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In vitro studies on the variations of biochemical metabolites in
Glycyrrhiza glabra by using various elicitors
THESIS
Submitted in partial fulfilment of the requirements for the award of the degree
of
Doctor of Philosophy
in
Biochemistry
by
Nancy Jaiswal
Department of Biochemistry & Biochemical Engineering,
Jacob Institute of Biotechnology & Bioengineering,
Sam Higginbottom University of Agriculture, Technology & Sciences
Allahabad-211007
2018
ID. No. 12PHCBC103
DECLARATION
I hereby declare that the thesis “In vitro studies on the variations of biochemical metabolites
in Glycyrrhiza glabra by using various elicitors” being submitted as the partial fulfilment for
the degree of Doctor of Philosophy in Biochemistry, Sam Higginbottom University of
Agriculture, Technology and Sciences, Allahabad (U.P.) is an original piece of research work
done by me under the supervision of Dr. (Mrs.) Yashodhara Verma, Assistant Professor. To
the best of my knowledge, no part or whole of the thesis has not been submitted elsewhere for
the award of any other degree or any other qualification of any University or examining body.
Nancy Jaiswal
Place: Allahabad
Date: 7/8/2018
ACKNOWLEDGEMENT
All glory goes to Almighty to whom the pride and perfection belong. It is all his blessing
and mercy that led me to know what is right. First of all I would like to express my deepest sense
of gratitude to the Almighty God.
I feel immensely happy to express my most sincere thanks and gratitude to owe my advisor
Dr. (Mrs.) Yashodhara Verma, Assistant Professor, Department of Biochemistry &Biochemical
Engineering, for her kind initiation, encouragement, sincere and rentless efforts and inspiration
throughout the course of investigation. It would not have been possible to present his report in its
present form without her help and support. I am extremely grateful to her for guiding me
through her meticulous thought during the investigation.
I am greatly indebted to my co-advisor Dr. (Mrs.) Pragati Misra, Assistant Professor,
Department of Molecular & Cellular Engineering for her noble guidance, untiring supervision,
encouraging and creative suggestion and authentic support in bringing up this work in status
throughout the period of my lab work and in the preparation of manuscript. I indeed feel
honoured to have worked with her who nurtured my academic capabilities and creativity in
gentle ways and provided operational freedom and healthy environment.
I would like to extend my gratitude to Prof. (Dr.) A.M. Lall (Head& Professor) and
Dr.(Mrs.) Reena Lawrence, (Associate Professor), Department of Biochemistry & Biochemical
Engineering, the erudite member of my advisory committee for their creative suggestion,
constructive criticism, motivating guidance and keen interest throughout my endeavour in
carrying out the experiment successfully.
I owe my sincere gratitude to Dr. (Mrs.) Sushma (Assistant Professor), Dr. Veeru
Prakash (Associate Professor), Dr. Shailendra Kumar Srivastava (Assistant Professor) and Er.
Akhilesh Bind (Associate Professor), Department of Biochemistry & Biochemical Engineering
for their selfless co-operation, outstanding support and guidance at every step of my work which
has made it a success.
I found no theoretical gems from the ocean of words to express my grateful appreciation to
my father Mr. Narendra Jaiswal, mother Mrs. Ranjana Jaiswal, husband Mr. Ichhanshu
Jaiswal, sisters Ritika, Riya and Sakshi, brother Naman and all other family members. It was
their dream and ambition that made me strong to pursue further studies. It is their endless
support, patience, inspiration, affection and encouragement that have made it possible.
Academic life without friends is worthless. I really thank God as I am blessed with a
number of friends who ungrudgingly took pain and spared time and energy for me. I express
profound and sincere sense of gratitude to my friends and colleagues Preeti Rajoriya, Aanisa
Zahoor, Krishna Ash, Deepshika Singh, Sarvesh Kumar Mishra, Vivek Kumar Singh and
Shubhendra Singh Chauhan for their cordial help, innovative ideas, prefectural
encouragement, moral and mental support during hard hours of work.
Atlast but not least I am extremely thankful to Mr. Ramsagar, Mr. Hemant, Mr. Sanjay,
Mr. Dilshad, Mr. Vimlesh, and Mr. Marshall, who have helped me incompletion of my work. I
owe my heartiest thanks to all those people who have helped me to complete this task.
Countless others have contributed to install in me the “Scientific Attitude” and I am
apologise that they don’t find a mention here. Nevertheless, they shall forever command the
deepest respect and highest gratitude. I am thankful to all those who have directly and
indirectly helped me steer through the work.
Place: Allahabad
Date: 7/8/2018
Nancy Jaiswal
CONTENTS
Chapter No. Particulars Page no.
List of Tables i – iv
List of Figures v – vi
List of Plates vii – ix
List of Abbreviation x – xii
Abstract xiii
1 Introduction 1 – 6
2 Review of Literature 7 – 48
3 Materials and Methods 49 – 75
4 Results and Discussion 76 – 131
5 Summary and Conclusion 132 – 135
6 References 136 – 189
Annexure xiv – xlv
i| P a g e
LIST OF TABLES
Table No. Title Page No.
2.1Pharmacological roles of the active components found in
licorice (Glycyrrhiza spp.)14
2.2Preparation of Glycyrrhiza species for tissue culture studies
with emphasis on G. glabra L. (chronological order)22 – 23
2.3In vitro conditions for tissue culture studies on Glycyrrhiza
species, with special emphasis on Glycyrrhiza glabra(chronological order)
31 – 36
2.4Elicitation of secondary metabolites in licorice through biotic
and abiotic elicitors44
3.1 Instruments used 50
3.2 Composition of MS medium stock solution 52
3.3Treatment of explants with different surface sterilizers of
different concentration55
3.4Hormonal combination of different growth regulators used for
shoot initiation57
3.5Hormonal combination of different growth regulators for shoot
proliferation58
ii| P a g e
3.6Hormonal combination of different growth regulators for
callus induction59
3.7Hormonal combination of different growth regulators for
organogenesis60
3.8Hormonal combination of different growth regulators for
rooting61
3.9 Preparation of Nutrient agar (NA) medium 72
3.10 Preparation of Potato dextrose agar (PDA) medium 73
3.11 The skeleton of two way ANOVA analysis 74
4.1Standardization of sterilization protocol for G. glabra using
different sterilants for different time duration77
4.2Effect of different combination of growth regulators on shootestablishment using nodal segment of G. glabra as an explant
81
4.3Effect of different combination of growth regulators on shoot
proliferation from the established shoot of G. glabra84
4.4Effect of different combination of growth regulators on callus
induction using leaves and stem of G. glabra as an explant90
4.5Effect of different additives on browning of callus regeneratedusing leaves and stem of G. glabra on MS media fortified with
2 mg/l BAP and 2,4- D93
iii| P a g e
4.6Effect of different combination of growth regulators on shoot
regeneration from the callus of G. glabra98
4.7Effect of different growth regulators on rooting of in vitro
regenerated shoot of G. glabra102
4.8Effect of different concentrations and combinations of
solutions on encapsulation107
4.9Effect of different substrate and storage period on the re-
growth frequency of encapsulated micro-shoots110
4.10Effect of different elicitors on biomass accumulation of in
vitro grown callus of G. glabra on MS media supplementedwith 2 mg/l BAP, 0.5 mg/l 2,4- D and 50 mg/l Ascorbic acid
111
4.11Phytochemical screening of active constituents in various
extract of plant115
4.12
Effect of different elicitors on the primary metabolites of invitro grown callus of G. glabra on MS media supplementedwith 2 mg/l BAP, 0.5 mg/l 2,4- D and 50 mg/l Ascorbic acid
and its comparison with field grown plant after 15 days
117
4.13
Effect of different elicitors on the secondary metabolites of invitro grown callus of G. glabra on MS media supplementedwith 2 mg/l BAP, 0.5 mg/l 2,4- D and 50 mg/l Ascorbic acid
and its comparison with field grown plant after 15 days
119
4.14
Effect of different elicitors on the antioxidant enzyme activityof in vitro grown callus of G. glabra on MS mediasupplemented with 2 mg/l BAP, 0.5 mg/l 2,4- D and 50 mg/lAscorbic acid and its comparison with field grown plant after15 days
124
iv| P a g e
4.15Anti-bacterial activity of root and leaves in different solvent
extract of G. glabra127
4.16Anti-fungal activity of root and leaves in different solvent
extract of G. glabra128
v| P a g e
LIST OF FIGURES
Figure No. Title Page No.
2.1(A) Glycyrrhiza glabra (B) G. inflate (C) G. uralensis and (D)
G. echinata9
2.2Chemical structure of some active constituents of Glycyrrhizaglabra
13
2.3Overall frequency of different in vitro culture systems used inchemical elicitation experiments for secondary metaboliteproduction
39
2.4 Classification of elicitors 41
2.5Diagrammatic depiction of elicitors and their mode of action
mimicking possible elicitation mechanism using elicited plantcell, tissue and organ cultures in vitro
42
2.6Chemical structures and the biosynthetic pathway forglycyrrhizin and related triterpenoids in licorice plants
45
4.1Effect of different sterilants on the contamination and survival of
explants of G. glabra78
4.2Effect of growth regulators on shoot emergence using nodal
segment of G. glabra as an explant81
4.3Effect of growth regulators on shoot number and shoot length of
G. glabra grown under in vitro condition85
4.4Effect of growth regulators on callus induction through different
explant (stem and leaves) of G. glabra89
vi| P a g e
4.5Effect of additives on browning and callus induction of G.
glabra96
4.6Effect of growth regulators on shoot number and shoot length of
in vitro regenerated G. glabra98
4.7Effect of different hormones on rooting of in vitro regenerated
shoot of G. glabra104
4.8Effect of elicitors on primary metabolites of in vitro grown
callus of G. glabra and its comparison with field grown plant118
4.9Effect of elicitors on secondary metabolites of in vitro growncallus of G. glabra and its comparison with field grown plant
119
4.10Effect of elicitors on antioxidant enzyme activity of in vitro
grown callus of G. glabra and its comparison with field grownplant
124
vii| P a g e
LIST OF PLATES
Plate No. Title Page No.
1.
Culture establishment from nodal segment of G. glabra wheninoculated in MS media supplemented with phytohormonecombination BAP, KIN and IAA at various concentration (a). 2mg/l KIN + 0.5 mg/l IAA (b). 2 mg/l BAP + 0.5 mg/l IAA(c). 4mg/l BAP + 0.5 mg/l IAA (d). 6 mg/l BAP + 0.5 mg/l IAA after20 days
82
2.
Shoot proliferation from the regenerated shoot of G. glabra wheninoculated in MS media supplemented with phytohormonecombination BAP, IAA, NAA and AdS at various concentration(a). 2 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS (b). 4 mg/l BAP +0.5 mg/l IAA + 40 mg/l AdS (c). 2 mg/l BAP + 0.5 mg/l NAA +40 mg/l AdS (d). 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l AdS
86
3.
Shoot proliferation from the regenerated shoot of G. glabra wheninoculated in MS media supplemented with phytohormonecombination BAP, NAA and GA3 at various concentration (a). 2mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (b). 4 mg/l BAP + 0.5mg/l NAA + 0.5 mg/l GA3 (c). 2 mg/l BAP + 0.5 mg/l NAA + 1mg/l GA3 (d). 4 mg/l BAP + 0.5 mg/l NAA + 1 mg/l GA3
87
4.
Callus induction using leaf as an explant of G. glabra wheninoculated in MS media supplemented with 2 mg/l BAP + 0.5 mg/l2.4-D + 50 mg/l Ascorbic acid (a). Curling of leaves (b). Swellingof leaves(c). Initiation of callus (d). Greenish yellow callus
94
5.
Callus induction using stem as an explant of G. glabra wheninoculated in MS media supplemented with 2 mg/l BAP + 0.5 mg/l2.4-D + 50 mg/l Ascorbic acid (a). Swelling of stem (b). Initiationof callus (c). Greenish yellow callus
95
6. Shoot regeneration from the callus of G. glabra when inoculatedin MS media supplemented with different phytohormone
99
viii| P a g e
combination (a). Shoot initiation (4 mg/l BAP + 0.2 mg/l IAA +50 mg/l Ascorbic acid) (b). Shoot elongation (4 mg/l BAP + 0.5mg/l IAA + 50 mg/l Ascorbic acid + 1 mg/l GA3) (c). Shootproliferation (4 mg/l BAP + 0.5 mg/l IAA + 1 mg/l GA3 + 50 mg/lAscorbic acid) and (d). Shoot multiplication (4 mg/l BAP + 0.2mg/l IAA +40 mg/l AdS + 50 mg/l Ascorbic acid)
7.
Shoot proliferation from the regenerated shoot of G. glabra wheninoculated in MS media supplemented with differentphytohormone combination (a). 4 mg/l BAP + 0.5 mg/l IAA + 50mg/l Ascorbic acid (b). 4 mg/l BAP + 0.2 mg/l IAA + 50 mg/lAscorbic acid (c). 4 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS + 50mg/l Ascorbic acid and (D.) 4 mg/l BAP + 0.2 mg/l IAA + 40 mg/lAdS + 50 mg/l Ascorbic acid
100
8.
(a). Rooting of regenerated shoot of G. glabra when inoculated inMS media supplemented with phytohormone combination IBAand IAA (b). 3 mg/l IBA (c). 3 mg/l IBA + 0.5 mg/l IAA (d). 3mg/l IBA + 1 mg/l IAA
103
9.Hardening and acclimatization of complete regenerated plantletsof G. glabra in bottles, cups and pots containing sterile sand, soiland vermiculite (1:1:1) mixture
105
10.
Re-growth of plantlet using synthetic seed containing nodalsegment of regenerated plantlet of G. glabra (a). Synthetic seed(b). Inoculation of seed in MS media (c - d). Shoot initiation (MSbasal media) (e - f). Shoot regeneration from seed using MS mediasupplemented with 4 mg/l BAP + 0.5 mg/l IAA + 40 mg/l Ads and4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads respectively
109
11.
HPLC chromatogram for glycyrrhizin interpretation in themethanolic extract of root and callus (a). Standard (Pureglycyrrhizin) (b). Root (in vivo) (c). Callus without elicitortreatment (d). Callus treated with adenine sulphate (e). Callustreated with biotin
120
ix| P a g e
12.
HPLC chromatogram for glycyrrhizin interpretation in themethanolic extract of callus (a). Callus treated with salicylic acid(b). Callus treated with putrescine (c). Callus treated withspermine (d). Callus treated with spermidine
121
13.
Anti-bacterial activity of root and leaves of G. glabra in differentsolvent extract [Aqueous (Aq), acetone (Ac), ethanol (Et) andmethanol (Mt)] against different bacterial strain (a). Bacillussubtilis (b). Proteus vulgaris and (c). Streptococcus mutans
129
14.
Anti-fungal activity of root and leaves of G. glabra in differentsolvent extract [Aqueous (Aq), acetone (Ac), ethanol (Et) andmethanol (Mt)] against different fungal strain (a). Candida albicanand (b). Aspergillus niger
130
15.
Anti-bacterial and anti-fungal activity of standard (Streptomycinand Bavistin) against different bacterial and fungal strainrespectively (a). Bacillus subtilis (b). Proteus vulgaris (c).Streptococcus mutans (d). Candida albican and (e). Aspergillusniger
131
x| P a g e
LIST OF ABBREVIATIONS
AdS : Adenine sulphate
ANOVA : Analysis of Variance
Avg. : Average
BAP : 6-Benzyladenine
C.D. : Critical Difference
cm : Centimetre (s)
cm2 Centimetre square
CWFT : Cool-white fluorescent tube
d.f. : Degree of freedom
2,4-D : 2,4-Dichlorophenoxyacetic acid
e.g. : For example
ESS : Error Sum of Square
et al. : And others
F (cal.) : F calculated
F (tab.) : F tabulated
Fig. : Figure
GA3 : Gibberellic acid
g : Gram (s)
h : Hour (s)
HCl : Hydrochloric acid
HgCl2 : Mercuric chloride
i.e. : That is
xi| P a g e
IAA : Indole acetic acid
IBA : Indole butyric acid
kg : Kilogram (s)
Kn : Kinetin
l Litre (s)
MSS : Mean sum of square
m : Metre (s)
mhz : Megahertz
μM : Micromolar
μl : Microlitre
mg : Milligram (s)
min. : Minute (s)
ml : Millilitre (s)
mm : Millimetre (s)
nm : Nanometre
NAA : α-Naphthaleneacetic acid
NaOH : Sodium hydroxide
NaOCl : Sodium hypochloride
no. : Number
NS : Non-significant
ppm : Parts per million
PP : Photoperiod
psi : Pound per square inch
RH : Relative humidity
rpm : Revolution per minutes
xii| P a g e
sec : Second (s)
S : Significant
SS : Sum of square
S.E. : Standard error
Temp. : Temperature
TSS : Total sum of square
via. : Through
viz. : Namely
w/v : Weight / volume
xiii| P a g e
ABSTRACT
The Glycyrrhiza glabra, of the Fabaceae family, is a medicinal and edible herbs that contain a
wide range of phytochemicals which are used pharmaceutically and commercially. Glycyrrhiza
glabra is under the threat of overexploitation and depletion therefore, there is an urgent need for
conservation. It is advantageous to develop in vitro techniques not only for propagation,
multiplication and preservation but also for elicitation of secondary metabolite production. The
present study provides information on the micropropagation of G. glabra related to the use of
different explants, the combination of plant growth regulators with different sterilization
strategies, the culture conditions, and additional factors influencing in vitro propagation (such as
light, temperature, humidity and pH). Conservation of germplasm from G. glabra, through
encapsulation have also been studied which avail the germplasm for commercial cultivation over
the long run. Qualitative and quantitative screening for phytoconstituents and the evaluation of
their antimicrobial activity have also been presented. Enhanced production of glycyrrhizin, main
bioactive component of G. glabra by different elicitors is deliberated. Successful multiplication
and elicitation will lead to the production of not only greater quantities of planting material with
improved quality but also commercially desired metabolites. This study will be helpful in future
studies on somaclonal variation, genetic transformation and drug discovery.
Keywords: Glycyrrhiza glabra, licorice, regeneration, callogenesis, encapsulation, elicitation
CHAPTER – 1
INTRODUCTION 1 |P a g e
INTRODUCTION
The widespread use of herbal remedies and healthcare preparations as those described in
ancient texts such as the Vedas, the Bible, and those obtained from traditional practices, has
been traced to the occurrence of natural products with medicinal properties (Hoareau and
DaSilva, 1999). Plants synthesize and store a wide variety of biochemical compounds called
secondary metabolites which are conventionally recognized as pharmaceuticals, flavours,
fragrances, dyes, pigments, pesticides, food additives and many more (Hussain et al., 2012).
Most of the secondary metabolites are metabolically induced in plants in response to
environmental stress and hence play defensive role enabling protection to plant as well as
humans from various biotic and abiotic factors (Mazid et al., 2011).
In most of the developing countries the use of medicinal plants has been observed as a
normative basis for the maintenance of good health. Furthermore, an increasing reliance on
the use of medicinal plants in the industrialised societies has been traced to the extraction and
development of several drugs and chemotherapeutics from these plants as well as from
traditionally used rural herbal remedies (UNESCO, 1998). Approximately 80% world
population relies on herbal medicines as over the counter herbal formulations and proprietary
herbal drugs. Industrialized societies are involved in extraction of bioactive constituents from
medicinal plants and use them directly or indirectly as new drugs (Farnsworth et al., 1985;
Saxena, 2002).
Secondary metabolites can be broadly classified as terpenoids, alkaloids and phenolic
compounds which are synthesized through their specific metabolite pathways and possess
specific structural and functional characteristics. Biochemical synthesis of these metabolites
for industrial use is often not feasible due to complex metabolic pathways, complicated
structures and chirality exhibited by these compounds (Namdeo, 2007). The commercial
demand of these compounds can only be met by obtaining them directly from field grown
plants. However, most of the plants accumulate secondary metabolites in small amounts in
specialized tissues probably after attaining a certain stage in their life cycle. Apart from this,
the yields of secondary metabolites extracted from field grown plants are influenced by many
factors like climate, pests and diseases which are difficult to control and in turn affect their
CHAPTER – 1
INTRODUCTION 2 |P a g e
consistent production, due to which the commercial exploitation becomes a challenging task
(Dixon, 2001; Oksman-Caldenteyl and Inze, 2004).
Efficient extraction of desired compounds may require complete harvesting of the plant
parts or whole plant. Blind harvesting of medicinal plants has led to extinction of several
valuable plant species (Rates, 2001). Based on the International Union for Conservation of
Nature and Natural Resources' (IUCN's) Red List Categories, the Indian government assessed
the status of 359 wild medicinal plants and 93 percent of plants were found to be either
threatened, vulnerable, endangered or critically endangered, primarily due to their
overexploitation (Singh et al., 2006a).
Biotechnological approaches, specifically plant tissue cultures, are found to be good
alternatives to overcome these demerits and offer consistent yield of secondary metabolites
for commercial use (Savitha et al., 2006). Plant cell and tissue cultures are capable of
producing specific phytochemicals at a rate similar or superior to that of intact plants.
Moreover, the biosynthetic capacity of cultured plant tissue can be enhanced by regulating
environmental factors, as well as by artificial selection or induction of variant clones for high
productivity. Several phytochemicals localized in morphologically specialized tissues or
organs of native plants have been produced in culture systems not only by inducing specific
organized cultures, but also by undifferentiated cell cultures (Aijaz et al., 2011).
In the process of plant tissue culture, explants are cultured under appropriate
physiological conditions and the desired product is extracted from the cultured cells/tissue.
Recent developments in plant tissue culture techniques and their processing have shown
promising results to improve the productivity to many folds and have made it possible to
gradually replace the whole plant cultivation as a source of useful secondary metabolites.
Today, various tissue culture techniques are being used to enhance yield of secondary
metabolites by invigorating plant defense and triggering stress response in plant cells with the
help of elicitors (Chattopadhyay et al., 2002).
Elicitors are being used as an enhancement strategy in plant secondary metabolite
synthesis as they play an important role in stimulating the biosynthetic pathways leading to
enhanced production of commercially important compounds. This provides an opportunity
CHAPTER – 1
INTRODUCTION 3 |P a g e
for intensive research in the field of plant sciences not only for exploitation of plant cells for
increased yield of secondary metabolites, but also for investigation of plant defense
mechanism and regulation of secondary metabolism (Radman et al., 2003).
Among the promising medicinal plants, Glycyrrhiza glabra has a good scope for
research on the production and enhancement of its secondary metabolites. Glycyrrhiza glabra
L. commonly known as ‘Licorice’ is an ancient ayurvedic medicinal plant which considers
being a “rasayana” with implicated use in treatment of respiratory and digestive disorders
(Meena et al., 2010). Licorice is one of the commercially important plant species from
theleguminosae family. It is referred to as Mulethi, Malahatti and Yastimadhu. The genus
Glycyrrhiza consists of 30 species native to the Mediterranean and certain areas of Asia
(Blumenthal et al., 2000). These species includes G. glabra, G. uralensis, G. inflata, G.
aspera, G. korshinskyi and G. eurycarpa. G. glabra also includes three varieties: Persian and
Turkish licorices are assigned to G. glabra var. violacea, Russian licorice is G. glabra var.
gladulifera, and Spanish and Italian licorices are G. glabra var. typical (Nomura et al.,
2002).
The conventional method for propagation of G. glabra is via seed. Limited seed set and
short span of seed viability restricted the commercial cultivation of licorice by seed (CIMAP
Newsletter, 1995). However, seed unavailability, seed dormancy and unfavourable
environment are the major obstacles in using seeds for the propagation of licorice.
Multiplication of plant is restricted due to poor seed germination potential (Sawaengsak et
al., 2011). The crop, however, is predominantly propagated through vegetative parts, mostly
rhizomes, stolons or other cuttings. This propagation method is destructive as it requires the
use of economically valuable part of the plant, slow due to the need to waiting for years until
rhizomes are ready and re-productivity of rhizomes is reduced by unfavourable climate and
soil conditions (Gupta et al., 1997; Duke, 1981).
The major constituents of licorice are triterpenoids and flavonoids. A number of other
components have also been isolated from licorice such as triterpene, saponins, flavonoids,
polysaccharides, pectins, simple sugars, amino acids, mineral salts, starches, gums, mucilage,
essential oil, fat, asparagines, tannins, glycosides, protein, resins, sterols, volatile oils and
various other substances (Fenwick et al., 1990; Blumenthal et al., 2000). Glycyrrhizin, a
CHAPTER – 1
INTRODUCTION 4 |P a g e
triterpenoid compound, accounts for the sweet taste of licorice root. This compound
represents a mixture of potassium-calcium-magnesium salts of glycyrrhizic acid
(Obolentsevaet al., 1999). Among the natural saponins, glycyrrhizic acid is a molecule
composed of a hydrophilic part, two molecules of glucuronic acid and a hydrophobic
fragment, glycyrrhetic acid. The yellow colour of licorice is due to the flavonoid content of
the plant (Yamamura et al., 1992).
Analytical methods such as TLC, GLC, HPLC and LC-MS were employed for the
separation of constituents of licorice. Further evaluation and determination were carried out
using spectrophotometric, mass spectrophotometric and NMR procedures. Various methods
employed to isolate the constituents of licorice were modified from time to time in
accordance with improvement in technology.
Glycyrrhizin (oleanane type terpenoid saponin), one of the active constituent of licorice
plant is a prescription drug used in the treatment of liver and allergic diseases. They are
exclusively obtained from the dried roots and stolons of licorice. Chemical analyses have
failed to detect them in the aerial part (Hayashi et al. 1988); the plants have been
indiscriminately exploited to meet the high demand, resulting in desertification of the habitat.
Glycyrrhizin is shown to be 50 times or more sweet than sugar and demands high prices in
the world market as a non-nutritive sweetener (Olukoga and Donaldson, 1998; Duke,
1981).It is manufactured in the form of injections (such as Stronger Neo-Minophagen®C)
and tablets (such as Glycyron®) which are available in India and many other countries
(Hayashi and Sudo, 2009). Glycyrrhetinic acid is also an active constituent of the
prescription drug used in the treatment of peptic ulcers. It has also been used as a cure to
atopic dermatitis, pruritis and cysts due to parasitic infestations of skin (Saeedi et al., 2003).
In modern medicine, licorice extracts are often used as flavouring agents to mask bitter
taste in tonics and as an expectorant in cough syrups (Kanimozhi and Karthikeyan, 2011).
Gels containing glycyrrhizin are used for the treatment of oral diseases and genital lesions
caused due to Herpes simplex virus (Segal et al., 1987, Varsha et al., 2009). Bioactive
constituents of Glycyrrhiza glabra are being explored for their potential used as anti-cancer
and anti HIV drugs. They are also used in the confectionery, tobacco and pharmaceutical
industry (Rastitel'nye Resursy, 1987). Licorice is certainly promising candidate for
CHAPTER – 1
INTRODUCTION 5 |P a g e
providing new derivatives of pharmaceutically active constituents which can be evaluated for
pharmacological use as future drugs for prevention and cure of large number of ailments.
In virtue of its importance in food and pharmaceutical industry, licorice was extensively
subjected to scientific investigations. Recent advents in molecular biology and biotechnology
led to the efforts to improve the yield of compounds which are of economic importance.
Attempts were made to understand the biochemistry of licorice and its derivatives. Efforts
were made to increase the yield in licorice, employing plant tissue culture techniques. Shah
and Dalal (1982) attempted for in vitro multiplication under various cultural conditions
employing modifications of MS medium. Their trails yielded successful establishment of
plantlets and found 15-20 fold increase in multiplication rate when compared to propagation
through stolon cuttings.
Attempts have been made for clonal and rapid propagation of licorice and for the in
vitro production of Glycyrrhizin, using root, stem (nodal segments), leaf and shoot tips as
explants (Ayabe et al., 1980; Kobayshi et al., 1985; Waithaka, 1992; Arias-Castro, et al.,
1993a; Dimitrova et al., 1994). These workers reported various optimum growth media.
Investigations were also carried out to derive commercially important phytochemicals
from licorice. Wu et al. (1974) reported the absence of glycyrrhizin in suspension cultures of
licorice. Hayashi et al. (1988) recorded similar observations in callus and cell suspension
cultures of G. glabra. The cells failed to produce detectable amounts of glycyrrhizin though
the intermediate compounds such as betulinic acid, beta-amyrin were detected. They
speculated the absence of glycyrrhizin production was to be due to interruption in the
biosynthetic pathway of glycyrrhizin. Later studies of G. glabra also revealed the failure of
suspension cultures to produce glycyrrhizin after exogenous supplementation of 18 β-
glycyrrhizic acid (Hayashi et al., 1992).
From the preceding part of this text, the economic potential of licorice is unequivocally
evident from its highly diverse intrinsic pharmacological properties apart from its use in food
industry. The commercial requirement of licorice will certainly increase in future. Poor
germination potential restricts its multiplication. To conserve foreign exchange and to meet
the increasing demand, it is highly necessary to focus our attention on licorice research. A
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rapid method of multiplication is necessary to overcome shortage of planting material of this
crop in our country and also for multiplying newly developed improved quality materials
which are available in small quantities. With a view to increase the rate of multiplication and
make the technique commercially viable, experiments will be conducted to standardize the
technique for rapid in vitro clonal propagation of licorice to obtain true type planting
materials in large quantity. Attempts on various aspects can be made to indigenize the
commercial production of licorice. The goal can be achieved, in a broad manner, by the
application of plant breeding techniques, micropropagation and metabolite production in
vitro.
The medicinal properties of plants are due to the presence of complex chemical
substances of varied composition present as metabolites in one or more parts of these plants.
In plant tissue culture, accumulation of metabolites can be enhanced by the treatment of
various kinds of elicitors, which can be biotic and abiotic. Previous studies have shown that,
the accumulation of different secondary metabolites can be efficiently induced by using
elicitors of various types. Many investigations manifest that a large number of food products
or medically important compounds of plant origin have been obtained through plant tissue
culture.
Keeping in mind the above considerations the experiments were conducted with the
following objectives.
OBJECTIVES:
To develop an efficient in vitro plant regeneration protocol in G. glabra.
To standardise the protocol for artificial seed production using nodal segment of G.
glabra.
To study the effect of various elicitors on the enhancement of biochemical metabolites
of G. glabra.
To study the effect of different elicitors on the antioxidant enzyme activity of G.
glabra.
To evaluate the antimicrobial activity of G. glabra in various solvents.
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REVIEW OF LITERATURE
2.1 Medicinal Plants
Medicinal plants have been the subjects of man’s curiosity since time immemorial (Constable,
1990). Medicinal plants include various plants in herbal with rich therapeutic value and are rich
source of ingredient that can be used in the development of various drugs. Medicinal plants have
the added advantage of being simple, effective, and offering a broad spectrum of activity with
well-documented prophylactic or curative actions. These are also proved to be useful in
minimizing the adverse side effects of various chemotherapeutic agents (Rasool Hassan, 2012).
Medicinal plants are used to treat illness and diseases for thousands of years and have gained
economic importance because of their useful application in pharmaceutical, cosmetic, perfumery
and food industries (Gomez-Galera et al., 2007; Leonard et al., 2009).
Approximately 80% of the people in the world’s developing countries rely on traditional
medicine for their primary health care, and about 85% of traditional medicine involves the use of
plant extracts (Vieira and Skorupa, 1993). India is a vast repository of medicinal plants that are
used in traditional medical treatments. Till now very few plants have been scientifically proved
by different researchers for their medicinal potential but the therapeutic ability of number of
plants are still unknown. The renaissance of medicinal potential of such plants is thus strongly
needed.
2.2 Current status of biodiversity of important medicinal plants in India
India is rich in medicinal plant diversity, one among the twelve mega diversity centers with all
the three levels of biodiversity (species diversity, genetic diversity, and habitat diversity). All
known types of agro climatic, ecologic, and edaphic conditions are met within India (Mukherjee
and Wahile, 2006). In India, medicinal plants comprise of approximately 8000 species and
accounts for about 50% higher flowering plant species (Sharma et al., 2010a). Forests are
estimated to harbour 90% of India’s total medicinal plants diversity; only about 10% of the
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known medicinal plants of India are restricted to non-forest habitats (Wakdikar, 2004). The
classic systems of medicines like Ayurveda, Siddha, and Unani make use of many medicinal
plants in various formulations. The world average stands at 12.5% while India has 20% plant
species of medicinal value (Schippmann et al. 1990; 2006). But according to Hamilton (2003),
India has about 44% of flora, which is used medicinally. Although it is difficult to estimate the
total number of medicinal plants present worldwide, the fact remains true that India with rich
biodiversity, which contain active medicinal ingredient (Mandal, 1999). According to
International Union for Conservation of Nature and Natural Resources (IUCN), 247 species are
threatened, 44 plant species are critically endangered, 113 endangered and 87 vulnerable (Singh
et al., 2006a).
