirayita seeds

have been reported. Raina et al. (1994) reported 91% germination in S. chirayita

seeds after a chilling treatment at –3 0C in comparison to 3.4% germination of

control seeds. In a more recent study, Basnet (2001) reported a maximum of

81% and a minimum of 76% germination in S. chirayita in the nursery

conditions. The high variations in germination percentage reported in these

different studies can be best explained by the variability in the genotypes of the

germplasm. Besides, factors such as storage conditions (ambient temperature

and humidity) and maturity of seeds also influence the germination proportion

(Colbach and Durr 2003, Wei et al. 2003). In the present study, seeds were pre-

treated with 400 ppm GA3 that resulted in 80% germination. Seeds without GA3

pre-treatment did not germinate at all. Wawrosch et al. (1999) also found the

treatment of seeds with GA3 essential for germination. The seeds lost their

viability and failed to germinate in the subsequent germination experiments

conducted after two months.

4.3.2 Culture establishment

4.3.2.1 Effect of different cytokinins on initial shoot bud induction

The role of cytokinins in axillary bud induction has been repeatedly illustrated in

the literature. Preliminary experiments were conducted to test the effect of

equimolar concentration (3 M) of BAP, Kn and 2iP with MS basal as control, on

shoot bud induction in nodal explants. The nodal explants inoculated on MS

basal and those inoculated on MS medium supplemented with 3 µM BAP, Kn

Micropropagation


and 2iP separately, showed a significant variation in terms of number of shoot

buds induced per explant. A maximum number of 5.9 shoot buds were induced

on 2iP supplemented MS medium, followed by 5.7 shoot buds / explant on MS

medium supplemented with BAP.

Shoot bud induction on basal medium remained almost negligible with only

1.6 shoot buds being formed per explant. The nodal explant formed shoot

clusters by the end of the third subculture on cytokinin containing medium. Our

results thus conclusively showed that cytokinins were essential for shoot bud

induction and multiplication.

The requirement of exogenous cytokinins for shoot bud induction during

culture establishment has also been illustrated in many other studies on

medicinal plants. For instance, shoot burst during culture establishment was

cytokinin dependent in Campotheca acuminata (Jain and Nessler 1996) as was

also the case in Nothapodites foetida (Satheeskumar and Seeni 2000). In fact,

the type of cytokinin also has a marked effect on shoot regeneration from nodal

explants. BAP was also found most effective for shoot bud induction in

Chlorophytum borivilianum (Purohit et al. 1994). In Gymnema elegans, BAP

showed better response over Kn in shoot bud induction (Komalavalli and Rao

1997). In Vitex negundo too, Sahoo and Chand (1998) reported BAP as most

effective for shoot bud induction and multiple shoot formation. In our study,

both 2iP and BAP were found to be equally conducive for initial culture

establishment and shoot bud induction.

However, in the present study, effect of cytokinins on shoot length did not

vary much and remained more or less similar to that observed on basal medium.

Hence, during the initial stages of culture establishment stunted shoot growth

was observed. Over a period of time the shoot length attained an average length

of 2 to 2.5 cm in four weeks time, as is evident in the experiments discussed

next. This can be attributed to the fact that explants get acclimatised to in vitro

conditions and hence respond more favourably. We can also attribute the lack of

elongation in in vitro cultures partially to the fact that S. chirayita is a slow

growing plant. In its natural conditions, the plant remains in a rosette form

during the vegetative phase of growth and elongates to 1-2 m length only prior to

flowering.

4.3.2.2 Effect of different sugar concentration on multiplication rate

and elongation

Sucrose, a disaccharide also synthesized and transported naturally by the plant,

is most commonly used as the carbon source in in vitro cultures. Sucrose

Micropropagation


present in the culture medium is rapidly broken down to fructose and glucose by

the extracellular enzymes. Since the culture conditions are deficient in light

energy and CO2 concentration, sucrose is indispensable for in vitro growth and

development (Tombolata and Costa, 1998). Sucrose concentration of 20 gl-1 and

30 gl-1 are most commonly used for plant tissue culture studies (Arditti 1974).

In the present study, the treatments containing different sucrose

concentration produced significant differences in plant height and

multiplication rate after the stipulated culture period of four weeks. Medium

supplemented with 3% sucrose proved most optimal for shoot bud induction as

well as shoot elongation and an average of 4.27 shoots with 2.43 cm length were

produced per explant.

