in vitro studies on the variations of biochemical metabolites in … · 2018-12-13 · declaration...

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


organogenesis60


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

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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


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


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


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


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

CHAPTER – 1


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 7 | P a g e

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

CHAPTER – 2


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

CHAPTER – 2


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).

CHAPTER – 2


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

CHAPTER – 2


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.

CHAPTER – 2


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.

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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).

CHAPTER – 2


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)


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)


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)

CHAPTER – 2



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)


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)


NR

Nodal segments

(1.0-1.5 cm)

Liquid detergent RTW

0.1% HgCl2 (3-5 min)

4-5X SDDW

Yadav and Singh

(2012)


NR

Fully expanded

young leaves

RTW 0.1% HgCl2 (5

min) 5X SDW

Gupta et al.

(2013)


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)


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)


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.

CHAPTER – 2


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).

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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


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).

CHAPTER – 2


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

CHAPTER – 2


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.

CHAPTER – 2


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)


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)


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)

CHAPTER – 2



G. glabra MS + 2 mg L−1 BAP + 2mg L−1 NAA

(SIM & RIM)


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)


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)


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)


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)

CHAPTER – 2



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)


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)


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)

CHAPTER – 2



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)


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)


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)

CHAPTER – 2



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)


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)


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)


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)

CHAPTER – 2




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)

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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,

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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)

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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

CHAPTER – 2


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).

CHAPTER – 3

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.

CHAPTER – 3


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

CHAPTER – 3


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.

CHAPTER – 3


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

CHAPTER – 3


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.

CHAPTER – 3


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.

CHAPTER – 3


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%

CHAPTER – 3


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

CHAPTER – 3


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.

CHAPTER – 3


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

CHAPTER – 3


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

CHAPTER – 3


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


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 (%),

CHAPTER – 3


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


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

CHAPTER – 3


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).

CHAPTER – 3


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.

CHAPTER – 3


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.

CHAPTER – 3


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)

CHAPTER – 3


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.

CHAPTER – 3


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.

CHAPTER – 3


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

CHAPTER – 3


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)

CHAPTER – 3


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)

CHAPTER – 3


- 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)

CHAPTER – 3


- 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

CHAPTER – 3


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

CHAPTER – 3


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:

CHAPTER – 3


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

CHAPTER – 4

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.

CHAPTER – 4


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

CHAPTER – 4


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


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.

CHAPTER – 4


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


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


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



a.

c. d.

b.

CHAPTER – 4



a.

c. d.

b.

CHAPTER – 4


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


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


CHAPTER – 4


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


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



a.

c. d.

b.

CHAPTER – 4



a.

c. d.

b.

CHAPTER – 4


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



a.

c. d.

b.

CHAPTER – 4



a.

c. d.

b.

CHAPTER – 4


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


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


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


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


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


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


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



a.

c. d.

b.

CHAPTER – 4



a.

c. d.

b.

CHAPTER – 4


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



a.

c.

b.

CHAPTER – 4



a.

c.

b.

CHAPTER – 4


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


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


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


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


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



a.

c. d.

b.

CHAPTER – 4



a.

c. d.

b.

CHAPTER – 4


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


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

---


CHAPTER – 4


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


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


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


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


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

-

-

-


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


(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


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



a.

c. d.

b.

f.e.

CHAPTER – 4



a.

c. d.

b.

f.e.

CHAPTER – 4


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


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


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


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).

CHAPTER – 4


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

CHAPTER – 4


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


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


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


CHAPTER – 4


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)

CHAPTER – 4


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


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



Callus(control)


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


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


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


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


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.

CHAPTER – 4


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


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



Callus(control)


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


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


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

CHAPTER – 4


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


CHAPTER – 4


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


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).

CHAPTER – 4


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.

CHAPTER – 4


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.

CHAPTER – 4


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.

CHAPTER – 5

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.

CHAPTER – 5


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


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).

CHAPTER – 5


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.

CHAPTER – 6

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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


ANNEXURE xxiii | P a g e

ANOVA table 10: Effect of different combination of growth regulators on shoot length (on the


ANNEXURE xxiv | P a g e

ANOVA table 11: Effect of different combination of growth regulators on rooting (on the basis


ANNEXURE xxv | P a g e

ANOVA table 12: Effect of different combination of growth regulators on root length (on the


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


ANNEXURE xxix | P a g e

ANOVA table 16: Effect of different elicitors on biomass accumulation after 20 days (on the


ANNEXURE xxx | P a g e

ANOVA table 17: Effect of different elicitors on biomass accumulation after 30 days (on the basis


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)

in vitro studies on the variations of biochemical metabolites in … · 2018-12-13 · declaration...

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