The plant used in the phyto-pharmaceutical preparations are obtained mainly from the
naturally growing areas. Over 70% of the plant collections involve destructive harvesting
because of the use of parts like roots, bark, wood, stem and the whole plant in case of herbs. This
possesses a definite threat to the genetic stocks and to the diversity of medicinal plants. Also,
extensive destruction of the plant-rich habitat as a result of forest degradation, agricultural
encroachment, urbanization etc. is other factors, thus challenging their existence (Gupta et al.,
1998). In view of the tremendously growing world population, increasing anthropogenic
activities, rapidly eroding natural ecosystem, etc. the natural habitat for a great number of herbs
and trees are dwindling and has resulted in unsustainable exploitation of Earth’s biological
diversity, exacerbated by climate change, ocean acidification, and other anthropogenic
environmental impacts (Rands et al., 2010). A large sum of money is pumped every year to
replenish the lost biodiversity and large numbers of protocols are available at present.
Unfortunately, we are not witnessing any improvement in the status of these plant species in
nature and the number of threatened plant species is increasing gradually (Tripathi, 2008).
2.3 Glycyrrhiza glabra
The growing interest in and improved extraction efficiency of products from plants has renewed
scientific research in drugs and natural products from medicinal plants. Among many promising
medicinal plants, Glycyrrhiza spp. serves as a good model for research on the production and
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enhancement of secondary metabolites, as they possess a wide range of important
phytoconstituents. Glycyrrhiza spp., commonly known as licorice are perennial leguminous herbs of
the Fabaceae family (Fig 2.1). The word Glycyrrhiza is derived from Greek words ‘glycy’ and
‘rhiza’ which means ‘sweet root’ (Crusheva and Parvanov, 1978). Since 500 BC licorice have
been used medicinally and recognized as ‘the grandfather of herbs’ due to its anti-stress and
anabolic properties. It is an amazing ancient ayurvedic medicinal plant used in the treatment of
respiratory, digestive, gastrointestinal disorders and also have age long therapeutic uses in cough,
hair fall, baldness, piles, gout, weakness in back and limbs, lumbar and cervical spondylitis,
constipation and allergic rhinitis (Meena et al., 2010).
Fig. 2.1 (A) Glycyrrhiza glabra (B) G. inflata (C) G. uralensis and (D) G. echinata
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2.3.1 Origin and Distribution
Commercial licorice is derived from three Glycyrrhiza species, G. glabra L., G. uralensis Fisch.,
and G. inflate Batal which are indigenous to the Mediterranean region and certain parts of Asia
(Shibata, 2000). Glycyrrhiza glabra is native to South Europe (specially Italy and Spain),
Turkey, Iran, Iraq, Central Asia and the north-western China, whereas G. uralensis is native to
Central Asia, Mongolia, and north-western and north-eastern parts of China, and G. inflata is
native to the north-eastern part of China. G. glabra is divided into two varieties: G. glabra var.
typical (Spanish licorice) and G. glabra var. glandulifera (Russian licorice) (Hayashi, 2009;
Hayashi and Sudo, 2009). Three varieties of G. glabra have been reported; the Spanish and
Italian licorice, assigned to G. glabra var. typica; Russian licorice to G. glabra var. glandulifera;
Persian and Turkish licorice to G. glabra var. violacea (Nomura et al., 2002).
Countries growing licorice for economic gain include Afghanistan, Azerbaijan, Iran, Iraq,
Pakistan, the People’s Republic of China, Turkey, Turkmenistan and Uzbekistan (Lim, 2016). In
India, there is no occurrence of the licorice yielding species (Singh et al., 2006b) but attempts
have been made to cultivate it in many places particularly in Budelkhand, Dehradun, Gujarat,
Haryana, Jammu and Kashmir, Madhya Pradesh, and Punjab (Pandey and Dixit, 1980; Singh et
al., 1984; Arya et al., 2009)
2.3.2 Agroecology
Licorice grows well in temperate, warm and subtropical climate. It thrives best in well-lined,
well-drained, composted, loose, friable, deep soil, preferably in full sun. Licorice is not bothered
by frosts, as it is dormant in winter and actually benefits by the defined cold period, which
induces the translocation of properties to the underground rhizomes. They are easily grown from
divisions or root cuttings (Lim, 2016).
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2.3.3 Plant description
Licorice is an herbaceous perennial, leguminous plant of Fabaceace family. It grows to a height
of 1-2 m with woody base and densely scaly glandular punctuate with stoloniferous roots. It has
dark green leaves which are impairpinnate with 9–17 leaflets, abaxially densely scaly glandular
punctate and pubescent on veins, adaxially glabrescent or pilose. Stipules are caduceus and
linear. Inflorescences are open, racemose and many flowered. The pea-like flowers arise from
the leaf axils in a spike-like cluster. Calyx is campanulate; corolla is purple or pale whitish blue;
ovary is glabrous. Fruits are oblong, flat, glabrous or sparsely hairy legume containing 2–8 dark
reniform green seed (2 mm), turning brown at maturity (Lim, 2016). The plant has a deep tap
root system, and produces horizontal stolons and rhizomes that spread out from the main plant
just under the soil surface. The plant produces new shoots from buds on the underground stolons
(Ross, 2001; Lim, 2016).
2.3.4 Phytoconstituents
Licorice is a powerful natural sweetener, 50–170 times sweeter than sucrose (Mukhopadhyay
and Panja, 2008). The chemical constituents include several bioactive compounds such as
starch, D -glucose and sucrose, glycyrrhizin and traces of flavonoids, saponoids, sterols, amino
acids, gums and essential oils etc. (Fenwick et al., 1990). Licorice contained phenol, amines,
amino acids, sterols, sugars and starch of dried root (Blumenthal et al., 2000). The roots of G.
glabra were reported to contain water soluble polysaccharides (1.6%; rhamnose, arabinose,
mannose, glucose and galactose) and total polysaccharides (9.7%) (Dzhumamuratova et al.,
1978). The mineral elements included potassium, calcium, sulphur, iron, nitrogen, phosphorus,
magnesium, sodium, silicon, aluminium, manganese, zinc, copper, titanium and arsenic (Ercisli
et al., 2008). Eight commercial licorice extracts used as food additive were found to contain ash,
glycyrrhizin, sodium, potassium and ammonium nitrogen and pH of 4.1–6.8 (Iida et al., 2007).
Rhizomes were reported to contain alkaloids, triterpenes, saponins, flavonoids, polysaccharides,
steroids and tannins (Meena et al., 2010). G. glabra was found to contain neutral and polar lipids
(triacylglycerides, free fatty acids and free sterols) (Denisova et al., 2007).
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Other constituents include amines, amino acids, bitter principles (asparagines, betaine,
glycyramarin, choline), coumarins (glycerol, glycerine, glycycoumarin, herniarin,
licopyranocoumarin, licoarylcoumarin, licocoumarin, umbelliferone, etc.), flavonoids and
isoflavonoids (glicoricone, glisoflavone, isoliquiritigenin, isoliquiritin, licoflavonol, licoricidin,
licoricone, liquiritigenin, liquiritin, etc.), chalcone and its glycosides (isoliquiritigenin,
isoliquiritin, isoliquiritoside, liquiritoside, neoisoliquiritin, rhamnoisoliquiritin, rhamnoliquiritin,
etc.), gums, licofuranone, lignin, resins, starch, sterols (sitosterol, stigmasterol, 22,23-
dihydrostigmasterol β-sitosterol, etc.), stilbenes, sugars (glucose, fructose, mannose, sucrose,
mannitol), tannins, triterpenes (amyrin, glabrolide, 18-glycyrrhetinic acid, glycyrrhetol,
isoglabrolide, liquirtic acid, etc.), volatile oil (acetylsalicylic acid, salicylic acid, and
methylsalicylate), and a wax (Hayashi et al., 1996; Ammosov and Litvinenko, 2007; Siracusa
et al., 2011; Wei et al., 2014).
2.3.5 Therapeutic Uses
The medicinal properties of Glycyrrhiza glabra and its chemically bioactive constituent have
been extensively studied and documented. Licorice has long been used worldwide as an herbal
medicine and natural sweetener. Licorice root is a traditional medicine used mainly for the
treatment of peptic ulcer, hepatitis C, and pulmonary and skin diseases, although clinical and
experimental studies suggest that it has several other useful pharmacological properties such as
anti-inflammatory, antiviral, antimicrobial, anti-oxidative, anti-cancer activities, anti-obesity,
immunomodulatory, hepatoprotective and cardioprotective effects (Asl and Hosseinzadeh,
2008). A large number of components have been isolated from licorice, including triterpene,
saponins, flavonoids, isoflavonoids and chalcones, with glycyrrhizic acid normally being
considered to be the main biologically active component. The medicinal value of licorice lies in
these bioactive components that produce a definite physiological action in the treatment of
various diseases (Wang et al., 2013a). (Table 2.1)
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Fig. 2.2 Chemical structure of some active constituents of Glycyrrhiza glabra
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Table 2.1 Pharmacological roles of the active components found in Glycyrrhiza glabra
Active component Class Effects References
18β-Glycyrrhetinic
acid
Triterpenoid
saponin
glycoside
Anti-inflammation Xiao et al. (2010)
Anti-cancer Kuang et al. (2013)
Anti-obesity Moon et al. (2012)
Inhibits cholestasis Zhai et al. (2007)
Anti-allergic effect Shin et al. (2007)
Glycyrrhizin
(Glycyrrhizic acid)
Triterpenoid
saponin
glycoside
Immunomodulatory Song et al. (2011)
Anti-ocular hypertension Shi et al. (2011)
Protective effect on
respiratory system
Sen et al. (2011)
Anti-diabetic effect Ogiku et al.(2011)
Liver protection Tu et al. (2012)
Anti-inflammation Ni et al. (2011)
Neuroprotection Kim et al. (2012a)
Inhibits cholestasis Zhai et al. (2007)
Anti-allergic effect Shin et al. (2007)
Liquiritigenin Flavonoid Anti-cancer Liu et al. (2012)
Anti-inflammation Wang et al. (2012)
Angiogenesis Yang et al. (2012)
Isoliquiritin Flavonoid Anti-genotoxicity Kaur et al.(2009)
Antidepressant Wang et al. (2008)
Anti-inflammation Kim et al. (2008)
Licochalcone A Chalcone Anti-obesity Quan et al. (2012)
Osteogenic activity Kim et al. (2012b)
Antiangiogenic effect Kim et al. (2010a)
Anti-tumor and anti-
metastatic effect
Kim et al. (2010b)
Anti-inflammation Funakoshi-Tago et al.
(2009)
Licochalcone E Chalcone Neuroprotection Kim et al. (2012c)
Antimicrobial effect Zhou et al. (2012)
Antidiabetic effect Park et al. (2012)
Induces apoptosis Chang et al. (2007)
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Roots and stolons are highly valued commercial products used in medicine (Anonymous,
2005). In modern era, it is considered an important crude drug for its various pharmacological
activities (Jatav et al., 2011) including anti-diabetic, anti-inflammatory (Finney and Somers,
1958), hepato-protective, anti-ulcer, anti-allergic (Park et al., 2004), antiviral activity (Fiore et
al., 2008; Rathee et al., 2010; De, 2000) and anti-carcinogen (Zhang et al., 2009a). Licorice
have both estrogenic and anti-estrogenic activity (Tamir et al., 2001), thus it is an important
herb for treating hormone-related female problems (Paul et al., 1994). It also serves as a brain
tonic to enhance memory (Dhingra et al., 2004). The clinically proven activities of licorice such
as anti-ulcer, anti-microbial, anti-asthmatic, anti-diuretic and anti-hepatotoxic activity are
attributed to licorice (Vispute and Khopade, 2011).
Glycyrrhizin (an oleanane-type triterpenoid glucuronide), the main active and important
constituent in licorice, is 50-times sweeter than sugar (Brielmann, 1999) and is used in large
quantities as a well-known natural sweetener and as a pharmaceutical (Shibata, 2000). It is a
conjugate of two molecules of glucuronic acid and glycyrrhetinic acid and is found chiefly in
roots and stolons but not in aerial parts (Hayashi, 2007). Glycyrrhizin possesses anti-allergic,
anti-diabetic, anti-inflammatory, anti-ocular hypertension, immunomodulatory, anti-cholestasis,
hepatoprotective, and neuroprotective pharmacological activities. It also has protective effect on
the respiratory system (Wang et al., 2013a).
2.3.6 Cultivation
Licorice plants are seasonal and growing intact plant is also confined to certain climate (Mousa
et al., 2007). Dry seasons are beneficial and therefore it thrive well in warm regions with annual
rainfall (<50 cm). The conventional method for propagation of G. glabra is via. seed. The floral
structures of this family pose the mode of cross-pollination, mainly by insect pollinators,
resulting in variability of offspring in successive generations with delayed or absent flowering in
some environment (Poehman, 1977; Duke, 1981). Normally growers do not use licorice seed
for propagation as the seed loses its viability over short storage and are dormant due to hard
testa, which require scarification prior to planting (Gupta et al., 1997). However, poor seed
availability and viability, seed dormancy and unfavourable environment remain amongst the
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major obstacles in using seeds for the commercial cultivation of licorice (CIMAP Newsletter,
1995). The crop is predominantly propagated through vegetative parts, mostly rhizomes, stolons
or other cuttings but this method is destructive as it requires the use of economically valuable
part of the plant which has slow rate of re-productivity (Gupta et al., 1997; Duke, 1981).
Vegetative propagation of the plant is annual with low germination percentage, being highly
under the influence of environmental conditions (Gupta et al., 2013).
Most of the pharmaceutically important secondary metabolites of Glycyrrhiza glabra are
synthesized and accumulated in their roots only after attaining certain years of maturity. In vivo
extraction of these metabolites from roots of plant is difficult and requires harvesting of matured
roots often involving complete uprooting of plant after which there are minimal chances of its
revival even if it is replanted, leading to complete loss of this plant. Wild licorice plants have
been a source of secondary metabolites but unsustainable harvesting (overexploitation to the
point of diminishing return or extinction) has reduced its supply and increased the need for
cultivation to meet market demands (Lange, 1998). A report of the Planning Commission, New
Delhi (2006) task force on conservation and sustainable use of medicinal plants listed
Glycyrrhiza glabra under the list of major plants required by Indian pharmaceutical industries
and is at the verge of being endangered due to over exploitation. In this regard, there is an urgent
need for an alternative towards conservation of this plant without posing threat to biodiversity.
2.4 Conservation of biodiversity
Although species conservation is achieved most effectively through the management of wild
populations and natural habitats but most of the medicinal plants either do not produce seeds or
seeds are too small and do not germinate in soils. Even plants raised through seeds are highly
heterozygous and show great variations in growth, habit and yield and may have to be discarded
because of poor quality of products for their commercial release. Likewise, majority of the plants
are not amenable to vegetative propagation through cutting and grafting, thus limiting
multiplication of desired cultivars.
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Moreover many plants propagated by vegetative means contain systemic bacteria, fungi
and viruses which may affect the quality and appearance of selected items (Murch et al., 2000).
Thus mass multiplication of disease free planting material becomes a general problem. In order
to overcome these barriers, ex situ techniques can be used to complement in situ methods and, in
some instances, may be the only option for some species (Sarasan et al., 2006; Negash et al.,
2001). Therefore, conservation of medicinal plants can be accomplished by cultivating and
maintaining plants through long-term preservation of plant propagules in plant tissue culture
repositories (Rands et al., 2010).
In vitro techniques have been increasingly applied for mass propagation and conservation
of germplasm as it has superiority over conventional method of propagation. Some of these are
as follows: (1) collection may occur at any time independent of flowering period (2) there is the
potential of virus elimination from contaminated tissue through meristem culture (3) clonal
material can be produced for the maintenance of elite genotypes, (4) rapid multiplication (5)
germination of difficult or immature seed or embryo may be facilitated for breeding programmes
and (6) distribution across the border may be safer, in terms of germplasm health status (7)
reduces the storage space. Storage facilities may be established at any geographical location and
cultures are not subject to environmental disturbances such as temperature fluctuation, cyclones,
insect, pests, and pathogen (Bhojwani and Dennis, 1999; Shibli et al., 2006). In this regard the
micro-propagation holds significant promise for true to type, rapid and mass multiplication under
disease free conditions. Besides, the callus derived plants exhibit huge genetic variation that
could be exploited for developing superior clones/varieties particularly in vegetatively
propagated plant species.
Tissue culture has emerged as a promising technique and is envisaged as a mean for
germplasm conservation to ensure the survival of endangered plant species, rapid mass
propagation for large-scale re-vegetation and for genetic manipulation studies under precisely
controlled physical and chemical conditions (Bhojwani and Razdan, 1983). Combinations of in
vitro propagation techniques and cryopreservation may help in conservation of biodiversity of
medicinal plants (Fay, 1992; Singh et al., 2006a).
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2.5 Plant tissue culture
Plant tissue culture refers to as micropropagation which involves in vitro aseptic culturing of
cells, tissues, organs and whole plant under controlled nutritional and environmental conditions
often to produce clones of a plant. The science of plant tissue culture takes its root from the path
breaking research leading to the discovery of cell followed by propounding of cell theory.
Schleiden (1838) and Schwann (1839) proposed that cell is the basic unit of organism and
visualized that cell is capable of autonomy therefore it should be possible for each cell to
regenerate into whole plant. This theory proved landmark in the development of plant cell study
and later give birth to totipotency, a term coined by Steward (1968).
Plant tissue culture was conceived and enunciated by the German physiologist,
Haberlandt’s (1902) (father of plant tissue culture) prophecy of totipotency, the deemed
inherent ability in every living cell of all the plants to the genesis of an entire plant. Plant
propagation via. tissue culture technique has been emanated over last 40-50 years as a spinoff of
in vitro studies on differentiation, blossomed into success as a technology without parallel and
the progress has been overwhelmed. The elucidation of the facsimile, the inherent totipotency
was empowered by the pioneering experiments of Laibach (1929), Gautheret (1934), White
(1939), Van overbeek et al. (1941), Skoog (1944) and Loo (1945) and Murashige and Skoog
(1962).
Micropropagation, most advanced application of biotechnology, exploits the morphogenic
potential of existing growing parts of the plants (Giles and Morgan, 1987). The exploration of
the doctrine of totipotency at conservation level brought the micropropagation of medicinal
plants to the fore, which revitalized pharmaceutical industries. After the pioneer work of Morel
(1960) on virus elimination and clonal propagation, much progress have been witnessed in the
large-scale propagation of many medical and aromatic plants. Acquisition of morphogenic
competence can occur with greater or less ease in different plant tissues i.e., their ability to
induce de novo a range of development patterns including embryogenesis.
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PGRs play an important role in regulation of morphogenesis at molecular, cellular, organ
and whole plant level (Gasper et al,. 1996; Dodeman et al., 1997; Charriere and Hahne
1998). The process leading to the formation of adventitious bud is thought to be under the
control of growth regulators, a relatively high ratio of cytokinin to auxin (Skoog and Miller,
1957). According to the critique of Skoog and Miller (1957), in vitro tissue morphogenesis is
controlled by the balance ratio of auxin and cytokinin added to the culture medium and the same
were well later documented by Gaspar et al. (1996), Arockiasamy and Ignacimuthu (1998)
and Sivakumar and Krishnamurthy (2000). In vitro morphogenesis falls into mutually
exclusive pathways: organogenesis and somatic embryogenesis (Hicks, 1980).
2.5.1 In vitro studies in Glycyrrhiza species
An endeavor to review the various aspects of in vitro studies performed on Glycyrrhiza species
of plant are curtailed below under the subtitles. Tissue culture offers large scale in vitro
propagation, multiplication and conservation of invaluable germplasm which would allow
pathogen-free and season independent production of clonal plants. Till date there are many
reports on the micropropagation of Glycyrrhiza species (special emphasis on Glycyrrhiza glabra
L.) (Table 2.2, 2.3). In order to establish a complete plant using tissue culture protocol, the
collection and selection of explants, surface sterilization along with choice of vessel, culture
establishment, maintenance and multiplication followed by rooting, acclimatization to ex-vitro
transfer and adaptation to field conditions are the desired steps and finally testing the genetic
fidelity of in vitro raised plantlets is also of great concern.
2.5.1.1 Collection and selection of explants
Collection and selection of explants is the first and foremost important step essential for
establishing in vitro raised plantlets through plant tissue culture, as summarized for Glycyrrhiza
species in Table 2.2. Licorice is a seasonal plant due to fluctuations in growing condition and
geographical variation (Mousa et al., 2007). The seeds are dormant due to delayed or absence of
flowering and invariability of offspring in successive generations. Licorice is not bothered by
frosts, as it is dormant in winter, and actually benefits by the defined cold period which induces
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translocation properties to the underground rhizomes (Lim, 2016). Seed dormancy and non-
viability restricts its germination potential (Gupta et al., 1997; Duke, 1981). Mature seeds for in
vitro culture were collected in the month of June (Shams-Ardakani et al., 2007).
Selection of explants involves consideration of certain factors such as physiological or
ontogenic age (young vs mature), explants source and size. For clonal propagation, the explants
have been taken from mature plant (Sawaengsak et al., 2011). Terminal and axillary buds
collected in late winter were found to be most responsive as explants (Thengane et al., 1998).
Seasonal difference influences the cell cycle thereby affecting morphogenesis (Anderson et al.,
2001). Middle order nodes collected between May to August were found to have highest bud
break and shoot length (Yadav and Singh, 2012).
Different explants were used to establish plant tissue culture in Glycyrrhiza species such as
seed, axillary bud, leaves, stem, nodes, etc as described in Table 2.2. Nodal segments from field
grown plant have been a popular choice for the micropropagation of Glycyrrhiza species (Yadav
and Singh, 2012; Gupta et al., 2014; Sarkar and Roy, 2014). Young leaves are used to
establish regenerative callus from different Glycyrrhiza species (Mousa et al., 2007;
Wongwicha et al., 2008). Cotyledon was found to be best in the establishment of callus culture
from G. glabra (Wawrosch et al., 2009). Seedling derived explants (such as hypocotyl, leaf,
cotyledon and stem segment) have been used for the optimization of embryogenic callus of G.
glabra. Hypocotyl was found to give rise to the highest frequency and intensity of callus
formation than any other explant (Fu et al., 2010). In vitro plant regeneration system through
stolon culture has been established for the mass and clonal propagation of Glycyrrhiza (Kojoma
et al., 2010; Gupta et al., 2013).Precise and exact age of explant used in tissue culture was not
reported for Glycyrrhiza species.
2.5.1.2 Surface sterilization and choice of culture vessel
For establishing aseptic plant tissue culture protocol surface disinfection is one of the most
crucial and critical step (Teixeira da Silva et al., 2015). Establishing aseptic cultures from the
field grown plants is always a challenge as there is always a high risk of internal and external
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contamination (Hennerty et al., 1988). Selection of surface disinfectant, concentration and
duration is critical and may vary depending on the explant used because during sterilization only
contaminants should be eliminated and the biological activity of living material should not be
lost. Low concentration of disinfectant for low duration was required by soft young or juvenile
explant as compared to old and mature explants.
In Glycyrrhiza species, disinfection of explants was generally done with1% Tween 20 (10-
15 min), 70% ethanol (30 s), 2-5% sodium hypochlorite (10-15 min) or 0.1% mercuric chloride
(3-5 min) followed by continuous rinse of explant with sterile distilled water after every single
step (3-5 times). Other variants sterilization protocol proposed by Shams-Ardakani et al. (2007)
who used 30% H2O2 (3 min) followed by 2 times rinse with sterile de-ionised water to sterilize
the seed of G. glabra. Gupta et al. (2014) used mild detergent (10 min), a combination of 0.2%
bavistin and 0.2% streptocyclin (60 min) followed by a rinse with sterile distilled water and
treatment with 0.05% HgCl2 (1-4 min) and finally rinsed with sterile distilled water (4-5times)
during surface disinfection.
Borosilicate glass bottles, test tube and Erlenmeyer flask were the most commonly used
culture vessel for Glycyrrhiza species. Test tube was generally used for the culture initiation
(Mousa et al., 2006; Kojoma et al., 2010). Glass bottles and Erlenmeyer flask was most
frequently used for callus induction, stolon proliferation and shoot multiplication (Wongwicha et
al., 2008; Sawaengsak et al., 2011; Gupta et al., 2013).
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REVIEW OF LITERATURE 22 | P a g e
Table 2.2 Preparation of Glycyrrhiza species for tissue culture studies with emphasis on G.
glabra L.
Species Explant source Explant type
and size
Surface sterilization and
preparation
References
G. glabra 1-mo-old plant
planted from
subterranean stem
(15-20 cm)
Axillary bud (1
cm)
RTW (1hr) 2% Tween
20 + 1% NaOCl (5 min)
3X SDW
Kohjyouma et al.
(1995)
G. glabra Age of mother plant
NR
Stem segment
with apical tips
and axillary buds
(1-2 cm)
RTW Tween 20 (1-2
drops) 0.05% HgCl2 (5
min) SDW
Thengane et al.
(1998)
G. glabra Age of mother plant
NR
Long stem with
nodes, axillary
bud and petiolar
base (2-3 cm)
RTW Tween 20 0.1%
HgCl2 (1 min) SDW
Kukreja (1998)
G. glabra,
G. uralensis,
G. echinata,
G. squamulosa
Age of mother plant
NR
Nodal segments
70% EtOH (30 s) 5%
NaOCl (15 min) SDW
Kakutani et al.
(1999)
G. uralensis Seed of 2- to 4-y-old
plant
Seeds, leaves,
cotyledon, root
SDW (24 hr) 70% EtOH
(30 s) 2% NaOCl + 1
drop Tween 20 (5 min)
3X SDW
Oyunbileg et al.
(2005)
G. glabra in vitro-raised plant
grown in greenhouse
for 3 mo, from
selected mother
plant (L58)
Single bud
cuttings (1.5-2
cm)
1.5% NaOCl (15 min)
3X SDW
Mousa et al.
(2006)
G. glabra Seed of wild plant
(Age NR)
Seeds 30% H2O2 (3 min) 2X
SDIW
Shams-Ardakani
et al. (2007)
G. glabra Age of mother plant
NR
Nodal segments
(1 cm)
RTW (30 min) 10%
detergent (5 min) DDW
0.1% HgCl2 (3min)
SDDW
Patel and Shah
(2007)
G. glabra 4-wk-old,
greenhouse-grown
plantlets
Young leaflets (1
cm2)
1.5% NaOCl (15 min)
3X SDW
Mousa et al.
(2007)
G. glabra,
G. uralensis,
G. inflate
Seed2-wk-old in
vitro raised plantlets
Leaf and stem
segments (0.5
cm)
SDW 10% NaOCl (15-
20 min) 3X SDW
70% EtOH (1 min)
Wongwicha et al.
(2008)
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REVIEW OF LITERATURE 23 | P a g e
G. glabra Age of mother plant
NR
Leaves and
stems (1cm)
RTW (15 min) 1%
Tween-60 DW 80%
EtOH (60 s) 0.1% HgCl2
(3-5 min) SDW
Jain et al. (2008)
G. glabra Age of mother plant
NR
Shoots and stems DW 0.1% HgCl2 (8 min)
75% EtOH (10 s) 3X
SDW
Parsaeimehr et al.
(2009)
G. uralensis Seed1-mo-old
seed culture
Single node with
stem segment
RTW (3 hr) 70% EtOH
(1min) 2% NaOCl +
0.02% Tween 20 (15 min)
3X SDW
Kojoma et al.
(2010)
G. glabra Fully matured seed Hypocotyl,
cotyledon, young
leaf, stem
segment
RTW (30 min) 98% oil
of vitriol (30-40 min) 5X
SDW 1% HgCl2 (8-10
min) 3X SDW
Fu et al. (2010)
G. glabra Single, mature
mother plant
Shoot tips (1-2
cm)
RTW (30 min) 10%
Clorox + 0.1% Tween 20
(15 min) 3X SDW
Sawaengsak et al.
(2011)
G. glabra Age of mother plant
NR
Nodal segments
(1.0-1.5 cm)
Liquid detergent RTW
0.1% HgCl2 (3-5 min)
4-5X SDDW
Yadav and Singh
(2012)
G. glabra Age of mother plant
NR
Fully expanded
young leaves
RTW 0.1% HgCl2 (5
min) 5X SDW
Gupta et al.
(2013)
G. glabra Age of mother plant
NR
Stolon segment
with atleast one
primordium
RTW (30 min) 5%
Teepol (5 min) 70%
EtOH 0.1% HgCl2 (2-5
min) 3X SDW
Srivastava et al.
(2013)
G. glabra Age of mother plant
NR
Nodal segments
(2-3 cm)
RTW mild detergent (10
min) RTW 0.2%
bavistin + 0.2%
streptocyclin (60 min)
DDW 0.05% HgCl2 (1-4
min) 4-5X SDDW
Gupta et al.
(2014)
G. glabra Age of mother plant
NR
Nodal segment
with axillary
buds
RTW (10–15 min) 0.1%
HgCl2 + 7.5% Teepol (10-
12 min) SDW
Sarkar and Roy
(2014)
NR not reported, RTW running tap water, SDW sterile distilled water, SDDW sterile double distilled water,
SDIW sterile deionized water, DW distilled water, DDW double distilled water.
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REVIEW OF LITERATURE 24 | P a g e
2.5.1.3 Culture establishment, maintenance and multiplication medium
Once a sterile environment and explant has been established through standard aseptic protocol,
the second step involves the standardization of medium which includes selection of essential
nutrients (macro and micro nutrients), amino acids, vitamins, carbon source, plant growth
regulators (PGRs), gelling agents, additives and pH, summarized for Glycyrrhiza species in
Table 2.3.
The different types of media have been used for the development and establishment of
callus and cell suspension culture of Glycyrrhiza. The media such as LS medium (Linsmaier
and Skoog, 1965) containing NAA (α-naphthalene acetic acid) and BAP (6- benzyl-adenine)
(Hayashi et al., 1988, 1992), MS medium (Murashige and Skoog, 1962) containing 2,4-D (2,4-
dichlorophenoxy acetic acid) and Kn (kinetin) (Yoo and Kim, 1976) and B5 medium (Gamborg
et al., 1968) containing 2,4-D and Kn and reduced concentration of sucrose (2%) (Arias-Castro
et al., 1993b). Kukreja (1998) tested MS and NB (Nitsch, 1969) medium for culture
establishment and shoot multiplication and found that MS medium was more effective for
axillary shoot multiplication from the nodes of G. glabra.
Sawaengsak et al. (2011) described different media formulation such as MS, B5 and
Woody Plant Medium (WPM; Lloyd and McCown, 1981) of different strength (such as 1, ½
and ¼ strength) for the development of shoot tip culture and found that ½ strength B5 salt base
was most suitable for the growth and development of tissue cultured Glycyrrhiza plant whereas
higher explant proliferation was noticed in MS medium. Gupta et al., (2014) employed six
different types of media i.e. MS, SH (Schenk and Hilderbrandt, 1972), WH (White, 1943),
NB, B5and LS for in vitro regeneration of nodal explants of G. glabra and found MS medium to
be most effective with 100% regeneration. Most of the tissue culture studies of Glycyrrhiza
species were employed on MS medium and found highly responsive (Thengane et al., 1998;
Mousa et al., 2007; Arya et al., 2009; Fu et al., 2010; Yadav and Singh, 2012; Gupta et al.,
2013; Sarkar and Roy, 2014).
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REVIEW OF LITERATURE 25 | P a g e
Some of the reported pathways for the complete regeneration of Glycyrrhiza species are
micropropagation by axillary and adventitious shoot multiplication, direct and indirect
organogenesis and somatic embryogenesis.
2.5.1.3a Axillary and adventitious shoot multiplication
Quiescent and actively dividing meristem are present at the axillary and apical shoot which are
capable of developing plants during in vitro multiplication. In order to reduce the risks of
somaclonal variability during multiplication, apical and axillary meristem were the preferable
explants (George et al., 1993). Many attempts have been taken to establish and develop a
standard protocol for the clonal and rapid regeneration of Glycyrrhiza species utilizing different
types of explants such as stem segments (Thengane et al., 1998; Kukreja, 1998), axillary bud
(Kohjyouma et al., 1995), shoot tip (Sawaengsak et al., 2011), nodal segment (Yadav and
Singh, 2012), leaves (Mousa et al., 2007) and seed (Shams-Ardakani et al., 2007) through
tissue culture. Direct regeneration of roots and stolon proliferation from leaf and nodal segment
has been reported which finally resulted into high throughput plantlet regeneration in
Glycyrrhiza species (Kojoma et al., 2010; Gupta et al., 2013).
