Saurashtra University - COnnecting REpositories · (tones/ha.) Basrai, Rasthali 40-50 Shrimanti 70 Grand Naine 65 Ardhapuri , Meanyham 55 Hirsal, Safed Velchi, Red banana, Lal Velchi

Saurashtra University Re – Accredited Grade ‘B’ by NAAC (CGPA 2.93)

Chariya, Lalitkumar D., 2012, “Physiological, Biochemical and Molecular

Marker Studies in Musu Spp.”, thesis PhD, Saurashtra University

http://etheses.saurashtrauniversity.edu/id/eprint/554 Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given.

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Physiological, Biochemical and

Molecular Marker Studies

in Musa Spp.

Thesis submitted to Saurashtra University,

For the Award of Degree of

Doctor of Philosophy in

Biosciences (Plant Science)

: Submitted By :

Lalitkumar D. Chariya

Registration No.: 3725, Dt. 26/11/2007

Centre for Advanced Studies in plant

Biotechnology & Genetic Engineering (CPBGE)

Department of Biosciences

Saurashtra University

Rajkot-360 005 (India)

March 2012

Ref.: SU/BIO/ Date: /March/2012

CERTIFICATE

This is to certify that the data embodied in this doctoral thesis entitled

“Physiological, Biochemical and Molecular Marker Studies In Musa Spp.” are the

original work, carried out by Mr. Lalitkumar D. Chariya (Ph. D. Registration No.

3725, 26/11/2007) in my laboratory under my supervision and guidance for the award of

Ph. D. degree in Biosciences (Plant Science). All the work was carried out at the Plant

Physiology and Molecular Biology Laboratory / Centre for Advanced Studies in Plant

Biotechnology & Genetic Engineering (CPBGE), Department of Biosciences, Saurashtra

University, Rajkot (India).

It is further certified that he has put ten terms of research work and the work

have not been submitted either partly of fully to any other University or institution for

the award of any diploma or degree.

V. S. Thaker

Professor & Guiding Teacher

Forwarded Through,

S. P. Singh

Professor & Head

Acknowledgements

Fortunate are those who live under the blessings of Almighty. My continuous

hardwork and efforts resulted into this Ph. D. work, which would not have been

possible without inspiring guidance of my esteemed guide Prof. V. S. Thaker. It

gives me immense pleasure to express my profound feeling of reverence and

heartfelt gratitude to Prof. V. S. Thaker for her inspiring guidance, dynamic

initiation, continuous encouragement, thoughtful discussions, and untiring

supervision throughout the work.

I am highly thankful to Prof. S. P. Singh the Head of Department of Biosciences,

Saurashtra University, Rajkot for providing necessary laboratory facilities during

the course of work period. I am also thankful to all teaching and non-teaching

faculties of the department for their timely help whenever needed. I specially thank

to Dr. Manish Vekaria and Dr. Virendra Patel, Vimal Reaserch Socity for Agro-

Biotech and Cosmic Powers, for providing me necessary research facilities.

I wish to acknowledge University Grant Commission, New Delhi, India for financial

support. And also thanks to Centre For advanced Studies in Plant Biotechnology &

Genetic Engineering, state Government for providing lab facility.

This Ph. D. work presents my humble effort in the field of Plant Biotechnology

especially on Musa Spp. which could not have been possible to get in present shape

without support and guidance of other faculty members and colleagues.

I extend my sincere thanks to Bhatt Kunjal, Pawar Vaishali, Chudasama Rita, Joshi

Madhvi, Pandya Rohan and Nakum Anil for their helping hand whenever needed. I

acknowledge my colleagues, Chudasama Kiran, Maharshi Anjisha, Tank Jigna,

Mandaliya Viral, Sheth Bhavisha and Jhala Vibhuti and also thankful to Yogesh,

Mayur, Ram, Jaydip, Kaushal, Parth, Pooja, Purvi, Smita, Khyati, Dipika for their

company and assistance during my work.

I am also thankful to Prabhudas, Pravin, Rajabhai, Bharat, Rajan, Dhaval, Vishal,

Ashok, Govindbhai, Jivanbhai and Vaghelabhai for their help during my work.

There are so many people who helped me in one or other way whose names may not

be mentioned here but I heartily thank them for their kind support.

I am not able to find words for the contribution of my family during my work,

without their caring support this work would have been impossible.

March 2012 Lalit Chariya

INDEX

No Contents Page No

1 General Introduction 1-7

2 Chapter 1 8-31

Micropropogation of Banana

3 Chapter 2 32-51

Molecular marker studies of Banana

4 Chapter 3 52-65

Biochemical studies of Banana

5 References 66-98

Abbreviations:

ABA : Abscisic Acid

AFLP : Amplified Fragment Length Polymorphism

BAP : N6 -benzylaminopurine

BBMV : Banana Bract Mosaic Virus

BBTV : Banana Bunchy Top Virus

BLAST : Basic Local Alignment and Search Tool

BSA : Bovine Serum Albumin

BSV : Banana Streak Virus

CI : Chloroform : Isoamyl Alcohol, 24:1, v/v

CMV : Cucumber Mosaic Virus

DMF : Dimethylformamide

DNA : Deoxyribose nucleic acid

DPPH : 2,2-Diphenyl-1-Picrylhydrzyl

Dw : Dry Weight

EDTA : Ethylenediaminetetraacetic acid

ELISA : Enzyme-Linked Immunosorbent Assay

EtOH : Ethanol

Fw : Fresh Weight

GA : Gibberellic Acid

GC-MS : Gas Chromatography-Mass Spectrometry

H2O2 : Hydrogen Peroxide

HPLC : High Performance Liquid Chromatography

HRP : Horseradish Peroxidase

IAA : Indol-3-Acetic Acid

IBA : Indole-3-Butyric Acid

IgG : γ-immunoglobulin

ISSR : Inter-Simple Sequence Repeat

KiN : Kinetin

MeOH : Methanol

MS : Murashige and Skoog

MSA : Multiple Sequence Alignments

NAA : Naphthaleneacetic Acid

OPD : o-Phenylenediamine

PAA : Phenyl Acetic Acid

PBS : Phosphate buffer Saline

PCI : Tris saturated Phenol : Chloroform : Isoamyl alcohol, 25 : 24 : 1, v/v

PCR : Polymerase Chain Reaction

PGRs : Plant Growth Regulators

PTC : Plant Tissue Culture

PVP : Polyvinlypyrrolidone-40 (soluble)

PVPP : PVP polymer (insoluble)

RAPD : Random Amplified Polymorphic DNA

RFLP : Restriction Fragment Length Polymorphism

RNA : Ribose Nucleic Acid

SCAR : Sequence Characterized Amplified Polymorphism

TDZ : Thidiazuron

TLC : Thin Layer Chromatography

UPGMA : Unweighted Pair-Group Method for Arithmetic averages analysis

General Introduction

Centre for Advanced Studies in Plant Biotechnology & Genetic Engineering, Department of Biosciences, Saurashtra University, Rajkot-360 005. Gujarat (India).

1

Banana (Musa spp. family Musaceae) is a fourth most important fruit crop in

the world. It is a rich source of carbohydrates and vitamins, particularly

vitamins B, potassium, phosphorus, calcium and magnesium. The fruit is free

from fat and cholesterol, easy to digest, and its powder is used as the first

baby food. Beside these it has great medicinal value. It helps in reducing risk

of heart disease when used regularly and is recommended for patients

suffering from high blood pressure, arthritis, ulcer, gastroenteritis, and kidney

disorders.

Banana is being cultivated in tropical and subtropical countries as a major

staple food for millions of people. They contribute to food security by

producing fruit year-around and provide incomes to rural populations (Roux et

al. 2008). The estimated current world production of banana is 97.5 million

tons per year, covering 10 million ha land (Sahijram et al 2003).

Developed countries are the usual destination for export bananas. Production,

as well as exports and imports of bananas, are highly concentrated in a few

countries. Ten major banana producing countries accounted for about 75% of

total production in 2003 with India, Ecuador, Brazil, and China accounting for

half of the total (Zhang et al. 2005). In 2009, among the major producers,

India alone accounts for 27.43 % (26.2 million tonnes) followed by Philippines,

producing 9.01 million tonnes and China, Brazil and Ecuador, with production

ranging from 7.19 to 8.21 million tones (Singh et al 2011).

In India, Gujarat is the leading producer of banana after Tamil Nadu and

Maharashtra in the country. During 2008-09, area and production of banana in

Gujarat were 60.9 thousand ha and 3571.6 ha per metric tons respectively

(Figure 1 and 2) (http://nhb.gov.in/).

Genus Musa includes 118 different species though out the world (USDA,

ARS, National Genetic Resources Program, http://www.ars-grin.gov). It shows

that Musa species are widely known with their varieties, synonyms and

cultivars.


2

Figure 1. The major banana producing states in India (2008-09). [Source Indian horticulture database 2009].

Figure 2. Production trend of banana in last two decade. [Source Indian horticulture database 2009].


3

Banana is known with their cultivars names in India. Around 20 cultivars viz.

Dwarf Cavendish, Robusta, Monthan, Poovan, Nendran, Red banana, Safed

Velchi, Basrai, Ardhapuri, Rasthali, Karpurvalli, Karthali and Grande Naine

etc. are commercially cultivated (Table -1).

Table 1. The important banana varieties cultivated in different states of India.

State Varieties grown

Andhra Pradesh

Dwarf Cavendish, Robusta, Rasthali, Amritpant, Thellachakrakeli, Karpoora Poovan, Chakrakeli, Monthan and Yenagu Bontha

Assam Jahaji (Dwarf Cavendish), Chini Champa, Malbhog, Borjahaji (Robusta), Honda, Manjahaji, Chinia (Manohar), Kanchkol, Bhimkol, Jatikol, Digjowa, Kulpait, Bharat Moni

Bihar Dwarf Cavendish, Alpon, Chinia , Chini Champa, Malbhig, Muthia, Kothia , Gauria

Gujarat Dwarf Cavendish, Lacatan, Harichal (Lokhandi), Gandevi Selection, Basrai, Robusta, Grand Naine, Harichal, Shrimati, Soneri, Desi, Red, Khasadi

Jharkhand Basrai, Singapuri

Karnataka Dwarf Cavendish, Robusta, Rasthali, Poovan, Monthan, Elakkibale

Kerala Nendran (Plantain), Palayankodan (Poovan), Rasthali, Monthan, Red Banana, Robusta

Madhya Pradesh Basrai

Maharashtra Dwarf Cavendish, Basrai, Robusta, Lal Velchi, Safed Velchi, Rajeli Nendran, Grand Naine, Shreemanti, Red Banana

Orissa Dwarf Cavendish, Robusta, Champa, Patkapura (Rasthali)

Tamil Nadu Virupakshi, Robusta, Rad Banana, Poovan, Rasthali, Nendran, Monthan, Karpuravalli, Sakkai, Peyan, Matti

West Bengal Champa, Mortman , Dwarf Cavendish, Giant Governor, Kanthali, Singapuri


4

Grand Naine, an imported variety, is gaining popularity and may soon

become the most preferred variety due to its tolerance to abiotic stresses and

good quality bunches. Fruit develops attractive uniform yellow colour with

better shelf life and quality than other cultivars therefore it is widely cultivated

in Gujarat and other state of India through sucker and micropropogation

techniques.

The following table shows the state-wise growing belts and variety wise

average yield (tones/ha.) of important variety grown though out the country

(http://nhb.gov.in/):

State Growing belts

Andhra Pradesh East Godavari, West Godavari, Kurnool, Cuddapah

Assam Goalpara, Nagaon, Sonitpur, foothills of Garo hills

Gujarat Surat, Vadodara, Anand, Kheda, Junagadh, Narmada, Bharuch

Jharkhand Ranchi , Sahebganj

Karnataka Bangalore, Chitradurga, Shioroga, Hassan, Chikka Mangloor

Kerala

Thiruvananthapuram, Kollam, Pathanamthitta, Alappuzha, Kottayam, Idukki, Ernakulam, Thrissur, Palakkad, Malappuram, Kozhikode , Wynadu, Kannur, Kasargod

Madhya Pradesh Khandwa, Badwani, Khargaon, Dhar

Maharashtra

Jalgaon, Ahmednagar, Buldhana, Pune, Wardha, Dhule, Nanded, Parbani, Nandurbar, Satara, Sangli, Osmanabad, Buldhana, Akola, Yeothmal, Amravati, Thane, Kulara, Alibag

Orissa Ganjam, Puri, Khurda, Gajpati, Cuttack , Dhenkanal, Angul, Sundargarh, Sambalpur, Bargarh, Deogarh, Koraput, Keonjhar, Raygada, Mayurbhanj

Tamil Nadu Thoothukudi, Tiruchirapalli, Coimbatore, Tirunelveli, Karur, Erode, Kanniyakumari

West Bengal Hooghly, Nadia, Parganas


5

Varieties Average yield

(tones/ha.)

Basrai, Rasthali 40-50

Shrimanti 70

Grand Naine 65

Ardhapuri , Meanyham 55

Hirsal, Safed Velchi, Red banana, Lal Velchi 45

Poovan 40-50

Monthan 30-40

Dwarf Cavendish , Robusta Champa and Chini desi 50-60

Nendran 30-35

Low adoption rates of agricultural biotechnology in developing countries have

been associated with factors such as the lack of credit, limited access to

information, aversion to risk, inadequate farm size, inadequate incentives

associated with farm tenure arrangements, insufficient human capital,

absence of equipment to relieve labor shortages (thus preventing timeliness

of operations), chaotic supply of complementary inputs (such as seed,

chemicals, and water), and inappropriate transportation infrastructure (Feder

et al., 1985). The rapid evolution and divergent perception of biotechnology

also affects its adoption (Qaim, 1999). The uncertainity about the long term

effects of genetic modification technology on health and the environment

coupled with incomplete or absent national legislations act as major barrier to

the safe and effective application of biotechnology in developing countries

(Muyanga 2009).

Banana is grown under diverse conditions and production systems and hence

selection of varieties is based on needs and propogation feasibility. Desi,


6

Red, Khasdi and Soneri varieties of Chorvad region [21°1'36"N 70°15'53"E] in

Gujarat is a popular cultivars to rural banana growers due to its higher

keeping quality, better taste and flavor, however, studies on these varieties

are rather scantes. For this reason there is an increasing demand of these

cultivars to be studied. In this thesis, banana varieties have been studied for

tissue culture, biochemical characterization, and molecular marker analysis.

General objectives of this study:

This study is organized in three main sections as tissue culture, molecular

marker analysis and biochemical characterization of Musa as follows:

In the first section, primarily hormonal concentration in media for in vitro

culture of Musa was reviewed. On the basis of this, it is concluded that prior

knowledge of endogenous hormones from the explant may help in

development of efficient and rapid protocol for in vitro propogation. Thus the

estimation of endogenous level of hormones from Musa varieties were done

to minimize the trial and error combinations of the used hormones. After the

achievement of profound healthy multiple clones, they were exceed for

rooting, and rooted plantlets made ready for acclimatization.

The second section was divided into three major subsections: Standardization

of high quality DNA isolation protocol, RAPD and ISSR molecular markers

analysis and nucleotide sequencing of molecular markers amplified

amplicons.

In first sub section, various DNA isolation protocols for banana (Musa

species) were tested, but these methods resulted in DNA with lot of impurities

and not suitable for RAPD and ISSR analysis. Therefore, the total genomic

DNA isolation protocol derived from a method originally developed for other

plants were tested (Mandaliya et al. 2010a). Modifications were made to

minimize phenols, polysaccharide co-isolation and to simplify the procedure


7

for processing of large number of DNA samples. The DNA which is isolated

by the present protocol is suitable for further downstream applications.

