Journal of Applied Pharmaceutical Science Vol. 5 (07), pp. 103-111, July, 2015
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2015.50716
ISSN 2231-3354
A simple cost effective method for mass propagation of Chrysanthemum
morifolium and antibacterial activity assessment of in vitro raised plantlets
Manu Pant*, Ankita Lal, Rashi Jain
Department of Biotechnology, Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun, India.
ARTICLE INFO
ABSTRACT
Article history:
Received on: 29/03/2015
Revised on: 23/04/2015
Accepted on: 14/05/2015
Available online: 27/07/2015
An efficient low cost procedure for in vitro propagation of Chrysanthemum morifolium has been developed with
subsequent assessment of antibacterial property of in vitro raised plantlets. Optimal axillary shoot multiplication
was achieved on MS medium supplemented with low concentration of BAP. Psyillium husk and market sugar
were standardized as suitable alternatives to the conventionally used agar and sucrose, cutting down the
production cost of tissue culture raised plantlets to over six times. Optimal in vitro rooting was obtained on half
strength MS medium containing IBA. Regenerated plantlets with well developed shoots and roots were
acclimatized successfully and transferred to field conditions where they flowered. The leaves of ex vitro growing
tissue culture raised plantlets were later assessed for activity against bacterial pathogens. The present protocol
ensures minimal cost input in large scale production of a commercially important ornamental plant and opens up
scope of scientific interventions directed at its allied therapeutic usage.
Abbreviations: MS: Murashige and Skoog (1962); HgCl2 : Mercuric chloride; PGR: Plant growth regulator,
TCR: tissue culture raised; BAP: 6, Benzylaminopurine; NAA: α-Naphthalene Acetic Acid; IBA: Indole-3
butyric acid; IAA: Indole-3 acetic acid; min: minutes; ***:significant at 99.9%
Key words:
Chrysanthemum morifolium,
in vitro axillary shoots, cost
effective, clonal
multiplication, antibacterial
activity
INTRODUCTION
Floriculture industry has emerged as one of the
important and escalating commercial trades in agriculture. A big
volume of cut flowers and potted plants are sold on daily basis
across the world. In terms of production value Netherlands, US ,
Japan, Italy, Germany and Canada are the largest producers and
exporters of cut flowers and plants while Germany, US, France
and UK are the major consumers (Getu, 2009). In India, with
over 161,00 ha land under flower cultivation (Starman et
al.,1995), suitable climatic conditions and commercial
floriculture displaying high potential per unit area than most field
crops, floriculture is being perceived as a lucrative business.
Chrysanthemum (commercially available as Chrysanthemum
morifolium Ramat.) is a well known name in the global
floriculture trade, just one position behind the top-ranked rose in
world markets (Texeira da Silva, 2003). Exceptional foliage,
* Corresponding Author
Manu Pant, Department of Biotechnology, Graphic Era University,
Dehradun, India. Email: himaniab@gmail.com
attractive flowers and wide range of hues make C. morifolium a
grower’s muse while an added feature of long vase life (Ilahi,
2007) accounts for its high value as cut flower in floral
arrangements. To add to it, C. morifolium is well recognized in
traditional system of medicine for treatment of common cold,
regulation of blood pressure, detoxification and improving eyesight
(Lin and Harnly, 2010). In India the significance of
chrysanthemum cultivation can be assessed from the fact that an
average of 16.19 tons of chrysanthemums was reported to be
produced in the year 2012-13 alone (www. apeda.gov.in). Since
conventional propagation methods of suckers and terminal cuttings
are unsuitable to support large scale production of
chrysanthemums (Nhut et al., 2005, Waseem et al., 2009) tissue
culture technology can be of much help to the growers as it
promises season-independent mass production of the chosen
variety. The technique has already been employed to study large
scale propagation of C. morifolium through different regeneration
pathways (Ben Jaacov and Langhans, 1972, Kaul et al., 1990, Lu
et al., 1990, Fay, 1992, Khan et al., 1994, Ilahi et al., 2007,
Hodson et al., 2008, Nalini, 2012, Karim, 2002, 2003, Shatnawi et
al., 2010, Keresa, 2012).