2.5.1.3b Direct and indirect organogenesis
Organogenesis starts with a distinct organization of a group of new meristematic cells either
directly or indirectly which were later transformed into shoots and roots meristem (Bhojwani
and Razdan, 1996; Thorpe, 1994). Direct and indirect (callus-mediated) organogenesis was
reported for Glycyrrhiza species (Mousa et al., 2007; Patel and Shah, 2007; Wongwicha et al.,
2008; Arya et al., 2009; Gupta et al., 2014; Sarkar and Roy, 2015). Kakutani et al. (2008)
reported the formation of adventitious roots from the friable and white or yellow callus of
G.glabra and G. uralensis on MS medium supplemented with 1or 5 mg/l NAA.
The root cultures can be easily manipulated and have potentially high regenerative capacity
(Franklin et al., 2004). Gupta et al. (2013) established in vitro root culture of G. glabra to
retrieve regenerated plant via., stolon proliferation. After 2 weeks well grown white roots were
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REVIEW OF LITERATURE 26 | P a g e
induced from the proximal end of the leaf cultured on MS medium supplemented with 1 mg/l Kn
with 1mg/l NAA or IBA and 1 mg/l IAA alone. Rapid proliferation of root culture was observed
within 3 weeks in liquid medium containing 0.01 mg/l NAA. Root grew vigorously on repeated
sub-culturing in medium enriched with IAA or IBA along with Kn and forms a white cottony
mass under dark incubation. Within 3 weeks these elongated roots were transformed into thick
stout pale brown colour stolon from which several shoot primordia (8 shoots of 3 cm per stolon)
aroused on MS medium with 1 mg/l NAA under light. Successful acclimatization of plantlets in
soil with 70 % survival was observed.
2.5.1.3c Somatic embryogenesis
Mousa et al. (2007) reported the regeneration of plantlets from embryogenic callus of different
selected clonal genotype of G.glabra using leaf as an explant. Plantlet regeneration through
somatic embryogenesis using leaf from four different species of licorice (G. echinata L., G.
glabra L., G. squamlosa F. and G. uralensis F.) has been reported by Kakutani et al., (2008).
Somatic embryo was formed from brown and compact calli of G. squamlosa and G. echinata on
MS medium incorporated with BAP (1 mg/l) + NAA (1 mg/l) and BAP (1 mg/l) + 2,4-D (0.5
mg/l) or 2,4-D (0.5 mg/l) respectively. Both somatic embryos developed into shoots (20
shoots/callus) and roots.
Wawrosch et al., (2009) reported that cotyledon explants from 7 days old seedlings were
best suited for callus induction. The growth regulator TDZ (thidiazuron) was found to be
superior to 2,4-D or picloram for the formation and vigorous growth of embryogenic callus.
Nutrient medium without growth regulators was best suited for embryo maturation and the
genotype did not significantly influence the embryogenic potential. Somatic embryogenesis of G.
glabra was evidenced by Fu et al., (2010) in the presence of BAP (0.5 mg/l) in combination of
0.5 mg/l KT (kinetin zeatin) and 0.1 mg/l IBA (indole 3-butyric acid) in MS medium. For the
further development and maturation, somatic embryos were transferred into same medium
enriched with 1000 mg/l malt extract. Few shoots were formed but unfortunately most of them
are weak and recalcitrant to re-differentiate into plantlets. Microscopic observation and
histological section of globular somatic embryo revealed low embryoid regeneration due to the
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REVIEW OF LITERATURE 27 | P a g e
unmature embryoid i.e., translucent calli was submerged with many small green globular
embryo.
2.5.1.4 Rooting, adaptation and ex-vitro transfer of plants
The successful in vitro regeneration protocol relies on the efficient and rapid rooting of shoots
followed by subsequent acclimatization, one of the most essential steps of complete
micropropagation protocol. Rooting of in vitro raised plantlets greatly depends on the strength of
medium with or without growth regulators. Various concentration of auxins (IAA, IBA and
NAA) supplemented in MS medium were found to effective for the induction of root in
Glycyrrhiza species as shown in Table 2.3.
Kukreja (1998) observed rooting on MS medium containing 1 mg/l IAA. Well rooted
plantlets of G. glabra were transferred into earthen pots filled with sand, soil and manure (1:1:1)
and finally irrigated with nutrient broth (Hoagland and Arnon, 1950) and showed 95% survival.
Thengane et al. (1998) used half strength liquid as well as semi solid MS medium enriched with
different concentration of IAA, IBA and charcoal but half strength semi solid MS medium with
2.85μM IAA, 4.90μM IBA and charcoal (0.25 g/l) was found to be most effective for rooting of
licorice. High mortality rate was observed during direct transfer of in vitro raised plants into soil
due to wilting. Therefore they were transferred into sterile pots filled with sterilized sand and soil
mixture resulted in 90% survival of plant. There are some more reports on the rooting of in vitro
regenerated shoots of licorice on half strength MS medium along with IAA or IBA (Arya et al.,
2009; Yadav and Singh, 2012; Gupta et al., 2014). Sawaengsak et al. (2011) observed in vitro
rooting (80%) of G. glabra on half strength B5 medium containing 5 mg/l IAA after 6 weeks.
Well rooted plants were then transplanted to plastic cups filled with sterile garden soil and
showed 95% survival.
Rooting of in vitro regenerated shoots of Glycyrrhiza species on full strength MS or B5
medium devoid of growth regulators has also been reported (Kakutani et al., 1999; Mousa et
al., 2007; Wongwicha et al., 2008) which later transferred to potted soil with vermiculite and
then acclimatized. NAA is also used in regenerating root from in vitro culture of Glycyrrhiza
CHAPTER – 2
REVIEW OF LITERATURE 28 | P a g e
species. (Kohjyoum et al., 1995; Mousa et al., 2006). Rooted plantlets were transplanted into
germinating trays containing sterilized top soil, manure and rock sand (1:1:1) and watered daily
which later transplanted into green house. Regeneration of adventitious roots from in vitro
regenerated stolon and shoots of G. uralensis F. was readily achieved on MS medium enriched
with 0.01μM NAA (Kojoma et al., 2010).
2.6 Germplasm conservation
In vitro conservation offers rapid multiplication of elite and rare plant species and also an
alternative method of ex situ conservation. There are different strategies for the germplasm
conservation viz., encapsulation (artificial or synthetic seed), cryopreservation and slow growth
system (Monette, 1995).
2.6.1 Synthetic seed production
The term synthetic seed means an artificially encapsulated vegetative propagule capable to
develop into a complete plant in vitro and ex vitro via., organogenesis and somatic
embryogenesis (Aitken-Christie et al., 1995). Encapsulation matrix serves as an artificial
endosperm (nutrient reservoir) and supply necessary nutrients to the encased embryo or shoots
(Pattnaik et al., 1995). Alginate encapsulation is a viable approach for in vitro germplasm
conservation (Standardi and Piccioni, 1998; Ara et al., 2000). Synthetic seeds designed as
genetically identical material with the ease of handling and transportation along with increased
efficiency of in vitro propagation in terms of space, time, labour and cost (Nyende et al., 2003).
Synthetic seed have been widely utilized for micropropagation and conservation of various
medicinal plant species (Singh et al., 2006c; Narula et al., 2007; Ray and Bhattacharya, 2008,
Lata et al., 2009).
2.6.2 Cryopreservation
Cryopreservation (Liquid nitrogen; -196°C) halts all metabolic processes, thereby effectively
maintaining plant material in stasis for decades (Engelmann, 2004; Kaczmarczyk et al., 2012).
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REVIEW OF LITERATURE 29 | P a g e
Cryopreserved germplasm requires significantly less maintenance, space and facilitate long term
preservation of diverse genetic lines, representative of in situ genetic diversity- with the capacity
to readily add new genetic material (Benson, 2008). This method relies on the use of
concentrated cryosolvent solution to protect the tissues during rapid freezing (vitrification)
(Benson, 2008; Engelmann, 2004). Plant Vitrification Solution 2 (PVS2; Sakai et al., 1991) is
the most well-known cryoprotective solution, resulting in more rapid cooling, reduced ice
formation at critical ice forming temperatures (Day et al., 2008) and promoting metastable glass
formation that enhances the survival of plant material following warming from cryogenic
temperatures (Sakai and Engelmann, 2007; Kaczmarczyk et al., 2011). Germplasm
conservation through cryopreservation has proven to be readily applicable on many plant species
(Menon et al., 2014; Funnekotter et al., 2013, 2015).
2.6.3 Slow growth system
Successful slow growth system for germplasm conservation had been developed for different
plant species (Engelmann, 2011). Slow growth in vitro may be obtained by low temperature,
required for minimum growth of plantlets. Usually 4-8°C storage temperature is required for
temperate crops and 10-15°C for tropical crops (Keller et al., 2006). Slow growth was generally
achieved either by the addition of osmotic agent (sucrose, sorbitol and mannitol) of varying
concentration or by the removal of growth promoters (cytokinin and auxin) (Lata et al., 2010;
Scherwinski-Pereira et al., 2010). Addition of osmotic agent in culture media significantly
increase the storage life of in vitro tissues (Sharaf et al., 2012) whereas Ancymidol and abssissic
acid were used as growth retardant (Yun-peng et al., 2012).
2.6.4 Germplasm conservation in Glycyrrhiza species
Verma et al. (2012a) successfully conserved the germplasm of G. glabra by encapsulating
nodes of highly proliferating in vitro grown shoot culture and found 50% germination of
synthetic seed. The availability of germplasm of in vitro grown encapsulated axillary micro-
shoots for long term
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REVIEW OF LITERATURE 30 | P a g e
storage and commercial cultivation has been demonstrated by Mehrotra et al. (2012). In above
study, the protocol used to induce 2 months in vitro shoots was evidenced by Mehrotra et al.
(2009). The axillary buds (3-5 mm long) were excised from in vitro shoots and cultured on MS
medium enriched with 0.1 mg/l IAA. Shoot tips and nodal segments were suspended in sterile
sodium alginate mixture (MS medium, 0.1 M sucrose and 3% sodium alginate) and were
dispensed drop wise into sterile 100 mM calcium chloride solution on magnetic stirrer under
continuous shaking. A complete ion exchange reaction takes place resulted into micro-shoots
encapsulation which was then rinsed with sterile distilled water thoroughly and stored for 6
months in moist environment at 25+2°C.The re-growth and development of shoot and root from
6 months stored encapsulated micro-shoots showed 98 % survival on its incubation in MS
medium supplemented with 0.1 mg/l IAA within 30 days. Acclimatization of complete plantlets
to glass house showed 95 % survival. RAPD and ISSR techniques were used to evaluate the
genetic fidelity of regenerated plants of G. glabra.
Srivastava et al. (2013) developed a protocol for preserving shoot apices of G. glabra
under slow growth conditions. Culture responded best when incubated at 10°C under low light
intensity (2.5 μmol m-2s-1quantum flux density). The optimized MS modified medium (MS
medium with 20 mg/l glutamine and 15 mg/l arginine) formulation to maintain slow growth
contained 5 mg/l ancymidol and 0.1 mg/l abscissic acid and high osmoticum was achieved by 1
mg/l polyethylene glycol where cultures could be conserved upto 6 months. MS medium with
0.1 mg/l BA and 0.05 mg/l IAA was found to be beneficial for 100% survival and retrieval of
conserved shoots. Half strength modified MS medium containing 0.25 mg/l BA, 1 mg/l IAA and
10 mg/l adenine sulphate proved to be beneficial for shoot growth, foliage development and
rooting as well. The in vitro raised plantlets showed 100% survival when transplanted into green
house.
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REVIEW OF LITERATURE 31 | P a g e
Table 2.3 In vitro conditions for tissue culture studies on Glycyrrhiza species, with special emphasis on Glycyrrhiza glabra
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra LS + 100 μM NAA + 1 μM BAP (CIM)
pH, sucrose, agar (NR)
Dark, Temp (25°C) Callus induced from various parts (hypocotyl, leaves,
stems and roots) and examined for triterpenoids
Hayashi et
al. (1988)
G. glabra B5 + 1 mg L−12,4-D + 0.1 mgL−1 Kn
(CIM)
pH 5.8, 2% sucrose, 0.8% agar
CWFT(0.22 Wm-2)
Temp (25°C)
After 3-4 wk lime-green or yellow, friable callus
observed from root explant. Shooting, rooting and
acclimatization NP
Arias-Castro
et al.
(1993b)
G. glabra MS + 5 mgL−1 BAP (CIM)
MS + 1 mg L−1 BAP (SIM)
MS + 0.10-0.50 mg L−1 NAA (RIM)
pH 5.5, 3% sucrose, 0.8% agar
PP (16 h)
CWFT (3000 lux)
Temp (25±2°C)
Morphogenetic changes observed after 40 d. Callus
induction, shoot (4.6 /explant), root (77.8%)
development observed. Acclimatization NP
Kohjyouma
et al. (1995)
G. glabra MS + 2mg L−1 BAP + 1 mg L−1 IAA
(SIM)
MS + 1 mg L−1 IAA (RIM)
pH 5.8, 3% sucrose, 0.8% agar
PP (16 h)
CWFT (3000 lux)
Temp (25±3°C)
RH (60-70 %)
6-8 adventitious shoots (2-4 nodes each) observed after
4 wks culture. Rooting frequency increased
significantly. Rooted plants were transferred to earthen
pots (sand, soil and organic manure in the ratio of 1:1:1)
and irrigated with nutrient broth. Plants acclimatized to
greenhouse showed 95% survival.
Kukreja
(1998)
G. glabra MS + 8.87μM BAP (SIM)
¾ MS + 4.44μM BAP (SMM)
½ MS + 2.85μM IAA + 4.90μM IBA
(RIM)
pH 5.7±0.1, 3% sucrose, 0.7% agar
PP (16 h)
CWFT
(35μmolm−2s−1)Temp
(25±2°C)
Greater number of healthy shoots (4/explant) induced.
Reduction in major salts of MS enhanced multiplication
ratio (1:10). Production of thick and healthy roots
(3.16/shoot) ±charcoal. Shoots growing on charcoal
media looked healthier. Rooted plants were transferred
to pots (sterilized sand and soil mixture 1:1) in
greenhouse showed 90% survival.
Thengane et
al. (1998)
G.glabra,
G.uralensis,
G. echinata,
G. squamulosa
1/3 MS (SIM)
MS + BAP/NAA/2,4-D (CIM)
[25 combination NR]
For G. glabra & G. uralensis
MS + 1 mg L−1 NAA (ARIM)
For G. echinata (SEM)
MS + 1 mg L−1 BAP + 1 mg L−1 NAA
For G. squamulosa
MS + 1 mg L−1 BAP + 0.5 mg L−1 2,4-D
or MS + 0.5 mg L−1 2,4-D (SEM)
pH 5.8, 1 % sucrose, 0.2 % gelrite
PP (16 h)
CWFT (4000 lux)
Temp (25°C)
Callus induced from leaf segment of in vitro-grown
shoot. Increased callus growth with increased PGRs.
Calluses from G. glabra &G. uralensis were friable,
white and formed adventitious root after 30 d. Calluses
from G. echinata & G. squamulosa were compact,
brown and formed somatic embryos after 30 d, which
developed into shoots (20 shoots/callus) after 60 d.
Shooting, rooting and acclimatization data NR
Kakutani et
al. (1999)
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REVIEW OF LITERATURE 32 | P a g e
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra MS + 2 mg L−1 BAP + 2mg L−1 NAA
(SIM & RIM)
pH 5.8, 3% sucrose, 0.8% agar
PP (16 h)
CWFT
(300 μmol m−2s−1)
Temp (23±2°C)
RH (70-90%)
Cloning in vitro possible after4 wks culture on MS
medium. Shoot cultures grow vigorously at 6 wk.
Increased in stem elongation (16.2 cm), leaves (15.8
leaves/ plantlets) and micro-nodes (9 nodes/ plantlets)
observed. Increase in root length (5.3 cm) and number
of main roots (4.8/ plantlets) observed. Rooted plantlets
transferred in germinating trays (sterilized top soil,
manure and rock sand in ratio of 1:1:1) and watered
daily; later transplanted into greenhouse. Data on
acclimatization NR
Mousa et al.
(2006)
G. glabra MS+ 1 mg L−1 2,4-D + 0.2 mg L−1 Kn
(CIM)
MS+ 1 mg L−1 NAA + 0.5 mg L−1 2,4-
D + 0.5 mg L−1 Kn (CIM)
pH 5.7, 3% sucrose, 1.2% agar
PP (16 h)
CWFT (5000 lux)
Temp (23°C)
Highest level of callus growth to date. Shooting and
rooting NP
Shams-
Ardakani et
al. (2007)
G. glabra MS + 1mg L−1 BAP + 0.05 mg L−1 NAA
(SIM)
MS + 0.5 mg L−1 IBA (RIM)
pH 5.8, 3% sucrose, 0.8% agar
PP (16h)
CWFT (1000
lux)Temp (25±2°C)
Earlier sprouting of explants (4 d);longer shoots (2.30
cm) and greater number of nodes (4.80/ shoots). In vitro
rooting better on full-strength MS, with greatest
reported number of roots (11.64) and root length (2.28
cm). Rooted plantlets hardened in pots (soil and leaf
mould in the ratio of 1:1) showed 72% survival
Patel and
Shah (2007)
G. glabra B5 + 1 mg L−1 2,4-D + 1 mg L−1 Kn
(CIM)
same media for further development.
B5 (no growth regulators) (SIM)
pH 5.8, 2% sucrose, 0.8% agar
PP (16 h),
CWFT (350 μmol
m−2s−1),
Temp (25±2°C)
Primary embryogenic callus developed and maintained
at globular stage by routinely subculturing. Secondary
embryogenic callus (4-5 mm in diameter) initiated in
same embryogenic cell suspension after 6-8 wk. Both
calluses readily morphogenic and regenerative, forming
vigorous, multiple shoots (80-90%). Regenerated plants
generally healthy and transferred successfully to
greenhouse. Data on acclimatization NR
Mousa et al.
(2007)
G. glabra B5 + 1 mg L−1 2,4-D + 0.5 mg L−1 Kn
(CIM)
B5 + 1 mg L−1 BAP + 0.5 mg L−1 NAA
(SIM)
B5 + 0.5 mg L−1 IBA (RIM)
PP, CWFT, Temp,
RH (NA)
Morphogenic calli formation was induced leading to
plant regeneration. Higher root proliferation with higher
number of root and root length was achieved. In vitro
raised plants were successfully acclimatized in field
Sharma et
al. (2008)
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REVIEW OF LITERATURE 33 | P a g e
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra
G. uralensis
G. inflate
MS basal (seed germination)
MS + NAA/2,4-D (0.5-1 mg L−1) +
BAP/Kn (0.5-1 mg L−1) (CIM)
MS + TDZ (0.1-1 mg L−1) (CIM)
For G. glabra & G. uralensis
MS + 0.1 mg L−1 TDZ (SIM)
For G. inflata
MS + 1 mg L−1 NAA + 0.5 mg L−1 BAP
(SIM)
pH 5.5, 3% sucrose, 0.9% agar
PP (16 h),
CWFT (70 W/m2),
Temp (25±1°C)
After 4 wks culture, callus induced (33–100 %).
Calluses cultured with NAA and BA grew well due to
loose texture, while calluses cultured in 2,4 D and
BA/Kn or TDZ alone were dwarfed with severe
necrosis. All 3Glycyrrhizaspp.regenerated shoots from
callus on MS with NAA and BA or only TDZ. From G.
inflata stem explant callus cultures, maximum shoot
induction (67%) and max. shoot per explant (2.0
shoots/explant). Data on rooting and acclimatization NR
Wongwicha
et al. (2008)
G. glabra MS + 2 mg L−1 BAP + 0.5 mg L−1 2,4-D
(CIMa)
MS + 2 mg L−1 BAP + 1 mg L−1 NAA
(CIMb)
pH 5.8, 3% sucrose, 0.6% agar
PP (12 h),
CWFT (1500-2000
lux),
Temp (25±1°C),
RH (80±10)
Light and compact callus induced CIMa while loose,
sponge and friable CIMb. Shooting and rooting NP
Parsaeimehr
et al. (2009)
G. glabra MS + 2 mg L−1 BAP (SIM)
MS + 2mg L−1 BAP + 0.5 mg L−1 Kn +
50 mg L−1 Ads (SMM)
½ MS + IAA (1.0 – 4.0 mg L−1) (RIM)
PP, CWFT, Temp,
RH (NA)
Bud break (85-90%) from nodal segment within 3 wks
of culture. One or two shoots were produced. 8- to 9-
fold increase in shoot multiplication rate, with
development of roots. Within 2 months, hardening and
acclimatization of tissue culture-raised plantlets
achieved in mist chamber and shade-out condition. Data
on acclimatization NR
Arya et al.
(2009)
G. uralensis MS basal (seed germination)
MS + 0.1 μM NAA (SIM)
pH 5.8, 3% sucrose, 0.2% gelrite
MS + 0.01 μM NAA (SPM)
pH 5.8, 6% sucrose
MS + 0.01 μM NAA/0.1 μM IBA (SIM
& RIM)
pH 5.8, 6% sucrose, 0.2% gelrite
For seed germination,
SIM & RIM –
PP (16 h),
CWFT (40
μmolphotons m−2s−1),
Temp (23°C)
For stolon
proliferation-
incubation in dark at
100 rpm,
Temp (26°C)
Seed germination and shooting achieved. Stolon
formation induced in single-node stems (with axillary
buds). Same NAA concentration produced high rates of
stolon proliferation (6.58-fold in 4 wk). 6% sucrose
enhanced stolon proliferation (6.34-fold in 4 wk).
Adventitious root and shoot regeneration from stolon
culture. Regenerated plants easily acclimatized. Data on
acclimatization NR
Kojoma et
al. (2010)
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REVIEW OF LITERATURE 34 | P a g e
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra B5 + 2 mg L−1 2,4-D (CIM)
B5 + 0.5 mg L−1 BAP + 0.5 mg L−1
NAA (SIM)
pH 5.8, 20 g L−1 sucrose, 7.5 g L−1 agar
B5 + 0.5 mg L−1 IBA (RIM)
pH 5.8, 10 g L−1 sucrose, 7.5 g L−1 agar
PP, CWFT, Temp,
RH (NA)
High frequency callus induction (65.93) with maximum
shoot proliferation (94.12), shoot per explant (9.32) and
mean shoot length (5.20 cm). Higher root proliferation
with maximum root length also induced. Rooted
plantlets successfully established in field after
hardening. Data on acclimatization NR
Sharma et
al. (2010b)
G. glabra MS + 2 mg L−1 BAP + 0.5 mg L−1 2,4-D
(CIM)
MS + 0.5 mg L−1 BAP + 0.5 mg L−1ZT
+ 0.1 mg L−1 IBA (SEM)
MS + 0.5 mg L−1 BAP + 0.5 mg L−1ZT
+ 0.1 mg L−1 IBA + 1000 mg L−1 ME
(SEGM)
MS + 3 mg L−1 BAP + 1 mg L−1 + 0.1
mg L−1 NAA (SIM)
pH 5.8, 3% sucrose, 0.8% agar
PP and CWFT (NR)
Temp (25°C)
Highest callus induction frequency and intensity
observed from hypocotyl (93.3%). Callus fresh weight
increased (1.5-to 2-fold) by repeated monthly
subculture. Many green globular somatic embryos
observed on the surface of callus after 15 d of culture.
Histological study revealed development of somatic
embryos. Multiplication index of embryo were 9.34 and
diameter was2-5 cm. After a few weeks, shoots
observed. Data on shooting NR
Fu et al.
(2010)
G. glabra MS + 0.5 mg L−1 BAP (SIM)
½ B5 + 5 mg L−1 IAA (RIM)
pH 5.7, 3% sucrose, 0.7% agar
PP (16 h),
CWFT (55 μmol
l.m−2s−1),
Temp (28±2°C)
1.5 shoots/explant induced, leading to extensive
proliferation rate (4.75/explant). High frequency root
formation (80%) observed. In vitro-raised plantlets
transferred to plastic cups (sterile garden soil) showed
95% survival
Sawaengsak
et al. (2011)
G. glabra MS + 2 mg L−1 BAP + 0.5 mg L−1 NAA
(SIM)
½ MS + 1 mg L−1 IAA (RIM)
pH 5.8, 30g L−1 sucrose, 8g L−1 agar
PP (16/8 h),
CWFT (4000 lux),
Temp (25±2°C)
Highest bud break (86.6%) with longest shoot length (8
cm) and maximum number of shoots (3) obtained when
middle nodes (3rd to 5th from apex) collected between
May to Aug. Multiple shoot formation increased from
first to fourth subculture. Early rooting (100%) and
maximum root growth observed after 16-17 d. Plantlets
acclimatized in pots (soil and sand in ratio of 3:1) in
greenhouse. Data on acclimatization NR
Yadav and
Singh (2012)
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REVIEW OF LITERATURE 35 | P a g e
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra MS + 1mg L−1 Kn + 1.0 NAA/IBA
(RIM)
MS + 1mg L−1 IAA (RIM)
MS + 0.01 mg L−1 NAA (SPM)
MS + 1 mg L−1 NAA (SIM)
pH 5.8, 3% sucrose
For rhizognesis and
stolon culture-
incubation in dark at
100 rpm
For stolon
proliferation from
stolon culture-
PP (16 h),
CWFT (40
μmolm−2s−1),
Temp (28±2°C),
RH (80-90%)
White roots (100%) formed from leaf after 2 wks
culture. Extensive roots proliferate rapidly in dark (mat-
like appearance). Root meristem grew vigorously as a
whitish cottony mass. Elongated roots slowly
transformed into thick and stout pale-white or light-
brown stolons. Several shoot primordia (>8 shoots/3-cm
stolon) observed on stolon within 3 wk culture. Shoots
rooted efficiently, resulting in complete plantlets which
were acclimatized (sand:soil,2:1, mist-irrigated)
showing 70% survival. ISSR used to confirm genetic
stability of regenerants
Gupta et al.
(2013)
G. glabra MS + 1.5 mg L−1 BAP (SIM)
½ MS + 2.0 mg L−1 IBA (RIM) pH 5.8,
30g L−1 sucrose, 8g L−1 agar
PP (16 h),
CWFT (100
μEm−2s−1),
Temp (25±2°C)
MS most effective, with 100% regeneration. 12
shoots/explant. BA more effective than Kn for shoot
multiplication. 60% rooting in RIM. Rooted shoots
transferred to pots (sand+soil+vermiculite;1:1:1)
showed 100% survival. Hardened plants transferred to
field
Gupta et al.
(2014)
G. glabra MS + 5mg L−1 BAP (SMM)
MS + 1.5mg L−1 2,4 D (CIM)
MS + 5mg L−1 BAP + 1mg L−1 NAA
(SIM)
MS + 3mg L−1 IAA (RIM)
pH 5.8, 3% sucrose, 0.6% agar
PP (16 h),
CWFT (3000 lux),
Temp (25±2°C),
RH (55-60%)
Proliferation of shoots (14.3±0.51/explant) achieved
after 30-35 d. Green and compact organogenic callus
formed from leaf explant after 15-20 d. Shoot buds
(10.3+0.47/g callus) from callus after 21-25 d. 68%
rooting. Complete plant mass-propagated in MS minus
PGR. Rooted plants transferred to pots (sterile
soilrite).Acclimatized plants transplanted to normal
environmental condition. Data on acclimatization NR
Sarkar and
Roy (2014)
G. glabra MS + 1.5 mg L−1 2,4 D (CIM)
MS (liquid) + 3% Maltose (SEM)
MS + 1.5 mg L−1 GA3 + 0.5 mg L−1
ABA + 3% Sorbitol (SEGM)
pH 5.8, 3% sucrose, 0.8% agar
PP (16 h),
CWFT (3000 lux),
Temp (25±2°C),
RH (55-60%)
Compact and greenish callus induced in CIM. Globular
and heart-shaped embryos after 25-30 d. Embryogenic
efficiency and embryo development promoted by high
maltose concentration (3%). After 10 d, 47 % somatic
embryo germination
Sarkar and
Roy (2015)
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REVIEW OF LITERATURE 36 | P a g e
Species Culture Medium, PGRs, additives Culture conditionsa Experimental outcomes Reference
G. glabra MS + 2.0 mg L−1 BAP (SIM)
MS + 2.0 mg L−1 BAP + 0.5 mg L−1
NAA (SMMa)
MS + 1.0 mg L−1 BAP + 0.25 mg L−1
NAA (SMMb)
MS + 1.0 mg L−1 IAA (RIM)
pH 5.8, 30g L−1 sucrose, 8g L−1 agar
PP (16 h),
Temp (24±2°C),
CWFT (NR)
Highest shoot forming frequency achieved (3.67
shoots/explants). Best regeneration frequency (86.67%)
was found in SMMa whereas largest no. of shoots in
SMMb after 4th wk. Maximum rooting frequency
(100%) with highest root no. (16 roots/explant) and root
length (2.33 cm) induced. Rooted plantlets transferred
to plastic cups (soil:sand:compost, 1:1:1) showed 87%
survival. Hardened plants transferred to field
Badkhane et
al. (2016)
MS Murashige and Skoog’s (1962) medium; LS Linsmaier and Skoog (1965) medium; B5 Gamborg medium (Gamborg et al. 1968); PGRs plant growth
regulators; BAP 6-benzyladenine; Kn kinetin; ZT zeatin; IAA indole-3-acetic acid; IBA indole-3-butyric acid; NAA α-naphthalene acetic acid; 2,4-D 2,4-
dichlorophenoxyacetic acid; TDZ thidiazuron; GA3 gibberellic acid; Ads adenine sulphate; ABA abscisic acid; ME malt extract; CIM callus induction medium;
SIM shoot induction medium; SMM shoot multiplication medium; RIM root induction medium; ARIM adventitious root induction medium SPM stolon
proliferation medium; SEM somatic embryogenesis medium; SEGM somatic embryo germination medium; PP photoperiod; CWFT cool-white fluorescent tube;
Temp temperature; RH relative humidity; NP not performed; NR not reported; NA not accessible (only abstract was accessed)
a The original light intensity reported in each study has been presented, since the conversion of lux to μmol m−2s−1 is different for different illumination: for
fluorescent lamps, 1μmol m−2s−1= 80 lux; the sun, 1μmol m−2s−1= 55.6 lux; high-voltage sodium lamp, 1μmol m−2s−1= 71.4 lux (Thimijan and Heins 1983)
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2.7 Secondary Metabolites
Plants are the complex living organisms; their constituents and nutritional value have been
intensively studied for decades and forms an important part of our everyday diet. In addition to
primary metabolites, higher plants are also able to synthesize a number of low molecular weight
compounds called the secondary metabolites. Two hundred years of modern chemistry and
biology have described the role of primary metabolites (carbohydrates, lipids and amino acids) in
basic life functions such as cell division, growth, respiration, storage and reproduction. In
biology the concept of secondary metabolite was attributed by Kossel (1891) who was the first to
define these metabolites as opposed to primary ones. Thirty years later Czapek (1921) defined
secondary metabolites by their low abundance usually occurring in dedicated cells or organs.
These metabolites have a restricted distribution than primary metabolites in the whole plant
kingdom i.e., they are often found only in one plant species or a taxonomically related group of
species. Plant secondary metabolites are produced to facilitate interaction with the biotic and
abiotic environment to establish the defense mechanism (Wink et al., 1988; Verpoorte et al.,
2002; Wang et al., 2013b; Murthy et al., 2014a).
Plant secondary metabolites are usually classified according to their biosynthetic pathway
and constitute large classes of compounds including terpenes, phenolics (coumarin, lignin,
flavonoids, isoflavonoids, tanins), nitrogen (alkaloids, glycosides, non-protein amino acids) and
sulphur (GSH, GSL, phytoalexins, defensins, thionins, lectins) containing compounds
(Harborne, 1999). Several plants are rich in secondary metabolites which are potential source of
drugs, agrochemicals, food additives, flavors, fragrances, pigments and essential oils (Murthy et
al., 2014a). Due to their large biological activities, plant secondary metabolites have been used
for centuries in traditional medicines (Mosihuzzaman, 2012). Now a days, they correspond to
valuable compounds such as pharmaceutics, cosmetics, fine chemicals or more recently
nutraceutics. Recent surveys have established that in western countries where chemistry is the
backbone of the pharmaceuticals industry, 25% of the molecules used are of natural plant origin
(Payne et al., 1991). Plants will continuously produce the novel products as well as chemical
models for new drugs in the coming centuries, because the chemistry of the majority of plant
species is yet to be explored and characterized (Cox and Balick, 1994). The advent of chemical
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REVIEW OF LITERATURE 38 | P a g e
analysis and the characterization of molecular structures have helped in precisely identifying
these plants as well as their compounds and correlating them with their activity under controlled
experimentation. Despite of advancements in synthetic chemistry, we still depend upon
biological sources for a number of secondary metabolites (Pezzuto, 1995).