In second sub section, DNA samples were subjected to RAPD and ISSR

based DNA amplification. RAPD and ISSR have demonstrated to be sensitive

in detecting DNA polymorphism, development of unique marker, and

establishment of phylogenetic relationship among selected four Musa

varieties.

The last subsection was based on novel approach to encase monomorphic

band pattern in ISSR analysis. To evaluate the power of this approach,

multiple sequence alignments (MSA) within the sequence of monomorphic

bands was performed. This approach used to test (i) to infer the nucleotide

sequence variation, (ii) the interrelationships of Musa varieties, and (iii) the

availability of restriction sites. Application of this technique for the selection of

variety is discussed.

The aim of the last section was to examine the changes in antioxidant

potential and phenolic content in fully expanded leaves of various Musa

varieties. The plant extracts of mature leaves were subjected to high-pressure

liquid chromatography for qualitative and quantitative analysis of various plant

phenols.

Chapter 1 Micropropogation

of Banana


8

Abstract

Banana (Musa sp.), is fourth most important fruit crop in the world, it has

great agriculture importance and therefore to propagate the plant species,

present work was carried out using plant tissue culture. Prior knowledge of

endogenous hormonal concentrations in explants reduces the time of

selection for best combinations of hormone in media. Therfore, immunoassay

is used to estimate endogenous hormones from the explants and the new

optimized protocol devised for rapid in vitro propagation of plant through

multiple shoot induction. The results achieved in MS medium where,

phytohormones BAP and NAA were tested in different concentrations. The

highest number of multiple shoot (8-10 shoots/explant) was obtained along

with 4-5 cm shoot length with BAP ranging from 1.0 to 6.0 mg/l and while

NAA 2.0 to 4.0 mg/l was suitable for better root induction in banana. The

hardened plantlets were successfully acclimatized in green house chamber

with survival rate of 40-45%. The present protocol provides rapid and efficient

micropropogation of Banana.


9

Introduction

About 70% of the farmers are using banana suckers as planting material

while the rest 30% of the farmers are using tissue culture seedlings. Sword

suckers with well developed rhizome, conical or spherical shape having

actively growing conical bud and weighing approximately 450-700 g are

commonly used as propagating material.

Suckers are generally infected with some pathogens and nematodes.

Similarly due to the variation in age and size of sucker, crop is not uniform,

harvesting is prolonged and management becomes difficult. Therefore, in

vitro clonal propagation i.e. tissue culture plants are recommended for

planting. They are healthy, disease free, uniform in growth and early yielding.

Micropropogation has become an important part of the commercial

propagation of many plants (Fiorino and Loreti 1987) because of its

advantages as a multiplication system (Debergh 1987, Razdan 2003). There

are several techniques for in vitro plant propagation like the induction of

auxilary and adventitious shoots, the culture of isolated meristem and plant

regeneration by organogenesis and/or somatic embryogenesis (Williams and

Maheswaran 1986). The common stages known for the micropropogation of

most plants are sterilization and in vitro aseptic culture, shoot multiplication,

root induction and acclimatization.

Depending on the type of explant, shoot formation may be initiated from

apical and auxilary buds from adventitious meristems that originate on

excised shoots, leaves, bulb scales, flower stems or cotyledons, or from

callus that develops at the cut surfaces of explants. Usually 4-6 weeks are

required to complete initiation stage and to generate explants that are ready

to be moved to shoot proliferation (Hartmann et al. 2002).

At shoot proliferation and multiple shoot production stage, each explant has

expanded into a cluster of small shoots. Multiple shoots are separated and

transplanted to new culture medium (Hartmann et al. 2002). Shoots are sub


10

cultured every 2-8 weeks. Material may be sub cultured several times to new

medium to maximize the quantity of shoots produced.

The rooting stage prepares the regenerated plants for transplanting from in

vitro to ex vitro conditions in controlled environment rooms, in the glasshouse

and, later, to their ultimate location. This stage may involve not only rooting of

shoots, but also conditioning of the plants to increase their potential for

acclimatization and survival during transplanting (De Klerk et al. 1997, 1999).

Transplantation of in vitro derived plants to soil is often characterized by lower

survival rates. Before transfer of soil rooted plants to their final environment,

they must be acclimatized in a controlled environment room or in the

glasshouse (Rohr et al. 2003). Plants transferred from in vitro to ex vitro

conditions, undergo gradual modification of leaf anatomy and morphology,

and their stomata begin to function (the stomata are usually open when the

plants are in culture). Plants also form a protective epicuticular wax layer over

the surface of their leaves. Regenerated plants gradually become adapted to

survival in their new environment (Donelly and Tisdall 1993).

There are several common problems associated with each stage in the

micropropagation of most plants: one is browning of culture and other is

microbial contamination. Explants placed on culture medium exude dark

colored compounds into the culture medium (phenols, pigments) that are

released from the cut ends of the explants. This can cause browning of tissue

and the medium, which is often connected with poor culture establishment

and reduced regeneration ability. Minimization of the wounding of explants

during isolation and surface disinfection may help to reduce this response.

Other approaches to prevent tissue browning include removal of these

compounds by washing of explants in sterile water for 2-3 h, frequent

subculture of explants to new medium with the excision of brown tissues,

initial culture in liquid medium with subsequent transfer to semi-solid medium,

culture on a porous substrate (paper bridges) and adsorption with activated

charcoal or PVP (polyvinylpyrrolidone) by addition of these compounds to the

culture medium. However, activated charcoal can also adsorb growth


11

regulators or be toxic to some tissues. The use of antioxidants, such as

ascorbic acid, citric acid, L-cysteine or mercaptoethanol, can also prevent

browning of the tissues in culture (George 1996).

Excessive browning may cause serious problems in the different stages of

shoot regeneration. Sometimes decline of vigor in culture with stagnancy in

shoot growth and proliferation is observed which may be caused by several

factors. These include unsuitable composition of the culture medium, lack of

some nutrients, calcium deficiency in the apices, which causes necrosis, the

presence of latent persistent microbial contaminants, cytokinin habituation,

loss of regeneration ability in long-term cultures (due to epigenetic variation)

and culture aging, including transition from the juvenile to a mature stage.

Tissue culture contamination frequently originates during any stages it

includes explants contaminated with endophytic micro-organisms or common

environmental micro-organisms. Heterotrophic plant tissue media are capable

of supporting the growth of many common environmental micro-organisms.

Cultivable micro-organisms may over-run the cultures killing the explants; or

inhibited by media components. Some micro-organisms may positively

influence plant growth and development by acidification of the medium or by

release of plant growth regulators. Conversely, some micro-organisms are

inhibited by acidification of culture media and by exudates from the plant

tissues and may be weakly expressed or latent as endophytes in the tissues.

The release of contaminated plants in the environment brings with it the risk

of large-scale disease or poor plant performance in the field. The best

strategy to control tissue culture contamination is to establish aseptic cultures

and to maintain good laboratory practice (Cassells, 2003).

BBTV (Banana bunchy top virus), CMV (Cucumber mosaic virus), BSV

(Banana streak virus) and BBMV (Banana bract mosaic virus) are the four

known viral diseases affecting bananas. In addition, banana is an attractive

host for nematodes, particularly Pratylenchus coffeae, Meloidogyne incognita,

Helicotylenchus multicinctus and Radopholus similis. The pests also spread

through transportation of non-quarantined planting material. Insect pests like,


12

banana weevils (Odoiporus longicollis, and Cosmpolites sordidus) which till

now had a limited presence in some states of India have now spread to

Bangladesh and beyond. Disease and pest pressure on Asian bananas is

unlikely to lessen in the foreseeable future (Gold and Messiaen 2000,

Padmanaban and Sathiamoorthy 2001).

The tissue culture technology has been the foundation of high quality, disease

free planting material production at a mass scale, particularly in vegetatively

propagated crops. The development of micropropogation techniques has

been a major focus of Musa research during the past two decades (Israeli et

al. 1995). The earliest reports of in vitro culture of bananas came from Taiwan

in the 70’s (Ma and Shii 1974). Till date, protocols have been standardized for

in vitro propagation of a wide range of Musa species and cultivars

(Sathiamoorthy et al. 1998). Since most banana produce seedless fruits, they

are vegetatively multiplied in vivo as well as in vitro. Conventional propagation

of Musa spp. depends on planting young suckers, sword suckers and corms

suffer slow multiplication rate. For large scale plantings sufficient quantities

are needed. Therefore, in vitro techniques have become most popular for

producing high number of plantings material for banana. The starting

materials used in vitro propagation of bananas were meristem derived from

corm tissue (Ho et al. 1993) and shoot tips (Morpurgo et al. 1994). On the

other hand, somatic embryogenesis has also been developed as an

alternative technique for mass propagation.

One of the most common problems associated with the in vitro establishment

of Musa is the deleterious effects of oxidized phenols (Zweldu and Ludders

1998). Banana plants are known to release large quantities of phenolic

compounds (Titov et al. 2006). The blackening was most probably caused by

oxidation of the phenolic compounds release by the roots regularly reported

for in vitro culture of banana plantlets (Banerjee and de Langhe 1985, Titov et

al. 2006). Blackening of roots could be reduced by adding activated charcoal,

antioxidants such as ascorbic acid or citric acid in the culture medium

(Banerjee and de Langhe 1985). George (1996) details the use of citric acid

and ascorbic acid combinations to delay browning. The successful prevention


13

of browning in explants of Musa by using a mixture of ascorbic acid, citric acid

and cysteine are reported by Mante and Tepper (1983). The behavior of the

citrate in citric acid works as a chelating agent binding to ions responsible for

activating polyphenol oxidative enzymes (George 1996). Ascorbate behaves

as a reducing agent and is converted to dehydro-ascorbic acid (Panaia 1998).

Ascorbate is able to scavenge oxygen radicals produced when tissue is

damaged and therefore cells are protected from oxidative injury. Oxygen

radicals are attributed to exacerbating oxidative injury. Antioxidants containing

citrate and ascorbate reduce browning of tissue by detoxifying these free

radicals.

The potential growth and development pattern of plant have been regulated

by several scientific efforts. These include numerous hormonal and

environmental stimuli with genetic setup of the plant. The prospective role of

agriculture depends on the main application of hormones at various phases

for rapid growth and development of plants.

Plant growth regulators are inevitable for in vitro regeneration of crop plants in

any artificial medium. It is well known that plant growth hormones like auxin,

cytokinin, gibberellins, abscissic acid and ethylene play important role in

various stages of plant growth and development. This growth regulators

include natural and as well as synthetic group which become very important

because of its potentiality. They are small, simple, molecules with diverse

chemical composition: indole compounds, terpens, adenine derivatives,

steroids, aliphatic hydrocarbons and derivatives of carotenoids or fatty acid,

and one is in gaseous form (Shrivastava 2002). Generally, cytokinin helps in

shoot proliferation and auxins helps in rooting of proliferated shoots (Al-Amin

et al. 2009). Cytokinins such as BAP and kinetin are generally known to

reduce the dominance of apical meristems and induce auxilary as well as

adventitious shoot formation from meristematic explants in banana

(Madhulatha et al. 2004). The application of higher BAP concentrations

inhibits elongation of adventitious meristems and the conversion into

complete plants (Zaffari et al. 2000).


14

Plant hormones are required in minute quantities and their concentrations at

site of activity are precisely regulated. Each of these brings about a variety of

growth, response and effect (Shrivastava 2002). There are variations in

concentration of each hormone between different species, types of organs,

tissue and different phase of plant growth and development (Sheldrake 1973,

Naithani et al. 1982). So growth regulators influence almost all metabolic

process of the cell and it has remained difficult to correlate physiological

process.

The crucial role of auxin in plant at growth and developmental stage including

cell division, expansion and dedifferentiation (Mercier et al. 2003).

Accumulation of endogenous auxin in plant tissue can induce root formation.

The reason could be inhibition of IAA oxidase activity by IBA and NAA. IAA

content increased with a decrease of IAA oxidase activity during root

formation (Liu et al. 1998).

Further the auxin and cytokinin appears as a major group of plant hormones

(Momtaz 1998). When it applied to plant they produce various effects. It also

includes cell division, cell dedifferentiation, dedifferentiation, and root and

shoot growth branching, chloroplast development (George et al. 2008), leaf

senescence, and pathogen resistance. Jouanneau (1975) suggest that

cytokinin might be required to regulate the synthesis of proteins involved in

the formation and function of the mitotic spindle.

Major study has been done on growth regulators in the various stages of plant

development that are seed germination (Leubner et al. 1996), cell elongation

(Patel and Thaker 2007), stem elongation (Zeevart 1983, Yang et al. 1996),

flower development (Nakayama et al. 2002) and also in fruit setting and

overcome seed and bud dormancy. Due to major investigation on gibberellins

(GAs), it is large group having 120 members (Murata et al. 2002). It present in

wide range of living organisms like bacteria and fungi to angiosperms (Bottini

et al. 1989, Chaube and Singh 2000).

Two or more hormones can interact synergistically or antagonistically in many

aspects. Endogenous level of hormones is affected equally and they may


15

affect the biosynthesis or metabolism of another (Palni et al. 1988). For

synergetic effect, it is apparent that not only auxin and cytokinin, but the level

of both hormone and proportion of one to the other are determinant for the

cell cycle, cell division and differentiation control. For example, auxiliary shoot

proliferation in some species may be promoted by the presence of an auxin

together with cytokinin and tissue from monocotyledons can often be induced

to form callus by culture in high level of auxin alone, and cytokinin may be

non essential componant (Skoog and Miller 1957).

Both auxin and cytokinin are usually required for growth or morphogenesis;

auxin can inhibit cytokinin accumulation (Hansen et al. 1985), while cytokinin

can inhibit at least some of the action of auxin. The requirement of cytokinin

and auxins depends on the variety of plant and culture conditions (Cronauer

and Krikorian 1984). The endogenous phytohormones concentration is

important for regulation of plant physiological and biochemical process. The

typical effect of auxin is to promote cell elongation and root initiation, while

cytokinin promote cell division and shoot differentiation. The ratio of auxin and

cytokinin play important role in morphogenesis or organogenesis. High auxin

in combination with low cytokinin induces root formation (Mala et al 2005);

while low auxin in combination with high cytokinin induces auxilary shoot

proliferation (George et al. 2008).

The nutrients and growth regulators requirement of culture medium are varied

by such factors like type of culture, sensitivity of culture cells, culture age,

types and concentration of medium supplements etc. (Omar et al. 2004). Due

to all these medium optimization is necessary to achieve optimum growth

however testing of range of these parameters in protocol establishments.

Solution to these problems is estimation of endogenous level of growth

regulators. Despite the recognized importance of endogenous hormone levels

on shoot organization in vitro (Peres and Kerbauy 1999, Peres et al. 1999)

and ex vitro (Sarul et al. 1995), very few exogenous hormone studies have

been reported in the case of banana micropropogation. Sub- and supra-

optimal levels of plant growth substances in the culture media have been

associated with somaclonal variation in this species (Smith 1988, Zaffari et al


16

1998). Primarily hormonal concentration in media for in vitro culture of Musa

was reviewed (Table -1.1). Rapid elongation and rooting in Musa shoots

regenerated in vitro by Srivatanakul et al. (2000), who suggested that kenaf

might contain high auxin levels that are favorable for rooting and might

overcome a “carry over” effect of TDZ from multiple shoot-induction medium

(Sakhanokho et al. 2008). Generally, adenine-based cytokinins are the most

commonly used in Musa spp. in vitro propagation, especially N6-

benzylaminopurine (BAP). The concentration of BAP cytokinin appears to be

main factor affecting multiplication rate in banana particularly to induce

multiple shoots (Wong 1986). On the other hand, there are reports on the use

of diphenylurea derivates, thiadiazuron (TDZ) in Musa spp. which had shown

significantly higher propagation rate for shoot proliferation compared to any

types of other adenine-based cytokinins particularly BAP hormone (Arinaitwe

et al. 2000).