4.3.2.3 Effect of different gelling agents on plant growth

Most cultures require a gelling agent to act as a support for the plant tissue.

Gelling agents influence the gel strength, water potential and contribute to

regulation of humidity, which in turn affects the availability of water (Debergh

1983) because the water content in semisolid medium exists in bound form.

Further, determining the better gelling agent for a particular species is

worthwhile as the effect of gelling agent is mostly considered to be genotype

dependent (Makunga et al. 2005).

Agar is the most commonly used gelling agent and has been used in many

protocols defining micropropagation of medicinal plants (Joshi and Dhar 2003,

Chaturvedi et al. 2004, Makunga et al. 2005). In comparison to agar, which is

derived from seaweed (red algae Gelidium amansii, Araki and Arai 1967),

gelrite, derived from bacterium Pseudomonas elodea, is a natural anionic

heteropolysaccharide, consisting of glucuronic rhamnose, glucose and O-acetil

molecules that form rigid, brittle agar like gel in the presence of soluble salts

(Kang et al. 1982). Agargel on the other hand is a blend of agar and gelrite and

was developed to help control vitrification in plant tissue cultures. It is superior

to gelrite where vitrification is a problem and is as an economical alternative to

agar.

In our study, response of explants in medium gelled with agar was found

superior as compared to medium gelled with agar gel and gelrite. However, it

was on gelrite medium that explants showed increase in shoot length. A few

scattered cases of hyperhydricity were also observed on gelrite medium. In fact,

gelrite is known to encourage hyperhydricity in some plant species (Debergh

1983, Thomas et al. 2000, Makunga et al. 2005). Hyperhydricity was also

evident in S. chirayita cultures as studied by Wawrosch et al. (1999), however,

Micropropagation


they attributed this behaviour to the influence of cytokinins. Eventually, we

opted for agar as the gelling agent, which gave a higher multiplication fold (5.9

shoot buds/explant).

4.3.3 Culture multiplication

The organogenic potential of an explant under in vitro conditions is closely

associated with content of natural and exogenously applied plant growth

regulators (PGRs). Hence, it becomes mandatory to work out the type and

concentration of different PGRs for deriving optimal multiplication results.

4.3.3.1 Effect of different cytokinins on culture multiplication

The regulatory action of cytokinin and apical dominance in in vitro shoot

induction and multiplication has been well documented (Wickson and Thiman

1958). Among the three cytokinins that were tested at varying concentration,

BAP at 4 M was most effective in promoting axillary shoot proliferation and

culture multiplication. In the presence of 4 M BAP the multiplication rate

increased to 3.6 (in terms of shoot clusters) as compared to 1.3 fold

multiplication observed on MS basal medium. Thus, the superiority of BAP over

other cytokinins was established in this experiment. Benzyladenine has often

been found to be more effective at stimulating axillary shoot development in

many medicinal plants such as Cholorophytum borivilianum (Dave et al. 2003),

Rotula aquatica (Martin 2003), S. chirata (Ahuja et al. 2003) and Orthosiphon

stamineus (Leng and Lai-Keng 2004).

It was observed that the optimal concentration of each of the cytokinin was

considerably low and an increase in concentration beyond a certain threshold

resulted in decrease in the multiplication rate for each of the cytokinin tested.

The reduction in number of shoots generated from each explant at cytokinin

concentration higher than the optimal level was also reported in Withania

somnifera (Sen and Sharma 1991), Kaempferia galanga (Vincent et al. 1992)

and Vitex negundo (Sahoo and Chand 1998). Decrease in shoot multiplication at

higher concentration has been variously attributed to callus induction and

adventitious bud formation (Zimmerman et al. 1980), inhibition of shoot

initiation (Sen and Sharma 1991) and hyperhydricity (Wawrosch et al. 1999).

An associated advantage of low concentration of cytokinin in culture medium

is that the occurrence of adventitious bud formation is reduced and this

eliminates the consequent risk of somaclonal variations (George 1996) thus

increasing probability of propagation of clonally uniform plants as was achieved

in our study (detailed in Chapter 5).