© 2015 Manu Pant et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike
Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
104
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
However, the technology is beset with major constraints
of high production cost (minimum sale price for each
chrysanthemum plant remains to be INR (Rs.) 30.00- 40.00) and
problems in ex vitro survival of tissue culture raised (TCR)
plantlets. Consequently, there is a need to develop a cost effective
system for large scale production of chrysanthemum plants
wherein the farmers can purchase the variety of their choice at
minimal rates hence ensuring monetary gain to the growers. In
addition, the potential of TCR plant parts can also be exploited to
determine their medicinal significance, if any. The present study
was taken up with an aim to develop a rapid and cost effective
micropropagation protocol of Chrysanthemum morifolium and
identify the antibacterial properties of tissue culture raised
plantlets. The findings of the study were endeavored at having
practical applications for researchers who can produce
chrysanthemums of choice with minimal investments besides
realizing the therapeutic potential of tissue raised plantlets and for
the growers who can purchase healthy TCR plantlets at subsidized
rates hence making overall chrysanthemum trade a commercially
and scientifically beneficial venture.
MATERIAL AND METHODS
Micropropagation
Culture initiation
Mother plants of Chrysanthemum morifolium were
procured from Choudhary nursery, Sahastradhara, Dehradun and
grown in polyhouse of Department of Biotechnology, Graphic Era
University. Nodal segments containing single axillary buds were
used as the source material for micropropagation. Explants were
washed with liquid detergent for 5 min (Cetrimide, Shalaks
Pharmaceuticals Pvt. Ltd, India 3 - 4 drops / 100 ml) followed by
washing under running tap water. Thereafter, the 2-3 cm nodal
segments (with at least one node in each sector) were excised and
subjected to surface sterilization with 0.1% (w/v) HgCl2 (Himedia)
for 4 minutes under laminar-air flow cabinet followed by thorough
washing ( four to five times) with autoclaved distilled water to
wipe out the traces of steriliant.
Culture establishment and multiplication
For establishment of cultures, surface sterilized explants
were inoculated on to full strength MS (Murashige and Skoog,
1962) basal medium containing BAP (0.0- 2.0 mg/l), sucrose
(30g/l) and solidified with agar (7g/l w/v) (HiMedia) .
Single shoots were separated from established cultures
and shifted to MS medium supplemented with varying
concentrations and combinations of BAP (0.5 mg/l – 1.0 mg/l),
IAA (0.25 mg/l – 0.5 mg/l) and NAA (0.25 mg/l - 0.5 mg/l) for in
vitro shoot multiplication. Basal MS medium devoid of any PGR
served as control. Most optimal PGR combination and
concentration was used for setting up low cost experiments. In the
next set of experiments solidifying agent agar was replaced with
psyllium husk (commonly known as isabgol) and market sugar
was used as a substitute for sucrose in the medium at a
concentration range of 20-30 mg/l. Double distilled water for
preparing culture medium was also replaced with RO water. MS
medium fortified with 7 g/l (w/v) agar and 30g/l sucrose was used
as control.
Data on average number of shoots formed and average
shoot length were scored after 2 weeks and 4 weeks of culture.
Routine subculturing was carried out at regular intervals of 2
weeks alternately on basal MS medium and MS medium
containing 0.5 mg/l BAP.
In vitro rooting
Experiments on in vitro rooting were attempted with 2-3
cm long shoots having 1 or 2 nodes. The microshoots were excised
and transferred to root induction medium comprising of 1/2
strength MS medium with psyllium husk or 0.6% agar and 20 g/l
market sugar and fortified with different concentrations of IAA,
IBA or NAA (0.25- 1.0 mg/l). Data on the number of roots per
shoot and root length were collected after a period of 2 weeks and
4 weeks.
Medium and culture conditions
The pH of the medium was adjusted to 5.8 using 1 N
NaOH or 1N HCl prior to adding the solidifying agent and
dispensed in conical flasks (250 ml, Borosil, India). The culture
medium was autoclaved at 120 0C and 1.0 x 105 Pa for 15 min. All
the chemicals used were of Himedia grade. Cultures in all
experiments were incubated in culture room at 23 ± 2 0C and 60 65% relative humidity under a 16/8 hr (light/dark) photoperiod
with light supplied by cool-white fluorescent tubes (Philips, India)
at an intensity of 50 µmol m-2 s-1.