In general, rooted clones kept, prior to potting, for the basal material widely

used soil, sand, dung and cocopit in desired manners. In some cases liquid

MS medium in absence of hormones enhance the survival percent noted

earlier by Karthikeyan et al. (2009). At the hardening stage the most viable

tool is maintenance of a high humid atmosphere during the initial period. Up

to 95% humidity can give desirable result (Ali et al. 2008). The success rate

depends on environmental condition as well as used materials and

concentration of various fertilizer applications (Ngamau 2001) for

acclimatization plant development.

The development of micropropogation techniques has been a major focus in

Musa Spp. The aims of the experimental design addresses the following

aspects of in vitro development were: Estimation of endogenous hormones to

minimize media designing. Sterilization procedure to be developed which will

optimize tissue survival in vitro. Antioxidant treatment minimizes phenolic

secretion. Cytokinin and auxin ratios will establish tissue regeneration.


17

Materials and Methods

Estimation of Phytohormones:

Raising of Antibody against PGR

Preparation of IAA-BSA and IAA-casein conjugate:

To raise antibodies against IAA, IAA-BSA and IAA-casein conjugates were

prepared as described by Weiler (1981) and Gokani and Thaker (2002),

respectively. IAA (52.3mg) was dissolved in 2 ml of DMF and reacted with 75-

µl tri-n-butyl amine and the solution was cooled to (0 oC). After this 40 µl

isobutyl chlorocarbonate was added and incubated for 8 min at room

temperature. This reaction mixture was added with constant stirring to 421 mg

BSA dissolved in 22 ml of DMF: water (1:1, v/v) and 420 µl 1M NaOH. After 1

h incubation at 0 oC another 0.2 ml of 1M NaOH was added and stirring was

continued for 5 h. The mixture finally dialyzed against 10% DMF for 24 h and

against distilled water for 4 day.

Preparation of PAA-BSA and PAA-casein conjugate:

To raise antibodies against PAA, PAA-casein and PAA-BSA conjugates were

prepared as described by Weiler (1981) and Gokani and Thaker (2002),

respectively. PAA (100mg) was dissolved in 2 mL of DMF and reacted with

75-µl tri-n-butyl amine and the solution was cooled to (0 oC). After this 40 µl

isobutyl chlorocarbonate was added and incubated for 8 min at room



h incubation at 0 oC another 0.2 ml of 1M NaOH was added and stirring was

continued for 5 h. The mixture finally dialyzed against 10% DMF for 24 h and


Preparation of GA-BSA and GA-casein conjugate:

To raise antibodies against GA3, GA3-BSA conjugate was prepared as

described by Weiler (1981). GA3 (106 mg) was dissolved in 2.5 mL of DMF


18

and reacted with 75-µl tri-n-butyl amine and the solution was cooled (0 oC), 40

µl isobutyl chlorocarbonate was added and incubated for 20 min at room



h incubation at 0 oC another 0.2 mL of 1M NaOH was added and stirring was

continued for 5 h. The mixture finally dialyzed against 10 % DMF for 24 h and


Preparation of Kinetin-BSA and Kinetin-casein conjugate:

Kinetin -BSA conjugation was prepared by modified protocol of Weiler (1980).

For preparation of Kinetin-BSA conjugate, 10 mg kinetin was suspended in

1ml of methanol and 0.01m Na2SO4 was added over a period of 7min. The

solution was stirred for another 13min and 0.3ml of 0.1M ethylene glycol

(30µmol) was added. After 5 min, the reaction mixture was added drop wise

to a stirred solution of 110 mg BSA in 5 ml distilled water. During addition of

the oxidized riboside the pH was kept constant between 9.2 to 9.4 by addition

of 5% K2CO3. After 60 min solid NaBH4 (5mg) was added and its repeated

addition was done after 40 min. Then, pH was adjusted to 6.5 by the addition

of 1M acetic acid and the solution was stirred for another 2 hr. The conjugate

was purified by dialysis against water for 3 days at 4°C and stored at 0°C.

Immunization and separation of IgG:

The IAA-BSA, PAA-BSA, GA-BSA, and Kinetin-BSA conjugates were mixed

with an equal volume of Freund’s complete adjuvant and injected into two

rabbits, respectively by intramuscular injection. Booster injections were given

periodically to raise the titer. Rabbits were bled periodically and every time

about 10-15 ml of blood was collected. Blood was incubated at 37 oC for 1 h

and serum was separated. IgG (γ-immunoglobulin) was collected by passing

the serum through DEAE cellulose pre-equilibrated with 0.01 M phosphate

buffer (pH 8.0). The purified IgG was concentrated to the original volume of

serum taken, by 0.01 M phosphate buffer (pH 8.0). Purified antibodies were

stored in glass vials at 0 oC and used for estimation after appropriate dilution.


19

Extraction of Hormones:

Meristematic tip of the three different banana varieties (Desi, Red and Khasdi)

banana suckers (500 mg) were crushed in 5ml of methanol and incubated for

48h in dark. Extract was centrifuged at 10,000g for 15 min. and supernatant

was collected. It was allowed to evaporate and residues remaining were

dissolved in 5ml of Phosphate buffer saline (pH 7.2). This was used for

estimation of phytohormones.

Enzyme Linked Immuno Sorbunt Assay (ELISA):

ELISA was performed as confirmation test by indirect ELISA assay as

described by Gokani et al. (1998).

(a) Preparation of conjugates:

In indirect ELISA assay antigen is absorbed on the wall of microtiter plate.

PGRs are haptens of low molecular weight and cannot be absorbed directly

on polystyrene plate wells. For this reason PGRs were conjugated with bigger

protein molecules to facilitate binding. Immunization was done in PGRs-BSA

conjugate therefore; it gives cross reaction with BSA. To avoid artifact of

assays, PGRs were conjugated with casein. The procedure was same as

PGRs-BSA conjugate. Casein was added instead of BSA.

(b) Adsorption of antigen

First step involved in indirect ELISA is adsorption of antigen on wall of wells of

microtiter polystyrene plate. PGRs-casein conjugate were diluted to different

concentration with freshly prepared coating buffer, which was made by

100mM carbonate buffer (Na2CO3, pH 9.7). Then, 300 µl of respective antigen

solution was added to each well and incubated at 37 0C for 3h or overnight at

4 0C to allow complete binding of antigen. The positive and negative controls

had to left for this procedure.

(C) Washing of plate

The plate was washed thoroughly thrice with phosphate buffer saline, pH 7.2,

containing 0.05% V/V tween-20 (PBS-T). The plate was flooded with PBS-T


20

and incubated for 5 min, which was then inverted and wrapped with filter

paper.

(D) Blocking of free protein binding sites

The next step involved was blocking of free protein binding sites of well in

order to check direct biding of antibodies on well. The blocking buffer was

made with egg albumin/PBS-T in mg/ml manner. 300 µl of blocking buffer was

added into each well (except positive and negative controls) and incubated for

2 h at 37ºC then the plate was washed thrice with PBS-T.

(E) Coating of antibody

After the blocking of free protein binding sites, next was coating antibodies

into the wells. The sample was diluted in different concentration with PBS, pH

7.2 and mixed with respective antibodies in 1:1 dilution. This was mixed well

and incubated overnight at 4ºC. This mixture was coated into the wells and

incubated for 3 h at 37ºC or overnight at 4ºC.

(F) Coating of secondary antibody

Anti-rabbit got IgG tagged with peroxidase enzyme was used as secondary

antibody. It was diluted with PBS (1:2500) and 300 µl was added into each

well. The plate was incubated for 2 h at 37ºC followed by three washes with

washing buffer (PBS-T). Color was developed by using O-phenylene diamine

(OPD) as a substrate and reaction was termined with 1M sulphuric acid

(H2SO4). The color developed was measured at 490 nm by ELISA Reader (µ

Quant, Biotek, USA). Endogenous level of hormones estimated from Banana

shoot tip was expressed, as µg/g FW.

In vitro culture:

For rapid in vitro multiplication of banana, shoot tips of desi variety were used

as explants.


21

Chemicals and Hormones:

6-Benzyl amino purine (BAP), naphthalene acetic acid (NAA), agar (1.2%),

activated charcoal, adenine sulphate, ascorbic acid, MS salts (Table -1.2),

sucrose (3%) and other all the chemicals and media components were

purchased from Duchefa Biochemical Netherlands.

Media preparation

MS (Murashige and Skoog 1962) media as described in Table -1.2 was used

as the basal medium. Different concentrations of hormones were applied based

on endogenous hormone concentration (Table -1.3) as described in Table -1.4.

For induction of root, full strength of MS media with activated charcoal and

different concentration of NAA were used Table -1.5.

Sterilization process

The suckers for micropropogation of Desi variety were soaked in 1-2%

Bavistin for 2 hours to remove the fungus and fungal spores. Then they

washed in running tap water, dipped into 0.2 % detergent (95 % laboline + 5

% tween 20) for 10 min and washed under tap water. Washed suckers

transferred to laminar air flow chamber, sterilized with 0.1% mercury chloride

(HgCl2), washed thrice using distilled water. Then, its dipped in antibiotic

mixture solution containing 100 ppm of each gentamycine, ofloxacine,

ceptazidine, ampicilline and rafempcine for 10 min, washed twice with distilled

water. The layers of sucker were removed with sterile scalpel and shoot tips

dipped into 70% ethyl alcohol for 30 second, and finally washed thrice with

sterile distilled water.

Explants Inoculation

The aseptically sterilized explants were transferred to the sterilized glass

plates under laminar air flow cabinet; they were further cut in 1-1.5 cm length

along with meristematic region, and each explant was placed erect position in

the early prepared autoclaved basal MS media containing macro nutrients,

micronutrients and vitamins (Table -1.2) and various concentration of plant


22

growth regulator for multiple shoot induction (Table -1.4 and -1.5). All medium

components were mixed and adjusted to pH 5.8 prior to autoclaving at 121 oC, 15 lbs for 20 minutes. Culture vessels were placed in the culture room

under the standard conditions of temperature (25 ± 2ºC) for 16 hours of

daybreak and 8 hours night break under cool white fluorescent light.

Shoot multiplication:

After approximately 10-15 days of incubation, the proliferation of shoot was

seen in some explants. For multiplication, the shoots were dissected to

remove undesirable blackening of some parts and decapitate and further

placed in a fresh medium. Further culture vessels were placed in the culture

room under the standard conditions of temperature (25 ± 2ºC) for 16 hours of

daybreak and 8 hours night break under cool white fluorescent light.

Root induction of shooted clones:

In vitro raised shoots were excised and transferred to medium containing the

early prepared autoclaved MS media containing macro nutrients,

micronutrients and vitamins (Table -1.2), various concentration of plant

growth regulator for root induction (Table -1.4 and -1.5) and 0.3% activated

charcoal. All medium components were mixed and sterilized according to the

conditions mentioned previously.

Hardening of rooted plantlets:

In vitro rooted shoots were carefully taken out of the culture bottle and gently

washed under sterile distilled water so as to remove all traces of agar. The

shoots were dipped in 1 % aqueous solution of bavistin, a systemic fungicide

for 5 min and then washed with sterile distilled water. Thereafter the treated

shoots were transferred in 8 cm thermocol pots containing a mixture of

unautoclaved soil: sand: wormiculite (1:1:1). For ensuring high humidity, pots

were kept under net house and plantlets were sprinkled with water as per

plant requirement for high humidity.


23

Results and Discussion

In vitro propagation of bananas provides excellent advantages over traditional

propagation, including a high multiplication rate, physiological uniformity, the

availability of disease-free material all the year round, rapid dissemination of

new plant materials throughout the world, uniformity of shoots, short harvest

interval in comparison with conventional plants, and faster growth in the early

growing stages compared to conventional materials (Vuylsteke 1989, Daniells

and Smith 1991). This study provides detailed information on

micropropogation procedures in banana along with endogenous hormone

detection with the objective of facilitating healthy banana seedling to banana

growers. The present study reports a very simple one step protocol using MS

with BAP for initiation, multiplication and elongation of shoot and MS with

NAA for rooting of banana. The protocol raised in the present attempt could

be used for the massive in vitro production of the plantlets of the banana.

Estimation of endogenous phytohormones

Endogenous hormone has been measured to minimize the efforts in

designing the hormonal combination. The endogenous hormonal level plays

an important role in developmental stages in vitro. This endogenous hormonal

analysis reduces time; labor and cost required for designing and testing the

hormonal combination suitable for efficient micropropogation (Ramanuj et al.

2010). Several authors reported the endogenous levels of cytokinins,

indoleacetic acid (IAA), abscisic acid (ABA) in banana leaves (Zaffari et al.

1998). In present study, the level of endogenous hormones in different

varieties of banana (Desi, red and Khasdi) i.e. IAA, PAA, kiN, and GA were

measured from meristematic shoot tips.

Estimation of endogenous level of hormones are presented in Table -1.3. It

was observed that amongst the three varieties studied for hormonal

estimation, Desi variety have more IAA (13.64) μg/g fresh wt, followed by Red

variety (3.26) μg/g fresh wt and Khasdi (0.5) μg/g fresh wt. similarly, PAA

levels were also studied in these varieties showed almost equal levels in Red


24

(21.92) μg/g fresh wt and Khasdi (21.90) μg/g fresh wt while it was low in Desi

(13.64) μg/g fresh wt. Thus IAA was more in Desi while PAA was found to be

more in other two varieties. These results are different from the result

obtained on cotton fiber varieties (Gokani and Thaker 2002) and on Cajanus

cajan (Chudasama and Thaker 2007) where PAA levels were more than IAA.

Levels of kinetin were more in Desi (59.66) μg/g fresh wt followed by Khasdi

(49.62) μg/g fresh wt and Red (36.05) μg/g fresh wt. However, gibberellic acid

acid was more in Red variety (24.17) μg/g fresh wt followed by Khasdi (21.71)

μg/g fresh wt and lowest was observer in Desi (15.5) μg/g fresh wt. Hormonal

balance play an important role in growth and development of plant is well

documented (Ammirato 1983, Gaspar et al. 1996, Mercier et al. 2003). Since

Desi variety is popularly growing around the area, micropropogation protocol

is designed for Desi variety for uniform growth of the plant and quality and

quantity of the fruit per unit area.

The prior knowledge of endogenous hormonal levels in explant helps to

minimize the number of attempts required to design the best suitable

hormonal combination for multiple shoot culture (Mercier et al. 2003). The

endogenous plant hormone is believed to be involved the regulation of cell

growth and development (White and Rivin 2000). This endogenous hormonal

concentration varies from cell to cell and from tissue to tissue (Gokani et al.

1998). The reports suggest that cell growth regulation may be controlled by

balance or ratio between hormones (White et al. 2000). The uptake of

exogenous hormones from the medium represented an essential

requirement, causing an increase of endogenous hormonal levels which

appear to be involved in the shoot organogenesis process (Mercier et al.

2003). Auxin like NAA induced multiple shoots or adventitious bud under in

vitro system in plant (Mala et al. 2005). There are some reports indicating that

cytokinin could induce adventitious shoot buds and/or multiple shoots from

cultures of shoots tips and nodal stem explant on MS medium in the presence

or absence of cytokinin like BAP, zeatin or kinetin (De Klerk et al. 2001).

Inclusion of BAP in the medium has been reported to form a clonal sector in

cotton meristems which causes the development of multiple shoots (Hazra et


25

al. 2000). The high performance of BAP over other cytokinins in the

multiplication of shoot tips has been reported in different cultivars of banana

(Wong 1986, Gilmar et al. 2000). In contrary, Priyono (2001) reported that

BAP has no positive effect on the cormlet development of giant cavendish

banana, which all concentration of BAP tested inhibited initial development. In

addition BAP also has an important role in stimulating and proliferating lateral

bud growth in other plants such as Centella asiatica, Kaempheria galanga

and Bacopa monerria (Shirin et al. 2000, Tiwari et al. 2000, 2001).