2.7.1 Production of secondary metabolites by plant cell culture
The production of these metabolites in plant is very low (less than 1% dry weight) and depends
greatly on the physiological and developmental stage of the plant (Dixon, 2001; Oksman-
Caldenteyl and Inze, 2004). Due to over-harvesting, many plants containing high value
compounds are difficult to cultivate or are becoming endangered (Rates, 2001). The chemical
synthesis of plant derived compounds is often not economically feasible because of their highly
complex structures and the specific stereo-chemical requirements of the compounds (Namdeo,
2007). Due to the limited availability and complexity of chemical synthesis, plant cell culture
becomes an alternative route for large-scale production of this desired compound (Savitha et al.,
2006). Plant tissue culture emerged as an escapable tool with the possibilities of complimenting
and supplementing the conventional method in plant breeding, plant improvement and
biosynthetic pathways of secondary metabolites (Anis et al., 2009, 2011). The biotechnological
production of secondary metabolites in plant cell and organ cultures is an attractive alternative to
the extraction of the whole plant material (Skrzypczak et al., 2014). Especially, plant cell and
organ cultures are promising technologies to obtain plant-specific valuable metabolites as it has
higher rate of metabolism (Verpoorte et al., 2002; Kehie et al., 2015) and leads to the rapid
proliferation and to a condensed biosynthetic cycle (Rao and Ravishankar, 2002).
Evidence that plant cell cultures are able to produce secondary metabolites came quite late
in the history of in vitro techniques. It is considered for a long time that undifferentiated cells,
such as callus or cell suspension cultures were not able to produce secondary compounds, unlike
differentiated cells or specialized organs (Krikorian and Steward, 1969). Zenk (1975)
experimentally demonstrated that this theory was wrong, as they could observe de-differentiated
cell culture of Morinda citrifolia yielding 2.5g of anthraquinones per litre of medium. This
finding opened the door to a large community of in vitro culturists who extensively studied the
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REVIEW OF LITERATURE 39 | P a g e
possible use of plant cultures for the production of secondary compounds of interest.
Unorganized plant cell cultures are known to synthesize a wide range of secondary metabolites
(Laurain-Mattar et al., 1999; Caruso et al., 2000). Production of secondary metabolites has
been reported in many medicinal plants eg. Anti-microbial flavonoids from Glycyrrhiza glabra
(Li et al., 1998), tropane alkaloids from Datura metel (Cusido et al., 1999), taxane from Taxus
cuspidata (Son et al., 2000), diterpenoids from Torreya nucifera (Orihara et al., 2002), etc.
The morphogenic differentiation such as shoot or/and root has been reported to enhance the
production of secondary compounds (Selles et al., 1999; Pepin et al., 1999; Ray and Jha, 2001;
Hussain et al., 2012). In some cases secondary metabolites are only produced in organ cultures
such as hairy root or shooty teratoma (tumor-like) culture (Spencer, 1993; Sevόn and
Oksmann-Caldentey, 2002; Georgiev et al., 2007; Dehghan et al., 2012; Danphitsanuparn et
al., 2012).
Fig. 2.3 Overall frequency of different in vitro culture systems used in chemical elicitation
experiments for secondary metabolite production [C callus, CS cell suspension, HR hairy
roots, AR adventitious roots, MS multiple shoots, MISLNUS miscellaneous] (Giri and
Zaheer, 2016)
The advantage of this method is that it can ultimately provide a continuous, reliable yield
of natural products under controlled environmental and nutritional conditions, ratio of cell
growth and biosynthesis in culture from a small amount of plant material is quite high,
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production of novel compounds through the recovery of new routes of synthesis from mutant cell
and cell culturing may be more economical for those plants, which take long periods to achieve
maturity. In recent years, various strategies have been developed for biomass accumulation and
synthesis of secondary compounds, such as strain improvement, optimization of medium, and
culture environments, elicitation, precursor feeding, metabolic engineering, permeabilization,
immobilization, and biotransformation methods, bioreactor cultures, and micropropagation
(Sarin, 2005).
Cell cultures have been established from many plants but often they do not produce
sufficient amounts of the required secondary metabolites (Rao and Ravishanker, 2002;
Oksmann-Caldentey and Hiltunen, 1996). Recent research in the in vitro culture systems, a
wide variety of elicitors have been employed in order to modify cell metabolism. These
modifications are designed to enhance the productivity of useful metabolites by the treatment of
undifferentiated cells with elicitors (Dicosmo and Misawa, 1985; Ebel and Cosio, 1994;
Poulev, 2003). The cultivation period in particular, can be reduced by the application of elicitors,
although maintaining high concentrations of product (Rao et al., 2002; Shilpa et al., 2010).
2.7.2 Elicitation of secondary metabolites
Stress is an important factor in determining the chemical composition and therapeutic activity of
medicinal plants. Actively stimulating, or eliciting, the plant stress response to induce the desired
chemical response is called elicitation, harnessing the connection between plant stress and
phytochemistry.
“Elicitor is a scientifically described term for stress factors that directly or indirectly
triggers the inducible defense changes in a plant system that results in an activation of array of
protection mechanisms, including induction or expansion of biosynthesis of fine chemicals
which do have a major role in the adaptation of plants to the stressful environment” (Goel et al.,
2011). Elicitation is the induced or enhanced biosynthesis of metabolites due to addition of trace
amounts of elicitors (Radman et al., 2003). Several biotechnological strategies have been
hypothesized and applied for the enhanced productivity, enhancement and elicitation is
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REVIEW OF LITERATURE 41 | P a g e
recognized as the most practically feasible strategy for increasing the production of desirable
secondary compounds from cell, organ, and plant systems (Poulev et al., 2003; Angelova et al.,
2006; Namdeo, 2007).
On the basis of nature, elicitors can be divided into two types: abiotic elicitors comprise of
substances that are of non-biological origin and are grouped in physical (UV radiation, osmotic
and thermal stress, salinity, drought), chemical (heavy metals, mineral salts, gaseous toxins) and
hormonal factors and biotic elicitors are the substances of biological origin that include
polysaccharides originated from plant cell walls (e.g. chitin, pectin, and cellulose) and micro–
organisms. Abiotic and biotic elicitors have a wide range of effects on the plants and in the
production of secondary metabolites.
Fig. 2.4 Classification of elicitors (Poornananda and Jameel, 2016)
Elicitors appear to be recognized by plant cells via. Interactions with specific receptors on
plant plasma membranes and activate certain gene expression through signals transduction
pathway thereby stimulating an array of defense responses (Yoshikawa et al., 1993). The
multitasking ability of such elicitors is unique as well as multidimensional (Gorelick and
Bernstein, 2014). Elicitor regulates large number of biochemical control points; trigger the
expression of key genes and transcription factors too. They also have the ability to control array
of cellular activities at biochemical and molecular level (Zhao et al., 2005; Baenas et al., 2014)
and increase the intensity of plant’s response to biotic and abiotic stresses with the enhanced
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synthesis of signal compounds and its subsequent influence on secondary metabolite production
(Sudha and Ravishankar, 2002). The manifestations of elicitor treatment with altered or
elevated genetic and biochemical activities in the cellular background is observed as enhanced
yield of target chemicals, higher gene expression and discovery of entirely novel biomolecules
(Caretto et al., 2011; Murthy et al., 2014b; Ramirez-Estrada et al., 2015).
Fig. 2.5 Diagrammatic depiction of elicitors and their mode of action mimicking possible
elicitation mechanism using elicited plant cell, tissue and organ cultures in vitro [ABA
abscisic acid, Ca2+ Calcium ion, cADPR cyclic adenosine diphosphoribose, cGMP cyclic
guanosine monophosphate, E elicitor, EDSPs enchanced disease susceptibility proteins,
ERP elicitor receptor perception, ET ethylene, ICSs isochorismate synthases, JA jasmonic
acid, JAZ jasmonate zim domain, MAPKs mitogen activated protein kinases, NO nitric
oxide, NPRI non-expressor of pothogenesis-related genes 1, PM plasma membarane, ROS
reactive oxygen species, SA salicylic acid, TF transcription factors, TGAs leucine zipper
transcription factors, 26SPD 26S proteasomal degradation] (Giri and Zaheer, 2016)
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2.7.3 Elicitation of secondary metabolites in Glycyrrhiza species
Since last few decades numerous strategies have been adopted for the production of secondary
metabolites from medicinal plants using plant tissue cultures. With the objective to improve
secondary metabolites production different strategies using cell culture systems have been
extensively studied which could be used for the large scale culturing of plant cells and secondary
metabolites extraction. The method is advantageous as it provides a continuous, reliable source
of natural products (Niraula et al., 2010).Glycyrrhiza is one such medicinal plant species which
has been known to have pharmaceutical importance. The pharmaceutical and other properties of
this plant are all due to the presence of secondary metabolites of varied composition, present in
one or more parts of these plants (Nomura and Fukai, 1998).
Cell and tissue culture of Glycyrrhiza plant species is a good source of phytoconstituents
and therefore can be seen as efficient systems for in vitro production of valuable secondary
metabolites but it can be justified only if it turns to be highly productive and cost effective.
Therefore elicitation is one of the most successful methods used for inducing or enhancing the
biosynthesis of metabolites in the plants due to the addition of trace amounts of elicitors
(Radman et al., 2003; Namdeo, 2007). Many elicitation studies have been done on Glycyrrhiza
plant species for the enhancement of pharmaceutically important metabolites (Table 2.4).
Various types of biotic elicitors such as yeast extract (Hayashi et al., 2005; Zhang et al., 2009b;
Wongwicha et al., 2011), chitosan (Wongwicha et al., 2011; Vijayalakshmi and Shourie
2015), arbuscular mycorrhizal fungi (Orujei et al., 2013), Aspergillus niger (Li et al., 2016b)
and abiotic elicitors such as UV light (Afreen et al., 2005), chemicals (Ayabe et al., 1986;
Hayashi et al., 2003; Shabani et al., 2009; Zhang et al., 2011; Wongwicha et al., 2011; Guo et
al., 2013; Li et al., 2016a) were used to enhance the secondary metabolites in Glycyrrhiza plant
species.
Stress induced formation of echinatin and 5- prenyllicodine in cultured cells of G.
echinata have been demonstrated by Ayabe et al. (1986). Hayashi et al. (2003) studied the
elicitation of soyasaponin biosynthesis by methyl-jasmonate in cultured cells of G. glabra. Yeast
extract was also found to be effective in promoting betulinic acid and soyasaponin accumulation
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in cell culture of G. glabra (Hayashi et al., 2005). Transgenic and wild culture of G. uralensis
was elicitated with PEG-8000 and yeast extract to enhance the accumulation of total flavonoid
content through a combined approach of elicitation and genetic engineering (Zhang et al.,
2009b).
Table 2.4 Elicitation of secondary metabolites in licorice through biotic and abiotic elicitors
Species Elicitor Product Reference
G. echinata Sodium-alginate Echinatin Ayabe et al. (1986)
G. glabra Methyl jasmonate Soyasaponin 5-deoxyflavonoid Hayashi et al. (2003)
G. uralensis UV-stress Glycyrrhizin Afreen et al. (2005)
G. glabra Yeast extract Betulinic acid and Soyasaponin Hayashi et al. (2005)
G. uralensis PEG8000 &Yeast
extract
Flavonoid Zhang et al. (2009b)
G. glabra Methyl jasmonate &
Salicylic acid
Glycyrrhizin Shabani et al. (2009)
G. uralensis Tween-80 Licochalcone A and total flavonoid Zhang et al. (2011)
G. inflata Chitosan, Methyl
jasmonate, Yeast extract
Glycyrrhizin Putalun et al. (2011)
G. inflata Methyl jasmonate,
Chitosan, Yeast extract
Glycyrrhizin Wongwicha et al.
(2011)
G. uralensis Methyl jasmonate &
Phenylalanine
Flavonoids & Polysaccharides Guo et al. (2013)
G. uralensis Molybdenum Glycyrrhizic acid Wang et al. (2013c)
G. glabra Glomus mosseae &
Glomus intraradices
Glycyrrhizin & total phenols Orujei et al. (2013)
G. glabra Chitosan Licochalcone, Liquirtigenin and
Licoisoflavone
Vijayalakshmi &
Shourie (2015)
G. uralensis Salicylic acid Glycyrrhizic acid, Glycyrrhetinic acid,
total flavonoids, Polysaccharide and
antioxidant enzymes
Li et al. (2016a)
G. uralensis Methyl jasmonate &
Phenylalanine
Glycyrrhetinic acid and total flavonoids Wang et al. (2016)
G. uralensis Aspergillus niger &
Salicylic acid
Total flavonoids, Polysaccharides,
enzymes incl. antioxidant
Li et al. (2016b)
Glycyrrhizin (oleanane-type triterpenoid saponin) is the main active component of licorice
which is 50 times sweeter than sugar (Brielmann, 1999) and possesses a wide range of
pharmacological properties (Fujisawa and Tandon, 1994; Jurgen, 1999). Methyl jasmonate
and salicylic acid are two key signal molecules used as elicitor to enhance the production of
glycyrrhizin in the G. glabra by 3.8 and 4.1 times respectively (Shabani et al., 2009). Attempts
to enhance the glycyrrhizin accumulation in hairy root culture of G. inflata have been studied by
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REVIEW OF LITERATURE 45 | P a g e
Wongwicha et al. (2011). Two fungal elicitors (Glomus mosseae and Glomus intraradices) were
also used to enhance the production of glycyrrhizin and total phenol in licorice by rising the
triterpenoid and phenolic metabolism (Orujei et al., 2013). UV-B stress also stimulates the
glycyrrhizin concentration of G. uralensis in hydroponic system (Afreen et al., 2005).
Production of other pharmaceutically important constituents of Glycyrrhiza plant such as
licochalcone, liquirtigenin and licoisoflavone were also enhanced by using different elicitors
such as tween 80 and chitosan (Zhang et al., 2011; Vijayalakshmi and Shourie, 2015).
Enhanced glycyrrhizic acid, glycyrrhetinic acid, total flavonoid, polysaccharide and antioxidant
enzymes in the roots of G. uralensis using either salicylic alone or in combination with
Aspergillus niger was also reported (Li et al., 2016a, 2016b).
Fig. 2.6 Chemical structures and the biosynthetic pathway for glycyrrhizin and related
triterpenoids in licorice plants (Kojoma et al., 2010)
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2.8 Antimicrobial assay of Glycyrrhiza glabra
Diethyl carbonate extracts of Glycyrrhiza glabra root from Astrakhan region (Russia) exhibited
maximum activity against Staphylococcus aureus, Escherichia coli and Bacillus subtilis than that
of root from Calabria region (Italy). Antibacterial activity was directly proportional to the
content of glycyrrhizin and 18 β-glycyrrhetinic acid (Astaf’eva and Sukhenko, 2014). Aqueous
and ethanolic licorice root extract were found to have antimicrobial activity against oral
pathogens, Streptococcus mutans and Lactobacillus acidophilus (25% and 12.5% MIC
respectively) (Ajagannanavar et al., 2014). The mixture of Capsella bursa- pastoris and
Glycyrrhiza glabra extracts was more effective against all oral pathogens (Streptococcus mutans,
S. sanguis, Actinomyces viscosus and Enterococcus faecalis) than the separate individual extracts
indicating synergistic effects between two plant extracts (Soleimanpour et al., 2013).The
hydromethanolic G. glabra root extract displayed in vitro antibacterial activities against
Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri Staphylococcus aureus,
Staphylococcus epidermidis and Bacillus substilis. Out of which Shigella flexneri was found to
be more sensitive (Varsha et al., 2013). The methanol licorice root extract exhibited moderate
antimicrobial activity. The extract was more potent against Staphylococcus aureus (at 500µg/ml;
13mm inhibition zone) among bacteria and Rhizopus spp. (at 500µg/ml; 11mm inhibition zone)
among fungi whereas was least active against Aspergillus awamori (Chopra et al., 2013). The
ethanolic extract of G. glabra showed good antifungal activity against Aspergillus niger,
Aspergillus fumigates, Candida albicans, Mucor sp. and Penicillium marneffei (Geetha and
Roy, 2013). Glabridin and licochalcone A extracted from G. glabra showed antifungal activity
against C. albicans while glycyrrhizic acid had no effect. Licochalcone A (0.2µg/ml) inhibits the
formation of biofilm by 35-60%. Glabridin or licochalcone A showed strong inhibitory effect
(>80%) on hypal formation (Messier and Grenier, 2011).
The ethanolic extract of leaves was most active extract against gram positive bacteria
whereas acetone, chloroform and ether extract of licorice root showed significant antibacterial
activities against two gram positive (Bacillus subtilis and Staphylococcus aureus) and two gram
negative (Escherichia coli and Pseudomonas aeruginosa) bacteria (Nitalikar et al., 2010). The
hydroalcoholic extract of licorice exhibited antifungal activity in vitro against Candida albicans
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REVIEW OF LITERATURE 47 | P a g e
and Aspergillus niger (Tharkar et al., 2010). Extracts of licorice root and leaf showed activity
against Candida albicans, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus in
a dose dependent manner (Irani et al., 2010). Glabridin from licorice root was found to be active
against both yeast and filamentous fungi and also showed resistance modifying activity against
drug resistant mutants of Candida albicans at 31.25-250 µg/ml (Fatima et al., 2009). The in
vitro growth of Candida albicans strains was reduced markedly in a pH dependent manner by
18-β glycyrrhetinic acid (6.2µg/ml) of G. glabra root. 18-β glycyrrhetinic acid is a biological
alternative for the tropical treatment of vulvovaginal candidiasis (Pellati et al., 2009). Raw
polysaccharides of Glycyrrhiza glabra act as an anti-adhesive agent against Porohyromonas
gingivalis, pathogen responsible for periodontal inflammation (Wittschier et al., 2009). The
methanolic extract of licorice roots showed antibacterial activities against Agrobacterium
tumefaciens, Bacillus cereus, B. subtilis and Pseudomonas syringae, but none of water extracts
showed any antibacterial activity against microorganism (Ercisli et al., 2008).
Antimicrobial activity of licorice root (at 500µg/ml) was assayed. Bioactivity guided
phytochemical analysis identified glabridin (29.16µg/ml)as potentially active against two
different strains of Mycobacterium tuberculosis (H37Ra and H37Rv) and also exhibited
antimicrobial activity against gram positive and negative bacteria (Gupta et al., 2008).
Glycyrrhiza glabra extract (>7.5%) exhibited inhibitory effects in vitro against Salmonella typhi,
S. paratyphi B, Shigella sonnei, S. flexneri and enterotoxigenic Escherichia coli (Shirazi et al.,
2007). The ether-water extract were found to have effective antibacterial activity against E. coli,
B. subtilis, E. aerogenes, K. peumoniae and S. aureus (Onkarappa et al., 2005). Extract of G.
glabra samples collected from Calabria and Italy exhibited antimicrobial activity against bacteria
(S. aureus, E. faecalis and Micrococcus luteus) and fungus (Trichophyton mentagrophytes) some
sample inhibited Pythium ultimum (Statti et al., 2004). Of various oriental herb extracts tested,
only G. glabra showed a remarkable activity against Propionibacterium acnes, similar to that of
erythromycin antibiotic (Nam et al., 2003). Rhizome of licorice exhibited antifungal activity
against Candida albicana in vitro with MIC value of 1.56mg/ml (Motsei et al., 2003).
Licochalcone A was effective against all gram positive bacteria especially against the
vegetative cell growth of Bacillus sp. with MIC 2-3µg/ml, but was not effective against gram
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negative bacteria or eukaryotes at 50µg/ml. Licochalcone A did not inhibit the germination of
heat treated spores of Bacillus sp. induced by L-alanine (Tsukiyama et al., 2002). Glabridin
exhibited antibacterial activity in vitro against both methicillin sensitive and resistant
Staphylococcus aureus whereas licochalcone A exhibited activity only against methicillin
resistant strain (Fukai et al., 2002). Several flavonoids with C5 aliphatic residues isolated from
licorice was effective against methicillin resistant Staphylococcus aureus and also restored the
effects of oxacillin and β-lactam antibiotic against methicillin resistant strain (Hatano et al.,
2000, 2005).
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MATERIALS & METHODS 49 | P a g e
MATERIALS AND METHODS
The present experiment was carried out in the Department of Biochemistry and
Biochemical Engineering and Department of Molecular and Cellular Engineering, Jacob
Institute of Biotechnology and Bioengineering, Sam Higginbottom University of
Agriculture, Technology & Sciences, Allahabad.
3.1MATERIALS
3.1.1 Sample Collection
The plants of licorice (Glycyrrhiza glabra) were procured from the nursery of Central
Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow. The nodal segments and
the leaves from single mother plant were used as explant for the in vitro plant regeneration of
licorice.
3.1.2 Culture Collection
The antimicrobial activity of licorice was examined against different bacterial viz., Bacillus
subtilis (MCCB0062), Streptococcus mutans (MCCB0084) and Proteus vulgaris
(MCCB0035) and fungal viz., Candida albicans (MCCB0290) and Aspergillus niger
(MCCB0201) culture. All mentioned organisms were procured from Department of
Microbiology and Fermentation Technology, Sam Higginbottom University of
Agriculture, Technology & Sciences, Allahabad.
3.1.3 Glassware & Miscellaneous items
All the glasswares i.e., beaker, brown bottles, conical flask, culture bottles with
pyropropylene lids, cuvette, glass rod, measuring cylinder, petri plates, separating funnel, test
tubes, volumetric flask, used during the investigation were of Borosil or Merck. Other
materials used during the investigation are alumininum foil, cotton, forceps, filter paper,
funnel, magnet, millipore filter, parafilm, scalpel, surgical blade, test tube stand, tissue paper,
etc.
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MATERIALS & METHODS 50 | P a g e
3.1.4 Chemicals and Equipments
All the chemicals used during the investigation were of analytical grade and procured from
Hi media and Sigma. Detail of the equipments used was given in Table 3.1.
Table 3.1Instruments used
S.No. Instrument used Source
1. Air conditioner Voltas, India
2. Analytical balance K-Roy, Europe Ltd.
3. Autoclave MAC, Macro Scientific works Pvt. Ltd, India.
4 Cold centrifuge Remi C-28, India.
5. Double distillation unit WDU2000, India.
6. Electronic balance Ohaus, India.
7. Hot Air oven MAC, Macro Scientific works Pvt. Ltd, India.
8. Heater Usha, Noida
9.Humidity and temperature
controllerVista, Biocell Pvt. Ltd., Noida
10. Incubator MAC, Macro Scientific works Pvt. Ltd, India
11. Laminar air flow MAC, Macro Scientific works Pvt. Ltd, India
12. Magnetic stirrer Icon, Scientific instrument, India
13. Micropipette Accupippet, Tarson Pvt. Ltd.
14. Microwave oven LG Electronics, India Pvt. Ltd.
15. pH meter TIMPL, Toshniwal Insts Mfg, Pvt. Ltd., Ajmer
16. Photoperiodic timer Vista, Biocell Pvt. Ltd., Noida
17. Refrigerator LG Electronics, India Pvt. Ltd.
18. Rotatory Evaporator Buchi, Switzerland
19. Spectrophotometer Systonics
20. Ultrasonicator Elma, Germany
21. Water bath Icon, Scientific instrument, India
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MATERIALS & METHODS 51 | P a g e
3.2 METHODOLOGY
REGENERATION
3.2.1 Washing and sterilization of glassware
All the glassware were thoroughly washed after overnight soaking in commercial liquid
detergents to avoid contamination followed by washing with running tap water to remove all
the adhere detergent residue and finally rinsed with double distilled water for two or three
times. After washing, the glassware were oven dried at 160ºC for 60 mins (dry heat
sterilization) prior to storage. Sterilization of glassware and metal instruments (forceps,
scissors, needle, scalpel, etc.) was done by dry heat sterilization in oven at 120ºC for 90 mins.
Whereas cotton, caps and filter papers were sterilized by autoclaving (wet sterilization) at
121ºC (15 psi) for 20 mins after wrapping in clean brown paper or aluminium foil.
3.2.2 Preparation of culture media
Precise media preparation was critical to the success of tissue culture. Media were generally
prepared by diluting concentrated stock solution. Stock solutions were prepared such that the
chemical included in the stock solution do not react among themselves and also do not
precipitate. (Table 3.4)
3.2.2.1 Preparation of stock solution
Detailed procedure for the preparation of stock solution of MS (Murashige & Skoog, 1962)
is given below in Table 3.4. All the stock solutions were prepared by dissolving the required
amount of chemicals in double distilled water. Each stock was poured in their respective
glass bottles with proper labeling and stored at 4ºC, in refrigerator for the further use. Iron
stocks should be stored in dark bottle. It was obligatory to shake the bottles before use and
any with contaminant or suspension in the form of precipitate must be discarded.
3.2.2.2 Stock preparation of plant growth regulators
Dissolved 10 mg of required auxin or cytokinin in minimum volume of ethanol or 0.1 N
NaOH and made up the volume to 10 ml with distilled water.
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Table 3.2 Composition of MS medium stock solution
Stock Chemicals Quantity for stocksolution (g/100ml)
Volumerequired/litre
medium
Major Salts (10X)
NH4NO3
KNO3
MgSO4.7H2O
KH2PO4
CaCl2.H2O
16.5
19.0
3.7
1.7
4.4
10 ml
Minor Salts(100X)
MnSO4.4H2O
ZnSO4.7H2O
H3BO3
Na2MoO4.2H2O
CuSO4.5H2O
CoCl2.6H2O
2.230
0.860
0.620
0.025
0.0025
0.002510 ml
Iron (100X)Na2EDTA.2H2O
FeSO4.7H2O
0.373
0.278 10 ml
Vitamins (100X)
Nicotinic acid
Thymine. HCl
Pyridoxine. HCl
Myo-inositol
0.050
0.050
0.010
10.00010 ml
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MATERIALS & METHODS 53 | P a g e
3.2.2.3 Preparation of MS media (1 litre)
500 ml distilled water was poured to 1 litre flask.
The required amount of stock solutions were added alongwith the required amount of
plant growth regulators, if required.
30 g sucrose was added as solid and this was allowed to dissolve.
Now the pH of the solution was adjusted to 5.8 with the help of 0.1N NaOH or 0.1N
HCl.
The volume of above solution was adjusted to 1 litre by adding distilled water.
Finally 8.0 g agar was added as a solidifying agent and kept in microwave or water
bath for heating so as to dissolve the agar properly.
Required amount of medium was disposed to culture bottles and the bottles were
capped.
Then the bottles were autoclaved for 20 mins at 121ºC (1.06 kg/cm2) or 15 psi and
were allowed to cooled and solidify.
3.2.3 Sterilization of media components
There are two methods of sterilizing media and its components viz. autoclaving and
membrane filtration under positive pressure. Culture media, distilled water and stable
mixtures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum
foil or plastic caps. However, solutions that contain heat labile components must be filter
sterilized.
Generally, nutrients of plant tissue culture media are autoclaved at 15 psi and 121ºC for
15-20 mins. The pressure should not exceed 20 psi as higher pressure may lead to the
decomposition of carbohydrates and other thermolabile components such as growth
hormones of medium. Minimum autoclaving time includes the time required for the liquid
volume to reach the sterilizing temperature (121°C) and it was 15 mins (Berger, 1988). Time
may be varied due to difference in size and shape of autoclaves.
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3.2.4 Sterilization of plant material (explant)
All tissue cultures are likely to end up contaminated if the inoculum or explant used was not
obtained from properly disinfected plant material. Getting sterile plant material was difficult
because in the process of sterilization living materials should not lose their biological activity,
only bacterial or fungal contamination should be eliminated. The explants taken from the
field carry a heavy load of microorganism and contamination therefore it needs to be
sterilized with the help of antimicrobial agent. However their indiscriminate use may lead to
phytotoxicity problem and development of resistant strain. The detailed surface sterilization
protocol is given below.
3.2.4.1 Pretreatment of explant (outside LAF)
The explants were removed carefully from healthy plants of G. glabra and subjected to
preliminary washing under running tap water for 20 mins followed by its treatment with 0.1%
antifungal agents (such as bavistin and indofill) for 2-3 mins. Explants were then rinsed with
distilled water for 4-5 times to remove the adhere residue. Now the explants were again
treated with Tween – 20 (2-3 drops) for 5 mins followed by continuous washing with distilled
water for 4-5 times until foam is removed. Finally the explants were taken to laminar airflow
(LAF) for further treatment.
3.2.4.2 Surface sterilization of explant (inside LAF)
Plant materials taken from field carry a wide range of contaminants. After the preliminary
washing outside the laminar air flow, an experiment was conducted to standardize the surface
sterilization procedure for the explants. For further disinfection, the explants were treated
with different sterilizing agents such as ethanol, sodium hypochloride (NaOCl) and mercuric
chloride (HgCl2) at different concentration for different time duration. The explants were
disinfected using 70% ethanol for 30 sec followed by 10-30% sodium hypochlorite for 5-10
mins and 0.1-0.2% mercuric chloride solution for 2-4 mins. Sterilization protocol for G.
glabra was standardized by using different steriliants at different concentration as shown in
Table 3.5, and each time the explants were rinsed with sterile autoclaved distilled water for
4-5 times to remove the traces of disinfectants. Finally the explants were sterilized i.e., free
from microbial load and dust particles.
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MATERIALS & METHODS 55 | P a g e
The contamination, lethal and survival percentage of explant was recorded through
visual observation. The best treatment was used to carry out the further experiments.
Table 3.3 Treatment of explants with different surface sterilizers of different
concentration
Treatment
Concentration and duration of surface sterilizer
Ethanol NaOCl HgCl2
(30 sec.) (5 mins.) (10 mins.) (2 mins.) (4 mins.)
ST1 70% 10% - - -
ST2 70% 20% - - -
ST3 70% 30% - - -
ST4 70% - 10% - -
ST5 70% - 20% - -
ST6 70% - 30% - -
ST7 70% - - 0.10% -
ST8 70% - - 0.20% -
ST9 70% - - - 0.10%
ST10 70% - - - 0.20%
ST11 70% 10% - 0.10% -
ST12 70% 20% - 0.10% -
ST13 70% 30% - 0.10% -
ST14 70% 10% - - 0.10%
ST15 70% 20% - - 0.10%
ST16 70% 30% - - 0.10%
ST17 70% - 10% 0.20% -
ST18 70% - 20% 0.20% -
ST19 70% - 30% 0.20% -
ST20 70% - 10% - 0.20%
ST21 70% - 20% - 0.20%
ST22 70% - 30% - 0.20%
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3.2.5 Inoculation
Before performing inoculation the laminar airflow was treated with UV for 20 mins and then
the working surface was swabbed with 70% alcohol using absorbent cotton. The hands were
also wiped with 70% alcohol before inoculation.
The following steps were observed during inoculation.
The sterilized explants were taken in a conical flask and placed near the burner in
laminar airflow.
Forceps or needles were sterilized by flaming. Instrument was sterilized each time
after handling the tissue.
The explants were placed one by one on a sterilized petri dish. The explants were held
tightly with the help of forceps and the scalpel was used to cut the dead end or to
scrap the tissue.
The explants were then inoculated on to the surface of the culture medium by pressing
on agar to ensure good contact. 2 explants per culture bottle were inoculated.
The culture bottles were covered and then sealed with parafilm to avoid any kind of
contamination.
3.2.6 Incubation of the culture plates
The culture bottles were incubated in culture room. All types of plant tissue are therefore
incubated under conditions of well controlled temperature, humidity, illumination and air
circulation. A typical culture room should have both light and temperature programmable for
a 24 hr period. The cultures were kept under the photoperiod of 16/8 hours of light and dark.
Usually air – conditioners and heaters are used to maintain the temperature around 25±2°C.
The light intensity (through white fluorescent tubes) was adjusted at 2500 – 5000 lux. The
period of incubation varied depending upon the nature of experiment. (i.e., callus induction
and regeneration).
3.2.7 Direct organogenesis
3.2.7.1 Standardization of growth regulators for shoot initiation
The effect of different growth regulators was studied on culture initiation and establishment
using nodal segments as an explant. The MS basal medium supplemented with following
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MATERIALS & METHODS 57 | P a g e
combinations of growth regulators were used for culture initiation (Table 3.6).From the
observation the number of explants showing shoot emergence was calculated out of the total
number of explants inoculated.
On the basis of visual observations, the number of explants showed shoot emergence
out of total number of culture inoculated and the number of days taken for shoot emergence
till final sprouting was recorded.