Hardening and acclimatization
For in vitro hardening, rooted shoots were transferred to
1/4 strength MS medium devoid of carbon source and any PGR for
5 days in flasks. Thereafter, they were transferred to poly-bags
containing a mixture of soil: sand: manure (1:1:1) and covered
with perforated polythene bags (that were periodically removed)
and kept under agronet-shade house conditions. Acclimatized
plants were subsequently shifted to soil in pots/ garden soil and
maintained for growth under ex vitro conditions.
Statistical Analysis
Data collected in the Completely Randomized Design
(CRD) of experiments (Compton, 1994) was analyzed using
Microsoft Excel ver. 2007 © Microsoft Technologies, USA.
Experiments were repeated thrice and data represents the mean of
three experiments. Each treatment consisted of minimum twelve
replicates. Data was subjected to one way Analysis of Variance
(ANOVA). Degree of variation was shown by Standard Error
(SE), Critical Difference (CD) at 5%. The significance level was
tested determined at 5% (p < = 0.05), 1% (p < = 0.01) and 0.1% (p
< = 0.001). The significance of the data as ascertained by F-test
and the CD values computed, were used for comparing differences
in means of various treatments.
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
105
Fig. 1: (A) In vitro culture establishment of Chrysanthemum morifolium via nodal explants. (B) In vitro shoot multiplication in low cost medium (C) Maintenance
of shoots via subculture (D) In vitro rooted plantles (E) flowering in TCR plantles (F1) Antibacterial activity of ethanolic extract against Bacillus subtilis (F2)
Antibacterial activity of ethanolic extract against Staphylococcus aureus (F3) Antibacterial activity of ethanolic extract against E. coli.
Table 1: Effect of cytokinin on axillary bud induction in C. morifolium.
Treatment
MS + BAP (mg/l)
T1(Control)
T2
T3
T4
T5
0.0
0.5
1.0
1.5
2.0
Significance
CD at 5%
Average Shoot Number
2 weeks
4 weeks
0.00±0.00
0.00±0.00
1.53±0.07
2.53±0.18
4.73±0.07
6.53±0.07
2.27±0.07
3.73±0.07
2.93±0.07
4.53±0.07
***
***
0.18
0.28
Table 2: Effect of PGRs on in vitro shoot multiplication in C. morifolium
MS + PGR (mg/l)
Treatment
BAP
IAA
NAA
T1(Control)
0.00
0.00
0.00
T2
0.50
0.00
0.00
T3
0.50
0.25
0.00
T4
0.50
0.00
0.25
T5
1.00
0.25
0.00
T6
1.00
0.00
0.25
T7
0.50
0.50
0.00
T8
0.50
0.00
0.50
T9
1.00
0.50
0.00
T10
1.00
0.00
0.50
Significance
CD at 5%
Average Shoot Number
2 weeks
4 weeks
0.00±0.00
0.00±0.00
10.73±0.07
17.27±0.24
7.60±0.12
9.93±0.07
4.67±0.18
6.40±0.12
7.27±0.07
9.20±0.12
6.73±0.07
9.73±0.27
6.33±0.13
9.27±0.27
6.13±0.07
8.60±0.12
7.67±0.07
9.47±0.18
4.53±0.07
6.27±0.13
***
***
0.28
0.50
Average Shoot Length (cm)
2 weeks
4 weeks
0.00±0.00
0.00±0.00
2.50±0.05
4.58±0.04
3.65±0.04
5.95±0.03
2.37±0.03
3.81±0.04
3.18±0.04
4.81±0.10
***
***
0.10
0.16
Average Shoot Length (cm)
2 weeks
4 weeks
0.00±0.00
0.00±0.00
6.44±0.08
8.67±0.09
4.98±0.04
6.16±0.06
4.85±0.04
5.74±0.06
5.94±0.03
7.16±0.02
5.65±0.03
7.09±0.01
5.67±0.03
6.85±0.02
5.91±0.01
6.98±0.02
5.06±0.03
6.23±0.05
4.87±0.04
5.79±0.04
***
***
0.12
0.13
106
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
Antibacterial Assay
Sample Collection
Leaves of in vitro developed plantlets of Chrysanthemum
morifolium were used to assess antibacterial activity. For the
purpose eight months old TCR plants growing under ex vitro
conditions were collected and dried under shade for 6-7 days
followed by drying in hot-air oven for 4-6 hours at 500C to remove
excess moisture.