Micropropagation


4.3.3.2 Synergistic effect of BAP along with 2iP and Kn on cluster


Synergistic effect of two or more cytokinins on promotion of shoot

multiplication is well documented for medicinal plants. Various synergistic

combinations have been reported such as BAP + Kn for Kaempferia galanga

(Vincent et al. 1992) and Feronia limonia (Hossain et al. 1994). Synergistic use

of BAP and Kn resulted in improvement in shoot elongation as well as

multiplication in Chlorophytum borivilianum (Purohit et al. 1994). Similarly, a

significant synergistic effect of cytokinins is well documented in a study on Piper

longum in which a combination of BAP and Kn produced significant increase in

culture multiplication (Philip et al. 2000). In the present study also it was

observed that BAP along with 1.5 M 2iP produced the highest multiplication

fold of 4.5 in S. chirayita explants followed by 3.9 multiplication fold obtained

on MS medium supplemented with 4 M BAP and 0.5 M 2iP.

4.3.3.3 Synergistic effect of 4 M BAP along with auxins on growth

of S. chirayita

Cytokinins, along with auxins also play important role in plant growth and shoot

regeneration. Sachs and Thimann (1964) illustrated that morphogenesis is

controlled by the interplay of auxins and cytokinins. Synergistic effect of

cytokinins with auxins has been reported to produce superior results for shoot

proliferation in many medicinal plants (Sudha and Seeni 1996, George and

Ravishankar 1997, Khan et al. 1997). Various successful combinations have been

reported, such as, BA + IAA for Thapsia garganica (Makunga et al. 2005); BAP

+IBA for Hagenia abyssinicia (Feyissa et al. 2005) and BA + NAA for

Phellodendron amurense (Azad et al. 2005).

Contrary to the above studies, no significant synergistic effect of any of the

tested auxins (namely IAA, NAA or IBA) along with 4 M BAP was established

in S. chirayita with respect to cluster multiplication. The multiplication rate

observed for all the treatments, was much lower than that recorded for the

medium with 4 M BAP alone. In fact, the multiplication fold reduced to values

equivalent to those observed on basal medium for the two concentrations of

NAA (0.5 and 1.0 M) tested. Cluster height was stunted and shoot clusters

obtained on these two medium combinations were the smallest among all the

medium combinations tested. Ahuja et al. (2003) on the other hand reported the

use of 1 mgl-1 IAA in their patented media composition for faster propagation of

S. chirayita. This variation in S. chirayita response might be due to the

Micropropagation


difference in the genotypes used in the two studies. Also, differnce in the

physiological state of the explants (mature and juvenile) used in the two studies

also explains the variations in their response.

Similar inhibitory influence of NAA on shoot multiplication was also

reported for Aegle marmelos (Varghese et al. 1993, Ajithkumar and Seeni 1998).

Inhibition of multiple shoot induction on auxin supplemented multiplication

medium was also reported for Gymnema elegans (Komalavalli and Rao 1997),

and is usually attributed to callus induction and in many cases to incidences of

rooting as well.

4.3.3.4. Effect of casein hydrolysate and adenine on cluster


Additives such as casein hydrolysate (CH) and adenine sulphate provide an

additional source of nitrogen and have been frequently used as supplements for

enhancing multiplication and elongation in micropropagation protocols. Casein

hydrolysate contains a mixture of essential amino acids and is an excellent

source of reduced nitrogen for many plant tissue culture systems. However in

our study, neither the use of adenine nor that of CH produced any increase in

multiplication of S. chirayita. Similar results were also evident in Bixa orellana

(Sharon and D’Souza 2000), where addition of casein hydrolysate did not result

in increased number of shoots. In Peganum harmala (Saini and Jaiwal 2000)

addition of adenine sulphate increased the efficiency of regeneration; however,

there was a definite reduction in the number of shoots regenerated per explant.

Thus, contrary to the expected result of an increase in multiplication, use of

additives resulted in decline of cluster growth in S. chirayita.

4.3.4 Rooting

4.3.4.1 Effect of different auxins and activated charcoal on root

induction

Among the different auxins tested for root induction, most optimal response was

recorded for NAA at 1 M concentration. In an earlier study on S. chirata,

Wawrosch et al. (1999) used considerably higher concentration of auxins (10

M) for rooting and this resulted in induction of short, tuberous roots

accompanied with prolific callusing. They subsequently altered the rooting

procedure and rooting was obtained on basal medium after a short pulse

treatment of 2 seconds in 15 ppm NAA. Ahuja et al (2003) on the other hand

reported 60-65% rooting in time duration of 8 weeks, on MS media

supplemented with IAA ranging between 1-5 mgl-1.