Table 3.4Hormonal combination of different growth regulators used for shoot initiation
TreatmentGrowth regulators(mg/l)
BAP KN IAA
SIM1 2 - 0.5
SIM2 4 - 0.5
SIM3 6 - 0.5
SIM4 8 - 0.5
SIM5 10 - 0.5
SIM6 - 0.5 0.5
SIM7 - 1 0.5
SIM8 - 1.5 0.5
SIM9 - 2 0.5
SIM10 - 2.5 0.5
3.2.7.2 Shoot proliferation
The micro shoots regenerated from nodal segment were sub-cultured on MS medium
supplemented with different combination of growth regulators for shoot proliferation and
elongation (Table 3.7). From the above the number of shoots per explant, shoot length and
number of leaves was recorded. The best shoot proliferation medium was used to carry out
further experiments.
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Table 3.5 Hormonal combination of different growth regulators for shoot proliferation
TreatmentGrowth regulators (mg/l)
BAP IAA NAA GA3 Ads
SP1 2 - 0.5 0.5 -
SP2 4 - 0.5 0.5 -
SP3 6 - 0.5 0.5 -
SP4 2 0.5 - 0.5 -
SP5 4 0.5 - 0.5 -
SP6 6 0.5 - 0.5 -
SP7 2 - 0.5 1 -
SP8 4 - 0.5 1 -
SP9 6 - 0.5 1 -
SP10 2 0.5 - 1 -
SP11 4 0.5 - 1 -
SP12 6 0.5 - 1 -
SP13 2 - 0.5 - 40
SP14 4 - 0.5 - 40
SP15 6 - 0.5 - 40
SP16 2 0.5 - - 40
SP17 4 0.5 - - 40
SP18 6 0.5 - - 40
SP19 2 - 0.5 - 60
SP20 4 - 0.5 - 60
SP21 6 - 0.5 - 60
SP22 2 0.5 - - 60
SP23 4 0.5 - - 60
SP24 6 0.5 - - 60
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3.2.8 Indirect organogenesis
3.2.8.1 Callus induction
Callus induction was initiated from the stem segments and young leaves of one month old
plant of licorice. Explants were gently cut or scraped using a scalpel and then inoculated on
the MS medium supplemented with different combinations of growth regulators BAP, 2,4-D
or NAA (Table 3.8). After 30 days, callus induction rate, type and quality were rated
visually. The best callus was selected for the regeneration of plants and elicitation.
Table 3.6 Hormonal combination of different growth regulators for callus induction
TreatmentHormone concentration (mg/l)
BAP 2,4D NAA
CIM1 - 0.5 -
CIM2 1 0.5 -
CIM3 2 0.5 -
CIM4 - 1 -
CIM5 1 1 -
CIM6 2 1 -
CIM7 - - 0.5
CIM8 1 - 0.5
CIM9 2 - 0.5
CIM10 - - 1
CIM11 1 - 1
CIM12 2 - 1
CIM13 - 0.5 1
CIM14 1 0.5 1
CIM15 2 0.5 1
CIM16 - 1 0.5
CIM17 1 1 0.5
CIM18 2 1 0.5
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3.2.8.2 Enzymatic browning
To study the effect of additives such as ascorbic acid and activated charcoal on the incidence
of lethal browning, the combination of hormone showing best callus percentage was amended
with different concentrations of ascorbic acid and activated charcoal (0, 25, 50, 75 and 100
mg/l). The effect of different additives was studied by observing the browning percentage and
biomass production.
3.2.8.3 Shoot regeneration
Six week old calli were transferred to MS medium containing various concentrations of BAP
and IAA for shoot regeneration (Table 3.9). Number of calli that regenerated shoots, number
of shoots per explant and shoot length was recorded. Sub-culturing was done at the intervals
of 2 weeks in the shoot proliferation medium and then finally to rooting medium.
Table 3.7 Hormonal combination of different growth regulators for organogenesis
TreatmentHormone concentration (mg/l)
BAP IAA
SRM1 2 0.2
SRM2 4 0.2
SRM3 6 0.2
SRM4 8 0.2
SRM5 10 0.2
SRM6 2 0.5
SRM7 4 0.5
SRM8 6 0.5
SRM9 8 0.5
SRM10 10 0.5
3.2.8.4 Rooting
Adventitious shoots regenerated from the direct and indirect organogenesis were excised and
transferred to the rooting medium containing half strength MS medium supplemented with
different concentrations of IBA and IAA (Table 3.10). From the observation, rooting (%),
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root length and root morphology was recorded. Rooted plantlets were transplanted to a sterile
pot mixture, acclimatized in the culture room and then finally transferred to a greenhouse.
Table 3.8 Hormonal combination of different growth regulators for rooting
TreatmentHormone concentration (mg/l)
IBA IAA
RIM1 1 -
RIM2 2 -
RIM3 3 -
RIM4 - 0.5
RIM5 - 1
RIM6 1 0.5
RIM7 2 0.5
RIM8 3 0.5
RIM9 1 1
RIM10 2 1
RIM11 3 1
3.2.8.5 Hardening
Well rooted plantlets with atleast two roots were transferred to ex vitro conditions. The plants
were rinsed with sterile distilled water to remove the adhering medium from the roots and
transplanted to a sterile pot mixture (sand + soil + vermiculite, 1:1:1). The pots were placed
in the culture room (Humidity 80-90 %; Temperature 25+2°C; Photoperiod 16/8 hours) and
then after 4 weeks the pots were finally transferred to a greenhouse.
ARTIFICIAL SEED PRODUCTION
3.2.9 Production of synthetic seed
The alginate solution was prepared in MS medium supplemented with different
concentrations of sodium alginate (2%, 4% and 6%). The calcium chloride solution was
prepared in the range of 50, 75, 100 and 125 mM concentration. Both the gel matrix and the
complexing agent were autoclaved for 20 minutes at 121ºC (1.06 kg/cm2) or 15 psi. Shoot
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MATERIALS & METHODS 62 | P a g e
tips and nodal segment of in vitro grown plant were suspended in the sterile sodium alginate
mixture, and was drop wise dispensed into the calcium chloride solution under continuous
shaking on a magnetic stirrer. The encapsulated beads were allowed to remain in the calcium
chloride solution for 15-20 mins to complete the ion-exchange reaction resulting into
polymerization. The resulting encapsulated micro-shoots were collected, rinsed thoroughly
with sterilized distilled water and stored in sterilized bottle (moist with distilled water) at
25+2oC. After the definite storage period, the encapsulated shoots were incubated on different
substrates and growth medium at 25+2oC. Data of encapsulated micro-shoots on re-growth
frequency and efficiency for different storage time period was recorded.
ELICITATION & BIOCHEMICAL STUDIES
3.2.10 Elicitation
The callus was used to study the effect of various abiotic elicitors such as adenine sulphate,
biotin, salicylic acid and polyamines (putrescine, spermine and spermidine) in three different
concentrations (25, 50, 75 and 100 mg/l). Elicitors were prepared as a stock solution and were
added to the best callusing media. Callus was subcultured on the elicitation media to study
the effects of elicitation on plant growth and biochemical metabolites.
3.2.11Qualitative determination of phytoconstituents
3.2.11.1 Preparation of plant extract
The callus of in vitro plants, leaves and roots of field grown plants were air-dried at room
temperature for 3 days and then grounded into a coarse powder by a grinder. The methanolic
extract of the sample was prepared using Soxhlet apparatus (Harborne, 1988).
3.2.11.2 Phytochemical screening of extract
Chemical tests were carried out on the methanolic extract using standard procedures to
identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and
Harborne (1988).
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MATERIALS & METHODS 63 | P a g e
a. Test for Carbohydrates (Molisch’s Test)
Reagents
- Molish’s reagent
- Sulphuric acid (H2SO4)
To the 2ml of extract, 2 drops of Molisch’s reagent were added, followed by the addition of
2-3 ml conc. H2SO4 along the sides of the test tube. The formation of the purple ring at the
interphase indicated a positive reaction.
b. Test for Protein (Biuret Test)
Reagents
- Copper sulphate (CuSO4)
- Sodium hydroxide (NaOH)
To the 1ml of extract, 2-3 drops of CuSO4 solution was added followed by the addition of
10% NaOH solution. A blue to purple colour indicated a positive reaction.
c. Test for Amino acids (Ninhydrin Test)
Reagents
- Ninhydrin solution
To the 1 ml of extract, 5 drops of ninhydrin solution was added. A blue or purple or yellow
colour indicated a positive reaction.
d. Test for Phenol (Ferric chloride test)
Reagents
- Ferric chloride
To the 2ml of extract add few drops of 5 % ferric chloride was added. A formation of violet
colour indicated the presence of phenolic compound.
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MATERIALS & METHODS 64 | P a g e
e. Test for Alkaloids (Mayer’s Test)
Reagents
- Hydrochloric acid
- Mayer’s reagent
Extracts were dissolved individually in dilute hydrochloric acid and filtered. Filtrates were
treated with Mayer’s reagent (Potassium mercuric iodide). Formation of buff coloured
precipitate indicated the presence of alkaloids.
f. Test for Flavonoids (Shinoda’s test)
Reagents
- Magnesium
- Conc. Hydrochloric acid
To the 2 ml of extract, a piece of magnesium was added followed by the addition of conc.
hydrochloric acid. On heating appearance of magenta colour showed the presence of
flavonoids.
g. Test for Saponins (Froth test)
The crude extract (0.5 gm) was dissolved in 10 ml distilled water and was uploaded in a
graduated cylinder. The above solution was shaken vigorously for 30 sec and the formation
of 1 cm foam layer indicated the presence of saponins.
h. Test for Terpenoids (Salkowski Test)
Reagents
- Chloroform
- Sulphuric acid (H2SO4)
5ml of each extracts was mixed in 2 ml of chloroform and concentrated H2S04 (3ml) was
carefully added to form a layer. A reddish brown colouration at the interface showed positive
results for the presence of terpenoids.
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3.2.12 Quantitative determination of phytoconstituents
3.2.12.1 Estimation of primary metabolites
a. Total carbohydrate estimation
Anthrone method was used for total carbohydrates determination given by Hedge and
Hofreiter (1962).
Reagents
- 2.5 N HCl
- Anthrone reagent (200 mg anthrone in 100 ml of ice-cold 95% H2SO4)
- Glucose stock (1mg/ml)
Procedure
100 mg of the plant sample was hydrolysed with 5 ml of 2.5 N HCl for three hours in a
boiling water bath, cooled at room temperature and neutralized with sodium carbonate. The
volume was made upto 100 ml and centrifuged to collect the supernatant. To the1ml of
supernatant, 4 ml of anthrone reagent was added and heated for eight minutes in a boiling
water bath. Now rapidly cooled it and read the green to dark green colour at 630 nm using
spectrophotometer.
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of glucose
solution. Using standard graph the carbohydrates was calculated and the unit of
carbohydrates was expressed in the terms of mg/g sample.
b. Total protein estimation
Total protein was determined according to the method given by Lowry et al., 1951.
Reagents
- Analytical reagents: 50 ml of 2% sodium carbonate in 0.1 N NaOH was mixed with
1ml of 0.5% copper sulphate solution in 1% sodium potassium tartarate solution.
- Folin - Ciocalteau reagent (FCR): Dilute commercial reagent (2N) with an equal
volume of water on the day of use.
- Bovine serum albumin (BSA) stock solution (1mg/ml)
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MATERIALS & METHODS 66 | P a g e
Procedure
Plant sample was centrifuged in distilled water (1g/10ml) at 10,000 rpm for 10 mins. The
supernatant collected was then used for protein estimation. To the above 0.2 ml supernatant,
0.8 ml distilled water and 5 ml of alkaline copper sulphate reagent (analytical reagent) was
added. The solution was incubated at room temperature for 10 mins. Now 0.5 ml Folin
Ciocalteau reagent was added to the above solution and then the solution was incubated for
30 mins. The optical density (absorbance) was measured at 660 nm in a spectrophotometer
for the colour development.
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of BSA solution.
Using standard graph the protein was calculated and the unit of protein was expressed in the
terms of mg/g sample.
c. Total phenol estimation
Total phenol was determined according to the method given by Bray and Thorpe (1954).
Reagents
- 80% Ethanol
- Folin - Ciocalteau reagent (FCR): Dilute commercial reagent (2N) with an equal
volume of water on the day of use.
- 20% Na2CO3
- Catechol (1mg/ml)
Procedure
Plant sample was centrifuged in 80% ethanol (i.e., 1g/10ml) at 10,000 rpm for 10 mins. The
supernatant collected and the residue was re-extracted and centrifuged five times using 80%
ethanol. The supernatant thus collected was evaporated till dryness and the dried residue was
then dissolved in 5ml of distilled water. To the above 0.2 ml solution, 2.8 ml distilled water
and 0.5 ml FCR solution was added. The solution was incubated for 3 min and then 2 ml of
20% Na2CO3 was added. Now the solution was slightly heated on boiling water bath for one
minute, cooled and then measured the optical density (absorbance) at 650 nm using
spectrophotometer.
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MATERIALS & METHODS 67 | P a g e
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of catechol
solution. Using standard graph the total phenol was calculated and the unit of total phenol is
expressed in the terms of mg/g sample.
d. Proline
Proline was determined according to the method given by Bates et al., 1973.
Reagents
- Acid ninhydrin: Warm 1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml
phosphoric acid, with agitation until dissolved.
- 3% aqueous sulphosalicyclic acid
- Glacial acetic acid
- Toluene
- Proline
Procedure
Extract 0.5g of plant material by homogenizing in 10 ml of 3% aqueous sulphosalicylic acid.
Filter the homogenate through whatman No. 2 filter paper. To the 2 ml of filtrate, 2 ml of
glacial acetic acid and 2 ml acid ninhydrin were added. The solution was then heated on the
boiling water bath for 1hr. The reaction was terminated by placing the tube in ice bath. 4 ml
of toluene was added to the reaction mixture and stirred well for 20-30 sec. Now the toluene
layer was separated and warm to room temperature and measured the red color intensity at
520 nm using spectrophotometer.
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of proline. Using
standard graph the proline content was calculated and the unit was expressed in the terms of
µmol/g sample. Proline content was calculated from the following formula
Proline content (µmol/g tissue) = µg proline/ml x ml toluene x 5 .115.5 sample (g)
Where, 115.5 is the molecular weight of proline.
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MATERIALS & METHODS 68 | P a g e
3.2.12.2 Estimation of secondary metabolites
a. Total alkaloid estimation
Total alkaloid was determined according to the method given by Fazel et al., 2008.
Reagents
- 2N HCl
- Phosphate buffer: Phosphate buffer solution (pH 4.7) was prepared by adjusting the
pH of 2 M sodium phosphate (71.6 g Na2HPO4 in 1 L distilled water) to 4.7 with 0.2
M citric acid (42.02 g citric acid in 1 L distilled water). The pH of phosphate buffer
solution was adjusted to neutral with 0.1 N NaOH.
- Bromocresol solution: Bromocresol green solution was prepared by heating 69.8 mg
bromocresol green with 3 ml of 2N NaOH and 5 ml distilled water until completely
dissolved and the solution was diluted to 1000 ml with distilled water.
- Chloroform
- Colchicine (1 mg/ml)
Procedure
Plant sample was centrifuged in methanol (i.e., 1g/10ml) at 10,000 rpm for 10 mins. The
supernatant collected and the residue was re-extracted and centrifuged. The supernatant thus
collected was evaporated till dryness and the dried residue was then dissolved in 5 ml 2N
HCl. One ml of this solution was transferred to a separating funnel and then 5 ml of BCG
solution along with 5 ml of phosphate buffer were added. The mixture was shaken and the
complex formed was extracted with chloroform by vigorous shaking. The extracts were
collected in a 10 ml volumetric flask and diluted to volume with chloroform. The absorbance
of the complex in chloroform was measured at 470 nm.
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of colchicine.
Using standard graph the total alkaloid content was calculated and the unit was expressed in
the terms mg/g
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MATERIALS & METHODS 69 | P a g e
b. Total flavonoid estimation
Total flavonoid was determined according to the method given by Chang et al., 2002.
Reagents
- Methanol
- 10% ammonium chloride
- 1M potassium acetate
- Quercetin (1 mg/ml)
Procedure
Plant sample was centrifuged in methanol (i.e., 1g/10ml) at 10,000 rpm for 10 mins. The
supernatant collected and the residue was re-extracted and centrifuged five times using
methanol. The supernatant thus collected was evaporated till dryness and the dried residue
was then dissolved in 5ml of distilled water. To the above 1 ml solution add 0.1 ml of 10%
ammonium chloride, 0.1 ml of 1M potassium acetate and 2 ml of distilled water. It remained
at room temperature for 30 mins. The absorbance of the reaction mixture was measured at
415 nm wavelength with a single beam systronics UV/ Visible spectrophotometer.
The standard curve was prepared by using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of quercertin
solution. Using standard graph the quercertin was calculated and the unit of protein was
expressed in the terms of mg/g sample.
c. Glycyrrhizin estimation
HPLC analysis of glycyrrhizin was performed according to the method reported by Hurst et
al. (1983).
Reagents
- Pure glycyrrhizin (HPLC-grade)
- Methanol (HPLC-grade)
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MATERIALS & METHODS 70 | P a g e
Ultrasound-assisted extraction of glycyrrhizin
The callus of in vitro plants, leaves and roots of field grown plants were weighed and crushed
using mortar, extracted for 10 mins using 2 ml of 50% methanol under ultra-sonication, for
30 mins at 45 MHz, at room temperature, twice. The extract was filtered (Millipore filter 0.45
μm) concentrated on a rotary-evaporator and 2 ml of solvent was added to the sample before
analysis. The resultant extracts were then used for subsequent HPLC analysis.
HPLC analysis
A 10 μl aliquot of the root extract was analyzed by HPLC at 30°C. The HPLC system
consisted of Waters HPLC 510 pump, a Nova-pak C18 column (3.9 × 150 mm, Waters,
United States), a Waters 2478 detector, and a Millennium chromatography data system
(Waters). The separation was performed with an isocratic elution using methanol–water–
acetic acid (60: 34: 6) at a flow rate of 1 ml/min with UV absorption detection at 254 nm.
Routine sample calculations were made by comparison of the peak area with that of the
standard.
3.2.12.3 Enzymatic antioxidants
Approximately 0.5 g fresh samples were homogenized in 50 mM PBS (pH 7.6) including 0.1
mM Na-EDTA. Samples were generally homogenized in 8 ml, and then centrifuge for 15
mins at 20,000 rpm and 4°C.
a. Superoxide dismutase (SOD)
The superoxide dismutase (SOD) activity was estimated by recording the decrease in optical
density of formazone made by superoxide radicals and nitrobluetetrazolium chloride (NBT)
dye by the enzyme (Cakmak and Marschner, 1992).
Reagents
- Ethylene diamine tetra acetic acid (EDTA, 0.1mM)
- L-methionine (12mM)
- Nitro blue tetrazolium chloride (NBT, 75μM)
- Potassium phosphate buffer (50 mM, pH 7.6)
- Riboflavin (2μM)
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MATERIALS & METHODS 71 | P a g e
- Sodium carbonate (50mM)
Procedure
For the assay of SOD, the reaction medium (5.0 ml) containing 50mM phosphate buffer, pH
7.6, 0.1mM Na-EDTA, enzyme aliquots (50-150μM), 50mM sodium carbonate (pH 10.2),
12mM L-methionine, 75μM NBT and 2μM riboflavin was maintained in glass vials.
Riboflavin was the last compound to be added. Reactions were carried out at room
temperature and under a light intensity of about 400μM m-2 s-1. One unit of SOD activity was
defined as the amount of enzyme required to cause 50% inhibition of the rate of NBT
reduction measured at 560 nm.
b. Peroxidase (POX)
Peroxidase (POX) activity was measured as described by Castillo et al., 1984.
Reagents
- Hydrogen peroxide (H2O2, 2mM)
- Potassium phosphate buffer (PBS, 50mM, pH 6.1)
- Guaiacol (16mM)
Procedure
The assay is based on the formation of tetra-guaiacol at 470 nm and the enzyme activity was
calculated as per extinction coefficient of its oxidation product, tetra-guaiacol (ϵ = 26.6mM-
1cm-1). The reaction mixture (1.0 ml) contains 50mM PBS (pH 6.1), 16mM guaiacol, 2mM
H2O2 and enzyme aliquots.
c. Ascorbate Peroxidase (APX)
Activity of ascorbate peroxidase (APX) was measured according to Cakmak (1994).
Reagents
- Ascorbic acid (AsA)
- Ethylene diaminetetraacetic acid (EDTA)
- Hydrogen peroxide (H2O2)
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MATERIALS & METHODS 72 | P a g e
- Potassium phosphate buffer (PBS, 50mM, pH 7.6)
Procedure
Activity of ascorbate peroxidase (APX) was measured by monitoring the rate of H2O2-
dependent oxidation of AsA at 290 nm. The reaction mixture (1 ml) contained 50mM PBS
(pH 7.6), 0.1mM EDTA, 12mM H2O2, 0.25mMAsA and enzyme aliquot.
STUDIES OF ANTIMICROBIAL ACTIVITIES
3.2.13 Antimicrobial assay
3.2.13.1 Preparation of plant extract
The fine dried (dried in shade) powder (1 g) of licorice leaves and root was used for the
extraction of active ingredient (5 ml). The organic solvents (aqueous, acetone, methanol,
and ethanol) were used for extraction. The above mixture was vortexed for 1 hrs and then
centrifuged at 10,000 rpm for 15 min at 25oC. The liquid faction were collected and used as
active ingredient for further applications. These extracts were dried under vacuum to obtain
the active ingredient and were re-suspended in solvent with a final concentration of 0.2
g/ml.
Table 3.9 Preparation of Nutrient agar (NA) medium
Chemical Used Quantity (g/l)
Beef extract 3.0
Peptone 5.0
Sodium chloride (NaCl) 5.0
Agar 20
The continuous shaking was done until all the solutes have dissolved. The pH was adjusted at
7.2 – 7.5 with 1N NaOH. The volume of the solution was adjusted to 1 litre with distilled
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MATERIALS & METHODS 73 | P a g e
water. The sterilization was done by autoclaving for 20 mins. at 15 lb/sq. Solid NA media
was used for streaking purpose. Liquid NA media was used for growing culture of required
strain.
Table 3.10 Preparation of Potato dextrose agar (PDA) medium
Chemical Used Quantity (g/l)
Potato 3.0
Dextrose 5.0
Agar 20
The continuous shaking was done until the solutes have dissolved. The pH was adjusted at
6.5 with 1N NaOH. The volume of the solution was adjusted to 1 litre with distilled water.
The sterilization was done by autoclaving for 20 mins. at 15 lb/sq. Solid PDA media was
used for streaking purpose. Liquid PDA media was used for growing culture of required
strain.
3.2.13.4 Preparation of inoculum
The stocks of cultures were maintained at 4oC on nutrient agar slants. The bacterial cultures
were inoculated on nutrient broth for overnight at 37oC, while fungal cultures were
inoculated on PDA (Potato Dextrose Agar). After appropriate growth the healthy cultures
were used for antimicrobial assay.
3.2.13.5 Agar-disc bioassay method
Antibacterial and antifungal activity of licorice was tested using agar disc bioassay. 24 hour
old cultures of test organisms (0.05 ml) were seeded onto nutrient agar plate and uniformly
spread with a spreader. What man paper discs were dipped in plant extract and were placed
on plates. These plates were incubated at 37oC for 24 hours. The growth of the bacterial and
fungal cultures was measured and compared with respective control plates. Antimicrobial
assay for each of the extracts against all microorganisms tested was performed in triplicates.
Diameters of inhibition zone formed in all the three replicates were measured in mm using
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MATERIALS & METHODS 74 | P a g e
measuring scale and average of three was determined. Controls contained streptomycin (for
bacteria) and bavistin (for fungi).
STATISTICAL ANALYSIS
All the experiments were conducted with a minimum of 10 explants in three replications. The
experimental data recorded during the course of investigation were statistically analyzed as
per the method of ‘Analysis of variance’ (Fisher, 1950).The significance and non-
significance of the treatment was judged with the help of ‘F’ variance ratio test. The
significant difference between the means was tested against the critical difference at 5%
level. For the hypothesis the following ANOVA table was used.
Table 3.11 The skeleton of two way ANOVA analysis
Source of Variation D. F. S.S. M.S.S. F (cal.) F (tab.) at 5%
Due to Replications (r-1) SS(R) SS(R)/(r-1) MSSR/MESS F(r-1), (r-1)(t-1)
Due to Treatments (t-1) SS(T) SS(T)/(t-1) MSST/MESS F (t-1), (r-1)(t-1)
Error (r-1)(t-1) ESS ESS/(r-1)(t-1)
Total (rt-1) TSS
Standard error due to mean
Standard error of mean was calculated by the following formula:
S.E. = √2 x MESS/r
Where,
EMSS = Error mean sum of squares
r = Number of replications
Critical difference (C.D.)
Critical difference was calculated by the following formula:
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MATERIALS & METHODS 75 | P a g e
C.D. = S.E. x tab 5%Where,
S.E. = Standard error due to mean
tab 5% = Table value at error degree of freedom at 5% level of significance
Significant ‘F’ value indicates that there is a significant difference among the treatment. But
to compare any two particular treatments, it is tested against C.D. value.
Test of Significance
If the variation ratio, f-calculated value of treatment was greater than the f-tabulated value at
5% and 1% level of significance, the variance between treatments was considered to be
significant. If the f-calculated value is less than the f-tabulated value, the differences between
treatments were considered to be non-significant.
Mean performance
Mean = ΣX/n
Where,
ΣX = sum of all observations for each character in each replication
N = number of observations
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RESULTS&DISCUSSION 76 | P a g e
RESULTS & DISCUSSION
In this chapter, the results and discussions of the present study entitled “In vitro studies on
the variations of biochemical metabolites in Glycyrrhiza glabra L. by using various
elicitors” have been compiled and portrayed in the form of tables, figures and plates. The
current investigation focused on in vitro propagation of Glycyrrhiza glabra, their
conservation through artificial seed formation, elicitation of metabolites, their qualitative and
quantitative analysis and antimicrobial assay. The overall findings obtained from the present
investigation as well as relevant discussion have been presented under the following headings
and sub-headings.
4.1 Standardization of in vitro protocol for rapid propagation
Plant tissue culture has a most promising potential and is an alternative source for the in vitro
propagation, multiplication and conservation of invaluable germplasm of Glycyrrhiza glabra
which would be pathogen-free and season independent. In our research, it was found that the
major problem in large scale multiplication of licorice plant was the high mortality rate, due
to microbial contamination and lethal browning caused by explant. The present study
therefore, aimed at rapid and efficient protocol for mass propagation of G. glabra L. by
eliminating contamination and browning problem.
4.1.1 Standardization of sterilization protocol
The present study was carried out to optimize the sterilization protocol for fast multiplication
of licorice plantlets. The explants (i.e., nodal segments and leaves) were procured from the
one month old healthy field grown plants of licorice and the prior washing with Bavistin and
Tween 20 was performed. To ensure the complete sterilization, explants were again treated
with various concentrations of different sterilizing agents for different time durations in
laminar air hood and their effect on the contamination as well as on survival percentage was
shown in Table 4.1 and Fig 4.1.
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RESULTS&DISCUSSION 77 | P a g e
Table 4.1 Standardization of sterilization protocol for G. glabra using different
sterilants for different time duration
Treatment
Concentration and duration of surface sterilizerContamination
(%)Survival
(%)Necrotic
(%)Ethanol NaOCl HgCl2
30 sec 5 mins. 10 mins. 2 mins. 4 mins.ST1 70% 10% - - - 89.34 6.28 4.38
ST2 70% 20% - - - 77.15 15.59 7.26
ST3 70% 30% - - - 62.57 20.42 17.01
ST4 70% - 10% - - 80.7 6.83 12.47
ST5 70% - 20% - - 71.26 8.75 19.99
ST6 70% - 30% - 62.39 5.24 32.37
ST7 70% - - 0.10% - 47.81 43.37 8.82
ST8 70% - - 0.20% - 41.65 36.89 21.46
ST9 70% - - - 0.10% 26.61 62.53 10.86
ST10 70% - - - 0.20% 20.37 39.5 40.13
ST11 70% 10% - 0.10% - 39.42 19.79 40.79
ST12 70% 20% - 0.10% - 31.33 19.34 49.33
ST13 70% 30% - 0.10% - 26.56 15.4 58.04
ST14 70% 10% - - 0.10% 21.39 15.88 62.73
ST15 70% 20% - - 0.10% 18.47 14.28 67.25
ST16 70% 30% - - 0.10% 12.3 16.04 71.66
ST17 70% - 10% 0.20% - 28.45 15.54 56.01
ST18 70% - 20% 0.20% - 20.73 14.55 64.72
ST19 70% - 30% 0.20% - 15.73 17.52 70.38
ST20 70% - 10% - 0.20% 12.1 7.82 76.45
ST21 70% - 20% - 0.20% 7.51 10.06 82.43
ST22 70% - 30% - 0.20% 4.88 5.71 89.41
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RESULTS&DISCUSSION 78 | P a g e
Fig 4.1 Effect of different steriliants on the contamination and survival of explants of G.
glabra
Present investigation highlighted the importance of different sterilizing agents for the
surface sterilization of explants of licorice. The surface sterilizing agents used were mercuric
chloride (HgCl2), sodium hypochloride (NaOCl) and ethanol, which were used at different
concentrations for different time intervals. Ethanol (70%) when used alone showed failure in
sterilizing the explants whereas NaOCl and HgCl2 alone at different concentration were far
much better than ethanol and also in line with the findings of Chen et al. (1988). The highest
percentage of survival (62.53%) was obtained by treating the explants with 70% ethanol (30
sec) and 0.1% HgCl2 (4 mins) whereas the lowest survival (5.24 and 5.71 %) was obtained by
treating the explants with 70% (30 sec) ethanol and 30% NaOCl (10 mins) or with 70% (30
sec) followed by 30% NaOCl (10 mins) and 0.5% HgCl2 (4 mins) respectively. Similarly
high necrotic percentage was found when explants were surface sterilized using 30% NaOCl
(10 mins) with 0.5% HgCl2 (4 mins) while low necrotic percentage was found on 10%
NaOCl (5 mins). Explants were found not responsive when treated with higher concentrations
of NaOCl and HgCl2. High period of exposure with NaOCl and HgCl2 in Mentha arvensis L.
results in the browning of explants which eventually leads to the death of explants (Johnson
et al., 2011). Similar finding were also reported by Tiwari et al. (2012) in sugarcane.
However, NaOCl along with HgCl2and ethanol at different concentration gives satisfactory
result. But at the same time survival (%) with respect to contamination (%) is low.
0102030405060708090
100
ST1
ST2
ST3
ST4
ST5
ST6
ST7
ST8
ST9
ST10
ST11
ST12
ST13
ST14
ST15
ST16
ST17
ST18
ST19
ST20
ST21
ST22
RE
SPO
NSE
(%
)
TREATMENTS
Contamination (%) Survival (%)
CHAPTER – 4
RESULTS&DISCUSSION 79 | P a g e
In vitro culture establishment from field grown plant is prone to contamination which is
the most important reason for losses during in vitro culture of plant. Avoiding contamination
and establishing aseptic cultures from the field grown plants is always a challenge due to the
high risk of internal and external contamination (Hennerty et al., 1988; Misaghi and
Donndelinger, 1990).Microbial contaminants (such as viruses, bacteria, yeast and fungi) are
found on surface as well as inside the plant body (Omamor et al., 2007). There is a
competition between microbes and the in vitro plants for nutrients which reduces the growth
of in vitro culture with the increase in mortality rate and necrosis of cultures (Kane, 2003).
The success of plant tissue culture protocol depends on the sterilization of explants (Dodds
and Roberts, 1985).
Selection of sterilizing agent, their concentration and time period of exposure is also
critical because the living material should not lose their biological activity and only
contaminants should be eliminated during sterilization (Tiwari et al., 2012). The sterilization
protocol varied depending upon the plant species as well as the type of explant used (Jan et
al., 2013). The exposure time of sterilant to explant is dependent on the type of tissue used
i.e., leaf tissue will requires a shorter time of exposure than that of seed due to hard seed coat
(Ndakidemi et al., 2013; Sharma and Nautiya, 2009). Excessive exposure of sterilants to
tissue resulted into necrosis leading to death of the tissue (Sharma and Nautiya, 2009).
Although surface contamination can be eliminated by suitable sterilization protocol but it is
difficult to remove the internal contamination which may arise at later stage. This can be
controlled to some extent by frequent sub-culturing in fresh medium or by the addition of
antibiotics in the medium.