Extraction of plant material
The dried leaves were softly crushed to prepare a powder
and 40 g of powdered leaves were successively extracted with 200
ml of ethanol at 56-600C and ethyl acetate at 40-500C in Soxhlet
extractor until the extract was clear. The extracted solvent was
removed under reduced pressure with rotary vacuum evaporator.
The sticky greenish-brown substance obtained was stored at 4°C in
storage vials for experimental use (Chessbrough, 2000).
Culture of microorganisms
Gram positive (Bacillus subtilis (MTCC No. 121),
Staphylococcus aureus (MTCC No. 6908)) and gram negative
bacteria (Escherichia coli (MTCC No. 1698) and Pseudomonas
aeruginosa (MTCC No. 4306)) were used for antibacterial assay.
Pure cultures were maintained on nutrient agar slants and plates on
regular basis. The cultures were streaked on sterile nutrient agar
plates and kept in incubator for 24 hours at 37°C. Bacterial
cultures were frequently subcultured to obtain pure cultures.
Inoculum was prepared by growing the pure bacterial culture in
nutrient broth over night at 37°C.
Determination of antibacterial susceptibility
Antibacterial activity was measured using disc diffusion
test (Bayer et al., 1966 with modification). 20ml of media was
transferred aseptically into each sterile petri dish and allowed to
solidify. 100µl of inoculum suspension was spread uniformly over
the agar medium using sterile glass rod for uniform distribution of
bacteria. The prepared sterile discs were loaded with different
concentrations (50mg/ml and 100mg/ml) of plant extract. The
paper discs were placed suitably apart on the medium and the
culture plates were kept in an incubator at 37ºC. After 24 hours of
incubation, the antibacterial activity was recorded by measuring
the width of the clear inhibition zone around the disc in millimetre.
RESULTS
Initiation and multiplication of shoot cultures
In the present study, axillary bud break in nodal explants
was observed within two weeks of culture in all media
combinations tried except the control. BAP at a concentration of
1.0 mg/l proved to be most efficient in initiating bud induction
giving an average of 4.73±0.07shoots per explant after 2weeks and
6.53±0.07 shoots after 4 weeks (Table 1). The regenerated shoots
were transferred onto fresh medium of same composition to
establish an initial stock of shoots (Figure 1A). This stock was
further used for conducting experiments to assess the effect of
various PGRs on shoot multiplication summarized in Table2.
Significant differences were observed in the effectiveness of PGRs
and their levels in MS medium. In control, the shoots showed
necrosis within a week of transfer. For all the parameters studied,
it was noted that treatment T2 proved to be most optimal giving a
mean number of 10.73±0.07shoots (mean shoot length 6.44 cm)
after 2 weeks and 17.27±0.24shoots (mean shoot length 8.67cm)
after 4 weeks. Synergistic effect of cytokinin and auxin proved
inefficient in inducing optimal shoot multiplication. Media
combinations containing auxins (IAA/NAA) at a concentration of
0.5 mg/l along with cytokinin BAP, callus initiation was observed
at the base of shoots towards third week of culture. With a
concept of developing a system for cost effective large scale
production of chrysanthemums, a media formulation comprising of
full strength MS medium (prepared with RO water), supplemented
with BAP(0.5 mg/l), psyllium husk (as solidifying agent) and
market sugar (as carbon source) was standardized in our study
(Table 3). In experiments conducted to determine the best
concentration of psyllium husk and market sugar for shoot
multiplication, MS medium fortified with 0.5 mg/l BAP (Hi
Media, INR 700.00/5gm), 7.0 g/l agar (HiMedia @INR
4,024.00/500 gm) and 30 g/l sucrose (Himedia @INR
455.00/500gm) served as control in which the most optimal results
were observed. However, of all other treatments tried, medium
containing 25 g/l psyllium husk (Sidhpur Sat Isabgol @INR