Micropropagation


In the present study, 1 M auxin (IAA, NAA or IBA) concentration was found

sufficient to induce rooting. However, thick swollen tuberous growth of roots

was evident. This was circumvented by addition of activated charcoal to the

rooting medium.

Addition of activated charcoal is known to result in adsorption of various

organic compounds, such as excess hormones, vitamins, abscisic acid, phenolic

metabolites and ethylene (Van Winkle et al. 2003) leading to the improved

growth of the cultures. The carry over effect of PGRs used during the

multiplication cycles can also lead to a difficulty in root induction and can affect

the quality of roots as well. In the present study, thick and tuberous roots were

induced in rooting medium without activated charcoal. However, addition of

activated charcoal in the rooting medium, improved the quality of roots and

thin, long roots developed directly from the shoot base without an intervening

callus. This can most likely be attributed to activated charcoal, which is known to

eliminate the residual effects of cytokinins applied during the multiplication

stage (Maene and Debergh 1985). Effect of activated charcoal on root induction

has been previously reported in different plant systems such as, Decalepis

hamiltonii (Reddy et al. 2001), Rollina mucosa (Figueiredo et al. 2001) and

Curcuma zedory (Loc et al. 2005).

4.3.4.2 Effect of different medium strengths on rooting

The influence of mineral concentration of culture medium on rooting can be

attributed to the participation of inorganic ions in processes regulating

hormonal balance (Amzallag et al. 1992). The salt strength concentration has

also been known to affect auxin stability thus altering the auxin’s concentration

(Dunlap et al. 1986). Further, the favourable effect of diluted mineral solution on

rooting can be explained by the reduction of nitrogen concentration (Driver and

Suttle 1987). In fact, in some plant species, dilution of salt concentration

prepares the plants for better adaptation to the acclimatization regime.

Therefore, it becomes mandatory to study the effect of varying salt strengths on

root induction besides optimising auxin concentration for root induction.

In many plant species shoots are transferred from high strength medium to

less concentrated solutions as a routine practice. The practice has been used for

most of the plant species irrespective of their being herbaceous, for instance

Saussurea obvallata (Joshi and Dhar 2003); or woody medicinal plants such as

Vitex negundo (Sahoo and Chand 1998) and Azadirachta indica (Chaturvedi et

al. 2004). In our study, the number of roots remained unaffected by diluting the

salt concentration to as low as one-tenth. However, a definite variation was seen

Micropropagation


in the length of the roots induced on medium with different MS salt strengths. It

increased as salt strength was reduced from MS full to MS half and one fourth;

albeit, further reduction in salt strengths resulted in decrease of root length.

Root induction response remained more or less similar on MS half and MS one-

fourth medium. The salt strength also affected the quality of shoots and

therefore, while determining the optimal dilutions, it is important to consider

quality of roots as well as shoots, as these have significant effect on

acclimatisation of the plant.

4.3.4.3 Effect of sugar concentration on root induction

Besides serving as a carbohydrate source, sucrose regulates the osmolarity of the

culture medium and also plays a definite role during morphogenesis (Sopory

1979). Results of the experiment on different sucrose concentration show that S.

chirayita shoots rooted well on medium containing sucrose concentration

ranging from 10 to 30 gl-1. However, at 2% concentration, optimal number of

roots (6.9) were induced without compromising the plant growth.

While considering cost economics, the presence of sugar in the culture

medium is one of the factors that contribute to a significant increase in

production cost of micropropagated plantlets (Kozai 1991). Also, according to

Debergh (1991), the absence / reduction of sugar in the culture medium reduces

contamination problems. Further, a decrease in sugar concentration in the

subsequent stages of micropropagation is usually favoured as it leads to increase

in hardening success during acclimatisation of the plants (Muller 2004).

Therefore, the decrease in sucrose concentration during the rooting stage augurs

well for commercial production of plants through tissue culture.

4.3.5 Acclimatisation and field transplantations

The success of a tissue culture protocol largely depends on ex vitro survival and

growth performance of micropropagated plants. In the present experimental

study, a hardening success of 94.5% (average of hardening achieved from

December to April, the most conducive months of the year for hardening of

chiretta at MTP at Gual Pahari, Haryana) and a transplantation success of 65.3%

were recorded.