Strosse et al. (2008) studied the effects of different cytokinins on banana

proliferation. In particular, cytokinins have been found to reduce the

dominance of apical meristems and induce axillary shoots, as well as

formation of adventitious shoot from meristematic explants (Madhulatha et al.

2004). Vidhya and Nair (2002) also reported the micropropagation of banana

which resulted from the high concentration of BAP in the medium. Pennazio

and Vecchiati (1976) recognized the fact that higher concentration of NAA

inhibit root and shoot growth. In vitro propagation of bananas using shoot tips

has been reported for many commercial cultivars (Kulkarni et al. 2004, 2006).

Meristem culture provides a reproducible and economically viable method for

producing pathogen free plants. As meristem tips are free from viruses,

elimation and generation of virus free plants are possible through meristem

culture (Jha and Ghosh 2005). Plantlets produced through micropropagation

method have been found to establish faster, healthier, stronger, shorter

production cycle and higher yields than those produced through conventional

methods (Ortiz and Vuylsteke 1996). Banana is probably the most intensely

micropropagated crop. However, a large number of banana genotypes need

to be screened for commercial micropropagation.

Based on this, in present experiment Desi variety was selected for the

establishment of protocol and the media was formulated with high

concentration of cytokinin (BAP) ranging from 2.0-6.0 mg/l and low

concentration of auxin (NAA) ranging from 2.0-4.0 mg/l Table -1.5.


26

Initiation of shoot:

The series of media containing different concentration of BAP ranging from 1

to 6 mg/l were studied. The initiation of shoot was observed after 12 days of

inoculation in each media. At subculture level the hardness of the tissue was

observed in each shoots. Sprouting of shoot was low in media containing 1.0

mg/l BAP this hormonal concentration (Figure 1.1a). Single leaf development

of shoot was observed in media containing 2.0 mg/l BAP, same as observed

in case of 1.0 mg/l BAP media (Figure 1.1b). In media containing 3.0 and 4.0

mg/l BAP, sprouted shoot was observed relatively better developed

conditions. At subculture level the hardness of the tissue was which rigidified

material to use for manipulations in in vitro condition is observed (Figure 1.1c

and 1.1d respectively). The initiation of 2-4 leaves with healthy development

of shoot was observed in media containing 5.0 mg/l BAP (Figure 1.1e), while

with 6.0 mg/l BAP in media two leaves showing slight healthy shoots was

observed (Figure 1.1f). Thus, in general, 5.0 mg/l BAP containing media was

sound better at initiation stages of culture growth.

Khanam et al. (1996) reported that high BAP concentration along with an

increase in the number of sub-cultures stimulated higher number of stunted

axillary bud proliferation from shoot tips explants of different banana cultivars.

Vidya and Nair (2002) reported the rate of shoot multiplication depends both

on the cytokinin concentration and the genotype of bananas.

Sub culture for multiplication:

After 12 days of initiation, the shoots were cut at the base, separated and sub

cultured to a fresh medium containing same hormonal concentration from

where they are subcultured. In major cases, after a week multiple shoots

were developed from the sub cultured shoot. Multiple shoot formation was not

observed in 1.0 and 2.0 mg/l BAP (Figure 1.2a and 1.2b, respectively). Media

containing 3.0 mg/l BAP concentration shown 4-5 shoots of 3-4 cm height

(Figure 1.2c) while 4.0 mg/l BAP concentration shown 6-7 shoots of 4-5 cm

height (Figure 1.2d). The higher numbers of shoots were observed in media

containing 5.0 mg/l BAP concentration i.e. 8-10 shoots of 5-7 cm height


27

(Figure 1.2e). In media containing 6.0 mg/l BAP concentration, 6-7 shoots of

3-4 cm height were observed (Figure 1.2f). Again subculture was done on a

new fresh medium.

Arinaitwe et al. (2000) reported that shoot proliferation from banana was

cultivar dependent. They stated that increasing of BAP concentration above

16.8 μM (~ 4 mg/L) did not significantly increase shoot proliferation in banana

cultivars. In addition, they reported that the cultivar responded significantly

when concentrations of BAP increased upto 28 μM (~ 7 mg/L). Similarly in the

present study also higher concentration of kinetin upto 5 mg/l showed healthy,

long and good number of the shoots/explant. Therefore it is concluded that

media containing 5 mg/l BAP showed optimum results with the variety

studied.

Shoot proliferation and multiple shoot development:

For multiple shoot development, shoots were further cut with two gentle cross

incisions upon apical meristem, separated and sub cultured in a fresh

medium as described above. After a week of second subculture the number

of auxiliary buds were developed (Table -1.4). In 1.0 mg/l BAP containing

media, slow growth and 3-5 shoots of 2-3 cm in length was recorded (Figure

1.3a). In a medium containing 2.0 mg/l BAP also showed slow growth and 4-5

shoots of 3-5 cm in length (Figure 1.3b) per explant. In a medium containing

3.0 mg/l BAP showed 6-7 shoots of 4-5 cm in length (Figure 1.3c) and 4.0

mg/l BAP showed 5-6 shoots of 4-5 cm in length (Figure 1.3d). The higher

numbers of shoots, 8-10, with 4-6 cm in length were observed with 5.0 mg/l

BAP containing hard ball like structure developed from the meristem (Figure

1.3e). The medium containing 6.0 mg/l BAP developed 6-8 shoots of 3-5 cm

in length (Figure 1.3f). Finally, 5.0 mg/l BAP concentration that gives higher

number of shoots was chosen to carry out further culture process.

Rahaman et al. (2004) had also observed hard ball like structure developed

from meristem explant in MS media supplemented with 5.0 mg/l BAP. They

noticed that single shoot regeneration from meristem explant was thinner than

shoot derived from shoot tip. Similar results were also obtained by Habib


28

(1994) and Ali (1996) in their experiments. They observed that some ball like

structures formed at the base of the shoot during shoot multiplication. These

balls like structures were suitable for in vitro germplasm conservation.

Final Multiplication:

Starting of shoot multiplication was observed after 7-8 days in final 5.0 mg/l

BAP concentration. After 12 days of sub culturing, clumps of 5-7 shoots were

observed with 4-5 cm height (Figure 1.4a). The results were recorded after 5

days intervals, 8-10 shoots observed with 5-6 cm height (Figure 1.4b), 10-12

shoots with 5-7 cm height (Figure 1.4c). 12-14 with 4-7 cm height (Figure

1.4d) and 12-14 shoots with 6-8 cm height (Figure 1.4e) respectively after 15,

20, 25 and 30-40 days time intervals. These shoots were suitable for root

development. BAP at 22.2 µM was optimal in investigation by Cronauer and

Krikorian (1984) and Jarret et al. (1985) and at 20 µM in a study by Vuyslteke

(1989). Wong (1986) stated that 44.4 µM BAP reduced shoot multiplication.

Arinaitwe et al. (2000) stated that shoot proliferation was cultivar dependent.

Root induction:

The auxins (NAA, IAA or IBA) are most frequently used to induce root

initiation in the banana (Vuylsteke 1989). Rooting is also achieved on basal

medium without any growth regulators (Cronauer and Krikorian 1984, Jarret

et al. 1985). The influence of activated charcoal on rooting is reported in the

literature. Cronauer and Krikorian (1984) reported that when NAA, IAA or IBA

was added to the medium in the presence of 0.025% (w/v) activated charcoal,

no difference in rooting was observed. Rooting can be stimulated when

individual shoots are transferred to basal medium alone (Cronauer and

Krikorian 1984, Jarret et al. 1985). However, auxins may induce further root

initiation (Vuylsteke 1989). Vuylsteke (1989) found that NAA (1 µM) was more

effective than IAA. The optimum IBA concentration was found to be 1 µM by

Vuylsteke and De Langhe (1985). Using activated charcoal alone for rooting

will reduce the cost of producing plantlets for the field. Cronauer and Krikorian

(1984) reported no differences in the root-inducing effects of NAA, IAA or IBA


29

in presence of 0.025% (w/v) activated charcoal. Hwang et al. (1984)

recommended 0.1-0.25% activated charcoal.

In this study, for root induction, shoots were separated from shoot clumps and

transferred to rooting medium containing different hormonal concentration

ranging from 2.0 to 4.0 mg/l NAA and activated charcoal (Table -1.5). After 10

days of transfer, 2.0 and 2.5 mg/l NAA containing medium shown poor rooting

(Figure 1.5a and 1.5b). The rooting medium containing 3.0 mg/l NAA shown

healthy growth of rooting with good quality numbers (Figure 1.5c). With

increase in hormonal concentration 3.5 and 4.0 mg/l NAA shoots were

healthy but poor in numbers of root was observed (Figure 1.5d and 1.5e).

Thus, 3.0 mg/l NAA was considered better for root induction.

Plantlet development:

For final rooting medium containing 3.0 mg/l NAA and activated charcoal

were used. Figure 1.6a shows observation of healthy growth of rooting from

elongated shoot after 15 days of transfer to rooting medium. While Figure

1.6b and 1.6c shows rooting and shoot elongation after 20 and 30 days. The

plantlets after 30 days time period were ready for hardening. Vessey and

Rivera (1981) reported root formation occurred 50 days after shoot

development. In contrast Berg and Bustamante (1974) noted that it needed 2-

3 months for root formation.

Hardening:

Al-Amin (2009) had transferred meristem derived plantlets to poly bags

containing 1:1 (ground soil: cowdung) mixture after 7 days hardening in room

temperature. Kalimuthu et al. (2007) had also transferred the elongated

shoots with roots (about 8-9 cm) to hardening and achieved a survival rate of

90-95% during the process. Similar to this in this study for hardening the

plantlets was prepared with removing of agar media. The plantlets from

culture vessels/bottles were moved from the laboratory to a room at ambient

temperature. Then plantlets were gently washed with sterile water to remove


30

agar and then planted in individual cup containing sand: soil: wormiculite

(1:1:1). Rooted plantlets were established for maintenance of humidity and

temperature in a small chamber. A good number of established plantlets were

shown in Figure 1.7. A survival rate of 40-45% was achieved during the

hardening.

Trouble shooting:

The formulation of plant tissue culture media for good number of plantlets

from the elite explants care need to be taken for sufficient micro and macro

nutrient with vitamins and carbon sources and a special supplement if

required. In addition adequate hormonal concentrations are designed by trial

and error method for desired results. In this study MS salts along with

vitamins and carbon source were tried. Endogenous level of hormones

estimated from number of the explants randomly selected from the field have

minimized the time for selection of proper hormonal concentration.

As reported in most published papers, the ability of various explants to

regenerate in culture varies not only with the cell and tissues themselves but

also with the species, cultivar and even the individual genotype (Venkataiah

et al. 2003). In addition, the developmental stage, location of plant tissue and

environmental factors such as temperature, light regime and intensity are also

critical in plant shoot regeneration (Szász et al 1995).

Contamination is quite frequent in the field grown explants and damage good

amount of culture if sufficient care is not taken. Bacterial and fungal

contamination was observed due to insufficient sterilization of explant or

entophytes after 3-5 days of inoculation in sterile medium. Plate 1c and 1d

shown the bacterial contamination observed during tissue, while figure 1.8e

and 1f shown the fungal contamination in culture. To overcome these

problems different sterilization treatments were used. Broad range of

antibiotic such as ampicillin, gentamycine, rifampicine, ofloxacine and

ceftaxime (50-100 ppm) were used to prevention of bacterial growth and

bavistine (0.1-1 %) was used for prevention of fungal contamination.


31

Initiated meristematic region appeared as a dark green after 8-9 days and 12-

14 days respectively (Figure 1.8a and 1.8b). Explants in culture grew to about

2-3 cm in length after 2 weeks of culture. Buds usually emerge from only one

cut end, of the younger tissue. In the subculture medium, leaves developed

from the adventitious buds. However, the shoot buds appeared abnormal

after transfer into the fresh medium. The leaves became pale green and they

broke readily at the point of attachment. Subsequently the buds turned brown

and failed to elongate into a normal shoot. The explant was greenish and

yellow in color after 8-10 days and become brown and black, resulted failed to

develop further after 2 weeks in subculture medium. To overcome browning

of media due to necrosis, explant is pretreated in ascorbic acid and cultured

in medium supplemented with activated charcoal.

Table 1.1. Review of in vitro investigation on Musa spp. (MS= Murashige and Skoog)

No. Medium Auxin Cytokinine

References NAA IAA IBA BAP KiN

1 MS - - - 4 mg/l - Mohammad Amiri 2008 2 MS - 1.0 mg/l - 1.0 mg/l - Zaffari et al. 2000 3 MS - 2.0 mg L - 2.0 mg L 2.0 mg L Titov et al. 2006 4 MS - 2.0 mg L - 4 mg/l - Sarder et al. 2006 5 MS - - 0.6 mg/l - - Molla et al. 2004 6 MS - - 1.0 mg/l 3.75 mg/l 2.0 mg/l Khan et al. 2001 7 MS 1.0 mg/l - - 1.0 mg/l - Mangi et al. 2000 8 MS - - 1.0 mg/l 5.0 mg/l 5.0 mg/l Rahman et al. 2006 9 MS - - - 8.0 mg/l - Darvari et al. 2010

10 MS - 0.2 mg/l - 2.25 mg/l - Koffi et al. 2009 11 MS 2.0 mg/l 2.0 mg/l 2.0 mg/l 5.0 mg/l - Bairu et al. 2006 12 MS 1.0 mg/l - - 2.0 mg/l - Kulkarni et al. 2007 13 MS 3.0 mg/l - 1.0 mg/l 6.0 mg/l - Jambhale et al. 2000 14 MS 0.2 mg/l - - 4.5 mg/l - Gubbuk and Pecmezci 2004 15 MS 2.75 mg/l 0.5 mg/l 0.5 mg/l 7.5 mg/l - Al-Amin et al. 2009 16 MS 2.0 mg/l - - 3.0 mg/l - Kalimuthu et al. 2007 17 MS - - - 2.0 mg/l - Youmbi et al. 2006 18 MS - - 1.0 mg/l 5.0 mg/l - Narayan et al. 2008 19 MS - - - 10 mg/l - Promsorn and Kanchanapoom 2006 20 MS - - - 5 mg/l - Nandi 1995 21 MS - 2.0 mg/l - 2.0 mg/l - Farahani et al. 2008 22 MS - 0.75 mg/l - 3.0-10.0 mg/l - Abeyaratne and Lathif 2002 23 MS - - - 5.0-8.0 mg/l - Subramaniam et al. 2008 24 MS 1.12 mg/l 2.6 mg/l - 2.25 mg/l - Ikram ul Haq and Dahot 2007 25 Knudsons 1 mg/l - 1 mg/l - - Berg and Bustamante 1974

Table 1.2. Composition of MS medium supplemented (Murashige and Skoog, 1962).

Sr no. Types of nutrient

Name of nutrients

Amounts (mg/l)

1 Macronutrients

NH4NO3 1650 KNO3 1900

CaCl2. 2H2O 440.0 MgSo4. 7H2O 370.0

KH2PO4 170.0

2. Micronutrients

MnSO4.4H2O 23.00 FeSO4. 7H2O 27.96 ZnSO4. 7H2O 8.600

H3BO3 310.0 KI 8.300

Na2MoO4. 2H2O 0.250 CoCl2.6H2O 0.020 CuSO4.7H2O 0.020

Na2EDTA. 2H2O 37.26

3. Vitamins

Nicotinic acid 0.500 Pyridoxine HCL 0.100 Thiamine HCL 0.500

Myo inositol 100.0 Glycein 2.000

4.