310.00/500gm) and 20g/l market sugar (INR 455.00/500gm)
proved to be the best (Figure1B) wherein a good number of
healthy shoots developed. In this combination 10.53±0.07shoots
with mean shoot length 6.36±0.07 cm and 16.87±0.18 shoots with
mean shoot length 8.62±0.05 cm were obtained after 2 weeks and
4 weeks, respectively. It was observed that on increasing the
concentration of psyllium husk in medium from 25 g/l to 30 g/l,
shoot multiplication declined while a still higher concentration of
30 g/l resulted in extremely hard media making shoot inoculation
difficult and damaged the shoots while being removed for
subsequent subculture. Replacing sucrose with market sugar
proved to be an economical choice without any negative affect on
in vitro shoot development. Henceforth, T3 was standardized to be
most favorable alternative to the high cost medium for shoot
multiplication. For long term maintenance of cultures, in vitro
multiplied shoots were periodically subcultured at an interval of 2
weeks. The subculture routine involved alternate subcultures on
basal MS medium and MS medium containing 0.5 mg/l BAP. By
this procedure, over 50 sub cultures have already been carried out
without any declination in multiplication rate over a period of two
years of maintenance of cultures (Figure1C). The modified
protocol resulted in deduction in production cost by over 6 times.
Figure 2 depicts a comparison in cost of a single flask of
chrysanthemum culture containing over 100 root-able shoots. The
input cost could also be reduced by using a Reverse Osmosis (RO)
water assembly (approx INR 8,000.00/ unit) for water supply for
media preparation instead of a double distillation unit (Borosil, @
31,455.00/unit) as the use of RO water did not intervene with the
growth characters in vitro.
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
Table 3: Effect of solidifying agent and carbon source on in vitro multiplication of regenerated shoots of C. morifolium.
Treatment
MS + BAP (0.5 mg/l)
Average Shoot Number
Agar (g/l)
Sucrose(g/l)
2 weeks
4 weeks
T1
7
30
10.67±0.07
17.07±0.27
Isabgol (g/l)
Sugar (g/l)
T2
20
20
8.73±0.07
14.40±0.12
T3
25
20
10.53±0.07
16.87±0.18
T4
30
20
5.40±0.12
6.87±0.18
T5
20
25
8.20±0.12
12.80±0.31
T6
25
25
7.60±0.12
9.53±0.18
T7
30
25
7.67±0.07
9.47±0.18
T8
20
30
4.73±0.07
6.53±0.18
T9
25
30
4.67±0.18
6.27±0.07
T10
30
30
5.47±0.07
6.87±0.18
Significance
***
***
CD at 5%
0.29
0.57
107
Average Shoot Length(cm)
2 weeks
4 weeks
6.37±0.09
8.57±0.07
5.91±0.05
6.36±0.07
4.95±0.03
5.67±0.04
5.03±0.05
5.07±0.02
4.88±0.03
4.85±0.03
5.08±0.03
***
0.15
7.97±0.06
8.62±0.05
5.81±0.03
7.81±0.02
6.29±0.08
6.31±0.09
5.85±0.06
5.81±0.02
6.31±0.09
***
0.18
Graph 1: Effect of auxins on in vitro rooting in C. morifolium
12.00
10.00
8.00
6.00
4.00
2.00
0.00
Control
-2.00
R1
0.25 mg/l IAA 0.25 mg/l NAA 0.25 mg/l IBA
R2
R2
Mean shoot number afer 2 weeks
0.50 mg/l IAA 0.50 mg/l NAA 0.50 mg/l IBA
R4
R5
Mean shoot number afer 4 weeks
R6
(in
1.0 mg/l NAA
1.0 mg/l IBA
R8
R9
R10
R7
Mean shoot length (cm) afer 2 weeks
14
Price per Shoot Culture Flask
INR)
1.0 mg/l IAA
Mean shoot length (cm) afer 4 weeks
Rs 12.55
12
10
Rs 8.45 (Agar)
8
HiMedia Make
6
Low Cost
Rs 4.1 (Sucrose)
4
2
Rs 1.86
(Isabgol)
Rs 1.98
Rs 0.12
(Market Sugar)
0
Gelling Agent
Make
Carbon Source
Total (150ml)
Components
Cost (in INR)
Agar
4,024.00 (per 500gm)
HiMedia
Sucrose
455.00 (per 500gm)
Isabgol
310.00 (per 500gm)
Low cost
Market sugar
20.00 (per 500gm)
Fig. 2: Comparative analysis of production cost in conventional tissue culture method versus low cost method (Total 150 ml depicts the amount of medium
required for production of over 100 rootable size shoots)
108
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
In vitro rooting
In the present study, shoots cultured in auxin-free
medium (control) failed to form roots while a varied effect of
auxins (IAA, NAA or IBA) was observed by incorporating them in
half-strength MS medium at different concentrations (Graph 1).