During the warmer months of May and August, when the initial hardening

trials were conducted, successful hardening was feasible only in growth room

under controlled conditions of temperature (22 2 0C) and humidity ranging

between 65-70%. Similar details have been reported for many other Himalayan

plants. Wawrosch et al. (1999) obtained hardening survival of 90-100% for

Micropropagation


Swertia chirata. Low temperatures (17-20 0C) were maintained stringently and

an increase in temperature even to 25 0C resulted in drastic mortality in their

study. In Picrorhiza kurroa, another medicinal plant of temperate Himalayas, a

high rate of plantlet survival (87.7%) was possible under the growth chamber

conditions of low temperature (15-20 0C) and constant relative humidity (60-

70%, Lal et al. 1988).

The potting mixes in which the plants are transferred also affect the survival

percentage of the plant. In Vitex negundo, of the four different potting mixes

examined, percentage survival of the plants was highest (93%) in vermin-

compost (Sahoo and Chand 1998). Similarly, in Melia azedarach, the plant

survival and growth were better in vermiculite, and it was reasoned that

lightweight and porous nature of the soil substitute prevents root damage and

thus allows for better root growth and hence healthier plants (Thakur et al.

1998). Contrary to these reports, survival percentage in S. chirayita plants

remained more or less similar (97-99%) for the different planting substrates that

were examined. Similarly, not much variation was noticed in the growth of the

plants on different planting substrates. The statistical variation in the data

recorded was not conspicuous when the plants were finally transferred to the

field.

The plants were eventually transferred to the field and 65.3% survival of the

micropropagated plants was recorded after the initial three months of growth.

The lower survival rate during field transplantations can be well explained by the

sudden change of climatic regime to which the plant is exposed during the

transportation of the plants from tropical conditions of Delhi to the temperate

environment of Himalayas. Despite the shock that the plants were exposed to, a

considerably high survival percentage (65.3%) was recorded and the plants

successfully completed their life cycles. In most in vitro studies conducted for

temperate plants, a low transplantation success has been recorded. For instance,

in Saussurea obvallata the highest percent survival recorded was 66.7 after the

initial 15 days of transfer to the field and this further reduced to 55.6% after the

first 30 days (Joshi and Dhar 2003). Wawrosch et al (1999) recorded 60% field

survival of micropropagated Swertia chirata after one year.

The post-transplantation growth of the micropropagated plants based on

visual observation was uniform. The uniformity of the plants raised via tissue

culture was also ascertained by ISSR assay as detailed in the next chapter. The

tissue culture protocol detailed above has been successfully utilised for large-

scale propagation of the endangered species and more than 100,000 plants have

Micropropagation


been produced and successfully transferred to the field at Mandal and

Mukteshwar.

4.3.6 Regeneration in Swertia chirayita from leaf explants

4.3.6.1 Differential regeneration response of leaf, root and shoot

explants

Morphogenetic potential of different explants has been regularly explored to

standardise regeneration protocols for different species. It is generally observed

that regeneration response of explants is genotype dependent and the number of

shoot buds regenerated also varies according to the species and type of explant.

For instance, Bhat et al. (1995) evaluated the morphogenetic potential of root,

leaf, node and internode explants of species belonging to genus Piperaceae.

Piper longum leaf explants formed shoot buds whereas those of P. belte and P.

nigrum did not. In the present study, most optimal regeneration response for S.

chirayita was obtained from leaf explants. Similarly, the leaves of Pothomorphe

umbellata showed greater organogenic potential for shoot induction (Pereira et

al. 2000). Soniya and Das (2002) also reported regeneration from leaf explant of

Piper longum using picloram instead of the regular auxins that are used for

regeneration. The use of leaf explants for shoot bud induction has also been

reported for medicinal plants, such as, Piper longum (Soniya and Das 2002),

Aconitum balfourii (Pandey et al. 2004), Phellodendron amurense (Azad et al.

2004) and Hypericum perforatum (Ayan et al. 2005).

In contrast to our results, Wawrosch et al. (1999) reported adventitious shoot

regeneration from root explants. Since the germplasm of S. chirayita for the two

studies was procured from different places, the variation in genotypes can likely

explain the difference in morphogenetic response. The root explants in our study

also induced shoot buds; however, this response was sporadic and non-

reproducible. The differential response of internodal segments and root and leaf

explants of S. chirayita on similar media might be related to the variations in

tissue and cell differentiation, as well as the endogenous amounts of

phytohormones in the explant.