Adenine sulphate*

10.00

5.

Ascorbic acid*

20.00

6.

Activated Charcoal*

3000

∗ Additional added to the MS composition

Table 1.3. Endogenous level of hormone in Musa varieties (Indoleacetic acid, phenyl acetic acid, kinetin and gibberellic acid) from meristematic shoot tip.

Hormone IAA PAA Kinetin GA

Variety µg / g Fwt

Khasdi - 21.90 49.62 21.71

Desi 13.64 8.36 59.66 15.50

Red 3.26 21.92 36.05 24.17

Table 1.4. Cytokinin concentrations supplemented to the basal medium for

multiple shoot induction.

Media PGR concentration Subculture

BAP (mg / L) No. of shoot Shoot length (cm)

MS

2.0 3-5 2-3

3.0 4-5 3-5

4.0 6-7 4-5

5.0 8-10 4-6

6.0 6-8 3-5

Table 1.5. Auxin concentrations supplemented to the basal medium for root induction.

Media PGR concentrationNAA (mg / L)

MS

2.0

2.5

3.0

3.5

4.0

Chapter 2 Molecular marker studies of Banana


32

Abstract

Molecular markers are popularly used to characterize plant genetic diversity.

High quality DNA extraction protocol is prerequisite for molecular marker

studies. In the present study, DNA isolation method M5 was optimal among

tested protocols for banana tissue which contains high amount of polyphenol

and polysaccharide. The DNA extracted by this method was subjected to

RAPD and ISSR based analysis. The results obtained using RAPD and ISSR

primers for four Musa varieties showed recordable monomorphic and

polymorphic band patterns. RAPD has shown 22.99 % polymorphism while

ISSR shown 23.08 % polymorphism. These primers were able to generate

sufficient markers to discriminate these varieties. Monomorphic and

polymorphic bands pattern of selected primers in all Musa varieties were

eluted from the gel and sequenced. The obtained sequences were submitted

to GenBank. The UPGMA relatedness was derived from nucleotide

sequences and protein sequences. The REN cutting sites were detected to

convert monomorphic band into polymorphic and unique marker of known bp

fragment for each varieties. These results promote the initiative to integrate

monomorphic bands in plant breeding applications, and DNA fingerprinting.

Bioinformatics tools such as tandem repeat finder used to locate and display

tandem repeats. The tandem repeats of GT rich region observed in nucleotide

sequence of Desi variety. This tandem repeats can be used as microsatellite

marker for Desi variety.


33

Introduction

Since Mendel (1866) started to formalize the empirical knowledge of farmers

and breeders into what later became known as genetics, the search for

“markers” has increased our knowledge of the genetic basis of inheritance.

Mendel used traits such as pea shape and color in order to trace the

hereditary process of segregation and assortment (Frankfurt et al. 1997).

Thus, traditional methods for testing genetic variability in fruit crops are based

on morphological or time consuming physiological assays (Scheliro et al.

2001). But the presently exist molecular assays are more advance to improve

these fruit crops (Williams et al. 1993, Smith et al 1994, Welsh et al. 1995,

Rafalski et al. 1996, Jain et al. 2007).

The botanical materials are characterized based on different types of markers

viz. morphological, biochemical and molecular markers (Kumar et al. 2009).

Morphological markers (such as plant height, leaf shape, colour, etc.) are

among the oldest markers used in the evaluation of genetic variability (Bhat et

al. 2010). However, they are not sufficiently specific and informative because

different gene expression in different environments causes wide variability of

phenotypic characters in individuals.

Similarly, biochemical markers (such isoenzymes or essential oil contents in

an individual organism) can be considered to be non-specific due to the wide

variability of biochemical characters, which are strongly influenced by an

individual's environment (Market and Moller 1959). The development of

recombinant DNA in the 1980s enabled the development and use of

molecular markers, thus providing a modern tool for determining genetic

variability (Mandaliya et al. 2010c).

Molecular markers relate directly to the plant’s genotype rather than its

phenotype and are based on differences in the nucleotide sequences of the

DNA in different individuals. Molecular markers have greater resolution than

phenotypic markers and, as they are based on physical DNA assays, require

more or less sophisticated technology. Genetic markers greatly facilitate the

selection of parents and assist in the development of new improvement


34

strategies based on marker-assisted breeding. As molecular markers can be

used at early stages of plant development, they have another big advantage

over most phenotypic markers. The ability to distinguish or identify one

individual or group of individuals from another has applications in many fields,

and has now been made much more efficient by the use of molecular

techniques (Frankfurt et al 1997).

Molecular markers techniques show variability among individuals at the DNA

level, which is not influenced by the environment. Different genetic markers

(e.g. RFLP, AFLP, RAPD, ISSR, SCAR) have different properties, (dominant

and codominant markers, different coverage of the genome) and different

advantages and disadvantages (e.g specificity, cost, ease of analytical

interpretation of the resulting data). However, they are highly informative

about genetic variability among individuals, populations and cultivars. Their

use is universal for all organisms. Molecular markers can be considered to be

essential tools in cultivar identification (DNA typing), assessment of genetic

variability and relationships, management of genetic resources and

biodiversity, studies of phylo-genetic relationships and in genome mapping

(Vural 2009). Thus, the molecular marker technologies have become a

powerful tool in crop improvement through their use in germplasm

characterization and fingerprinting, genetic analysis, linkage mapping, and

molecular breeding (Sheidai et al. 2010).

Random Amplified Polymorphic DNA (RAPD) requires only small amount of

DNA sample without involving radioactivity tests and are simpler as well as

faster. RAPD has proven to be quite efficient in detecting genetic variations

(Williams et al. 1990), even in closely related organisms such as two near

isogenic lines (NIL). At present, RAPD markers have been successfully

applied to detect the genetic similarities or dissimilarities in micropropagated

material in various plants (Carvalho et al. 2004). Lakshmanan et al. (2007)

had monitored the genetic stability of banana using the RAPD technique.

Microsatellites or simple sequence repeats are 1-6 based DNA sequences

that is tandem repeated (Joshi et al. 2000). Although, SSR is new generation


35

powerful marker for eukaryotic system, hampered by the requirement for

sequence information from flanking region from which primer are designed for

PCR amplification (Powell 1996). A modification of SSR-based marker

systems, i.e. ISSR analysis (Wolfe and Liston 1998), circumvents this

requirement for flanking sequence information. ISSR technique is also very

simple, fast, cost-effective, highly discriminative and reliable (Pradeep et al.

2002), thus has found wide application in plant systems. This analysis

involves PCR amplification of genomic DNA using single primer that targets

the repeat per sequence with 1-3 based that anchor the primer at the 3’ or 5’

end. In addition, optimization of amplicons can be achieved by changing

number and type of anchored base in primer design and by judicious

combining of primers.

The ISSR analyses published to date were based on monomorphic and

polymorphic band patterns. Frequently in such analyses the most

distance/similarity index are often used to construct phylogenetic tree. The

controversy remains over whether or not particular monomorphic band in an

amplicon (i.e. similar molecular weighted band amplified in each samples

tested) has similar nucleotide sequence and such assignments are not

appropriately taken into account. In this study, an innovative strategy to

exploit monomorphism rather than identifying polymorphic band with marker

technology, problem was designed to develop a sequence data set which

comprised exclusively of monomorphic bands.

A tandem repeat in DNA is two or more adjacent, approximate copies of a

pattern of nucleotides. It has been shown to cause disease or mutation, may

play a variety of regulatory and evolutionary roles and is important laboratory

and analytic tools (Benson 1999). Extensive knowledge about pattern size

copy number mutation history etc. for tandem repeats has been limited by the

inability to easily detect them in genomic sequence data. This can be easily

detected by a program called tandem repeat finder. In this study, tandem

repeat in nucleotide sequences of monomorphic and polymorphic were

identified to develop a variety specific marker.


36

Isolating high quality DNA is essential first step for molecular research.

According to Ramavat et al. (2010) to accommodate the need for the

molecular marker technologies, a rapid, simple, and reliable DNA preparation

method is required to provide high quality and quantity DNA for the analyses.

A reliable method should meet the following criteria: (i) require only a small

amount of tissue; (ii) involve simple procedures; (iii) use minimal number and

amount of chemicals; (iv) yield high-purity DNA; (v) yield large quantities of

DNA; (vi) should be inexpensive; (vii) applicable to various varieties.

Banana leaf contains exceptionally high amounts of polysaccharides,

polyphenols and secondary metabolites such as alkaloids, flavonoids and

phenols, and terpenes which interfere with DNA isolation procedure. The

problem encountered due to these compounds include co isolation of highly

viscous polysaccharides degradation of DNA due to endonuclease, inhibitor

compounds like polyphenols and other secondary metabolites which directly

or indirectly interfere with enzymatic reactions. Moreover, the contaminating

RNA that precipitates along with DNA causes suppression of PCR

amplification and other many problems (Pikkart and Villeponteau 1993,

Padmalatha and Prasad 2006) interference with DNA amplification involving

random primers e.g. RAPD analysis (Padmalatha and Prasad 2006) and

improper priming of DNA templates during DNA cycle sequencing.

Various protocols for DNA extraction have been successfully applied to many

plant species (Doyle and Doyle 1987, Sharma et al. 2002), though DNA

isolation by using one general protocol could not possible to achieve optimal

DNA yield from different plant taxa. For example, some closely related

species of the same genus require different isolation protocols (Das et al.

2009). DNA extraction from plant is preferentially performed from young

tissues due to the lower content of polysaccharides, Polyphenols and other

secondary metabolites which co-precipitate with DNA in the extraction

procedure, inhibit DNA digestion and PCR (Zang and Mc Steward 2000),

presumably by irreversible interactions with DNA (Dabo et al. 1993).


37


Plant Material

Musa varieties viz. Desi, Red, Khasdi and Soneri were collected from

Visanvel, Junagadh (Gujarat) and maintained at Botanical Garden,

Saurashtra University, Rajkot. Fresh, young, and expanded leaves were

collected for DNA extraction.

DNA extraction

Method-1 (M1)

Genomic DNA extraction was carried out first following the protocol reported

by Mohamed (2007), this method was termed as Method-1 (M1).

Method-2 (M2)

In later stage certain modification was done in M1 of Mohamed (2007) to

improve the DNA purity suitable for PCR based analysis, this modified method

was termed as Method-2 (M2). In brief, in initial step 0.5 g of leaves was

grinded in liquid nitrogen and homogenized in extraction buffer as described

by Mohamed (2007). The tubes were incubated at 60ºC for 20 min followed by

centrifugation at 10,000 x g for 5 min at room temperature. Then after directly

RNase treatment were given instead CI treatment and DNA precipitation. The

PCI and CI both treatments were given twice instead of single. Subsequent

DNA extraction steps were carried out according to Mohamed (2007).

Method-3 (M3)

In this method, genomic DNA extraction was carried out following the protocol

reported by Mandaliya et al. (2010a) for high yield and quality DNA extraction,

this method was termed as Method-3 (M3). In brief, in initial step one g of

leaves was grinded, instead of two g, in liquid nitrogen along with 500 mg

polyvinyl pyrrolidone polymer (PVPP) instead of 200 mg PVPP. Eight ml of

freshly prepared and pre-heated Triton-X-100 extraction buffer as described


38

by Mandaliya et al. (2010a) were added and mixed well. Following DNA

extraction steps were carried out according to Mandaliya et al. (2010a).

Method-4 (M4)

In M3 certain modification tested and this modified method was termed as

Method-4 (M4). In brief, one g of leaves was grinded in liquid nitrogen along

with 500 mg polyvinyl pyrrolidone polymer (PVPP). Eight ml of freshly

prepared and pre-heated Triton-X-100 extraction buffer as described by

Mandaliya et al. (2010a) were added and mixed well. The tubes were

incubated at 65 oC in water bath for 30 min rather than 10 min. The

successive DNA extraction steps were carried out according to Mandaliya et

al. (2010a) where the CI treatment was given twice instead of single.

Method-5 (M5)

This is slight modified protocol based on M4 method in which the CI treatment

was given single instead of twice to test the DNA yield loss.

DNA quantification

The DNA concentration was determined at 260 nm and purity was determined

by calculating the ratio of absorbance at 260 nm to that of 280 nm (Matasyoh

et al. 2008) using the μQuant microplate reader, Bio-Tek instruments

incorporation, USA. DNA concentration was determined using following two

equations. Water has virtually no absorbance between 200 and 900 nm, but it

has a small but significant peak at 977 nm. The microplate reader determines

the pathlength of the sample by measuring the absorbance of the water in the

microplate sample at 977 nm, blanks that value using a reference wavelength

of 900 and compares it to the absorbance of water at 1 cm. The ratio of those

two values was the pathlength of the sample (Eq. 1).

( A977 - A900 )sample / ( A977 - A900 )1.0cm water = Pathlength of sample

Eq.1


39

As background absorption of the microplate is generally quite high in the UV

range (below 340 nm), it is particularly important with UV absorbance

measurements to blank prior to pathlength correction in order to obtain an

accurate corrected value. Correction to 1 cm for DNA samples, which was

determined using the absorbance at 260 nm, would need to accommodate

this (Eq. 2).

{ ( A260sample) - ( A260blank) }/ Pathlength of sample

= Absorption of sample corrected to 1.0 cm

Eq.2

In terms of quantitation, different nucleic acid species have different average

extinction coefficients. By converting these known extinction coefficients to a

specific concentration for a particular absorbance value at a defined

wavelength, the nucleic acid concentration of solutions can be determined by

a simple absorbance measurement. For example, the extinction coefficient for

dsDNA (1 mg/ml) at 260 nm is 20 OD units; this can be rearranged to mean

that a DNA sample that returns an absorbance 1 OD unit has a concentration

of 50 μg/ml. Similar calculations have been performed for ssDNA

(oligonucleotides) and RNA species. Indicated values are accepted average

values for absorbance measurements at 260 nm with a 1 cm pathlength. The

DNA purity is measured using the Eq. 3 and 4 (Sharma et al. 2008).

A260 / A280 = Purity of DNA

Eq.3

A260 / A230 = Purity of DNA

Eq.4


40

The 96-well clear UV transparent Microplate was used. Distilled water in

triplicate, TE buffer in triplicate, and DNA sample solution, each 100 μl

aliquots were pipetted into microplate wells and absorbance measurements at

260, 280, 977, and 900 nm were then made using the μ Quant Microplate

Spectrophotometer, Bio Tek Instruments Incorporation, Winooski, VT. The

subsequent pathlength corrected absorbance was then transformed using the

appropriate conversion factor indicated in Table -2.1 for determination of the

concentration, and purity for samples in Microplate was carried out.

PCR amplification

The extracted DNA was subjected to RAPD (OPA and OPB series) and ISSR

analysis using selected primer enlisted in Table -2.3. Primers were selected

for PCR amplification. Primers were purchased from Operon Biotechnologies

GmbH, Germany. RAPD analysis was performed according Mandaliya et al

2010b. Each reaction mixture consists of total 12.5 μL. PCR amplification was

carried out in 3 stages in a Veriti (96 Well Fast Thermal Cycler), Applied

Biosystems, USA.

Agarose Gel Electrophoresis

Electrophoresis loading samples were prepared according to Sheidai et al.

(2007). Electrophoresis was carried out on 2 % agarose gel at constant

voltage of 50 V in 1x TAE buffer. High Range Ruler and Supermix DNA

Ladder, both from Bangalore Genei, India, were used as molecular size

standards.

Statistical Analysis

Jaccard’s similarity coefficient values for each pairwise comparison between

Musa varieties were calculated and a similarity coefficient matrix was

constructed with the help of Free Tree software (Rana et al. 2005). This

matrix was subjected to Unweighted Pair-Group Method for Arithmetic

averages analysis (UPGMA) to generate dendrograms (Hussein et al. 2007)


41

using CLC Main Workbench5 for Musa varieties. This software can be

obtained from http://www.clcbio.com.