The highest percentage of survival (62.53%) was obtained by treating the explants with
0.1% HgCl2 for 4 min. Therefore HgCl2 (0.1%) was found to be more effective for
sterilization with maximum survival of explant and minimum tissue injury and as well as for
further in vitro response of explants. The past findings also suggest that HgCl2 is an effective
and good sterilizing agent for the explants of licorice (Hayashi et al., 1988; Mousa et al.,
2007). Similar findings related to the use of 0.1% HgCl2 (4 mins) for the sterilization of
explant have also been reported (Dalal et al., 1992; Modgil et al., 1994, 2008; Gautam et
al., 2001; Rattanpal et al., 2011). Therefore, all the experiments were carried out with 0.1%
HgCl2.
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RESULTS&DISCUSSION 80 | P a g e
4.1.2 Direct organogenesis
4.1.2.1 Optimization of media for shoot initiation
After standardizing the sterilization protocol, the nodal segments and leaves were inoculated
on MS medium fortified with different combination and concentration of cytokinin (BAP and
KIN) and auxins (IAA) to evaluate their effect on shoot initiation. The detail related to the
combination of hormones used and their effect on shoot initiation and time taken for shoot
emergence was shown in Table 4.2and Fig 4.2.
The results revealed that no shoot formation occurred on MS medium without
cytokinin. Shoot initiation response varied in MS medium supplemented with different plant
growth regulators. Leaves do not take part in shoot induction. Of the various concentrations
tried, MS media supplemented with BAP (4 mg/l) + IAA (0.5 mg/l) was found to be most
effective as this concentration favoured early sprouting (7 days) with maximum shoot
emergence (%) i.e., 92.03% whereas minimum shoot emergence percentage was found on
MS media containing 10 mg/l BAP or 0.5 mg/l KIN with 0.5mg/l IAA. Combination of BAP
and IAA gave better response than KIN and IAA. Addition of IAA also decreased the number
of days required for bud break.
In vitro shoot bud proliferation is usually considered as a convenient route for
micropropagation (Altman and Loberant, 1998). Organogenesis starts with distinct
organization of a group of new meristematic cells, directly within the explants, which later
transformed into a shoot or root meristem (Street, 1969; Thorpe, 1994). The composition of
nutrient medium, use of appropriate plant growth regulators, additives and culture conditions
are the important factors for the successful establishment of tissue culture. To reduce the risks
of somaclonal variability during multiplication, apical and axillary meristems were preferred
as an explant for organogenesis (George, 1993).
In general, the axillary buds of the higher plants are dormant due to apical dominance
and the mechanism of apical dominance was under the control of various growth regulators
specially auxins (Cline, 1996). Cutting of nodal segment and culturing them on medium
supplemented with suitable growth regulators can break the dormancy of the bud (Dai et al.,
2006; Punyarani and Sharma, 2010).
CHAPTER – 4
RESULTS&DISCUSSION 81 | P a g e
Table 4.2 Effect of different combination of growth regulators on shoot establishment
using nodal segment of G. glabra as an explant
Treatment
Growth
regulators(mg/l)
Growth response
BAP KIN IAANo. of days for shoot
emergence
Shoot
emergence (%)
SIM 1 2 - 0.5 10 83.26b
SIM 2 4 - 0.5 7 92.03a
SIM 3 6 - 0.5 15 66.84d
SIM 4 8 - 0.5 20 42.10g
SIM 5 10 - 0.5 20 39.62h
SIM 6 - 0.5 0.5 14 40.39h
SIM 7 - 1 0.5 14 51.84f
SIM 8 - 1.5 0.5 12 66.66d
SIM 9 - 2 0.5 9 70.27c
SIM 10 - 2.5 0.5 20 63.47e
G. Mean
S.E.
C.D. (5%)
61.65
0.46
1.37
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Fig 4.2 Effect of growth regulators on shoot emergence using nodal segment of G. glabra
as an explant
0
20
40
60
80
100
120
SIM1 SIM2 SIM3 SIM4 SIM5 SIM6 SIM7 SIM8 SIM9 SIM10
SHO
OT
EM
ER
GE
NC
E (
%)
TREATMENTS
CHAPTER – 4
RESULTS&DISCUSSION 82 | P a g e
Plate 1. Culture establishment from nodal segment of G. glabra when inoculated on MSmedia supplemented with phytohormone combination BAP, KIN and IAA at variousconcentrations (a). 2 mg/l KIN + 0.5 mg/l IAA (b). 2 mg/l BAP + 0.5 mg/l IAA (c). 4 mg/lBAP + 0.5 mg/l IAA (d). 6 mg/l BAP + 0.5 mg/l IAA after 20 days
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 82 | P a g e
Plate 1. Culture establishment from nodal segment of G. glabra when inoculated on MSmedia supplemented with phytohormone combination BAP, KIN and IAA at variousconcentrations (a). 2 mg/l KIN + 0.5 mg/l IAA (b). 2 mg/l BAP + 0.5 mg/l IAA (c). 4 mg/lBAP + 0.5 mg/l IAA (d). 6 mg/l BAP + 0.5 mg/l IAA after 20 days
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 82 | P a g e
Plate 1. Culture establishment from nodal segment of G. glabra when inoculated on MSmedia supplemented with phytohormone combination BAP, KIN and IAA at variousconcentrations (a). 2 mg/l KIN + 0.5 mg/l IAA (b). 2 mg/l BAP + 0.5 mg/l IAA (c). 4 mg/lBAP + 0.5 mg/l IAA (d). 6 mg/l BAP + 0.5 mg/l IAA after 20 days
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 83 | P a g e
Shoot induction and development is a function of cytokinin activity (Sahoo and
Chand, 1998). BAP was crucial for stimulating the growth and development of explant
(Barless and Skene, 1980). This is in accordance with the earlier reported findings of G.
glabra by Sarkar and Roy (2014) and Badkhane et al. 2016. The stimulatory effect of BAP
in bud breaking and multiple shoot formation has been reported earlier in other medicinal
plants such as Melia azedarach (Sen et al., 2010), Aegle marmelos (Yadav and Singh,
2011), and Bacopa monnieri (Gurnani et al., 2012).It has been reported that a combination
of cytokinin and auxin is well suitable for the shoot regeneration and morphogenesis
(Smolenskaya and Ibragimova, 2002).
As the present study, Lal et al. (2010) also noted the synergistic effect of BAP in
combination with an auxin for efficient shoot regeneration. In consistent with our results, the
combinations of cytokinin (BAP or KIN) with low level of auxin (IAA) have also been used
to induce shoot formation in numerous other plants (Patnaik and Debata, 1996; Chen,
2001; Sivanesan and Jeong, 2007; Sunil, 2009; Band et al., 2011).
4.1.2.2Effect of different growth regulators and additives on shoot proliferation
The regenerated micro-shoots derived from nodal segments were sub-cultured on shoot
proliferation medium fortified with different combinations and concentrations of growth
regulators in amalgamation with additives to improve the proliferation rate. The detail related
to the combination of hormones used and their effect on shoot proliferation and shoot length
is shown in Table 4.3 and Fig 4.3.
Shoot proliferation alongwith callusing was found to occur at the proximal cut end of
the nodal segments. Data showed that highest shoot proliferation was achieved on MS
medium containing 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads (36.6 shoots/explant)
followed by 2 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads (30.3 shoots/explant). But the
highest shoot length was found on MS media supplemented with 2 mg/l BAP + 0.5 mg/l
NAA + 1 mg/l GA3 (6.8 cm) followed by 2 mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (6.2
cm). The minimum number of shoot/explant and shoot length was found on MS medium
containing 6 mg/l BAP. From the results it was concluded that the no. of shoots/explant were
enhanced by the addition of adenine sulphate whereas shoot length was improved by the
addition of GA3 in MS medium.
CHAPTER – 4
RESULTS&DISCUSSION 84 | P a g e
Table 4.3 Effect of different combination of growth regulators on shoot proliferation
from the established shoot of G. glabra
TreatmentGrowth regulators (mg/l) Growth response
BAP IAA NAA GA3 Ads No. of shoots/explants Shoot length (cm)
SP 1 2 - 0.5 0.5 - 12.3ij 6.2ab
SP 2 4 - 0.5 0.5 - 17.0fg 5.6cd
SP 3 6 - 0.5 0.5 - 7.0mn 3.5fg
SP 4 2 0.5 - 0.5 - 9.9kl 4.9e
SP 5 4 0.5 - 0.5 - 10.3jk 4.0f
SP 6 6 0.5 - 0.5 - 5.3n 3.0ghi
SP 7 2 - 0.5 1 - 19.9de 6.8a
SP 8 4 - 0.5 1 - 26.4c 6.0bc
SP 9 6 - 0.5 1 - 14.0hi 3.9f
SP 10 2 0.5 - 1 - 9.7kl 5.3de
SP 11 4 0.5 - 1 - 15.1gh 4.9e
SP 12 6 0.5 - 1 - 8.0lm 3.5fg
SP 13 2 - 0.5 - 40 30.3b 4.0f
SP 14 4 - 0.5 - 40 36.6a 3.5fg
SP 15 6 - 0.5 - 40 17.9ef 4.0ijk
SP 16 2 0.5 - - 40 21.2d 3.5fg
SP 17 4 0.5 - - 40 29.2b 3.1gh
SP 18 6 0.5 - - 40 7.9 lm 2.2k
SP 19 2 - 0.5 - 60 10.7jk 3.5fg
SP 20 4 - 0.5 - 60 18.8ef 2.8hij
SP 21 6 - 0.5 - 60 5.8mn 2.2jk
SP 22 2 0.5 - - 60 9.5kl 3.0ghi
SP 23 4 0.5 - - 60 10.5jk 2.6hijk
SP 24 6 0.5 - - 60 5.5n 2.0k
G. Mean
S.E.
C.D. (5%)
14.96
0.79
2.26
3.86
0.23
0.63
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
CHAPTER – 4
RESULTS&DISCUSSION 85 | P a g e
Fig 4.3 Effect of growth regulators on shoot number and shoot length of G. glabra
grown under in vitro condition
Formation of multiple shoots along with considerable amount of callusing at the basal
cut ends of explants was also reported in Azadirachta indica (Arora et al., 2010). It may be
due to the action of accumulated auxins at the basal cut ends which stimulates cell
proliferation, especially in the presence of cytokinins (Marks and Simpson, 1994).
Proliferation of shoots were significantly influenced by the concentration of adenine sulphate
in the medium (Singh and Patel, 2014).These findings are also in close conformity with the
previous reports in Bougainvillea (El-Shamy, 2002), pomegranate (Singh and Khawale,
2006), Picrorhiza scrophulariiflora (Bantawa et al., 2009) and common bean (Gatica et al.,
2010). Adenine sulphate (cytokinin like activity) can boost cell growth and greatly enhanced
the shoot formation, as it is the additional source of nitrogen to the cell, which can be taken
up more readily than inorganic nitrogen (Harry and Thrope, 1994). The role of adenine
sulphate on shoot proliferation was found more effective when it combined with cytokinins
such as BAP (Staden et al., 2008).Addition of higher concentration of adenine sulphate in
the medium does not always enhance the growth of shoot. Due to the unbalance of
indigenous hormonal level in culture with the higher level adenine sulphate the declined trend
in shoot growth was also reported in Ziziphus spina-christi (Al-Sulaiman, 2010).
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
SP
1
SP
2
SP
3
SP
4
SP
5
SP
6
SP
7
SP
8
SP
9
SP
10
SP
11
SP
12
SP
13
SP
14
SP
15
SP
16
SP
17
SP
18
SP
19
SP
20
SP
21
SP
22
SP
23
SP
24
GR
OW
TH
RE
SPO
NSE
TREATMENTS
No. of shoots/explants Shoot length (cm)
CHAPTER – 4
RESULTS&DISCUSSION 86 | P a g e
Plate 2. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, IAA, NAA and AdSat various concentration (a). 2 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS (b). 4 mg/l BAP+ 0.5 mg/l IAA + 40 mg/l AdS (c). 2 mg/l BAP + 0.5 mg/l NAA + 40 mg/l AdS (d). 4 mg/lBAP + 0.5 mg/l NAA + 40 mg/l AdS (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 86 | P a g e
Plate 2. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, IAA, NAA and AdSat various concentration (a). 2 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS (b). 4 mg/l BAP+ 0.5 mg/l IAA + 40 mg/l AdS (c). 2 mg/l BAP + 0.5 mg/l NAA + 40 mg/l AdS (d). 4 mg/lBAP + 0.5 mg/l NAA + 40 mg/l AdS (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 86 | P a g e
Plate 2. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, IAA, NAA and AdSat various concentration (a). 2 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS (b). 4 mg/l BAP+ 0.5 mg/l IAA + 40 mg/l AdS (c). 2 mg/l BAP + 0.5 mg/l NAA + 40 mg/l AdS (d). 4 mg/lBAP + 0.5 mg/l NAA + 40 mg/l AdS (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 87 | P a g e
Plate 3. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, NAA and GA3 atvarious concentration (a). 2 mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (b). 4 mg/l BAP +0.5 mg/l NAA + 0.5 mg/l GA3 (c). 2 mg/l BAP + 0.5 mg/l NAA + 1 mg/l GA3 (d). 4 mg/lBAP + 0.5 mg/l NAA + 1 mg/l GA3 (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 87 | P a g e
Plate 3. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, NAA and GA3 atvarious concentration (a). 2 mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (b). 4 mg/l BAP +0.5 mg/l NAA + 0.5 mg/l GA3 (c). 2 mg/l BAP + 0.5 mg/l NAA + 1 mg/l GA3 (d). 4 mg/lBAP + 0.5 mg/l NAA + 1 mg/l GA3 (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 87 | P a g e
Plate 3. Shoot proliferation from the regenerated shoot of G. glabra when sub-culturedon MS media supplemented with phytohormone combination BAP, NAA and GA3 atvarious concentration (a). 2 mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (b). 4 mg/l BAP +0.5 mg/l NAA + 0.5 mg/l GA3 (c). 2 mg/l BAP + 0.5 mg/l NAA + 1 mg/l GA3 (d). 4 mg/lBAP + 0.5 mg/l NAA + 1 mg/l GA3 (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 88 | P a g e
Gibberellins (GAs) have been demonstrated to promote cell division and cell elongation
(Kende and Zeevaart, 1997). GA3, a well-known shoot growth active GA (Hedden and
Coker, 1992; Jones, 1973; Zeevaart et al., 1993), significantly stimulated shoot growth
(both elongation and number of new nodes) of Hancornia seedlings (Caldas et al., 2009).
GA3did not show any much difference in the mean number of shoots produce, but it was
observed that shoot elongation occurred when GA3 was included in the medium. The
presence of GA3 induced shoot elongation resulting in prominent nodal segments which can
be utilized for further multiplication during the subculture (Gonad et al., 2014). Similar
response of GA3 on shoot elongation was also reported in Ficus carica L. (Fraguas et al.,
2004) and Macadamia tetraphylla L. (Mulwa and Bhalla, 2015).
In contrast, BAP was found to be necessary for shoot multiplication. Increased
concentration of BAP increased the number of shoots produced attaining a maximum number
at 3 mg/l BAP but further increase in BAP concentration (5-10 mg/l) reduced the number of
shoots produced, showed necrosis and had shoot fasciation (Gonad et al., 2014). The reduced
number of shoots could be due to the inhibition of adventitious meristem elongation due to
the use of higher BAP concentration as stated by Brochetia et al. (2009).Reduction in the
number of shoots and shoot lengths were found at higher concentration of BAP in several
other medicinal plants (Kukreja et al., 1990; Sen and Sharma, 1991; Hosoki and
Katahira, 1994).
4.1.3Indirect organogenesis
4.1.3.1 Optimization of media for callus induction
Stem segments and leaves as an explant was inoculated onto MS medium supplemented with
different combination and concentration of phytohormones to optimize the callus induction
medium on the basis of callus induction rate, growth, type and quality is shown in Table 4.4.
The best callus was selected for the regeneration of plants.
The nature of explant and growth hormone plays an important role in callus induction.
Explants such as stems and leaves were used to produce callus. Explant undergoes de-
differentiation and formed callus which was an unorganised mass of tissue. Callus tissue is a
good source of genetic variability and adventitious shoot formation. Within 4 week of
CHAPTER – 4
RESULTS&DISCUSSION 89 | P a g e
culture, amorphous callus tissue was started proliferating from the cut edges and surface of
the explants. Callus was composed of parenchymatous cells differing in size and vacuolation.
Successful induction of callus was achieved with varying induction rate of both the explants.
Callus thus formed are green and organogenic but the leaf as an explant was found to be
better source for callus induction than that of the stem segment as the former produced
consistently higher percentage of callus than the latter (Fig 4.4).
Soft white fragile type of watery callus was induced on MS medium supplemented with
2,4-D alone whereas no callus induction was observed on MS medium supplemented with
BAP or NAA alone. MS medium containing BAP (1-2 mg/l) in combination of 2,4-D or
NAA produced green and compact type of callus whereas soft friable callus was produced in
MS medium containing BAP (1-2 mg/l) in combination of 2,4-D and NAA (0.5-1 mg/l).
Among the different concentrations and combinations of plant growth regulators tested, best
callus induction from both leaf and stem explants (97.32% and 89.49%) was achieved when
MS medium was supplemented with 2.0 mg/l BAP and 0.5 mg/l 2,4-D (Table 4.4).
Fig 4.4 Effect of growth regulators on callus induction through different explants (stem
and leaves) of G. glabra
0.00
20.00
40.00
60.00
80.00
100.00
120.00
CA
LL
US
IND
UC
TIO
N (
%)
TREATMENTS
Leaves Stem
CHAPTER – 4
RESULTS&DISCUSSION 90 | P a g e
Table 4.4 Effect of different combination of growth regulators on callus induction using
leaves and stem of G. glabra as an explant
Treatment Hormone
concentration
(mg/l)
Callus induction
(%)
Nature of callus
BAP 2,4D NAA Leaves Stem
CIM 1 0.0 0.5 0.0 65.32j 61.63i soft watery fragile
white callus
CIM2 1.0 0.5 0.0 91.67b 86.56b hard compact nodular
greenish yellow callusCIM3 2.0 0.5 0.0 97.32a 89.49a
CIM4 0.0 1.0 0.0 50.47m 41.92m soft watery fragile
white callus
CIM5 1.0 1.0 0.0 53.91k 45.71k hard compact nodular
greenish yellow callusCIM6 2.0 1.0 0.0 66.28i 53.33j
CIM7 0.0 0.0 0.5 0.00o 0.00o no callus
CIM8 1.0 0.0 0.5 71.39h 65.53h hard compact nodular
whitish green callusCIM9 2.0 0.0 0.5 82.50d 76.93d
CIM10 0.0 0.0 1.0 0.00o 0.00o no callus
CIM11 1.0 0.0 1.0 47.51n 38.49n hard compact light
green callusCIM12 2.0 0.0 1.0 52.15l 43.49l
CIM13 0.0 0.5 1.0 0.00 o 0.00o no callus
CIM14 1.0 0.5 1.0 79.63e 72.23e soft friable nodular
greenish callusCIM15 2.0 0.5 1.0 84.26c 79.72c
CIM16 0.0 1.0 0.5 0.00o 0.00o no callus
CIM17 1.0 1.0 0.5 77.31g 68.35g soft friable nodular
greenish callusCIM18 2.0 1.0 0.5 78.27f 70.99f
G. Mean
S.E.
C.D. (5%)
55.44
0.29
0.83
49.69
0.32
0.91
-
-
-
All the value are calculated as mean. Means followed by different alphabetwithin a column are significantlydifferent (p<0.05). For more information refer to annexure.
CHAPTER – 4
RESULTS&DISCUSSION 91 | P a g e
The production of callus at the cut edges of explant may be due to the wound caused
during the process of cutting which resulted in a synchronous cell division and considered as
a process of de-differentiation of organised tissue (Qin et al., 2005; Xing et al., 2010). The
success of in vitro clonal propagation largely relies on the selection of suitable explant which
can be used as the starting material for the experiment. Similar responses of explants for
callus induction were reported by earlier worker in in vitro micropropagation of Hybanthus
floribundus (Bidwell et al., 2001). Their data revealed that both leaf and stem were capable
of producing callus but the callus induction rate of leaf is much higher than that of stem. Leaf
explants were more suitable for in vitro micropropagation of Viola uliginosa (Slazak et al.,
2015).
Callus induction, growth and quality vary with the difference in explants, species,
cultivars and growth regulators. Callus (an unorganised mass of cell) formation is controlled
by regulating growth substances (auxins and cytokinin) in the medium (Aloni et al., 2006)
and their concentrations varies from species to species and even depends on explant source
(Charriere et al., 1999). The concentration of individual auxin and cytokinin or in
combination will determine the efficiency of callusability and organogenesis (Kohlenbach,
1977). The combination of auxin and cytokinin in the medium leads to rapid cell division
resulted into relatively large number of small undifferentiated cells (Hassani et al., 2008).
Auxins are known to involved in rapid cell division, elongation, vascular tissue
differentiation, rhizogenesis, embryogenesis and inhibition of axillary shoot growth whereas
cytokinin involved in promotion of cell division, expansion and plant growth (Chawla, 2002;
George et al., 2008; Park et al., 2010). Auxins seems to cause more methylation of DNA
than usual which is necessary for the reprogramming of differentiated cells to begin the
division while cytokinin seems to be required for the regulation of protein synthesis involved
in the formation and function of mitotic spindle apparatus (Chawla, 2002; George et al.,
2008).
2,4 D classified as an auxin plant hormone derivative, is used in plant cell cultures as a
dedifferentiation (callus induction) hormones (Endress, 1994). Assem et al. (2014) reported
that MS medium augmented with 2,4 D was most effective in inducing callus from sorghum.
Parsaeimehr and Mousavi (2009) found that none of the explants in Glycyrrhiza glabra
produce callus on MS medium with single BAP. The effectiveness of 2,4 D and in
combination with cytokinins in inducing callus might be due to their role in DNA synthesis
CHAPTER – 4
RESULTS&DISCUSSION 92 | P a g e
and mitosis (Skoog and Miller, 1957). The combination effect of 2,4-D and BAP played a
significant role as plant growth regulator and has a noticeable effect on callus induction
(Nikolaeva et al., 2009; Verma et al., 2012b). High concentration of BAP combined with
2,4-D is necessary for the callus induction whereas poor callus induction was recorded in the
low BAP concentration combined with 2,4 D. In Catharanthus roseus, higher level of BAP
showed greater prominent swelling of tissue in the proximal half of the midrib towards the
petiolar end (Verma and Mathur, 2011). The similar interaction effect of 2,4-D and BAP on
callus induction of other medicinal plants were reported on Tridax procumbens (Wani et al.,
2010), Falcria vulgaris (Hamideh et al., 2012), Clitoria ternatea (Zafar and Humayun,
2012) and Scrophularia striata (Lalabadi et al., 2013).
Although not significant but the addition of BAP and NAA to the medium also induces
callus to some degree as well. The combination of BAP and NAA proved to be effective in
initiating callus formation in other medicinal plant species (Sylvère Siéet al., 2010).
4.1.3.2 Effect of different additives on browning
Callus thus formed showed stunted growth and slowly changes to brown colour releasing
dark substances into medium due to necrosis. This may be due to the production of phenol-
like substances which either inhibit or slows down the growth of callus. Similar findings were
also reported by Wongwicha et al., 2008. Licorice cells produce enormous quantity of
flavonoids and polyphenols which is responsible for the browning of medium (Kovalenko
and Kurchii 1998). Browning is significantly reduced or eliminated by the addition of
different additives in the medium and also by repeated subculturing. Therefore, the MS
medium along with the combination of phytohormones showing best callus induction rate
(MS + 2mg/l BAP + 0.5 mg/l 2,4-D) was supplemented with different concentration of
ascorbic acid or activated charcoal(Table 4.5).
The explants in the control group (0 mg/l) showed browning after 12 - 15 days which
decreases the growth rate of callus. Ascorbic acid and activated charcoal not only inhibits the
browning of the callus but also enhance the biomass production; the most favourable level
was 50 and 70 mg/l respectively, which brought maximum inhibition in browning with higher
biomass (fresh wt.). In general, ascorbic acid turned out to be most effective in controlling
browning with higher rate of callus induction (92.55%) and biomass production (2812 mg).
CHAPTER – 4
RESULTS&DISCUSSION 93 | P a g e
The subsequent higher level of ascorbic acid and activated charcoal not only decreased the
degree of browning (30-40%) but at the same time suppressed the callus growth rate (%) and
biomass (mg).
Table 4.5 Effect of different additives on browning of callus regenerated using leaves
and stem of G. glabra on MS media fortified with 2 mg/l BAP and 2,4- D
Additives (mg/l) Browning (%) Callus induction (%) Fresh wt. (mg)
Control 0 98.17a 29.08e 462
Ascorbicacid
25 63.86b 79.57b 845
50 42.25de 92.55a 2812.0
75 39.96e 65.04c 1873.7
100 32.25f 53.00d 1056.7
Activatedcharcoal
25 59.70b 61.79c 706.0
50 51.69c 78.24b 939.3
75 47.31cd 82.15b 1156.0
100 31.14f 63.24c 839.3
G. Mean
S.E.
C.D. (5%)
51.81
0.19
0.55
67.18
0.18
0.55
-
-
-
All the value are calculated as mean. Means followed by different alphabetwithin a column are significantlydifferent (p<0.05). For more information refer to annexure.
Browning of excised plant tissues and nutrient media occurs frequently and remains a
major basis for recalcitrance in vitro. Tissue culture techniques for mass multiplication of
plant involve the excision of explants which elicits the production and release of phenolic
compounds which oxidize to form phytotoxic products (Bhojwani and Razdan, 1996;
Poudyal, 2008). This layer of polyphenol is formed around the damaged and wounded plant
part which prevents the entrance of pathogens. This defense mechanism of plant in turn
creates hindrance in the uptake of nutrients and growth of plant resulting in browning of
tissue and medium which eventually leads to the explant death (Vuylsteke, 1989; Strosse et
al., 2004; Chikenzie, 2012; Jones and Saxena, 2013).
CHAPTER – 4
RESULTS&DISCUSSION 94 | P a g e
Plate 4. Callus induction using leaf as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Curling of leaves (b). Swelling of leaves (c). Initiation of callus (d). Greenish yellowcallus (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 94 | P a g e
Plate 4. Callus induction using leaf as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Curling of leaves (b). Swelling of leaves (c). Initiation of callus (d). Greenish yellowcallus (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 94 | P a g e
Plate 4. Callus induction using leaf as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Curling of leaves (b). Swelling of leaves (c). Initiation of callus (d). Greenish yellowcallus (15 days)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 95 | P a g e
Plate 5. Callus induction using stem as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Swelling of stem (b). Initiation of callus (c). Greenish yellow callus (15 days)
a.
c.
b.
CHAPTER – 4
RESULTS&DISCUSSION 95 | P a g e
Plate 5. Callus induction using stem as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Swelling of stem (b). Initiation of callus (c). Greenish yellow callus (15 days)
a.
c.
b.
CHAPTER – 4
RESULTS&DISCUSSION 95 | P a g e
Plate 5. Callus induction using stem as an explant of G. glabra when inoculated on MSmedia supplemented with 2 mg/l BAP + 0.5 mg/l 2.4-D + 50 mg/l Ascorbic acid (a).Swelling of stem (b). Initiation of callus (c). Greenish yellow callus (15 days)
a.
c.
b.
CHAPTER – 4
RESULTS&DISCUSSION 96 | P a g e
Fig 4.5 Effect of additives on browning and callus induction of G. glabra
Browning of plant tissue accompanied by darkening of culture medium was due to the
accumulation and oxidation of phenolic compounds by oxidative enzyme (such as
polyphenols oxidase (PPO), phenylalanine ammonia lyase (PAL) and peroxidase) (Krishna
et al., 2008; Ahmad et al., 2013a) resulted in the formation of quinines which is highly
reactive and toxic to plant tissue (Chawla, 2002). Phenolic compounds have been shown to
be involved in providing resistance of some host plants to pathogens (Nicholson and
Hammerschmidt, 1992). Antioxidants and adsorbent added in tissue culture media affect the
growth, colour and texture of callus cultures as reported in earlier studies (Babaei et al.,
2013; Jones and Saxena, 2013).
Activated charcoal often used in tissue culture for the adsorption of inhibitory
substances in the culture medium which drastically decreases the phenolic oxidation and
brown exudates accumulation (Thomas, 2008). Activated charcoal is known to promote
morphogenesis by adsorbing the phenolic like toxic substances secreted by cultured tissues
(Pierik, 1987; George, 1996; Abdelwahd et al., 2008).Addition of ascorbic acid to MS
medium was found as one of the best methods to control browning of explants of Pyrus
bretschneideri (Poudyal et al., 2008). Ascorbic acid not only prevents the occurrence of
lethal browning in subsequently produced plantlets but also stop the progress of browning in
affected plantlets (Ko et al., 2009). Role of ascorbic acid in inhibiting oxidative browning,
0.00
20.00
40.00
60.00
80.00
100.00
120.00
Control Ascorbicacid (25)
Ascorbicacid (50)
Ascorbicacid (75)
Ascorbicacid
(100)
Activatedcharcoal
(25)
Activatedcharcoal
(50)
Activatedcharcoal
(75)
Activatedcharcoal
(100)
RE
SPO
NSE
(%
)
ADDITIVES (mg/l)
Browning (%) Callus induction (%)
CHAPTER – 4
RESULTS&DISCUSSION 97 | P a g e
promoting explant survival and stimulating growth of calli has been reported (Poleschuk and
Gorbatenko, 1995; Siddiqui and Farooq, 1996; Abdelwahd et al., 2008). Incorporation of
ascorbic acid or activated charcoal into the medium evoked better response in terms of callus
induction which may be due to its antioxidant activity that prevented the formation of
oxidative by products responsible for browning (Jain et al., 2008). Other studies have also
proved ascorbic acid and activated charcoal as an effective growth promoter and adsorbent to
control browning (Lars, 1983; Murata et al., 2001).
4.1.3.3Effect of different growth regulators on shoot regeneration
After 14 days on respective callus inducing medium, the calluses were cut into pieces and
transferred onto MS medium augmented with BAP (2.0, 4.0, 6.0, 8.0 and 10.0 mg/l) in
combination with IAA (0.2 and 0.5 mg/l) and Ascorbic acid (50 mg/l) for the indirect
organogenesis i.e., shoot regeneration from callus (Table 4.6and Fig 4.6)
The undifferentiated callus thus formed from explant, was capable of undergoing re-
differentiation and regeneration. Shoot regeneration was observed after 3 weeks of culture.
The highest frequency of regenerating callus (98.35%) and the maximum number of shoots
per callus (11.2) were achieved on MS medium supplemented with 4.0 mg/l BAP and 0.2
mg/l IAA whereas lowest regenerating frequency (28.14%) with minimum number of shoots
per callus (4.2) was found on MS medium supplemented with 8.0 mg/l BAP and 0.5 mg/l
IAA. But the highest shoot length (4.3 cm) was observed on MS medium supplemented with
2.0 mg/l BAP and 0.2 mg/l IAA (Table 4.6). No shoot regeneration takes place on MS
medium fortified with 10.0 mg/l BAP and 0.2 or 0.5 mg/l IAA. The adventitious shoots
produced with well-developed leaves were sub-cultured after every two weeks. Repeated sub-
culturing of shoots on to the shoot proliferation medium increases the number of shoot and
shoots length resulting into shoot multiplication.
It is well known that cytokinin stimulate plant cell division and participate in the
release of lateral bud dormancy and growth, adventitious bud formation and in controlling the
cell cycle whereas auxins exert a strong influence in initiation of cell division, meristem
organization, cell expansion, cell wall acidification, apical dominance, vascular
differentiation and root formation (Gasper et al., 1996, 2003).
CHAPTER – 4
RESULTS&DISCUSSION 98 | P a g e
Table 4.6 Effect of different combination of growth regulators on shoot regeneration
from the callus of G. glabra
Treatment
Hormoneconcentration
(mg/l)Regeneration
(%)No. of shoots
per callusShoot length
(cm)BAP IAA
SRM 1 2 0.2 87.68b 9.7b 4.33a
SRM 2 4 0.2 98.35a 11.2a 3.83ab
SRM 3 6 0.2 69.27c 6.9c 2.70c
SRM 4 8 0.2 32.53d 4.8d 3.20bc
SRM 5 10 0.2 0.00e 0.00e 0.00d
SRM 6 2 0.5 80.34b 7.3b 4.13a
SRM 7 4 0.5 92.74a 10.3a 4.00ab
SRM 8 6 0.5 61.43c 5.4c 3.50b
SRM 9 8 0.5 28.14d 4.2d 2.40c
SRM 10 10 0.5 0.00e 0.00e 0.00d
G. Mean
S.E.