During optimisation of regeneration protocol for most species, such as,

Panax notoginseng (Shoyama et al. 1997), Withania somnifera (Manickam et al.

2000) and Aconitum balfouri (Pandey et al. 2004) to name a few, auxins, such

as, IAA, IBA, NAA and 2,4-D are routinely used. In our study 2,4,5-T (0.5 mgl-1

concentration), a less frequently used auxin, produced the most optimal results

in terms of callus and shoot bud induction. However, at higher concentration,

Micropropagation


2,4,5-T resulted in browning of explants. In corroboration with our study, 2,4,5-

T has been successfully employed for organogenesis studies in Eleusine

coracana (Kumar et al. 2001), Sorghum bicolor (Nirwan and Kothari 2004) and

Cicer arietinum (Kiran et al. 2005).

4.3.6.2 Effect of varying concentration of BAP on callus induction

and shoot regeneration from leaf explants

The effect of varying concentration of BAP

Among the different cytokinins regularly used for inducing regeneration, BAP

was employed in the present study. In the preliminary experiments MS ½ + 0.5

mgl-1 BAP + 0.5 mgl-1 2,4,5-T was found to induce regeneration response from

leaf explants. BAP concentration was tested in the range of 0.25 mgl-1 to 4 mgl-1

and it was revealed that 2 mgl-1 of BAP with 0.5 mgl-1 of 2,4,5-T induced

regeneration in maximum number of explants (76%). However, browning of

explants was noticed as BAP concentration was increased beyond 2 mgl-1,

resulting in a decline in regeneration response.

Wawrosch et al. (1999) also observed that BAP at 5 M concentration was

most optimal for regeneration in S. chirata. In their study, the use of 2iP

resulted in 40% hyperhydrated shoots. The authors, therefore, opted for two-

step regeneration protocol, which consisted of, an initial 3-week cultivation on

modified MS medium supplemented with 3 M BAP followed by another period

of three weeks on hormone free medium. This two-step protocol helped in

reducing hyperhydricity as well as led to elongation of shoots. In our study, the

transfer of explants from regeneration medium on to MS PGR-free medium

displayed increased proliferation in number of shoot buds as well as shoot

length.

The effect of additives on callus growth and shoot bud induction

Addition of additives, such as, casein hydrolysate, adenine and glutamine is

usually effective in increasing the regeneration response. In the present study,

the addition of adenine or glutamine to the regeneration medium led to

browning of callus. On the other hand, the addition of CH was not inhibitory as

was the case with adenine and glutamine. However, response of explants on

medium with CH did not supersede that of explants inoculated on regeneration

medium, which lacked CH. Similar results were also evident for axillary

multiplication of S. chirayita from nodal explants as discussed in section

4.3.3.4.

Micropropagation


4.3.6.3 Effect of different gelling agents on explant growth and

callus induction

Gelling agents are considered to be inert components of a culture medium, with

a limited role of providing support to the growing cultures. However, these

gelling substrates have different origin and therefore differences exist in their

quantitative and qualitative organic fractions. In fact, a definite variation has

been observed in response of explants of different species with respect to explant

growth and callus induction on different gelling agent. For instance, in Siberian

elm, proliferation in gelrite medium was greater than that on agar-solidified

medium (Cheng and Shi 1995), whereas Corchete et al. (1993) had reported

success of Siberian elm regeneration on agar medium. The variations reported

can be explained by the sensitivity of the explants of different species and

genotypes to varying organic fractions of the gelling agent. In our study, gelrite

and agar gel completely inhibited the regeneration response from leaf explants,

whereas medium solidified with agar promoted regeneration. Wawrosch et al.

(1999) also used agar as gelling agent for shoot regeneration from root explants

in S. chirata.

4.3.6.4 Effect of MS salt strength on the regeneration response of

leaf explants

Basal medium are known to influence growth response of various plants in

different ways. This is considered to be a consequence of differences in their salt

components and proportions (Bhojwani and Razdan 1996). Since MS salt

composition is considered very rich in terms of the total nitrogen available (60

M of nitrogen ions), usually the MS composition is modified with reduction in

major salt to ½ or ¼ of the original strength for inducing morphogenetic

responses, such as, somatic embryogenesis and shoot and root induction. In our

study, the best regeneration response of leaf explants with 78% explant

responding to shoot bud induction, was obtained on modified MS salt with

major and iron reduced to half the original strength. Further reduction in the

MS salt composition, however, resulted in a decrease in regeneration response.