Nucleotide sequencing

The primer products of I-8, I-16 and I-26 were synthesized once again and run

on 2 % low melting agarose gel at constant voltage of 50 V in 1x TAE buffer.

The selected bands were eluted and purified using Qiagen make QIAquick

Gel Extraction kit for nucleotide sequencing. The purified products were cycle

sequenced using Applied Biosystems BigDye Terminator v3.1 Cycle

Sequencing Chemistry on ABi Genetic Analyzer 3130 (Fernandez-Carvajal et

al. 2009). Sequences from the ISSR bands were compared with NCBI

database using BLAST program (Ghany and Zaki 2003). Then, multiple

sequence alignment (MSA) was performed on CLC Main Workbench5 to

construct phylogenetic relationship and to confirm availability of restriction

sites in selected sequences.

Prediction of protein sequences

The protein sequences were derived from the ISSR band sequences using

CLC Main Workbench5. Then, multiple sequence alignment (MSA) was

performed in protein sequences derived from monomorphic and polymorphic

ISSR band patterns.

ProtParam analysis

ProtParam a tool which allows the computation of various physical and

chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for

a user entered sequence (Gasteiger et al. 2005). The computed parameters

include the molecular weight, theoretical pI, amino acid composition, atomic

composition, extinction coefficient, estimated half-life, instability index,

aliphatic index and grand average of hydropathicity (GRAVY).

Aliphatic index

The aliphatic index of a protein is defined as the relative volume occupied by

aliphatic side chains (alanine, valine, isoleucine, and leucine). It may be

regarded as a positive factor for the increase of thermostability of globular


42

proteins. The aliphatic index of a protein is calculated according to the

following formula (Kyte and Doolittle 1982).

Aliphatic index = X(Ala) + a * X(Val) + b * ( X(Ile) + X(Leu) )

where X(Ala), X(Val), X(Ile), and X(Leu) are mole percent (100 X mole

fraction) of alanine, valine, isoleucine, and leucine. The coefficients a and b

are the relative volume of valine side chain (a = 2.9) and of Leu/Ile side chains

(b = 3.9) to the side chain of alanine.

GRAVY (Grand Average of Hydropathy)

The GRAVY value for a peptide or protein is calculated as the sum of

hydropathy values (Guruprasad et al. 1990) of all the amino acids, divided by

the number of residues in the sequence.

Instability index (II)

The instability index provides an estimate of the stability of your protein in a

test tube. Statistical analysis of 12 unstable and 32 stable proteins has

revealed (Guruprasad et al. 1990) that there are certain dipeptides, the

occurence of which is significantly different in the unstable proteins compared

with those in the stable ones. The authors of this method have assigned a

weight value of instability to each of the 400 different dipeptides (DIWV).

Using these weight values it is possible to compute an instability index (II)

which is defined as:

i=L-1

II = (10/L) * Sum DIWV(x[i]x[i+1])

i=1

where: L is the length of sequence

DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide starting in

position i.


43

A protein whose instability index is smaller than 40 is predicted as stable, a

value above 40 predicts that the protein may be unstable.

Tandem repeats finder

A tandem repeat in DNA is two or more adjacent, approximate copies of a

pattern of nucleotides. Tandem Repeats Finder is a program to locate and

display tandem repeats in DNA sequences. In order to use the program,

FASTA format is used. There is no need to specify the pattern, the size of the

pattern or any other parameter. The output consists of two files: a repeat table

file and an alignment file. The repeat table contains information about each

repeat, including its location, size, number of copies and nucleotide content.

Clicking on the location indices for one of the table entries opens a second

web browser that shows an alignment of the copies against a consensus

pattern.


44


DNA isolation and purification

Most procedures for DNA isolation and purification from plant species

containing high levels of polyphenolic and polysaccharide compounds are

modified versions of the standard protocols. Changes have been made to the

extraction buffer composition and to the purification steps (Permingeat 1998).

In this study with minor or major modifications five different protocols were

tested for good quality and quantity of DNA extraction from four varieties of

banana leaf tissue.

Mohamed (2007) had optimized conditions for DNA extraction from banana

and with extracted DNA reported that it shown reproducible PCR

amplification. Following this, in the first method (M1), genomic DNA extraction

was carried out using the protocol reported by Mohamed (2007). In M2, a few

changes were made in the M1 like RNase treatment was given before CI

treatment and DNA precipitation to reduce the extraction steps. The PCI and

CI both treatments were given twice instead of single to remove excess

protein contamination. M2 method has yielded double the amount of DNA with

better quality (Table -2.2).

The M3 was based on protocol described by Mandaliya et al. (2010a) for high

polyphenol and polysaccharide containing cotton tissues. This protocol was

tested on high polyphenol and polysaccharide containing banana tissues to

check its suitability for DNA extraction. Several changes had been made in

original method to develop simpler extraction of Banana DNA. In this method,

one g of leaves was used instead of two g to reduce the requirement of plant

material, while 500 mg PVPP used instead of 200 mg PVPP to improve DNA

purity. The M4 and M5 were slight modification of M3 to improve the DNA

purity and concentration. The M5 is one day protocol and involves simple

procedures and applicable to various banana varieties.


45

DNA Yield and Purity

Mohamed (2007) had reported that extracted DNA was suitable for

reproducible PCR amplification, while with same protocol M1, DNA yield was

16.69 μg/gm of fresh wt (Table -2.2) and DNA purity was 1.13. It indicated

excess protein contamination, so in M2, slight modification had done and it

achieved DNA yield between 30.93 μg/gm of fresh wt and DNA purity was

1.46. This result had indicated improvement in DNA concentration and purity,

but to achieve high quality DNA other protocols were also tested.

In M3, for high quality DNA extraction from Banana, recently reported protocol

by Mandaliya et al. (2010a) were tested with certain modification. The DNA

purity was 1.65 and DNA yield was 10.99 μg/gm of fresh wt achieved that

suggested this protocol was suitable for Banana tissue. The M4 and M5 were

processed to improve the DNA yield. In M4, DNA yield was 11.59 which was

slightly improved from M3, but in M5, DNA yield was 16.81 – 25.64 μg/gm of

fresh wt with DNA purity between 1.59 – 1.67. This protocol had provided high

quality of DNA. The extracted DNA samples were shown in Figure 2.1. The

M5 is simple, one day protocol and provide high quality DNA (Figure 2.1).

Each plant posses its own chemical compositions lead technique problem for

DNA isolation protocols. In this study, higher concentration of PVPP in

addition to the normally prescribed chemical yielded better quality and

quantity DNA in banana plants. PVPP removes phenolic groups from crude

DNA extracts via hydrogen bounding and formation of PVPP-phenolic

complexes (Coyer et al. 1995, Sutlovic et al. 2007). Similarly good quality and

quantity DNA was also obtained from microorganism using PVPP in isolation

protocol (Trochimchuk et al. 2003).

RAPD and ISSR Analysis:

The identification of desired varieties in Musa is of prime importance for crop-

improvement programs. However, the existences of numerous synonyms, the

long vegetative phase, and ambiguities in scoring morphologic traits


46

complicate the process of identification even of known varieties. These

limitations are surmountable by the DNA-fingerprinting approach. Using

molecular markers, it is now possible to make direct inferences about genetic

variability and interrelationships among organisms at the DNA level without

the confounding effects of environment. The number of studies reported in the

literature suggests ISSR analysis of plants (Pharmawati et al. 2005). The

present study was emphasized on RAPD and ISSR analysis based on

monomorphic and polymorphic band patterns in Musa.

Lakshmanan et al. (2007) compared and first reported the use of genetic

markers to establish the genetic fidelity of RAPD and ISSR patterns of 11

plants chosen from 4000 Musa plantlets with a control plant. A total of 377

scorable bands were generated out of 30 RAPD primers and 47 scorable

bands were generated from 5 ISSR primers. They had observed only

monomorphic patterns across all the plantlets analyzed. Venkatachalam et al.

(2007) also observed total of 16,875 bands from micropropagated plants and

14,175 bands from regenerated plants, which is the highest compared to all

other such studies reported so far (Carvalho et al. 2004, Martins et al. 2004,

Bennici et al. 2004, Ray et al. 2006). The primers they had selected based on

earlier report(s) where such primers were found to be of great use in

differentiating the varieties and developing phylogenetic relationships in

banana (Pillay et al. 2001, Ude et al. 2003, Onguso et al. 2004). The band

profile in their studies shows monomorphic pattern.

In present study, the DNA extracted by M5 was suitable for PCR based

analysis. For the screening of selected four varieties, 16 selected primers

were used, out of which 13 gave optimum RAPD bands with all the varieties

studied. OPB 2, OPA 5, and OPA 10 had not amplified any bands among

selected varieties. Hence, 13 primers were chosen for the analysis. A total of

187 bands were generated using these 13 primers (Table -2.4). The RAPD

amplification profile generated 22.99 % polymorphism (Table -2.5). The 30

ISSR primers including di, tri, tetra and penta nucleotide repeat are also

tested in this study. These have amplified DNA fragments ranging from 100 to

5000 bp length from the Musa varieties genomic DNA. Amongst the primers


47

studied (Table -2.3), 16 ISSR primers generated sharp, clear and reproducible

bands in all four Musa varieties (Table -2.6). Figure 2.2 and 2.3 shown

reproducible amplification product of Musa varieties viz. (i) Desi, (ii) Red, (iii)

Khasdi and (iv) Soneri produced by RAPD and ISSR primers, respectively.

The total number of bands, generated from 16 primers were 182 which

include 42 polymorphic and 140 monomorphic (Table -2.7); thus, percentage

of polymorphic bands observed 23.08 and of monomorphic 76.92. The

average 2.06 polymorphic amplicon per primer was observed. The maximum

number of amplified product was 20 (I-16) and minimum 2 (I-3).

The dendrogram was constructed based on RAPD and ISSR Jaccard

distance/similarity matrix (Table -2.8 and 2.9, respectively) using UPGMA

method (Figure 2.5). The dendrogram based on RAPD, ISSR, and combined

data of RAPD and ISSR were constructed that represented in Figures 2.5a,

2.5b, and 2.5c, respectively. Cluster dendrograms represents the genetic

distance and similarity by grouping the varieties in clusters and sub-clusters

(Esmail et al. 2008). The all dendrograms indicates Khasdi and Red varieties

were close from the diversity point of view, while Sonery was genetically

dissimilar from all varieties.

In numerous studies the primers either did not produce recordable profile or

amplification products in repeated experiments. Failure of several primers to

give informative banding patterns may be because technical problems like

those primers require special amplification condition such as alternative

chemical stabilizers of different annealing temperatures (Pharmawati et al.

2005). The type of gel electrophoresis and staining method used can also

influence the number of scorable bands and the level of polymorphism

obtained (Wiesner and Wiesnerova 2003). In studies, where polyacralamide

gel is used with silver staining instead of agarose gel with ethidium bromide

have resulted higher resolutions (Charters et al. 1996, Mathew et al. 1999).

Godwin et al. (1997) suggested polyacralamide gels visualized by

autoradiography of radiolabelled samples yielded good results.

Bands with same mobility were considered as identical irrespective of their

band intensity (Rout et al. 2009). Thus, PCR based amplification of gel helps


48

to presume that somewhat similar bp (length) monomorphic band pattern with

the identical primer among the varieties may express similar genomic region.

However, the reliability and efficiency of molecular markers in detecting large

scale genome arrangements have been frequently questioned and it is difficult

to conclude about the variations in amplified genomic region with limitations in

the technique. So, controversy remains over whether or not particular

monomorphic band observed by various authors (Lakshmanan et al. 2007,

Venkatachalam et al. 2007) as discussed above in each amplicon has similar

nucleotide sequence?

To address this problem, one of the monomorphic bands (Figure 2.4a) that

shown sharp and clear band pattern in all Musa varieties were eluted from the

gel and sequenced. The obtained sequences were 557-565 bp long. The

sequences were submitted to GenBank

(http://www.ncbi.nlm.nih.gov/Genbank/) and their accession number obtained

are JN232197, JN247634, JN247635 and JN247636 (Table -2.10). Further

these sequences were analyzed by MSA (Figure 2.7a). The sequences were

near to similar in length but no 100% similarity in nucleotide sequence was

observed.

The UPGMA relatedness derived from the sequences in CLC were presented

(Figure 2.8a). The pattern of UPGMA relatedness as described earlier was

similar to that obtained from scoring monomorphic and polymorphic bands

using RAPD, ISSR, and combine RAPD and ISSR primers (Figure 2.5a –

2.5c). However, it was interesting to note that similar trend also obtained

using sequences of single monomorphic band, using only one (I-16) primer

(Figure 2.8a). Both dendrogram indicates that the four varieties divided them

into two clusters. The varieties Khasdi, Red, Desi were clustered in the same

group where as Soneri was separated from them. The amplification products

could result from changes in either the sequences of the primer binding sites

or from changes that could have altered the sizes of the DNA fragments (of

template). Variation could also result due to the prevention of the successful

amplification of a target DNA fragment (e.g., insertions, deletions, and

inversions).


49

Further analysis with CLC also revealed that each sequence have unique

cutting site for RENs and hence can be converted to polymorphic bands

(Figure 2.9). Cutting site for Eco RI was present only on Soneri, while Hind III

was present on Khasdi. Bam HI will give 3 fragment of monomorphic band of

Desi and two in Red variety. Thus, these monomorphic bands can be

converted into polymorphic using appropriate RENs to obtain known bp

fragments. The polymorphism observed in monomorphic bands based on

sequence difference can be explored to precise identification of varieties

using RENs cutting sites. This has demonstrated the usefulness of this

approach for the identification of the Musa varieties. This technique could be

used in future for genetic mapping studies of plant species.

Similar to monomorphic band sequencing, the polymorphic marker bands of

all four varieties were also sequenced. These bands (total 8) were selected on

the bases of distinct polymorphism in studied varieties. It was observed that

sequences ranged from 100 to 291 bp with I-8 and I-26 primer. The obtained

sequences showed very poor similarity with each other (Figure 2.7b). The

dendogram prepared with these sequences (Figure 2.8b), also showed that

variety Soneri is distinct from other three.

Nucleotide sequence derived from the polymorphic and monomorphic bands

using I-8, I-26 and I-16 primers are translated to protein for further analysis

using CLC workbench and ProtParam Expasy proteomic tools. The frames

are further proceed in ProtParam to compare the properties of the derived

proteins. The predicted protein from the monomorphic and polymorphic bands

showed marked variation in their protein properties as described below.

The sequences derived from the monomorphic bands are translated into

protein sequences and are further proceed in CLC workbench (Figure 2.10a).

From multiple sequence alignment and from tree generated using protein

sequences further confirmed the relatedness of the variety studied (Figure

2.11a). It was found that Soneri variety has distinct clade. The protein analysis

revealed that these sequences have higher percentage of leucine except Red

variety followed by proline, serine, glycine and tryptophan as compare to other


50

amino acids (Table -2.11). However, in Soneri variety, cystenine, glutamic

acid and phenylenine were higher and isoleucine was low as compare to

other three varieties. The aliphatic index of Red and Soneri was low (85 and

87.56, respectively) as compare to Desi and Khasdi (115.26 and 116.78,

respectively) (Table -2.13). Instability index was more than 40 of all the

sequences suggesting there by that all are unstable proteins.

Similarly all four varieties were also studied for polymorphic band analysis.