Consistent rooting was noted in all the three auxins tried within 15
days of culture. Emergence of root primordia was observed within
10 days of culture following which rapid root development
occurred. Overall, treatment R7 (0.50 mg/l IBA (Hi Media, INR
664.00/ 5gm)) was most optimal in all rooting parameters and
significantly better than all the treatments tested. In this
combination an average number of 6.53 roots per shoot after 2
weeks, 10.40 roots after 4 weeks and average root length 5.17 after
2 weeks and 8.00 after 4 weeks was observed. The roots so formed
were long and healthy. NAA proved to be comparatively less
efficient in inducing root formation while the roots developed on
IAA supplemented medium were long but very thin.
The optimized PGR concentration was subsequently used
in half strength MS medium supplemented with 0.6% agar and 20
g/l market sugar for in vitro root formation (Figure 1D). This
medium resulted in formation of a healthy and viable root system.
Replacing agar with psyllium husk in rooting medium proved to be
detrimental for safe removal of rooted shoots from culture tubes.
Also, psyllium husk stuck to the roots and could not be properly
washed off. Contrastingly, lower concentrations of psyllium husk
resulted in liquefied medium that caused shoot hyperhydration.
Hardening, acclimatization and field transfer
Plantlets developed via axillary bud culture were
hardened in-vitro by placing them on liquid 1/4 MS medium
without PGR and carbon source for 5 days (or more in case of
climate constraints for shifting of plantlets to ex vitro conditions).
These plantlets were subsequently transferred to a rooting mixture
of soil: sand: manure (1:1:1) and maintained in net house for next
4-5 weeks where they exhibited enhanced growth. When
transferred to soil in pots and garden soil, over 96% survival rate
was obtained. These plantlets were maintained in areas with plenty
of air circulation, water drainage and optimal sunlight. The plants
were periodically checked for any kind of insect or fungal
infection. The tissue culture raised plantlets exhibited flowering in
the month of October (Figure 1E) proving the developed protocol
to be a successful cost effective package.
Antibacterial study
The current research work ethanol and ethyl acetate
extracts of C. morifolium leaves were prepared and antimicrobial
activity was assayed against bacterial pathogens by disc diffusion
method. As per data obtained (Table 4, 5), the ethanolic extract
showed minimum degree of inhibition against Bacillus subtilis
(Figure1: F1) but effective degree of inhibition against
Staphylococcus aureus (Figure1: F2) and E. coli (Figure1: F3).
The ethyl acetate extract showed minimum degree of inhibition
against Bacillus subtilis (only at high concentration) and moderate
degree of inhibition against Staphylococcus aureus. This indicated
ethanol to be a better solvent for extracting antimicrobial
substances from the leaves of C. morifolium than ethyl acetate. No
zone was observed against Pseudomonas aeruginosa in either of
the extracts.
Table 4: Antibacterial activity of ethanol extract of in vitro raised C.
morifolium leaves
Zone of inhibition by Ethanol
Extract (mm)
S. No
Microorganism
50 mg/ml
100 mg/ml
1.
Bacillus subtilis
4
5
2.
Staphylococcus aureus
6
9
3.
Escherichia coli
5
7
4.