The lowest regeneration response (24.7%) was obtained on MS full medium,

thus corroborating the fact that lower MS salt concentration favoured

regeneration in S. chirayita.

4.3.6.5 Effect of different sugar concentration on regeneration

frequency

Micropropagation


Sucrose is the main carbohydrate source for in vitro culture and is necessary to

sustain growth and also serves as an osmoticum. Sucrose concentration has also

been known to regulate shoot bud induction in different plant species.

For instance, on increasing sugar concentration from 1 to 3%, an increase in

regeneration percent was observed in quince (Baker and Bhatia 1993). Chevreau

et al. (1989) found positive linear effect on shoot regeneration in pear as sucrose

concentration was increased from 1 to 5%. In case of S. chirayita, in the present

study, 3% sucrose concentration proved most optimal for shoot bud induction

inducing a 78% regeneration response from the leaf explants. However, further

increase in sucrose concentration to 4% and 5% led to browning of the leaf

explants.

Figure 4.1 a) Nodal explant of Swertia chirayita derived from 4-week-old in vitro germinated seedlings and (b-d ) nodal explant developing into clusters after the initial establishment stages on MS medium supplemented with 3 M BAP and 3% sucrose for a culture period of 4 weeks.

a

c

b

d

1.7

5.84.9

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1.26 1.281.25

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basal 2iP Kn BAP

cytokinins

shoot buds

0.00

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2.00

shoot heig

ht (cm

)

sh oot bu ds shoot length

Figure 4.2 The effect of BAP, Kn and 2iP at equimolar concentration of 3 M on shoot bud induction and shoot elongation in Swertia chirayita after a culture period of 4 week each. A significant variation was visible in explants cultured on MS basal medium as compared to medium supplemented with 3

M each of the three cytokinins, namely 2iP, Kn and BAP with respect to shoot buds/explant.

Figure 4.3 The growth of S. chirayita shoot-clusters as obtained on MS medium supplemented with 3 M BAP + 3% sucrose and solidified with (a) agar (b) agarose and (c) gelrite. The difference in shoot length is visible in the three medium with gelrite showing the maximum elongation after 4 wk of culture period.

a b

c

0

0.5

1

1.5

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con cen tr a t ion (m icr om ola r s)

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ltip

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tio

n fo

ld

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1

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sh

oo

t len

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(cm

)

BA P sh oot bu ds kin et in sh oot bu d 2 iP sh oot bu ds

BA P sh oot len g th Kin et in sh oot len g th 2 iP sh oot len g th

Figure 4.4 Pictorial representation of the variations in cluster multiplication and cluster height in S. chirayita as obtained for the varying concentrations of BAP, Kinetin and 2iP after 4 wk of culture period. Among the eighteen different combinations tested, 4

M BAP produced the most desirable results, both in terms of multiplication fold (3.6) and cluster elongation (2.6 cm).

Figure 4.5 Effect of different auxins on root induction in S. chirayita shoots when inoculated on (a) basal medium (MS half +3% sucrose) and that supplemented with (b) 1 M IAA (c) 1 M NAA and (d) 1 M IBA as observed after 4 wk of culture period.

a

b

d c

Fig4.6 (a) Roots induced on rooting medium (MS ½ + 3% sucrose + 1 M NAA) without activated charcoal remain thick and small along with basal callusing as compared to (b) thin slender roots induced on rooting medium supplemented with 500 mgl-1 of activated charcoal.

b

a

Figure 4.7 In vitro hardening of micropropagated S. chirayita plantlets in growth room. (a) Plantlets transferred to culture vessels containing coir peat with polypropylene caps which were gradually opened over a period of three weeks for successful hardening and (b) successfully hardened plantlets 3 weeks after hardening.

b

a

Figure 4.8 Ex vitro hardening of Swertia chirayita plantlets representing (a) ex-agar plants transferred to poly bags kept in green house for acclimatization for a period of two weeks and (b) 4 week-old hardened plants shifted to shade net after acclimatization.