The nucleotide sequence of selected polymorphic band were also translated

for protein prediction and analyzed in ProtParam proteomics tools, revealed

that in amplification of I-8 primer, leucine and proline was higher as compare

to other amino acids in all varieties studied (Table -2.12). The bands

generated by the same primer showed little or no similarity in the same variety

and amongst the all four varieties studied. This study suggests that the

primers are binding at more than one place on genome and amplification have

no similarity and thus can be exploited as marker for the molecular

identification of the varieties. Instabilities index was higher in first four bands

from Soneri. The aliphatic index ranged from 42.9 to 106.36 from the proteins

studied. (Table -2.14). Thus from this study it is concluded that the sequences

are different from the each other at DNA and protein levels and can be

exploited for the molecular marker designing.

Designing of molecular markers specially SSR markers help to identify and

detect the desired variety as well as contamination of other variety in the gene

lot. Desi variety makes up nearly 90-95% of the total plantation in VERAVAL

and nearby area. Therefore, defined, accurate and reliable markers are

needed for the varietal discriminations. From the above study, an attempt is

made to define molecular marker for the most potential variety Desi using

sequence data.

Microsatellite or short tandem repeats or simple sequences repeats are

monotonous repetition of very short (one to five) nucleotide motifs, which

occurs as interspersed repetitive elements in all eukaryotic genomes (Tautz

and Renz 1984). Variation in the number of tendamly repeated units is mainly


51

due to strand slippage during DNA replication where the repeats allow

matching via excision or addition of repeats (Schlotterer and Tautz 1992). As

slippage in replication is more likely than point mutations, microsatellite loci

tend to be hyper variable. Microsatellite assay shows extensive inter-

individual length polymorphism during PCR analysis of unique loci using

discriminatory primers sets.

In the present study, Desi variety is a target for marker study since it is being

cultivated dominantly. It is imperative to have markers for this variety to avoid

contamination from other variety, better yield and uniform growth and

development of plant to increase the economic value. Polymorphic bands

generated using I-8 primer was distinctly demonstrating in the four different

varieties studied. A band from Desi variety was isolated, sequenced and also

analyzed using bioinformatics tools which showed distinct pattern. This

sequence is further analyzed in tandem repeat finder (Benson 1999) to locate

and display tandem repeats. It was observed that this polymorphic band

sequence with length of 277 bp showed 17.5 copy number of GT rich region

between 57-91 bp (Figure 2.12 and Table -2.15). Thus this tandem repeats

can be used as microsatellite marker for Desi variety. Microsatellites are

highly popular genetic markers because of their co dominant inheritance, high

abundance, enormous extent of allelic diversity, and the ease of assessing

SSR size variation by PCR with pair of flanking primers. The reproducibility of

microsatellites is such that, they can be used efficiently by different research

laboratory to produce consistent data (Saghai Marrof et al. 1994). Locus

specific microsatellite based markers have been reported from many plant

species (Wu and Tanksley 1993, Saghai Marrof et al. 1994, van de Wiel et al.

1999).

Table 2.1. Commonly accepted absorbance to concentration conversion for nucleic acids.

Nucleic Acid Species Concentration for 1 A260 Unit (μg/ml)

dsDNA 50 ssDNA

(oligonucleotides) 33

ssRNA 40

Table 2.2. Comparison of DNA purity and yield between M1-M5.

Method Varieties 260/280 260/230 Conc. µg/ml

Conc. µg

DNA/gm tissue

M1 Desi 1.13 - 41.72 16.69

M2 Desi 1.46 1.17 77.32 30.93

M3 Desi 1.65 1.05 54.95 10.99

M4 Red 1.59 1.22 57.94 11.59

M5 Khasdi 1.59 1.17 84.06 16.81

Soneri 1.67 1.37 128.18 25.64

Table 2.3. ISSR primers.

No. Sequence Name I-1 (AT)8C UBC803 I-2 (AG)8C UBC808 I-3 (CT)8A UBC814 I-4 (CA)8A UBC817 I-5 (TC)8A UBC822 I-6 (AC)8C UBC826 I-7 (AG)8TT UBC834 I-8 (GA)8TT UBC840 I-9 (AGC)6 UBC862 I-10 (ATG)6 UBC864 I-11 (GGC)6 UBC867 I-12 (GAA)6 UBC868 I-13 (GATA)4 UBC872 I-14 (GACA)6 UBC873 I-15 (GATA)2(GACA)2 UBC876 I-16 (GGAGA)3 UBC880 I-17 (GGGGT)3G UBC881 I-18 HBH(AG)7 UBC884 I-19 BHB(GA)7 UBC885 I-20 VDV(CT)7 UBC886 I-21 BDB (CA)7 UBC888 I-22 DBD(AC)7 UBC889 I-23 HVH (TG)7 UBC891 I-24 (AGC)5GC ISSR1 I-25 (CA)7AC ISSR2 I-26 (GT)7AC ISSR3 I-27 GCA(GA)7 ISSR4 I-28 (GA)9C ISSR5 I-29 (GA)9A ISSR6 I-30 (CG)8C ISSR7

(Where, V= AGC; H= ACT; D= AGT; B= GCT)

Table 2.4. RAPD amplification profile.

Primer Amplicon Desi Red Khasdi Soneri

OPB-1

1 1 1 0 0 2 0 0 1 0 3 0 0 1 0 4 0 0 1 0 5 0 1 1 1 6 0 0 0 1

OPB-4

1 0 1 0 0 2 0 1 1 1 3 0 0 0 1 4 0 1 0 1 5 0 1 0 1

OPB-5

1 0 0 0 1 2 0 0 0 1 3 0 0 0 1 4 1 0 0 0 5 0 1 1 0 6 0 1 1 0 7 0 1 1 0

OPB-7

1 0 1 0 0 2 1 1 1 0 3 1 1 1 1 4 0 0 0 1 5 0 1 1 0

OPB-8

1 0 0 0 1 2 0 0 0 1 3 1 1 1 0 4 1 0 0 0 5 1 1 1 1 6 0 0 1 1 7 1 0 0 0

OPB-6

1 1 1 1 0 2 0 1 1 1 3 0 0 0 1 4 1 0 1 0 5 0 0 0 1 6 1 1 1 1 7 0 0 0 1

OPB-16

1 0 0 1 0 2 0 1 0 1 3 0 1 1 1 4 0 0 0 1

OPB-9

1 0 1 0 0 2 1 1 1 0 3 0 1 0 0 4 1 1 1 1 5 0 1 1 1 6 0 0 0 1 7 0 1 1 1 8 0 0 0 1 9 1 0 1 0 10 0 0 0 1 11 1 1 1 1

OPB-17

1 1 1 1 0 2 0 0 0 1 3 0 0 1 1 4 1 1 0 0 5 1 1 1 1 6 1 1 1 1

OPB-18

1 0 0 1 1 2 1 1 1 1 3 1 1 1 0 4 0 0 0 1 5 0 0 0 1 6 1 1 1 1 7 1 1 1 1

OPA-3

1 1 1 1 1 2 1 0 0 1 3 1 0 0 1 4 0 1 1 0 5 0 0 0 1 6 1 1 1 1 7 0 1 0 0 8 0 1 1 1 9 0 1 1 1 10 1 1 1 0 11 1 1 1 0 12 0 0 0 1 13 0 1 0 0

OPA-4 1 0 1 1 0 2 0 0 0 1 3 1 1 1 1

OPA-9

1 0 1 0 0 2 1 1 0 1 3 0 1 0 0 4 0 1 0 0 5 0 0 0 1 6 0 1 0 0 7 0 1 0 0 8 0 1 0 0 9 1 1 1 0 10 0 1 1 0 11 0 0 0 1 12 1 0 0 0

Table 2.5. RAPD Amplicon profile, % monomorphism and % polymorphism.

Primer Total Amplicon

PolymorphicAmplicon

Monomorphic Amplicon

Total Band

Polymorphic Band

MonomorphicBand

% Poly.

% Mono.

OPA-3 13 4 9 30 4 26 13.33 86.67

OPA-4 3 1 2 7 1 6 14.29 85.71

OPA-9 12 9 3 17 9 8 52.94 47.06

OPB-1 6 4 2 9 4 5 44.44 55.56

OPB-4 5 2 3 9 2 7 22.22 77.78

OPB-5 7 4 3 10 4 6 40.00 60.00

OPB-6 7 3 4 15 3 12 20.00 80.00

OPB-7 5 2 3 11 2 9 18.18 81.82

OPB-8 7 4 3 13 4 9 30.77 69.23

OPB-9 11 5 6 24 5 19 20.83 79.17

OPB-16 4 2 2 7 2 5 28.57 71.43

OPB-17 6 1 5 16 1 15 6.25 93.75

OPB-18 7 2 5 19 2 17 10.53 89.47

Total 93 43 50 187 43 144

Average 7.15 3.31 3.85 22.99 77.01

Table 2.6. ISSR amplification profile.

Primer Amplicon Desi Red Khasdi Soneri

I-2

1 0 0 0 1 2 0 1 0 1 3 1 1 1 1 4 0 1 1 0

I-3 1 1 0 0 0 2 1 0 0 0

I-4 1 1 1 1 1 2 1 1 1 1 3 1 1 1 1

I-5

1 1 1 1 1 2 1 1 1 1 3 0 0 1 0 4 0 1 1 0 5 1 0 0 1 6 1 1 1 1

I-7 1 1 1 1 1 2 0 0 1 1 3 0 1 0 0

I-8

1 0 0 0 1 2 1 1 1 1 3 1 1 0 0 4 1 0 0 0 5 1 1 0 0 6 1 1 0 0 7 1 1 0 0

I-10

1 0 1 1 1 2 0 1 1 1 3 0 1 1 1 4 0 1 0 0 5 0 0 0 1 6 0 1 1 1 7 0 1 1 0 8 0 0 0 1 9 0 1 1 1 10 0 1 0 0

I-12 1 1 1 1 1 I-13 1 0 1 1 0

I-15

1 0 0 0 1 2 0 1 1 1 3 0 0 0 1 4 0 1 1 0 5 0 0 0 1 6 0 0 0 1 7 1 0 0 1 8 0 1 0 1

I-16

1 0 0 0 1 2 0 0 1 1 3 0 0 0 1 4 0 1 0 0 5 0 0 0 1 6 0 1 0 1 7 0 1 0 0 8 1 1 1 1 9 1 1 1 0 10 1 1 1 1

I-18 1 1 1 1 1 2 1 1 1 1 3 1 1 1 1

I-21

1 1 1 1 0 2 0 0 1 0 3 1 1 1 1 4 1 1 1 1

I-22

1 1 1 1 0 2 1 1 1 0 3 0 0 1 0 4 1 1 1 1 5 1 1 1 1

I-24

1 1 0 0 0 2 0 0 0 1 3 1 1 0 0 4 1 0 0 1 5 1 1 1 0

I-26

1 1 1 1 1 2 1 1 0 0 3 0 0 0 1 4 1 1 1 1 5 1 1 1 0 6 0 1 1 0

Table 2.7. ISSR Amplicon profile, % monomorphism and % polymorphism.

Primer

Total Amplicon

Polymorphic Amplicon

Monomorphic Amplicon

Total Band

PolymorphicBand

MonomorphicBand

% Poly.

% Mono.

I-2 4 1 3 9 1 8 11.11 88.89 I-3 2 2 0 2 2 0 100.00 0.00 I-4 3 0 3 9 0 9 0 100 I-5 6 1 5 17 1 16 5.88 94.12 I-7 3 2 1 7 3 4 42.86 57.14 I-8 7 6 1 14 10 4 71.43 28.57 I-10 10 4 6 21 4 17 19.05 80.95 I-12 1 0 1 4 0 4 0.00 100.00 I-13 1 0 1 2 0 2 0.00 100.00 I-15 8 4 4 13 4 9 30.77 69.23 I-16 10 5 5 20 5 15 25.00 75.00 I-18 3 0 3 12 0 12 0.00 100.00 I-21 4 1 3 12 1 11 8.33 91.67 I-22 5 1 4 15 1 14 6.67 93.33 I-24 5 2 3 9 2 7 22.22 77.78 I-26 6 4 2 16 8 8 50 50

Total 78 33 45 182 42 140 Average 4.88 2.06 2.81 23.08 76.92

Table 2.8. Jaccard distance/similarity matrix based on RAPD analysis among four Musa varieties.

Varieties Desi Red Khasdi Soneri

Desi 0.39683 0.43636 0.22857

Red 0.39683 0.57813 0.3012

Khasdi 0.43636 0.57813 0.32

Soneri 0.22857 0.3012 0.32

Table 2.9. Jaccard distance/similarity matrix based on ISSR analysis among four Musa varieties.

Varieties Desi Red Khasdi Soneri

Desi 0.55 0.47368 0.375

Red 0.55 0.67241 0.42254

Khasdi 0.47368 0.67241 0.46032

Soneri 0.375 0.42254 0.46032

Table 2.10. Nucleotide sequences of four Musa varieties submitted to gene bank.

No. Accession no. Definition Primer

detail Band

property

1 JN232197 Musa acuminata AAA Group cultivar Desi microsatellite SUP0001 sequence

I-16 Mono morphic

2 JN247634 Musa acuminata AAA Group microsatellite SUP0002 sequence I-16 Mono morphic

3 JN247635 Musa ABB Group microsatellite SUP0003 sequence I-16 Mono morphic

4 JN247636 Musa ABB Group microsatellite SUP0004 sequence I-16 Mono morphic

5 JN415132 Musa AAB Group microsatellite SUP0006 sequence I-8 Poly morphic

6 JN415133 Musa acuminata AAA Group microsatellite SUP0007 sequence I-8 Poly morphic


8 JN415135 Musa ABB Group microsatellite SUP0009 sequence I-8 Poly morphic

9 JN415136 Musa AAB Group microsatellite SUP0010 sequence I-8 Poly morphic




Table 2.11. Predicted protein profile of monomorphic sequences of four Musa varieties performed.

Amino acid JN232197 JN247634 JN247635 JN247636

Ala (A) 6 5 10 8

Arg (R) 6 8 12 8

Asn (N) 7 8 8 6

Asp (D) 8 6 6 5

Cys (C) 2 4 2 7

Gln (Q) 4 3 4 3

Glu (E) 4 2 3 8

Gly (G) 8 10 16 9

His (H) 7 3 4 5

Ile (I) 14 19 22 7

Leu (L) 28 12 28 26

Lys (K) 4 2 3 5

Met (M) 4 5 0 5

Phe (F) 3 7 3 11

Pro (P) 13 19 16 13

Ser (S) 23 22 17 23

Thr (T) 9 11 9 8

Trp (W) 4 5 5 3

Tyr (Y) 10 14 12 10

Val (V) 11 7 3 6

Pyl (O) 0 0 0 0

Sec (U) 0 0 0 0

(B) 0 0 0 0

(Z) 0 0 0 0

(X) 0 0 0 0

Table 2.12. Predicted protein profile of polymorphic sequences of four Musa varieties performed.

Amino acid JN415132 JN415133 JN415134 JN415135 JN415136 JN415137 JN415138 JN415139

Ala (A) 2 2 5 4 4 9 6 4 Arg (R) 9 4 4 3 4 6 12 3 Asn (N) 9 1 2 2 0 0 3 4 Asp (D) 1 1 1 1 2 0 1 3 Cys (C) 1 1 1 0 0 6 6 8 Gln (Q) 4 1 2 0 0 1 3 1 Glu (E) 4 2 4 3 4 4 5 7 Gly (G) 1 0 3 2 6 5 7 3 His (H) 6 0 2 1 2 13 1 2 Ile (I) 4 2 3 1 0 2 0 5

Leu (L) 11 14 13 9 1 5 5 3 Lys (K) 9 3 3 1 4 0 9 9 Met (M) 2 1 2 1 2 3 2 5 Phe (F) 7 7 10 5 1 10 7 2 Pro (P) 17 11 11 8 1 3 5 6 Ser (S) 8 4 9 4 3 4 7 9 Thr (T) 10 4 6 5 1 15 8 15 Trp (W) 1 0 0 0 0 0 1 2 Tyr (Y) 2 4 2 1 0 4 4 2 Val (V) 2 2 0 0 5 13 6 11 Pyl (O) 3 0 0 1 0 1 1 3 Sec (U) 0 0 0 0 0 0 0 0

(B) 0 0 0 0 0 0 0 0 (Z) 0 0 0 0 0 0 0 0 (X) 1 2 1 2 1 2 1 1

Table 2.13. Protein physico chemical analysis of monomorphic band from ProtParam.