Pseudomonas aeruginosa NZ
NZ
NZ- No Zone
DISCUSSION
With an aim to integrate scientific know-hows with
societal benefits, the present study was taken up to develop a
protocol for low cost micropropagation of a commercially
significant ornamental plant-Chrysanthemum morifolium and also
assess the antibacterial property of its leaves. The aim was to
deliver a cost effective package of plant production that will result
in maximal production with minimal expenditure and also assess
the probable role of otherwise unused plant parts in therapeutics.
Culture of nodal segments carrying axillary buds
involves the exploitation of buds already present on the parent
stock plant, hence providing an efficient means of rapid clonal
propagation allowing production of genetically stable and true-toparent type progeny (Hu and Wang, 1983). In the present study,
best results of axillary bud induction from nodal explants were
obtained on MS medium supplemented with 1.0 mg/l BAP. The
results are in corroboration with previous findings on
micropropagation of C. morifolium wherein efficacy of BAP for
axillary shoot induction was established (Khan et al., 1994, Haq et
al., 1998, Karim et al., 2002, 2003). This is in support of the fact
that plant tissues metabolize BAP more readily than other
synthetic growth regulators and has also been known to induce
production of natural hormones as zeatin within the tissue (Malik
et al., 2005). Contrastingly, BAP in combination with IAA
(Waseem et al., 2011) and BAP with GA3 (Keresa et al., 2012)
have also been reported to give maximal shoot induction in C.
morifolium.
In vitro shoot multiplication is the major criterion for
successful commercial micropropagation. In our study, BAP at a
concentration of 0.5 mg/l gave most optimal results of shoot
multiplication while a combination of cytokinin and auxin proved
to be comparatively less efficient for the same. Superiority of BAP
in axillary shoot multiplication has also been reported in C.
cinerariaefolium (Lindiro et al., 2013). These observations on
efficacy of low concentrations of BAP alone in inducing shoot
proliferation/ multiplication and calli formation in presence of
auxins reflect high levels of endogenous hormones in the mother
plants. In the present study, use of psyllium husk and market sugar
was standardized as suitable substitutes to agar and sucrose in
culture medium with an aim to lacerate the overall production cost
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
of TCR plantlets. Psyllium husk is a mucilaginous and sticky
material that can be used as a gelling agent in culture media
(Bhattacharya and Bhattacharya, 1994, Babbar et al., 1998, Tyagi
et al., 2007, Atici et al., 2008). In our study, psyllium husk at a
concentration of 2% proved to be the best while higher
concentrations resulted in decline in shoot multiplication which
could be due to ineffective diffusion of media nutrients at high
concentration of solidifying agent. Our results are in consonance
with earlier studies reporting positive effect of psyllium husk on in
vitro shoot induction, multiplication and long-term survival of
cultures (Fischer, 2004, Yusuf et al., 2011).
Market sugar contains 96-97% sucrose and 0.7-1%
reducing sugar while sucrose is made up of 99.98% sucrose and
0.01% reducing sugar (Sahu and Sahu, 2013). Considering this
similarity, market sugar was tried as an alternative to sucrose
which turned out to be a beneficial choice. In our study, 2%
market sugar with 2% psyllium husk gave the best results of shoot
multiplication. A higher sucrose concentration hampered shoot
development which could be attributed to destabilization of
osmosis in plant cells at elevated sugar levels. Role of sugar in in
vitro propagation have also been confirmed in some other plants
(Kaur et al., 2005, Goel et al., 2007, Agarwal et al., 2010).
Increased levels of BAP in the medium have been
reported to increase the number of shoots but suppress their
growth (Arora, 1990). Henceforth, in vitro multiplied shoots were
maintained by periodic subculturing alternately at regular intervals
of 2 weeks each on basal MS media in alternation with MS media
with 0.5 mg/l BAP. The process resulted in development of
healthy shoots without any basal callusing or decline in
multiplication rate, a condition often observed in case of excess
PGR provided to the cultured tissue.
The system so developed resulted in slashing down major
production cost of tissue culture plantlets by over six times the
expense in conventional micropropagation protocols.