C.D. (5%)
55.05
0.18
0.53
5.98
0.14
0.41
2.81
0.18
0.52All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Fig 4.6 Effect of growth regulators on shoot number and shoot length of in vitro
regenerated G. glabra
0.02.04.06.08.0
10.012.014.0
GR
OW
TH
RE
SPO
NSE
(%
)
TREATMENTS
No. of shoots/callus Shoot length (cm)
CHAPTER – 4
RESULTS&DISCUSSION 99 | P a g e
Plate 6. Shoot regeneration from the callus of G. glabra when inoculated on MS mediasupplemented with different phytohormone combination (a). Shoot initiation (4 mg/lBAP + 0.2 mg/l IAA + 50 mg/l Ascorbic acid) (b). Shoot elongation (4 mg/l BAP + 0.5mg/l IAA + 50 mg/l Ascorbic acid + 1 mg/l GA3) (c). Shoot proliferation (4 mg/l BAP +0.5 mg/l IAA + 1 mg/l GA3+ 50 mg/l Ascorbic acid) and (d). Shoot multiplication (4 mg/lBAP + 0.2 mg/l IAA +40 mg/l AdS + 50 mg/l Ascorbic acid)
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 100 | P a g e
Plate 7. Shoot proliferation from the regenerated shoot of G. glabra when inoculated onMS media supplemented with different phytohormone combination (a). 4 mg/l BAP +0.5 mg/l IAA + 50 mg/l Ascorbic acid (b). 4 mg/l BAP + 0.2 mg/l IAA + 50 mg/l Ascorbicacid (c). 4 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid and (d). 4mg/l BAP + 0.2 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 100 | P a g e
Plate 7. Shoot proliferation from the regenerated shoot of G. glabra when inoculated onMS media supplemented with different phytohormone combination (a). 4 mg/l BAP +0.5 mg/l IAA + 50 mg/l Ascorbic acid (b). 4 mg/l BAP + 0.2 mg/l IAA + 50 mg/l Ascorbicacid (c). 4 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid and (d). 4mg/l BAP + 0.2 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 100 | P a g e
Plate 7. Shoot proliferation from the regenerated shoot of G. glabra when inoculated onMS media supplemented with different phytohormone combination (a). 4 mg/l BAP +0.5 mg/l IAA + 50 mg/l Ascorbic acid (b). 4 mg/l BAP + 0.2 mg/l IAA + 50 mg/l Ascorbicacid (c). 4 mg/l BAP + 0.5 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid and (d). 4mg/l BAP + 0.2 mg/l IAA + 40 mg/l AdS + 50 mg/l Ascorbic acid
a.
c. d.
b.
CHAPTER – 4
RESULTS&DISCUSSION 101 | P a g e
Different aspects of cell growth, differentiation and organogenesis in organ cultures
have been found to be controlled by cytokinin and auxin interactions (Ammirato, 1983;
Rout and Das, 1997).Although cytokinin and auxin interactions are responsible for the plant
morphogenesis but their requisite concentration varies depending on the plant species and
need to be estimated accurately to achieve the effective rate of multiplication (Gomes et al.,
2010). Auxin – Cytokinin ratio governed the fate of the explants and was also responsible for
organogenesis (Skoog and Miller, 1957). Combination of auxin and cytokinin favoured
shoot bud differentiation in many other plants (Sudha et al., 2005; Rathore et al., 2005).
Similar advantageous effect on shoot proliferation of cytokinin and auxin addition to a
medium has also been reported for E. planum (Thiem et al., 2013).
From the present study it was observed that BAP proved to be the most effective plant
growth regulator for induction and proliferation of shoots in G. glabra. Similar finding was
also reported by Ahmad et al. (2013b). Our results confirmed the positive effect of BAP and
IAA on adventitious shoot formation of G. glabra. This results are in agreement with other
findings showing the synergetic and beneficial effect of BAP and IAA to induce indirect
organogenesis was reported in E. foetidum (Arockiasamy et al., 2002), Hypericum
perforatum L. (Wojcik and Podstolki, 2007), Adhatoda vasica (Dinesh and
Parameswaran, 2009), Dioscorea spp. (Felicia et al., 2012) and Eryngium maritimum L.
(Kikowska et al., 2014).
4.1.3.4 Effect of different growth regulators on rooting
The in vitro regenerated healthy shoots of direct or indirect organogenesis were excised from
the base and transferred onto the half strength MS medium supplemented with different
combination of IBA (1.0, 2.0 and 3.0 mg/l) and IAA (0.5 and 1.0 mg/l) and 50 mg/l Ascorbic
acid for the induction of root as shown in Table 4.7and Fig 4.7. The observations were
recorded in terms of rooting percentage, mean length of roots (cm), no. of roots and root
morphology.
Rooting was observed within 2 to 3 weeks of culture. Among the various combination
of hormones used, the highest rooting frequency (100%) was achieved on ½ MS medium
supplemented with 3.0 mg/l IBA or 3.0 mg/l IBA with 0.5 and 1.0 mg/l IAA also. But the
CHAPTER – 4
RESULTS&DISCUSSION 102 | P a g e
highest root length (4.23 cm) and no. of roots (11.00) was observed on ½ MS medium
supplemented with 2.0 mg/l IBA (Table 4.7).
Well rooted plantlets with atleast two roots were transplanted into sterile pots
containing sterile sand, soil and vermiculite (1:1:1) mixture, acclimatized in the culture room
and then transferred to green house after 30 days. The survival percentage of such plants was
90%.
Table 4.7 Effect of different growth regulators on rooting of in vitro regenerated shoot
of G. glabra
Treatment
Hormoneconcentration
(mg/l)Rooting
(%)Root length
(cm)Numberof roots
RootMorphology
IBA IAARIM 1 1 0 75.54c 3.10bc 2.50f
thin long
RIM 2 2 0 94.12b 4.23a 11.00athin long
RIM 3 3 0 100.00a 3.27b 5.50ethin long
RIM 4 0 0.5 53.28e 2.73cd 1.67gfragile long
RIM 5 0 1 62.03d 2.40d 1.17 gfragile long
RIM 6 1 0.5 75.19c 3.23b 5.33ethick long
RIM 7 2 0.5 96.47b 4.13a 9.17bthick long
RIM 8 3 0.5 100.00a 3.97a 7.67cthick long
RIM 9 1 1 78.40c 3.00bc 2.33fthick short
RIM 10 2 1 96.01b 4.20a 9.83bthick short
RIM 11 3 1 100.00a 3.80a 6.13dthick short
G. Mean
S.E.
C.D. (5%)
84.64
0.16
0.49
3.46
0.15
0.44
5.66
0.21
0.72
---
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
CHAPTER – 4
RESULTS&DISCUSSION 103 | P a g e
Plate 8 (a – d). Rooting of regenerated shoot of G. glabra when inoculated on MS mediasupplemented with phytohormone combination IBA and IAA (b). 3 mg/l IBA (c). 3 mg/lIBA + 0.5 mg/l IAA (d). 3 mg/l IBA + 1 mg/l IAA
c. d.
a. b.
CHAPTER – 4
RESULTS&DISCUSSION 104 | P a g e
Fig 4.7 Effect of different hormones on rooting of in vitro regenerated shoots of G.
glabra
Roots of many medicinal plants (approximately 60%) are used in medicine for drug
preparation. For the production of root drugs in the laboratory, development of fast growing
root culture system is essential as it offers unique opportunities (Murthy et al., 2008).
Application of auxins for micropropagated shoots may increase rooting percentage by
mounting the endogeneous contents of enzymes (Asghar et al., 2011). The stimulatory effect
of auxins in root initiation and multiplication had been reported previously in several other
plant species (Giridhar et al., 2001; Bhadra and Hossain, 2004; Nongdam and
Chongtham, 2012; Julkiflee et al., 2014). Liu et al. (2002) reported that auxin induced the
complicated process of lateral root formation through repetitive cell division. George et al.
(2008) suggested that auxins were essential for the maintenance of polarity of the plants.
IBA is known to plays an important role in the formation and development rooting.
Root formation and plant regeneration with IBA has been reported by Agastian et al. (2006)
and Naika and Krishna (2008). In this experiment, high concentration of IBA was effective
for root induction and root length. This result is in close conformity with that of Salehi et al.
(2014) in Carum copticum L. Many researchers have obtained similar results in some other
herbaceous plants such as Chlorophytum borivlianum (Purohit, 1994), Withania somnifera
(Rani, 1999), Catharanthus roseus (Dhandapani, 2008), Hypericum spectabile (Akbas et
al., 2011) and Altheae officinalis (Naz et al., 2015).
0.00
20.00
40.00
60.00
80.00
100.00
120.00
RO
OT
ING
(%
)
TREATMENTS
CHAPTER – 4
RESULTS&DISCUSSION 105 | P a g e
Plate 9. Hardenning and acclimatization of completely regenerated plantlets of G. glabra
in (a). bottles, (b). cups and (c). pots containing sterile sand, soil and vermiculite (1:1:1)
mixture
a.
b.
c.
CHAPTER – 4
RESULTS&DISCUSSION 106 | P a g e
4.2 Production of synthetic seed
4.2.1 Effect of different concentration of solutions on encapsulation
Nodal segment of in vitro regenerated plantlets were cut into small segments and transferred
into sodium alginate solution which were then dispensed into calcium chloride (CaCl2.2H2O)
solution to form alginate beads (synthetic seed containing nodal segment). Alginate beads
formation was influenced by the different concentrations and combinations of sodium
alginate (2, 4 and 6 %) and calcium chloride (50, 75, 100 and 125 mM) solutions (Table 4.8).
An optimal ion exchange between sodium ion (Na+) and calcium ion (Ca+) takes place
so that the beads must be sufficiently durable to resist manipulation upto planting. The
morphology of beads with respect to shape, texture, transparency and rigidity varied with
different concentrations of sodium alginate and calcium chloride solution. Formation of firm,
clear and iso-diameteric beads was achieved using 4% sodium alginate and 100mM
CaCl2.2H2O solution. Therefore these concentrations of sodium alginate and calcium chloride
were found to be the best combination for hydrogel complexion. Higher concentration of
sodium alginate and calcium chloride solution solutions leads to the formation of dark-colour,
hard and oval shaped beads resulted into late germination whereas lower concentration leads
to the formation of white, fragile and irregular beads which were difficult to handle.
Development of synthetic seed producing technology is currently considered as an
effective and efficient alternate method of mass propagation of elite plant species with high
medicinal value. In general synthetic seed is defined as artificially encapsulated somatic
embryos, shoot tips, axillary buds or any other meristematic tissue, used for sowing as a seeds
and possess the ability to convert into whole plant under in vitro and in vivo conditions and
keep its potentials after storage (Capuano et al., 1998). However, in vitro studies for mass
clonal propagation as well as in long term conservation of germplasm by using alginate
encapsulation techniques have been reported in many other plant species (Piccioni, 1997;
Pattnaik and Chand, 2000; Lata et al., 2009; Sharma et al., 2009; Mehrotra et al., 2012;
Islam and Bari, 2012).
CHAPTER – 4
RESULTS&DISCUSSION 107 | P a g e
Table 4.8 Effect of different concentrations and combinations of solutions on
encapsulation
Sodium
alginate (%)
Calcium
chloride (mM)
Formation of
beads (%)Bead Morphology
2 50 36.19c
white, fragile and irregular2 75 40.51c
2 100 59.20b
2 125 68.32a
4 50 53.56c clear, fragile and iso-diametric
4 75 69.17b
clear, firm and iso-diametric4 100 82.10a
4 125 85.70a clear, hard and iso-diametric
6 50 61.97c
dark,hard and oval with tailed end6 75 74.02b
6 100 87.55a
6 125 91.29a
G. Mean
S.E.
C.D. (5%)
67.46
1.31
3.82
-
-
-
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Lower concentration of sodium alginate and calcium chloride resulted in the formation
of soft and fragile beads that were difficult to handle, while at higher concentrations the beads
were iso-diametric but were hard enough to cause delay in sprouting (Lata et al., 2009).
Exposure of lower concentrations of sodium alginate to high temperature during autoclave
reduces its gelling ability (Larkin et al., 1998). The use of agar as a gel matrix was
deliberately avoided as it has been described inferior than alginate in terms of long storage
(Bapat et al., 1987). Sodium alginate was most suitable and accepted hydrogel for
immobilization of plant cell and was frequently used as a matrix for the production of
artificial seeds because of it is available in large quantities, inert, non-toxic, cheap, easily
handled and bio-compatibility characteristics (Endress, 1994; Ara et al., 2000; Saiprasad,
2001). This findings were in accordance with that of Kavyashree et al. (2006), Swamy et al.
CHAPTER – 4
RESULTS&DISCUSSION 108 | P a g e
(2009), Sundararaj et al. (2010) who reported that 4 % sodium alginate with100mM
calcium chloride was most suitable concentration for bead formation in Morus alba,
Pogostemon cablin and Zingiber officinale respectively whereas in most of the other cases
3% sodium alginate with 100 mM calcium chloride was proved to be ideal combination
(Tabassum et al., 2010; Mishra et al., 2011; Hung and Trueman, 2012). This variation
may be due to the different commercial source of chemicals.
4.2.2 Effect of different substrates on the conversion of encapsulated nodal segment into
plantlets
For the conversion of encapsulated nodal segments (synseed) into complete plantlets, the
encapsulated nodal segments were cultured on different medium. The effect of these
substrates on the shoot re-growth frequency was evaluated as shown in Table 4.9.
Encapsulated explants exhibited shoot development on each of the different planting
substrates at different rates. Encapsulated beads showed only 57.94 % sprouting when placed
on MS basal medium (control) for 4 weeks. Shoot development was induced within 2 weeks
on MS medium augmented with different phytohormone whereas this period was longer (5-6
weeks) in cotton. The maximum shoot re-growth frequency (82.39%) was observed on MS
medium augmented with 4 mg/l BAP+0.5 mg/l IAA+40 mg/l Ads. The minimum re-growth
frequency (19.27 %) was found on the usage of cotton as a substrate alongwith the emergence
of weak shoots which later failed to continue and died immediately.
The capability of these encapsulated explants to preserve their viability in terms of re-growth
and their conversion into complete plantlets after encapsulation is one of the most desirable
features (Micheli et al., 2007; Adriani et al., 2000). The results corroborates with the
findings of Rathore and Kheni (2017) who reported that maximum sprouting of
encapsulated nodal segment of Withania coagulans was achieved on MS medium
supplemented with BAP and IAA. Adenine sulphate alongwith BAP and IAA also proved to
be most efficient combination of phytohormone reported for the conversion of encapsulated
seed into complete plantlets (Hassan, 2003; Sharma and Shahzad, 2012). Ganapathi et al.
(2001) and Lata et al. (2009) also reported that the frequency of conversion of encapsulated
explant into complete plantlets was much lower and weaker in cotton than in MS medium.
CHAPTER – 4
RESULTS&DISCUSSION 109 | P a g e
Plate 10. Re-growth of plantlet using synthetic seed containing nodal segment ofregenerated plantlet of G. glabra (a). Synthetic seed (b). Inoculation of seed in MS media(c - d). Shoot initiation (MS basal media) (e - f). Shoot regeneration from seed using MSmedia supplemented with 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads and 4 mg/l BAP +0.5 mg/l IAA + 40 mg/l Ads respectively
a.
c. d.
b.
f.e.
CHAPTER – 4
RESULTS&DISCUSSION 109 | P a g e
Plate 10. Re-growth of plantlet using synthetic seed containing nodal segment ofregenerated plantlet of G. glabra (a). Synthetic seed (b). Inoculation of seed in MS media(c - d). Shoot initiation (MS basal media) (e - f). Shoot regeneration from seed using MSmedia supplemented with 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads and 4 mg/l BAP +0.5 mg/l IAA + 40 mg/l Ads respectively
a.
c. d.
b.
f.e.
CHAPTER – 4
RESULTS&DISCUSSION 109 | P a g e
Plate 10. Re-growth of plantlet using synthetic seed containing nodal segment ofregenerated plantlet of G. glabra (a). Synthetic seed (b). Inoculation of seed in MS media(c - d). Shoot initiation (MS basal media) (e - f). Shoot regeneration from seed using MSmedia supplemented with 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads and 4 mg/l BAP +0.5 mg/l IAA + 40 mg/l Ads respectively
a.
c. d.
b.
f.e.
CHAPTER – 4
RESULTS&DISCUSSION 110 | P a g e
Table 4.9 Effect of different substrate and storage period on the re-growth frequency of
encapsulated micro-shoots
Substrates Shoot re-growthfrequency (%)
Moist cotton 19.27g
MS basal medium 57.94f
MS+4mg/l BAP 68.83e
MS+4mg/l BAP+0.5 mg/l IAA 76.04b
MS+4mg/l BAP+0.5 mg/l NAA 71.25d
MS+4mg/l BAP+0.5 mg/l IAA+40 mg/l Ads 82.39a
MS+4mg/l BAP+0.5 mg/l NAA+40 mg/l Ads 74.56c
G. MeanS.E.
C.D. (5%)
64.330.290.87
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
4.3Elicitation of biomass and metabolite production
4.3.1 Effect of different concentrations of elicitors on biomass accumulation
After 15 days callus were sub-cultured on to the best callusing media (MS + 2 mg/l BAP +
0.5 mg/l 2,4-D) fortified with different elicitors (biotin, adenine sulphate, salicylic acid,
putrescine, spermine and spermidine) at different concentrations to optimize the
concentration of different elicitors on biomass accumulation. Biomass accumulation was
recorded at different concentrations (25, 50, 75 and 100 mg/l) for different time intervals (10,
20 and 30 days) as shown in Table 4.10.
Biomass accumulation in control was 3.15, 5.66 and 8.21 g/flask after 10, 20 and 30
days of incubation respectively. Elicitation with different elicitors increased the biomass in
callus culture of G. glabra at different rates. The implementation of elicitors has remarkable
effectson biomass accumulation of G. glabra callus culture. Out of different concentrations
studied, the optimum concentration of biotin (B), salicylic acid (SA) and spermidine (SD)
was 75 mg/l whereas that of adenine sulphate (AdS), putrescine (P) and spermine (SP) was
50 mg/l on the 20th day of incubation.
CHAPTER – 4
RESULTS & DISCUSSION 111 | P a g e
Table 4.10 Effect of different elicitors on biomass accumulation of in vitro grown callus of G. glabra on MS media supplemented with 2
mg/l BAP, 0.5 mg/l 2,4- D and 40 mg/l Ascorbic acid
Elicitors
concentration
(mg/l)
Biomass Accumulation (g/flask)
After 10 days After 20 days After 30 days
B AdS SA P SP SD B AdS SA P SP SD B AdS SA P SP SD
25 4.18c 4.89c 4.48c 6.99ab 4.15c 4.06b 7.52c 10.35d 7.94c 9.62c 6.19b 5.61b 5.28c 8.50c 5.59d 7.41b 5.14b 3.23b
50 6.36b 9.56a 5.57b 7.43a 6.52a 3.83b 8.72b 16.79a 9.37b 14.23a 6.87a 5.82ab 6.71b 12.37a 6.73b 10.61a 6.02b 3.36a
75 6.99a 6.35b 7.14a 6.41b 5.22b 4.95a 11.02a 13.91b 11.64a 10.74b 6.24b 6.37a 8.94a 9.92b 9.83a 7.18b 4.72a 4.34a
100 4.44c 5.13c 4.65c 5.24c 4.11c 3.10c 6.92d 11.04c 7.89c 8.03d 4.92c 4.57c 5.01c 8.28c 6.00c 6.00c 4.24c 2.23c
G. Mean
S.E.M.
C.D. (5%)
5.49
0.15
0.49
6.48
0.22
0.71
5.46
0.20
0.65
6.52
0.20
0.64
5.00
0.15
0.49
3.98
0.13
0.42
8.55
0.14
0.45
13.02
0.15
0.49
9.21
0.12
0.39
10.66
0.12
0.40
6.05
0.13
0.41
5.59
0.17
0.57
6.48
0.16
0.54
9.77
0.17
0.56
7.04
0.12
0.40
7.80
0.14
0.44
5.03
0.13
0.42
3.29
0.10
0.32
All the value are calculated as mean. Means followed by different alphabet within a column are significantly different (p<0.05). For more information refer to annexure.
CHAPTER – 4
RESULTS & DISCUSSION 112 | P a g e
Maximum biomass production was found in adenine sulphate (16.79 g/flask) elicitation
followed by putrescine (14.23 g/flask) at 50 mg/l. When compared to the control culture, a 2-
6 fold increase in biomass was achieved with elicitation. Increase in production by elicitors
varied from 1st day to the 30th day i.e., increased from 1st day of incubation, reached a
maximum on 20th day and then decreased. Growth period (20th day) produced the maximum
biomass accumulation.
It was observed that the change in biomass production induced by different elicitors
was dependent not only on their concentration but also on the incubation time. The biomass
accumulation was affected by high concentration of elicitors (75-100 mg/l) in the medium as
well as by prolonged incubation period (beyond 20 days) resulting into cell browning with
decreased viability and cell death. The results indicated that enhanced callus growth was
induced in the G. glabra when an optimum concentration of a suitable elicitor was added in
the callusing medium and incubated for a definite time period.
Elicitation is an effective strategy to enhance the metabolites production in low yielding
cell culture. Elicitation studies in callus culture using a variety of elicitors were reported to be
effective in enhancing the biomass accumulation and metabolite production (Brooks et al.,
1986; Sudha and Ravishanker, 2003; Parast et al., 2011; Ram et al., 2013). The age of
culture during the addition of elicitors is considered as an important parameter in the
production of biomass and secondary metabolites (Namdeo, 2007). The response of cells to
elicitors is dependent on the growth stage of the culture which indirectly affects the biomass
and secondary metabolite production (Deepthi and Satheeskumar, 2016). For elicitation,
the optimum age of the culture differs between various plant cell systems (Kang et al., 2009;
Ahmed and Baig, 2014).
Adenine in the form of adenine sulphate (AdS) can enhance the growth of the cell as
well as the proliferation of the shoot (Murashige, 1974). AdS was found to reinforce the
effect of other PGRs which may be due to the fact that it act as a precursor for cytokinn
synthesis and also enhance the biosynthesis of natural cytokinins (Bantawa et al., 2009).
They may act synergistically as a cytokinin and are therefore added in the culture media to
improve the growth as well as to strengthen the response normally attributed to cytokinin
action such as somatic embryogenesis and caulogenesis alongwith the proliferation of
axillary and adventitious shoots (Van Staden et al., 2008; Gatica et al., 2010). AdS was
CHAPTER – 4
RESULTS & DISCUSSION 113 | P a g e
found to be most effective in callus induction of Cichorium intybus (Nandagopal and
Ranjitha Kumari, 2006), Phoenix dactylifera (Sane et al., 2012), Rosa hybrid (Ram et al.
2015) and Dendrocalamus hamiltonii (Zhang et al., 2016). It was observed that in vitro
callogenesis, multiplication as well as biosynthetic activity of cultured cells can be enhanced
by optimizing medium components (Bhojwani and Razdan, 1996). Adenine sulphate boost
the cell growth as it is a nitrogen source which can be taken up more rapidly by the cell than
the inorganic nitrogen (Singh and Patel, 2014). Nitrogen being essential for plant, its
availability to plant tissue according to its source and concentration may affect different
physiological processes that control growth and morphogenesis rate (Ramage and Williams,
2002).
The exogenous supply of vitamins, in combination with other media constituents have
been reported to have direct and indirect effects on callus growth, somatic growth, rooting
and embryonic development (Abrahamian and Kantharajah, 2011). Biotin as a water
soluble B complex vitamin was a source of nitrogen found to be effective for callus induction
and growth (Al-Khayri, 2001; El-Shiaty et al., 2004). Biotin also plays an important role in
directing and transporting cytokinins necessary for plant growth and development as well as
for metabolism (Alban, 2000). Biotin promoted the callus induction and proliferation in
several other plant species such as Bryum coronatum (Kumra and Chopra, 1982), Capsicum
annuum (Kintzios et al., 2001), Curcuma mangga (Tamil et al., 2012) and Phoenix
dactylifera (Pervin et al., 2013; Diab, 2015).
Salicylic acid (SA) is widely used as a plant hormones (Hayat et al., 2007) in
regulating a wide range of plant’s physiological and metabolic processes thereby affecting
their growth and development (San Vicente and Plasencia, 2011; Yusuf et al., 2012; Hayat
et al., 2013; Pacheco et al., 2013; Khan et al., 2015). The effective concentration of SA
differs among species in the promotion of callus under normal condition (Arfan et al., 2007;
Al-oubaidi and Ameen, 2014). The stimulatory effect of SA on the production of callus and
plant development have been reported in C. officinalis L. (Bayat et al. 2012), Ziziphus spina
christii (Galal, 2012) and Vigna mungo (Lingakumar et al., 2014). This may be due to the
fact that SA alter the synthesis and signalling pathways of other plant hormones including
jasmonic acid, ethylene and auxin (Vlot et al., 2009).
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RESULTS & DISCUSSION 114 | P a g e
Polyamines (PAs) are small aliphatic amines (particularly, putrescine, spermine and
spermidine) present in all plant cells (Galston, 1983; Sawhney et al., 2003). They are
involved in stress reaction triggering cell division and differentiation (Bais and
Ravishankar, 2002; Amri et al., 2011; Liu et al., 2015). It may act as an endogenous plant
growth regulators or secondary hormonal messengers (Galston and Kaur-Swahney, 1987;
Gasper et al., 1996; Davis, 2004). Numerous reports were available showing a good
correlation between polyamine level and a variety of fundamental processes such as
macromolecular biosynthesis, cell division, cell and tissue differentiation, organogenesis and
somatic embryogenesis (Sawhney et al., 2003; Thiruvengadam et al., 2012; Arun et al.,
2014; Aydin et al., 2016). PAs have been shown to interact with other phytohormones
enhancing the production of callus in C. canephora (Kumar et al., 2008), M. charantia (Paul
et al., 2009; Thiruvengadam et al., 2012), P. gerardiana (Ravindra and Nataraja, 2013)
and Phoenix dactylifera (Ibrahim et al., 2014).
The efficiency of elicitors on cell growth and product yield varied depending on its
concentration, incubation time, genotypes and type of culture which may be attributed to the
difference among plant cell species, cell lines within species and cellular physiological state
(Zhao et al., 2005, 2010). The changes in product accumulation pattern with the incubation
time after the addition of elicitors have been reported in many studies (Karwasara et al.,
2010; Ahmed and Baig, 2014). Browning, retardation of growth and decrease in viability by
inhibiting cell division due to increased concentration of elicitors was observed in many plant
cultures (Lu et al., 2001; Saiman et al., 2014; Deepthi and Satheeshkumar, 2016). This
may be due to excessive availability of stress ion which induced osmotic imbalance with
reduced growth as reported in several other investigated plants (Shibli et al., 2007;
Elmaghrabi et al., 2013).
4.3.2 Screening of leaves, root and callus for the phytochemicals
The medicinal value of licorice lies in their bioactive components that produce a definite
physiological action in the treatment of various ailments. Phytochemical screening helps to
identify the presence and absence of these components in the methanolic extract of root and
callus which are responsible for their medicinal properties. Different conventional methods
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RESULTS & DISCUSSION 115 | P a g e
were followed to determine the presence of phytochemical constituent qualitatively and the
result of preliminary phytochemical analysis was summarized in Table 4.11.
Table 4.11Phytochemical screening of active constituents in various extract of plant
Phytoconstituents Test Leaves Root Callus
Carbohydrates Molisch test + + +
Proteins Biuret test + + +
Amino acids Ninhydrin test + + +
Phenols Ferric chloride test + + +
Alkaloids Mayer test + + +
Flavonoids Shinoda test + + +
Saponins Froth test + + +
Terpenoids Salkowski Test - + +
‘+’ denotes the presence and ‘-‘ denotes the absence of phytoconstituents
The results of preliminary phytochemical analysis for the methanolic extract of root and
callus indicated the presence of carbohydrates, proteins, amino acids, phenols, alkaloids,
flavonoids, saponins and terpenoids. Our findings are in consonance with the results obtained
by Meena et al. (2010). Flavonoids, saponins and sugars were found in the methanolic root
extract of Glycyrrhiza glabra whereas alkaloids, proteins and tannins were not detected
(Chopra et al., 2013). Ranganathan and Punniamurthy (2013) and Rekha and Paravathi
(2012) revealed the absence of saponin in the methanolic extract whereas Vashist et al.
(2013) showed the absence of alkaloid and phenols in the ethanolic extract of the plant.
Vijayalakshmi and Shourie (2013) identified one hundred and twenty six compounds
including flavonoids, terpenoids, saponins, essential oils, amino acids, and other nitrogen
containing compounds, hydrocarbons, fatty acid and their esters in the ethanolic extract of
licorice root. Husain et al. (2015) revealed the presence carbohydrates, phenols, flavonoids,
alkaloids, proteins, saponins, lipids, sterols and tannins in various solvent extract.
CHAPTER – 4
RESULTS & DISCUSSION 116 | P a g e
4.3.3 Effect of different elicitors on the production of different biochemical metabolites
The effect of different abiotic elicitors which were exogenously given to the callus, on the
variation of various phytoconstituent present in licorice was evaluated and summarized in
Table 4.12 and Table 4.13. The concentration at which elicitors (biotin, adenine sulphate,
salicylic acid, putrescine, spermine and spermidine) were showing highest biomass
accumulation was selected further for biochemical analysis. The time of incubation of callus
with different elicitors was of 15 days.
Elicitation with different elicitors seems to enhance the production of different
metabolites in G. glabra at different rates (Table 4.12 and Table 4.13). The abiotic elicitors
applied chemical stress to the callus culture triggering the production of different primary and
secondary metabolites at different rates that are normally not produced. Seven different
biochemical parameters viz. carbohydrates, proteins, proline, phenols, alkaloids, flavonoids
and glycyrrhizin were found to be higher in the callus culture as compared to the leaves and
roots of in vivo plants. This leads to the assumption that the in vitro raised plantlets have
higher content of metabolites than the in vivo plants. It was also observed that all the elicitors
were capable of enhancing the metabolites content at different concentration with varying
rates. The data revealed that the callus treated with elicitors had higher metabolite content
than the untreated callus (control).
The increase in the content of various biochemical parameters of in vitro regenerated
plants may be due to the effect of different phytohormones in in vitro raised plants
(Mohapatra et al., 2008). Yadav and Singh (2012) also observed significantly higher
chlorophyll, total sugars, reducing sugars and protein content of in vitro regenerated plants
than the natural plants of Glycyrrhiza glabra. The incorporation of increasing concentration
of biotic and abiotic elicitors in the medium resulted in higher carbohydrate, protein,
flavonoid and phenol accumulation than the in vivo and untreated in vitro raised plants of
Marsilea quadrifolia (Manjula and Mythili et al., 2012).
4.3.3.1 Effect of different elicitors on the production of primary metabolites
The variable concentrations of primary metabolites were observed in the elicitors treated and
untreated callus of studied plants (Table 4.12).Callus treated with different elicitors showed
CHAPTER – 4
RESULTS & DISCUSSION 117 | P a g e
higher primary metabolite content as compared to the untreated callus. Among the different
elicitors, adenine sulphate and putrescine were proved to be most effective in enhancing
primary metabolite content in callus culture of licorice. It was observed that the callus treated
with putrescine (50mg/l) showed the highest carbohydrates content (54.82 mg/g) followed by
adenine sulphate (50 mg/l) treated (49.71 mg/g) whereas highest protein (28.41 mg/g), phenol
(36.46 mg/g) and proline (0.082 µmol/g) content were found in adenine sulphate (50 mg/l)
treated callus. The least primary metabolite content was found in callus treated with
spermidine (75 mg/l). (Fig 4.7)
Table 4.12 Effect of different elicitors on the primary metabolites of in vitro grown
callus of G. glabra on MS media supplemented with 2 mg/l BAP, 0.5 mg/l 2,4- D and 50
mg/l Ascorbic acid and its comparison with field grown plant after 15 days
ElicitorsCarbohydrates
(mg/g)Protein(mg/g)
Phenol(mg/g)
Proline(µmol/g)
Leaves(field grown plant) 10.22i 4.45i 10.65i 0.019i
Root (field grown plant) 14.63h 7.66h 16.06h 0.022h
Callus (control) 19.74g 11.41g 20.53g 0.043g
Callus + Adenine sulphate (50 mg/l) 49.71b 28.41a 36.46a 0.082a
Callus + Biotin (75 mg/l) 38.79d 20.52d 28.44d 0.063d
Callus + Salicylic acid (75 mg/l) 45.99c 24.38c 33.35c 0.070c
Callus + Putrescine (50 mg/l) 54.82a 26.35b 34.10b 0.076b
Callus + Spermine (50 mg/l) 35.31e 18.07e 25.06e 0.057e
Callus + Spermidine (75 mg/l) 27.29f 15.81f 23.14f 0.051f
G. Mean
S.E.