a

b

Figure 4.9 Swertia chirayita plantlets from rooting stage to a well acclimatized plant. (a) micropropagated shoots in rooting medium (MS ½ + 2% sucrose + 1

M NAA) b) an ex-agar, well rooted plantlet before hardening c) few plantlets after hardening in green house d) a bare root micropropagated chiretta plant ready for transplantation.

a b

c d

Figure 4.10 (a) 2-month old hardened plants immediately after transplantation in field at Mandal, Dist. Chamoli, Uttaranchal, India (b) Micropropagated plants after six months of growth in the same field. An average height of 15 cm, growth area of 240 square inch and biomass of 106 gm was recorded for the plants six months after transplantation to the field.

a

b

Figure 4.11 In vitro multiplied plants of Swertia chirayita after ( a) 3-months, (b) 6-months and (c) one year of growth in field (Mandal, Uttaranchal). The plants show rosette growth with a maximum height averaging between 12-18 cm during the vegetative phase of growth (a and b). The plants bolt before flowering and attain an average height of 1-1.5 m ©.

a

c b

Figure 4.12 The chiretta plant during flowering stage after two years of vegetative growth. a) The shoot apex immediately before bearing flowers b) a well developed root after two years of growth c) A flowering twig d) flower of chiretta showing the four yellow-green petals, also visible are the two glands which are the identifying feature of Swertia chirayita.

a b

d c

Figure 4.13 Sporadic shoot regeneration from root explants (a) root explants after incubation period of six weeks on NAA + IBA (0.75 mgl-1 each) supplemented media (b) shoot elongation of regenerants on MS ½ basal medium after 4 weeks of culture period.

a

b

0

30

36

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16.59

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17

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nt re

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se

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nu

mb

er o

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oo

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ud

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er e

xp

la

nt

Figure 4.14 The effect of varying BAP concentrations (0-4 mgl-1 ) on the regeneration response of leaf explants and the number of shoot buds induced per explant. Maximum regeneration response of 76% was observed at BAP concentration of 2 mgl-1 with an average of 11 shoots buds/explant.

Figure 4.15 (a) Leaf explants on regeneration medium showing initial swelling and expansion due to water absorption and cell proliferation in the initial six weeks of culture followed by (b) profuse callus formation after another 6 weeks of culture period on regeneration medium (MS ½ supplemented with 2.0 mgl-1 BAP and 0.5 mg l-1 2,4,5-T with 3% sucrose).

a b

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10.3 10.7

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r eg en er a t ion m ediu m

nu

mb

er o

f s

ho

ot b

ud

s p

er e

xp

lan

t

r eg en er a t ion m edia ba sa l m edia

Figure 4.16 Comparative graphical illustration of shoot buds induced on the three best regeneration medium (among the different tested combinations) numbered as 1, 2 and 3 and the increase in their numbers after transfer to basal medium at the end of 4 week culture period. The regeneration medium numbered 1, 2 and 3 represent MS ½ supplemented with 0.5 mg/l 2,4,5-T each and BAP at 0.5, 1.0 and 2.0 mg/l, respectively.

4 3 .0

3 4 .8

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3 .02

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mb

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ot b

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sh

oo

t l

en

gth

(cm

)

Sh oot bu ds sh oot len g th

Figure 4.17 The increase in number of shoots and elongation in shoot length after the transfer of explants from the regeneration medium, numbered as 1 (MS ½ + 0.5 mgl-1 2,4,5-T + 0.5 mgl-1 BAP ), 2 (MS ½ + 0.5 mgl-1 2,4,5-T + 1.0 mgl-1 BAP) and 3 (MS ½ + 0.5 mgl-1 2,4,5-T + 2 mgl-1 BAP) to MS1/2 basal medium after 4 weeks of culture period.

Figure 4.18 Shoot elongation and proliferation after transfer of explants from regeneration medium (MS ½ supplemented with 2.0 mgl-1 BAP and 0.5 mg l-1 2,4,5-T with 3% sucrose) to MS ½ basal medium after 4 weeks.

Figure 4.19 a) Callus growth and shoot buds induction on control medium (MS ½ medium supplemented with 2.0 mgl-1 BAP+ 0.5 mgl-1 2,4,5-T) (b) browning of callus browning in case of explants cultured on control medium supplemented with 500 mgl-1 CH (c) control + 125 mgl-1 adenine and (d) control + 125 mgl-1 glutamine.

a b

c d

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