Present study on in vitro rooting suggested efficacy of
IBA supplemented half strength MS medium. Reduced
macroutrient concentration is known to be favorable for in vitro
rooting considering the fact that nitrogen ions concentration
requirement for root formation is much lower than of shoot
formation (Driver and Suttle, 1987). Half strength medium for
rooting in C. morifolium has already been reported (Kaul et al.,
1990, Waseem et al., 2009). In our study, IBA at a concentration
of 0.25 mg/l proved to be most optimal for in vitro rooting. The
results are in agreement with previous studies on chrysanthemum
reporting superiority of IBA over other auxins (Khan et al., 1994,
Singh and Arora, 1995, Hoque and Fatima, 1995, Karim et al.,
2002, Waseem et al., 2009, Waseem et al., 2011, Nalini, 2012,
Lindiro et al., 2013). Contrastingly, basal MS medium has also
been reported to be efficient for in vitro rooting in C. morofolium
(Vantu et al., 2005).
Plants growing under tissue culture conditions are semiautotrophic (Hazarika, 2003) and leaves may be photosynthetically
incompetent. Besides, micropropagated plants are also generally
susceptible to transplantation shock due to delicate root system,
109
reduced amount of epicuticular wax and reduced or abnormal
stomata. This can result in excessive dehydration, poor control of
gaseous exchange and also poor absorption of nutrients by the root
system. In the present study, a decrease in medium strength (onefourth) for in vitro hardening was done to create favourable stress
conditions to enable the TCR plantlets perform well under field
conditions. The plantlets so developed when raised under ex vitro
conditions showed healthy growth and blossomed in flowering
season.
The present research work also dealt with the evaluation
of two different extracts of in vitro grown leaves of C. morifolium
for antibacterial susceptibility. Previous reports on phytochemical
screening of C. morifolium suggested presence of flavonoids,
sesquiterpenoids, chlorogenic acids, vitamins, and amino acids
(Zhang and Zhang 2007, Zhao et al., 2009) and subsequently led
to exploitation of plant parts to assess their antimicrobial property.
However, most of the research on the antimicrobial assay of C.
morifolium has been focussed on the flower extracts. Such studies
indicated enhanced antimicrobial activity of yellow flowers as
compared to purple and white flowers (Urzua and Mendoza, 2003,
Voon et al., 2011). Methanolic extract of C. morifolium
inflorescence has also been reported to show antimicrobial activity
against B. cereus, L. monocytogenes, E. coli and Salmonella
anatum (Shan et al.,2007) while petroleum ether and ethyl acetate
extracts were found to be effiicient against S. aureus and
methicillin resistant S. aureus (MRSA) (Zhao et al.,2009).
Ethanolic leaf extract of Chrysanthemum indicum have
been reported to exhibit antimicrobial activity against K.
pneumonia, E. coli, S. mutans, P. aeruginosa and B. subtilis
(Rajalakshmi et al., 2013). Besides, other studies have also
reported efficiency of ethanolic extracts of Chrysanthemum sps
(Sassi et al., 2008, Pitinidhipat and Yasurin, 2012). These reports
support of our findings wherein superority of antibacterial activity
of ethanolic extracts of C. morifolium leaves against Gram
negative (E. coli) and Gram positive (B. subtilis and S. aureus)
bacteria was established. The results are suggestive of broad
spectrum antibiotic compounds in the leaves of tissue culture
raised C. morifolium that need to be isolated and identified for
scientific confirmation of the findings.
CONCLUSION
The findings of this research overcome the major hurdles
in application of tissue culture technology to the field i.e. high
production cost and failure to survive under ex vitro conditions.
The protocol is a low cost strategy for mass production of a
commercial flower variety C. morifolium that ensured successful
flowering in TCR plantlets after shifting to field conditions besides
confirming the antibacterial properties in field grown plants. The
study is therefore a complete package for large scale production of
chrysanthemum plantlets at subsidized cost that can be easily and
equally adopted by both farmers and researchers. The findings also
open up scope for appraisal of chrysanthemum leaves as a
potential component in medicinal preparations.
110
Pant et al. / Journal of Applied Pharmaceutical Science 5 (07); 2015: 103-111
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How to cite this article:
Manu Pant, Ankita Lal, Rashi Jain. A simple cost effective method
for mass propagation of Chrysanthemum morifolium and
antibacterial activity assessment of in vitro raised plantlets. J App
Pharm Sci, 2015; 5 (07): 103-111.