C.D. (5%)
32.94
0.26
0.76
17.45
0.33
0.97
25.31
0.20
0.60
0.054
0.001
0.003
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
CHAPTER – 4
RESULTS & DISCUSSION 118 | P a g e
Fig 4.7 Effect of elicitors on primary metabolites of in vitro grown callus of G. glabra
and its comparison with field grown plant
4.3.3.2 Effect of different elicitors on the production of secondary metabolites
Elicitors not only enhanced the production of primary metabolites but also enhanced the
secondary metabolite in the callus culture of licorice (Table 4.13).All the elicitor treated and
untreated calluses were found to have higher secondary metabolites concentration than that of
root and leaves of field grown plant. Among the different elicitor tried, putrescine (50 mg/l)
showed highest alkaloid content (13.71 mg/g) whereas adenine sulphate (50 mg/l) showed
highest flavonoid content (16.29 mg/g). The least alkaloid and flavonoid content (4.69 and
7.12 mg/g) was found in spermidine treated callus (Fig.). Glycyrrhizin content was found to
be absent in the leaves of the field grown plant. The highest glycyrrhizin (35.44µg/g) content
was found in adenine sulphate treated callus whereas least (8.02 µg/g) was found in untreated
callus.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Leaves(fieldgrownplant)
Root (fieldgrownplant)
Callus(control)
Callus +Adeninesulphate
Callus +Biotin
Callus +Salicylic
acid
Callus +Putrescine
Callus +Spermine
Callus +Spermidine
CO
NC
EN
TR
AT
ION
ELICITORS
Carbohydrates (mg/g) Protein (mg/g) Phenol (mg/g)
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RESULTS & DISCUSSION 119 | P a g e
Table 4.13 Effect of different elicitors on the secondary metabolites of in vitro grown
callus of G. glabra on MS media supplemented with 2 mg/l BAP, 0.5 mg/l 2,4- D and 50
mg/l Ascorbic acid and its comparison with field grown plant after 15 days
ElicitorsAlkaloid(mg/g)
Flavonoid(mg/g)
Glycyrrhizin(µg/g)
Leaves (field grown plant) 0.73h 3.99h 0.00h
Root (field grown plant) 1.89g 6.66g 8.02g
Callus (control) 3.47f 7.01f 9.55f
Callus + Adenine sulphate (50 mg/l) 11.51b 16.29a 35.44a
Callus + Biotin (75 mg/l) 6.78d 10.60d 11.46d
Callus + Salicylic acid (75 mg/l) 9.02c 13.95c 12.69c
Callus + Putrescine (50 mg/l) 13.71a 15.03b 15.03b
Callus + Spermine (50 mg/l) 6.49d 8.50e 10.47e
Callus + Spermidine (75 mg/l) 4.69e 7.12f 8.11g
G. Mean
S.E.
C.D. (5%)
6.48
0.30
0.90
9.90
0.08
0.23
12.31
0.04
0.10
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Fig 4.8 Effect of elicitors on secondary metabolites of in vitro grown callus of G. glabra
and its comparison with field grown plant
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
Leaves(fieldgrownplant)
Root (fieldgrownplant)
Callus(control)
Callus +Adeninesulphate
Callus +Biotin
Callus +Salicylic
acid
Callus +Putrescine
Callus +Spermine
Callus +Spermidine
CO
NC
EN
TR
AT
ION
ELICITOR
Alkaloid (mg/g) Flavonoid (mg/g) Glycyrrhizin (µg/g)
CHAPTER – 4
RESULTS & DISCUSSION 120 | P a g e
Plate 11. HPLC chromatogram for glycyrrhizin interpretation in the methanolic extractof root and callus (a). Standard (Pure glycyrrhizin) (b). Root (in vivo) (c). Callus withoutelicitor treatment (d). Callus treated with adenine sulphate (e). Callus treated withbiotin
d.
e.
b.
c.
a.
CHAPTER – 4
RESULTS & DISCUSSION 121 | P a g e
Plate 12. HPLC chromatogram for glycyrrhizin interpretation in the methanolic extractof callus (a). Callus treated with salicylic acid (b). Callus treated with putrescine (c).Callus treated with spermine (d). Callus treated with spermidine
c.
b.
d.
a.
CHAPTER – 4
RESULTS & DISCUSSION 122 | P a g e
Adenine sulphate as nitrogen source can be taken up more rapidly by the cell than the
inorganic nitrogen (Singh and Patel, 2014). Chandler and Dodds (1983) noticed that
phosphorus, nitrogen and carbohydrates had influenced the in vitro production of secondary
products of Solanum laciniatum. Adenine sulphate (a nucleoside bases) provide assistance to
sulphate assimilation and amino acid biosynthesis necessary for the production of secondary
metabolites (Sharma et al., 2014). The mode of action of adenine has not been fully
explained; however, in many cultures it acts as a synergist of cytokinin, a substrate for the
cytokinin synthesis and could retard the degradation of cytokinins by feed-back inhibition, or
by competing for the enzymes involved in cytokinins metabolism (Van Staden et al., 2008).
Adenine sulphate was found to enhance the different metabolite contents in several other
plant species such as Vaccinium myrtillus L. (Bolda et al., 2011), Ruta gravoelens L.
(Mohamed and Ibrahim, 2011),Merwilla plumbea (Baskaran et al., 2012), Swertia
chirayita (Kumar et al., 2014), Centellaasiatica L. (Sharma et al., 2014), Tectona grandis
L. (Akram and Aftab, 2015), Withania somnifera L. (Sivanandhan et al., 2015).
In tissue culture, growth media was supplemented with arbitrarily selected vitamins at
variable concentrations (thiamine, nicotinic acid, pyridoxine, pantothenate, folic acid and
biotin) (Gamborg and Shyluk, 1981; Omar et al., 1992). Vitamin as a cofactor of several
enzymes considered essential for the metabolism of proteins, fats and carbohydrates
(Hildebrand, 2005). Biotin, water soluble B complex vitamin, was a source of nitrogen and
plays an important role in directing and transporting cytokinins necessary for plant growth
and development as well as for metabolism (Alban, 2000). Biotin, a heterocyclic compound,
acts as a cofactor for a small number of enzymes involved in several reactions (carboxylation,
decarboxylation, transcarboxylation and transamination) concerned with the fatty acid and
carbohydrates metabolism and protein synthesis (Knowles, 1989; Alban et al., 2000).Till
date, no reports are available on metabolite production from callus culture treated with biotin.
Recent studies suggest that SA can modulate the physiological as well as biochemical
function of plant such as Cistus heterophyllus (Orenes et al., 2013). SA enhances both the
primary as well as secondary metabolite in plant (Babel et al., 2014). It plays an important
role in systemic acquired resistance to pathogens and is able to induce pathogen resistance
protein (George et al., 2008).SA potentially alters the metabolic pathways leading to the
accumulation of phytoconstituents during in vitro culture have been reported (Sudha and
CHAPTER – 4
RESULTS & DISCUSSION 123 | P a g e
Ravishankar, 2003; Ram et al., 2013). This may be due to the fact that SA alter the
synthesis and signalling pathways of other plant hormones including jasmonic acid, ethylene
and auxins (Vlot et al., 2009). Salicylic acid was found to enhance the different metabolite
contents in several other plant species such as Zingiber officinale (Ghasemzadeh and
Jaafar, 2012), Jatropha curcas L. (Mahalakshmi et al., 2013), Hypericum perforatum
(Gadzovska et al., 2013), Conium maculatum L. (Meier et al., 2015), Andrographis
paniculata L. (Zaheer and Giri, 2015), Bacopa monnieri (Largia et al., 2015), Achillea
millefolium L. (Gorni and Pacheco, 2016).
Polyamines (PAs) act as an endogenous plant growth regulators or secondary hormonal
messengers (Galston and Kaur Swahney, 1987; Gasper et al., 1996; Davis, 2004). PAs are
also known to involve in several macromolecular biosynthesis (Galston and Kaur Swahney,
1995; Sawhney et al., 2003). They plays an important role in secondary metabolism which
may be caused by their influence on membrane transport due to the regulation of proton
pumps (Garufi et al., 2007; Janicka-Russak et al., 2010). PAs have been shown to interact
with other phytohormones enhancing the production of secondary metabolites in several
other plant species such as Glycine max (Shetty et al., 1989), Beta vulgaris and Tagetes
patula (Bais et al., 2000), Cichorium intybus L. (Bais and Ravishankar, 2003), Psoralea
corylifolia L. (Shinde et al., 2009), Nepeta cataria L. (Yang et al., 2010), Panax ginseng
(Marsik et al., 2014).
4.3.4 Effect of different elicitors on the antioxidant enzyme activity
To obtain some insights of the cellular antioxidant responses caused by elicitation, the
activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD)
were evaluated. The antioxidant enzyme activities of callus increased rapidly after elicitation
(Table 4.14). The enzyme activities of calluses treated with elicitors were found to be higher
than that of leaves, root and control. Superoxide dismutase (1.382 unit/mg protein) and
ascorbate peroxidase (0.531 unit/mg protein) activity was maximum in callus treated with
adenine sulphate while peroxidase activity (0.733 unit/mg protein) was highest in putrescine
treated.
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RESULTS & DISCUSSION 124 | P a g e
Table 4.14 Effect of different elicitors on the antioxidant enzyme activity of in vitro
grown callus of G. glabra on MS media supplemented with 2 mg/l BAP, 0.5 mg/l 2,4- D
and 50 mg/l Ascorbic acid and its comparison with field grown plant after 15 days
Elicitors SOD(unit/ mg protein)
APX(unit/ mg protein)
POD(unit/ mg protein)
Leaves (field grownplant)
0.913h 0.186h 0.337g
Root (field grown plant) 1.169g 0.338g 0.581f
Callus (control) 1.232f 0.360f 0.604e
Callus + Adenine sulphate(50 mg/l)
1.382a 0.531a 0.727a
Callus + Biotin (75 mg/l) 1.305c 0.434c 0.679c
Callus + Salicylic acid(75 mg/l)
1.316bc 0.453b 0.714b
Callus + Putrescine (50mg/l)
1.323b 0.458b 0.733a
Callus + Spermine (50mg/l)
1.286d 0.415d 0.673c
Callus + Spermidine (75mg/l)
1.265e 0.379e 0.657d
G.Mean
S.E.
C.D. (5%)
1.24
0.005
0.02
0.40
0.003
0.10
0.64
0.004
0.01
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Fig 4.9 Effect of elicitors on antioxidant enzyme activity of in vitro grown callus of G.
glabra and its comparison with field grown plant
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Leaves(fieldgrownplant)
Root (fieldgrownplant)
Callus(control)
Callus +Adeninesulphate
Callus +Biotin
Callus +Salicylic
acid
Callus +Putrescine
Callus +Spermine
Callus +Spermidine
CO
NC
EN
TR
AT
ION
(Uni
t/m
g pr
otei
n)
ELICITOR
SOD APX POD
CHAPTER – 4
RESULTS & DISCUSSION 125 | P a g e
In the presence of elicitors, there is an immediate cellular response to trigger plant
defense signals with increased accumulation of reactive oxygen species (ROS) such as
hydrogen peroxide (H2O2), superoxide anions (O2-) and hydroxyl free radicals (HO-).
Different cell compartments may activate different defensive systems to reduce ROS excess,
using antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate
peroxidase (APX), peroxidases (POD), and glutathione reductase (GR) (Martinez et al.,
2016). The main sources of ROS production are chloroplast and peroxisomes in the light and
mitochondria in the dark (Foyer and Noctor, 2005). Oxidative stress is defined as a serious
imbalance between ROS production and the antioxidant defenses. This situation can cause
cellular damage and an increase in the secondary metabolites (Zhao et al., 2005). Hence, it
has been also reported that application of elicitor induced the ROS burst in dark-grown
cultured cells of Parsley (Kauss et al., 1994) and Taxus chinensis (Wang and Wu, 2005), as
well as in protoplasts of Arabidopsis (Sasaki-Sekimoto et al., 2005). Therefore, H2O2 may
function as a signal activating defense genes and, as part of the coordinate antioxidant
response, could enhance phytoalexin production (Ramos-Valdivia et al., 2012).
During the reactive oxygen species (ROS) detoxification process, the primary reaction
was catalyzed by the SOD which provide defense against the toxic effects of ROS (Ali et al.,
2006). Evidences showed that abiotic elicitor increased the SOD, CAT, APX and POD
activities (Martinez et al., 2016). SOD is the first line to scavenging off toxic O2− level, and
other antioxidant enzymes such as CAT, POD, APX and GR convert H2O2 to water and
molecular oxygen, and prevent the cellular damage under unfavorable condition (Chaitanya
et al., 2002; Scandalios, 2005). During elicitation, increased SOD activity under various
stresses was observed in several investigated plant genera (Samar et al. 2011). The CAT,
POD and APX activity also showed similar pattern with added levels of elicitors, although
tissue and dose specific variation was also not uncommon (Elkahoui et al. 2005).
SA induces the development of systemic acquired resistance (SAR) by reactive oxygen
species (ROS) production (Kawano and Muto, 2000). Subsequently, SA changes catalase
(CAT) and peroxidase (POX) activities (increases or reduces depending on
H2O2concentration) (Guan and Scandalios, 2006). CAT and POX are known as a defensive
team, targeted at protecting cells from oxidative damage (Mittler, 2002). Salicylic acid was
reported to enhance the antioxidant enzyme activity in many other plant species (Agarwal et
al., 2005; Ali et al., 2006; Ghasemzadeh and Jaafar, 2013; Gholamnezhad et al., 2016;
Rehman et al., 2017). Adenine sulphate was also reported to elicit different antioxidant
CHAPTER – 4
RESULTS & DISCUSSION 126 | P a g e
enzyme activities in other plant species (Misra and Kochhar, 2008; Jana and Shekhawat,
2012; Sharma et al., 2016; Ahmad et al., 2017). Polyamines was effective in improving
both enzymatic (Radhakrishnan and Lee, 2013) and non-enzymatic (Asthir et al., 2012)
antioxidants activities. Abundant studies have emphasized interaction between PAs and the
ROS when plants are under stress (Gill and Tuteja, 2010; Velarde-Buendia et al., 2012;
Pottosin et al., 2014).
4.4 Antimicrobial assay
The biological evaluation of different solvent (aqueous, acetone, ethanol and methanol)
extract of the leaves and root of G. glabra was carried out and the extracts were screened for
its antimicrobial activity against three bacteria (Bacillus subtilis, Streptococcus mutans and
Proteus vulgaris) and two fungi (Candida albicans and Aspergillus niger). The anti-microbial
activities and their potency were assessed by determining the inhibition zone diameter.
Screening of antimicrobial activity of different plant parts (leaves and root) against both
bacteria and fungi were shown in Table 4.15 and Table 4.16.
The analysis showed positive inhibitory activity against both microbes in all the
different solvent extracts of leaves and root. The acetone extract showed significantly higher
activity as compared to other extract followed by ethanol, methanol and aqueous. Similarly
root of G. glabra was more effective against microbes than that of leaves. The extracts were
most potent against S. mutans amongst bacteria and showed maximum potency against C.
albicans amongst fungi. The data revealed that the acetone extract of root exhibited highest
zone of inhibition against S. mutans (23.7 mm) while aqueous extract of leaves showed least
zone of inhibition against P. vulgaris (3 mm). In some cases the microbial activity of acetone
and ethanol extract was higher than that of standard.
Licorice used in traditional Chinese medicine have more than 20 triterpenoids and
nearly 300 flavonoids which possess many pharmacological activities such as anti-viral, anti-
microbial, anti-inflammatory, anti-tumour and other activities (Adianti et al., 2014;
Chandrasekaran et al., 2011; Choi et al., 2014; Ahn et al., 2012; Bordbar et al., 2013).
The potential of G. glabra in therapeutic effects as an antimicrobial agent is documented
(Gupta et al., 2008). This antimicrobial activity is due to the phytoconstituent such as
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RESULTS & DISCUSSION 127 | P a g e
alkaloid, saponins, flavonoids, glycosides and phenols found in the licorice. These
phytochemical groups are known to possess anti-microbial compounds (Meghasri and
Shubha, 2009). Among them glycyrrhizin, 18 β-glycyrrhetinic acid, liquiritigenin,
licochalcone A, licochalcone E and glabridin are the main active component which possess
anti-microbial activities (Wang et al., 2015).
Table 4.15 Anti-bacterial activity of root and leaves in different solvent extract of G.
glabra
Microorganism→
Extract ↓
Zone of Inhibition (mm)
B. subtilis S. mutans P.vulgaris
AqueousRoot 7.7e 10.0e 6.7e
Leaves 5.0f 4.0f 3.0f
AcetoneRoot 21.7a 23.7a 17.0a
Leaves 13.3d 16.0c 13.7bc
EthanolRoot 16.7c 18.7b 15.0ab
Leaves 9.0e 12.0d 12.3c
MethanolRoot 12.3d 15.3c 9.7d
Leaves 7.7e 11.0de 7.3e
Streptomycin (standard) 18.7b 11.3de 12.7c
S.E.
C.D. (5%)
12.44
0.59
1.75
13.56
0.65
1.93
10.815
0.745
2.215
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
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RESULTS & DISCUSSION 128 | P a g e
Table 4.16 Anti-fungal activity of root and leaves in different solvent extract of G. glabra
Microorganism→
Extract ↓
Zone of Inhibition (mm)
C. albicans A. niger
AqueousRoot 5.3e 0.0e
Leaves 2.0f 0.0e
AcetoneRoot 18.7a 13.7a
Leaves 13.3b 7.7c
EthanolRoot 15.0b 11.0b
Leaves 10.0cd 4.3d
MethanolRoot 11.3c 0.0e
Leaves 9.3d 0.0e
Bavistin (standard) 14.7b 10.7b
G. Mean
S.E.
C.D. (5%)
11.07
0.67
1.98
5.26
0.48
1.44
All the value are calculated as mean. Means followed by different alphabet within a column are significantlydifferent (p<0.05). For more information refer to annexure.
Increasing antibiotic resistance has resulted in an urgent need for alternative therapies
to treat diseases. In recent years, many studies have shown that different licorice extract (Al-
Turki et al., 2008; Park et al., 2008)have potent effects in inhibiting the activities of both
Gram-positive and Gram-negative bacteria such as Staphylococcus aureus (Long et al.,
2013), Escherichia coli (Awandkar et al., 2012), Pseudomonas aeruginosa (Yoshida et al.,
2010), Bacillus subtilis (Irani et al., 2010). These extracts are also being considered as
potential alternatives to synthetic fungicides (Irani et al., 2010). Based on the above
inhibitory activities, licorice may serve as an alternative therapy for treating dental caries,
periodontal disease, digestive anabrosis and tuberculosis (Wang et al., 2015).
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RESULTS & DISCUSSION 129 | P a g e
Plate 13. Anti-bacterial activity of root and leaves extract of G. glabra in differentsolvent [Aqueous (Aq), acetone (Ac), ethanol (Et) and methanol (Mt)] against differentbacterial strain (a). Bacillus subtilis (b). Proteus vulgaris and (c). Streptococcus mutans
Streptococcus mutans
Mt
Mt
AqAc
Et
Aq
EtAc
Leaves extract
Mt
MtAq
Ac
Et
Aq
Et
Ac
Proteus vulgaris
Mt
Mt
Aq
Ac
Et
AqEt
Ac
Bacillus subtilis
Root extract
Root extract
Root extract
Leaves extract
Leaves extract
a.
b.
c.
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RESULTS & DISCUSSION 130 | P a g e
Plate 14. Anti-fungal activity of root and leaves extract of G. glabra in different solvent[Aqueous (Aq), acetone (Ac), ethanol (Et) and methanol (Mt)] against different fungalstrain (a). Candida albican and (b). Aspergillus niger
Candida albican
Et
AcMt
Aq
Aq
Mt
Ac
Et
Aspergillus niger
Et
Ac
Mt
Aq
Aq
Mt
Ac
Et
Leaves extractRoot extract
Root extract Leaves extract
a.
b.
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RESULTS & DISCUSSION 131 | P a g e
Plate 15. Anti-bacterialand anti-fungal activity of standard (Streptomycin and Bavistin)against different bacterial and fungal strain respectively (a). Bacillus subtilis (b). Proteusvulgaris (c). Streptococcus mutans (d). Candida albican and (e). Aspergillus niger
Bacillus subtilis Proteus vulgaris
Streptococcus mutans Candida albican
Aspergillus niger
c. d.
e.
b.a.
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SUMMARY & CONCLUSION 132 | P a g e
SUMMARY & CONCLUSION
The salient findings of this present investigation entitled “In vitro studies on the variations
of biochemical metabolites in Glycyrrhiza glabra L. by using various elicitors” are
summarized below:-
1) In vitro study:-
a) Sterilization: The explants (i.e., nodal segments and leaves) procured from field
grown plants of licorice was kept under running tap water for 30 mins and then
washed with 0.1 % Bavistin (2-3 mins) and Tween 20 (2-3 drops) for 5 mins. To
ensure the complete sterilization, the explant was again treated with 70% ethanol (30
sec) followed by 0.1% HgCl2 (4 mins) and showed highest percentage of survival
(62.53%) with the lowest contamination percentage (26.61%).
b) Shoot initiation: MS media supplemented with BAP (4 mg/l) + IAA (0.5 mg/l) was
found to be most effective as this concentration favoured early sprouting (7 days) with
maximum shoot emergence (92.03%).
c) Shoot proliferation: The highest shoot proliferation was achieved on MS medium
containing 4 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads (36 shoots/explant) followed
by 2 mg/l BAP + 0.5 mg/l NAA + 40 mg/l Ads (30 shoots/explant). But the highest
shoot length was found on MS media supplemented with 2mg/l BAP + 0.5 mg/l NAA
+ 1 mg/l GA3 (6.8 cm) followed by 2mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l GA3 (6.2
cm).
d) Callus induction: Leaf as an explant was found to be the better source for callus
induction than that of the stem segment based on the percentage response. Best callus
induction from both leaf and stem explants (97.32% and 89.49%) was achieved when
MS medium was supplemented with 2.0 mg/l BAP and 0.5 mg/l 2,4-D.
e) Browning: Ascorbic acid (50 mg/l) turned out to be most effective in controlling
browning with higher rate of callus induction (92.55%) and biomass production (2812
mg).
f) Shoot regeneration: The highest frequency of regenerating callus (98.35%) and the
maximum number of shoots per callus (11.2) were achieved on MS medium
supplemented with 4.0 mg/l BAP and 0.2 mg/l IAA. But the highest shoot length
(4.33 cm) was observed on MS medium supplemented with 2.0 mg/l BAP and 0.2
mg/l IAA.
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SUMMARY & CONCLUSION 133 | P a g e
g) Rooting: The highest rooting frequency (100 %) was achieved on ½ MS medium
supplemented with 3.0 mg/l IBA or 3.0 mg/l IBA with 0.5 and 1.0 mg/l IAA also. But
the highest root length (4.23 cm) was observed on ½ MS medium supplemented with
2.0 mg/l IBA.
h) Hardening: Well rooted plantlets with atleast two roots were transplanted into sterile
pots containing sterile sand, soil and vermiculite (1:1:1) mixture, acclimatized in the
culture room and then transferred to green house after 30 days. The survival
percentage of such plants was 90%.
2) Artificial seed production:-
a) Encapsulation: Formation of firm, clear and iso-diameteric beads through
encapsulation was achieved using 4% sodium alginate and 100 mM CaCl2.2H2O
solution.
b) Shoot re-growth: The maximum shoot re-growth frequency of encapsulated seed
(82.39%) was observed on MS medium augmented with 4 mg/l BAP + 0.5 mg/l IAA
+ 40 mg/l Ads.
3) Elicitation:-
a) Biomass accumulation: The optimum concentration of biotin, salicylic acid and
spermidine was 75 mg/l whereas that of adenine sulphate, putrescine and spermine
was 50 mg/l on the 20th day of incubation. Maximum biomass production was found
in adenine sulphate (16.79 g/flask) elicitation followed by putrescine (14.23 g/flask)
at 50 mg/l.
b) Phytochemical screening: The results of preliminary phytochemical analysis for the
methanolic extract of leaves, root and callus indicated the presence of carbohydrates,
proteins, amino acids, phenols, alkaloids, flavonoids, saponins and terpenoids.
c) Primary metabolites: Primary metabolites were found to be higher in the callus
culture as compared to the leaves and roots of in vivo plants. Callus treated with
putrescine (50 mg/l) showed the highest total sugar content (54.82 mg/g) whereas
highest protein (28.41 mg/g), phenol (36.46 mg/g) and proline (0.082 µmol/g) content
were found in adenine sulphate (50 mg/l) treated callus. The least primary metabolite
content was found in callus treated with spermidine (75 mg/l).
d) Secondary metabolites: Secondary metabolites were found to be higher in the callus
culture as compared to the leaves and roots of in vivo plants. Putrescine (50 mg/l)
CHAPTER – 5
SUMMARY & CONCLUSION 134 | P a g e
showed highest alkaloid content (13.71 mg/g) whereas adenine sulphate (50 mg/l)
showed highest flavonoid content (16.29 mg/g). The least alkaloid and flavonoid
content (4.69 and 7.12 mg/g) was found in spermidine treated callus. Glycyrrhizin
content was found to be absent in the leaves of the field grown plant. The highest
glycyrrhizin (35.44 µg/g) content was found in adenine sulphate treated callus
whereas least (8.02 µg/g) was found in untreated callus.
e) Enzyme activity: The enzyme activities of calluses treated with elicitors were found
to be higher than that of leaves, root and control. Superoxide dismutase (1.382
unit/mg) and ascorbate peroxidase (0.531 unit/mg) activity was maximum in callus
treated with adenine sulphate while peroxidase activity (0.733 unit/mg) was highest in
putrescine treated.
4) Anti-microbial assay:-
a) Anti-bacterial activity: The acetone extract of root exhibited highest zone of
inhibition against B. subtilis (21.7 mm), S. mutans (23.7 mm) and P.vulgaris (17.0
mm) while aqueous extract of leaves showed least zone of inhibition. In some cases
the anti-bacterial activity of acetone and ethanol extract was higher than that of the
standard (Streptomycin).
b) Anti-fungal activity: The acetone extract of root exhibited highest zone of inhibition
against C. albicans (18.7 mm) and A. niger (13.7 mm) followed by ethanol extract
against C. albicans (15.0 mm) and A. niger (11.0 mm). The zone of inhibition
exhibited by acetone and ethanol extract was higher than that of the standard
(Bavistin).
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SUMMARY & CONCLUSION 135 | P a g e
Conclusion
The present investigation concluded that the efficient and highly reliable protocol has been
developed provides rapid and large scale multiplication of G. glabra. A practicable protocol
has been optimized for synthetic seed formation in G. glabra using nodal segment and with
subsequent re-growth on various planting media. This research study on micropropagation of
G. glabra is beneficial for future work on protoplast culture, somaclonal variation, genetic
transformation and secondary metabolites production. Collection of wild material for in vitro
studies and propagation opens fresh avenues towards conservation and resource management.
Application of abiotic elicitors were found to be effective in elicitating callus biomass at
certain concentration. Accumulation of biochemical metabolite and enzyme activity was also
influenced by the elicitors bringing about its enhancement and variation. Elicitation of higher
levels of the bioactive constituents of licorice plants is a step towards the development of
novel drugs which can be used in the field of therapeutics to treat various ailments and
improvement in the productivity of desired compounds, to overcome the problems related
with chemical synthesis. The potential of G. glabra as an anti-microbial agent is documented
which may be serve as an alternative therapy for treating several ailments.
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ANNEXURE xiv | P a g e
ANNEXURE
ANOVA table 1: Effect of different combination of growth regulators on shoot establishment (on
the basis of data obtained from table 4.2)
ANNEXURE xv | P a g e
ANOVA table 2: Effect of different combination of growth regulators on shoot/explant (on the
basis of data obtained from table 4.3)
ANNEXURE xvi | P a g e
ANOVA table 3: Effect of different combination of growth regulators on shoot length (on the basis
of data obtained from table 4.3)
ANNEXURE xvii | P a g e
ANOVA table 4: Effect of different combination of growth regulators on callus induction using
leaves as an explant (on the basis of data obtained from table 4.4)
ANNEXURE xviii | P a g e
ANOVA table 5: Effect of different combination of growth regulators on callus induction using
stem as an explant (on the basis of data obtained from table 4.4)
ANNEXURE xix | P a g e
ANOVA table 6: Effect of different additives on browning (on the basis of data obtained from table
4.5)
ANNEXURE xx | P a g e
ANOVA table 7: Effect of different additives on callus induction (on the basis of data obtained
from table 4.5)
ANNEXURE xxi | P a g e
ANOVA table 8: Effect of different combination of growth regulators on regeneration frequency
(on the basis of data obtained from table 4.6)
ANNEXURE xxii | P a g e
ANOVA table 9: Effect of different combination of growth regulators on shoot/callus (on the
basis of data obtained from table 4.6)
ANNEXURE xxiii | P a g e
ANOVA table 10: Effect of different combination of growth regulators on shoot length (on the
basis of data obtained from table 4.6)
ANNEXURE xxiv | P a g e
ANOVA table 11: Effect of different combination of growth regulators on rooting (on the basis
of data obtained from table 4.7)
ANNEXURE xxv | P a g e
ANOVA table 12: Effect of different combination of growth regulators on root length (on the
basis of data obtained from table 4.7)
ANNEXURE xxvi | P a g e
ANOVA table 13: Effect of different concentrations and combinations of solutions on
encapsulation (on the basis of data obtained from table 4.8)
ANNEXURE xxvii | P a g e
ANOVA table 14: Effect of different combination of growth regulators on shoot re-growth
frequency (on the basis of data obtained from table 4.9)
ANNEXURE xxviii | P a g e
ANOVA table 15: Effect of different elicitors on biomass accumulation after 10 days (on the basis
of data obtained from table 4.10)
ANNEXURE xxix | P a g e
ANOVA table 16: Effect of different elicitors on biomass accumulation after 20 days (on the
basis of data obtained from table 4.10)
ANNEXURE xxx | P a g e
ANOVA table 17: Effect of different elicitors on biomass accumulation after 30 days (on the basis
of data obtained from table 4.10)
ANNEXURE xxxi | P a g e
ANOVA table 18: Effect of different elicitors on carbohydrates (on the basis of data obtained
from table 4.12)
ANNEXURE xxxii | P a g e
ANOVA table 19: Effect of different elicitors on protein (on the basis of data obtained from table
4.12)
ANNEXURE xxxiii | P a g e
ANOVA table 20: Effect of different elicitors on proline (on the basis of data obtained from table
4.12)
ANNEXURE xxxiv | P a g e
ANOVA table 21: Effect of different elicitors on phenol (on the basis of data obtained from table
4.12)
ANNEXURE xxxv | P a g e
ANOVA table 22: Effect of different elicitors on flavonoid (on the basis of data obtained from
table 4.13)
ANNEXURE xxxvi | P a g e
ANOVA table 23: Effect of different elicitors on alkaloid (on the basis of data obtained from
table 4.13)
ANNEXURE xxxvii | P a g e
ANOVA table 24: Effect of different elicitors on glycyrrhizin (on the basis of data obtained from
table 4.13)
ANNEXURE xxxviii | P a g e
ANOVA table 25: Effect of different elicitors on ascorbate peroxidase (on the basis of data obtained
from table 4.14)
ANNEXURE xxxix | P a g e
ANOVA table 26: Effect of different elicitors on peroxidase (on the basis of data obtained from
table 4.14)
ANNEXURE xl | P a g e
ANOVA table 27: Effect of different elicitors on superoxide dismutase (on the basis of data
obtained from table 4.14)
ANNEXURE xli | P a g e
ANOVA table 28: Anti-bacterial activity of root and leaves in different solvent extract against B.
subtilis (on the basis of data obtained from table 4.15)
ANNEXURE xlii | P a g e
ANOVA table 29: Anti-bacterial activity of root and leaves in different solvent extract against S.
mutans (on the basis of data obtained from table 4.15)
ANNEXURE xliii | P a g e
ANOVA table 30: Anti-bacterial activity of root and leaves in different solvent extract against P.
vulgaris (on the basis of data obtained from table 4.15)
ANNEXURE xliv | P a g e
ANOVA table 31: Anti-fungal activity of root and leaves in different solvent extract against C.
albicans (on the basis of data obtained from table 4.16)
ANNEXURE xlv | P a g e
ANOVA table 32: Anti-fungal activity of root and leaves in different solvent extract against A.
niger (on the basis of data obtained from table 4.16)