JN232197 JN247634 JN247635 JN247636Instability index 44.16 62.59 50.11 49.76 Aliphatic index 115.26 85 116.78 87.56 Grand average of hydropathicity (GRAVY) 0.209 0.037 0.139 0.086

Theoretical pI 6.38 8.37 9.48 7.09

Table 2.14. Protein physico chemical analysis of polymorphic band from ProtParam.

JN415132 JN415133 JN415134 JN415135 JN415136 JN415137 JN415138 JN415139Instability index 55.07 49.87 47.25 66.62 -5.94 25.26 43.36 34.81 Aliphatic index 58.16 106.36 80.24 79.63 54.63 69.81 42.9 62.13 Grand average of hydropathicity (GRAVY) -0.918 0.336 0.069 -0.026 -0.72 0.337 -0.69 -0.167

Theoretical pI 11.05 9.65 9.05 6.77 9.52 8.35 10.22 8.17

Table 2.15 Tandem repeats detected in JN415137 (SUP0011 Desi Musa variety).

Indices PeriodSize

Copy Number

ConsensusSize

PercentMatches

PercentIndels Score A C G T Entropy

(0-2) 57--91 2 17.5 2 93 0 61 2 0 48 48 1.16

Figure 2.1. Agarose gel electrophoresis of the DNA samples of four Musa varieties [M=Supermix DNA ladder, 1 to 4 are DNA samples of Desi, Red, Khasdi and Soneri, respectively].

OPB-8

OPA-9

OPB-17

OPB-18

Figure 2.2. The amplification pattern of four Musa varieties produced by the

various RAPD primers [M=Supermix DNA ladder, 1 to 4 are DNA samples of Desi, Red, Khasdi and Soneri, respectively].

I-5

I-15

I-16

I-26

Figure 2.3. The amplification pattern of four Musa varieties produced by the various ISSR primers [M=Supermix DNA ladder, 1 to 4 are DNA samples of Desi, Red, Khasdi and Soneri, respectively].

Figure 2.4a. The amplification product of Musa varieites viz. (1) Desi, (2) Red, (3) Khasdi and (4) Soneri produced by primer I-16 [The aero indicates monomorphic bands selected for nucleotide sequencing].

I-8

Figure 2.4b. The amplification product of Musa variety (1) Desi produced by primer I-8 [The aero indicates polymorphic bands selected for nucleotide sequencing].

(a) RAPD

(b) ISSR

(c) RAPD+ISSR

Figure 2.5. UPGMA relationship amongst four Musa varieties.

Figure 2.6a Electrophorogram of monomorphic amplification product of Musa varieties viz. (1) Desi, (2) Red produced by primer I-16

Figure 2.6b. Electrophorogram of monomorphic amplification product of Musa varieties viz. (3) Khasdi and (4) Soneri produced by primer I-16

Figure 2.7a. Multiple Sequence Alignment (MSA) of monomorphic band pattern of four Musa varieties performed on CLC Main Workbench5.

Figure 2.7b. Multiple Sequence Alignment (MSA) of polymorphic band pattern of four Musa varieties performed on CLC Main Workbench5.

Figure 2.8a. UPGMA relationship of nucleotide sequence derived from monomorphic band pattern of four Musa varieties.

Figure 2.8b. UPGMA relationship of nucleotide sequence derived from polymorphic band pattern of four Musa varieties.

Figure 2.9. Assessments of restriction sites on monomorphic bands of four Musa varieties performed on CLC Main Workbench5.

Figure 2.10a. Multiple Sequence Alignment (MSA) of protein sequence derived from monomorphic band pattern of four Musa varieties performed on CLC Main Workbench5.

Figure 2.10b. Multiple Sequence Alignment (MSA) of protein sequence derived from polymorphic band pattern of four Musa varieties performed on CLC Main Workbench5.

Figure 2.11a. UPGMA relationship of protein sequence derived from monomorphic band pattern of four Musa varieties.

Figure 2.11b. UPGMA relationship of protein sequence derived from polymorphic band pattern of four Musa varieties.

Figure 2.12. Tandem repeats detected in JN415137 (SUP0011 Desi Musa variety).

Chapter 3 Biochemical

studies of Banana


52

Abstract

The amounts and composition of phenolic compounds is diversified in the

plant leaves. In this study, antioxidant potential, total phenol content,

monophenol content and diphenol content were measured in four Musa

varieties in fully expanded leaves. The DPPH assay was found to be quick,

reliable, and reproducible parameter to search antioxidant potential of plant

extract. The present method of RP-HPLC is simple, easy to use, and effective

enough for the identification and quantification of major phenolic compounds

in Musa. It is established that phenolic compounds are commonly distributed

in plant leaves. Tannic acid, quercetin, chlorogenic acid, syringic acid and

caffeic acid was detected in all four Musa varieties, while Gentisic acid

detected only in one sample of Desi variety.


53

Introduction

Banana plant is a rich source of phenolics. Plant polyphenols are aromatic

hydroxylated compounds commonly found in vegetables, fruits and many food

sources. They form a significant portion of our diet, and are among the most

potent and therapeutically useful bioactive substances. The health benefits of

fruit and vegetables are mainly from the phytochemicals and a range of

polyphenolics (Wang et al. 1996). Significant antioxidant, antitumor, antiviral

and antibiotic activities are frequently reported for plant phenols. They have

often been identified as active principles of numerous folk herbals. In recent

years, the regular intake of fruits and vegetables has been highly

recommended, because the plant phenols and polyphenols they contain are

thought to play important roles in long term health and reduction in the risk of

chronic and degenerative diseases (Manach et al. 2004).

The phenolic compounds are essential to plant life. They provide defense

against microbial attacks by making food unpalatable to herbivorous predators

(Harborne 1982). They have been associated with diverse functions, including

nutrient uptake, protein synthesis, enzyme activity, photosynthesis, and

structural components (Einhellig et al. 1996, Wu et al. 1999). Thus, they are

important for normal growth, development and defense against infection and

injury in the plant.

An antioxidant by definition is a substance that significantly delays or prevents

oxidation of its oxidizable substrate when present at low concentrations

compared to those of its substrate (Halliwell and Gutteridge 1989, Halliwell

1990). Potential sources of antioxidant compounds have been searched in

several types of plant materials such as vegetables, fruits, leaves, oilseeds,

cereal crops, barks, roots, spices, herbs and crude plant drugs (Ramarathnam

et al. 1997). Free radical damages the structural and functional components

of the cell such as lipid, protein, carbohydrates, DNA, and RNA. Banana peel

contains high content of micronutrient compared to fruit pulp (Davey et al.

2007). It attracts great attention because of their nutritional and antioxidant

properties, especially the compounds, ascorbate, catechin, gallocatechin, and


54

dopamine. Due to the importance of these compounds, it is necessary to

understand its initial production and losses during fruit development, ripening,

and maturation (Shanthy et al. 2011).

It is well established that phenolic compounds are commonly distributed in

plant leaves, flowering tissues and woody parts such as stem and bark. The

antioxidant potential of plant materials strongly correlates with their content of

the phenolic compounds (Velioglu et al. 1998). In plants, these antioxidant

phenolics play a vital role for normal growth and protection against infection

and injuries from internal and external sources (Ara and Nur 2009, Tibiri et al.

2010).

Different parts of the same plant may synthesize and accumulate different

compounds or different amounts of a particular compound due to their

differential gene expression, which in turn affects the antioxidant potential and

other biological properties of the plant extracts produced (Jaffery et al. 2003,

Rafat et al. 2010). Many studies have confirmed that the amounts and

composition of phenolic and flavonoid compounds is diversified at the sub-

cellular level and within plant tissues as well (Macheix et al. 1990, Randhir et

al. 2004). Plant phenolics, such as phenolic acids, stilbenes, tannins, lignans,

and lignin, are especially common in leaves, flowering tissues, and woody

parts such as stems and barks (Larson 1988).

A universally define acceptable solvent, 80 % MeOH and 70 % EtOH are

generally preferred solvents for phenolics extraction from plants (Prior et al.

2005). Phenolic compounds are known for its radical scavengers, therefore, it

is worthwhile to determine the phenolic content in the plant chosen for the

study (Pracheta et al. 2011). Many available methods of quantification of total,

mono and di phenolic content in food products or biological samples are

based on the reaction of phenolic compounds with a colorimetric reagent,

which allows measurement in the visible portion of the spectrum. The DPPH

(2,2-Diphenyl-1-picrylhydrazyl radical) radical is widely utilized to evaluate the

free radical scavenging capacity of antioxidants (Tung et al. 2007).

Antioxidant potential can be determined by monitoring the decrease in the

absorbance.


55

The monohydroxy benzoic acids act as very weak antioxidants owing to the

electronegative potential of a single carboxyl group only m-hydroxy bezoic

acid has antioxidative potential. This activity increases considerably in the

case of dihydroxy substituted benzoic acids, whose antioxidant response is

dependent on the relative positions of the hydroxyl groups in the ring. Gallic

acid (3,4,5-trihydroxy benzoic acid) is the most potent antioxidant of all

hydroxybenzoic acids (Rice-Evans et al. 1996).

The analysis of phenolic compounds is very challenging due to the great

variety and reactivity of these compounds (Bronze and Boas 1998). The

introduction of enhanced resolution and increased automation has resulted in

HPLC, the most popular analysis method for plant phenolics (Robards and

Antolovich 1997, Waksmundzka-Hajnos 1998). Separation is commonly

achieved by HPLC although GC is used in some instances. In the first role,

screening of plant extracts for biologically active natural products played an

important role in the phytochemical investigation of crude extracts (Saleem et

al. 2001, Gorinstein et al. 2002). Methods of characterization and identification

of plant phenols followed those in general use for natural products (Zhou et al.

2001).

The most studied bioactivity of the phenols is their antioxidant status. The

action of phenols as antioxidants is viewed in plant parts where phenols are

oxidized in preference to other food constituents or cellular components and

tissues. Thus, measurement of antioxidant potential of a phenol or mixture of

phenols has been applied. The need for profiling and identifying individual

phenolic compounds has seen traditional methods replaced by high-

performance chromatographic analyses. The limited volatility of many phenols

has restricted the application of GC to their separation. Merken and Beecher

(2000) have presented a comprehensive review on the analytical chemistry of

food flavonoids in which they present detailed tabulations of columns and

mobile phases used in HPLC. The most common mode of separation exploits

reversed-phase systems typically with a C18 column and various mobile

phases.


56

The phenolic compounds of natural origin have the positive property of being

soluble in polar solvents. This leads to the possibility of using reversed phase

HPLC (RP-HPLC) in their analysis, sufficient retention being achieved by

using acidic conditions in order to avoid the presence of ionised forms of the

analytes (Waksmundzka and Hajnos 1998).

Routine detection in HPLC is typically based on measurement of UV

absorption (Molyneux et al. 2002, Khokhar and Magnusdottir 2002) or, less

commonly, visible radiation (Vlahov 1992, Bengoechea et al. 1995). No single

wavelength is ideal for all classes of phenols since they display absorbance

maxima at distinctly different wavelengths (Pietta and Mauri 2001). Indeed,

there are significant differences in absorption maxima and molar absorptivities

(Tsimidou et al. 1992) of even the major phenols identified in a single fruit.

This creates problems in quantification as discussed by Tsimidou et al. (1992)

who classified the various phenols into four groups and used a single

calibration standard for the members of each group. The most commonly

used wavelength for routine detection has been 280 nm which represents a

suitable compromise (Zunin et al. 1995, Martınez-Valverde et al. 2002,

Khokhar and Magnusdottir 2002) although detection at other wavelengths

(Park et al. 1983, Tutour and Guedon 1992, Brenes et al. 1995) and dual

wavelength detection (Montedoro et al. 1992) has been applied.


57


Preparation of leaf extract

Fully expanded leaves of four Musa varieties viz. Khasdi, Desi, Red, and

Soneri were collected and transferred immediately to laboratory. Leaves were

washed thoroughly with tap water to remove dust particles and rinsed with

distilled water. One cm2 leaf disc as a plant material was crushed with liquid

nitrogen in mortal and pastel and preserved in methanol. Material was

homogenized in methanol and purity of homogenate was increased by

centrifugation at 2740x g for 10 minutes at room temperature. Final volume of

5 mL of supernatant was prepared with same solvent (Pandya 2010). For the

antioxidant assay samples were evaporated and final volume of residue was

prepared in methanol.

Microplate assay for DPPH free radical scavenging potential

Radical scavenging potential of different variety of banana leaf extracts was

determined using stable free radical DPPH, according to Velazquez et al.

(2003), with alterations for micro plate assay. The DPPH accepts the proton

and gets reduced and decolorized, therefore it can be utilized for the

evaluation of free radicals scavenging potential. The reaction mixture

contained 100 μL, 1 mM DPPH and 100 μL leaf extract in microplate well and

it was incubated at room temperature for 20 minutes. The decrease in

absorbance of DPPH due to the extract was measured at 517 nm using Bio-

Tek microplate reader μQuant, USA. Range of various concentrations of

ascorbic acid was used for the preparation of standard curve. Antioxidant

potential of the plant extract was calculated as μg equivalent ascorbic acid per

milligram dry weight of different leaf weight. The blank sample was prepared

using methanol.

Microplate estimation of phenolic content

Mono, di- and total phenol content of leaf of four banana variety was

estimated according to Rabadia (1993). Reactions were scaled down


58

proportionally for microplate estimation. Samples were estimated in seven

replicate and the amount of phenolic content was calculated as equivalent to

μg standard compound per milligram dry weight of leaf (1 cm2 leaf disc). Total

phenols were estimated from 90 μL of sample. The sample was mixed with

10 μL Folin-Phenol reagent (1 N) and 500 μL saturated sodium carbonate.

Reaction was developed in dark by incubating samples for 45 minutes at

room temperature. Total 300 μL of this mixture was taken on microplate for

estimation. Range of different concentrations of chlorogenic acid was used,

for the preparation of standard curve. The absorbance of samples was

measured on microplate reader at 750 nm.

For estimation of o-dihyeroxy phenol, 20 μL of 0.5 N hydrochloric acid was

added in 10 μL of sample. Then, 10 μL Arnow’s reagent (10 g Sodium Nitrite

+ 10 g Sodium Molibdate in 100 mL Water) and 20 μL of 1 N sodium

hydroxide was added. The readings were taken at 525 nm on microplate

reader. Pyrocatechol was used as standard.

Monohydroxy phenols were estimated from 10 μL of sample extract; by

mixing the sample with 8 μL of 0.5 N sodium hydroxide, 10 μL 4-

aminoantipyrine (0.6 %), 12 μL of 0.5 M sodium carbonate and 10 μL of 2.4

% Potassium ferricyanide. Calibration curve was prepared using different

concentrations of hydroxybenzene and the absorbance of reaction was

measured at 520 nm.

Qualitative and Quantitative analysis of plant Phenols

A Shimadzu LC-20AD Prominence Chromatograph HPLC system

(Shimadzu, Japan) with a binary solvent delivery system (LC-20AD), a

column temperature controller (CTO-20A Prominence Column Oven),

Rheodyne injector with 20ul sample loop and a PDA detector (SPD-M 20A

Prominence) coupled with LC-solution V. 2 SP2 software were used for

analysis. A reverse phase (RP) column (Phenomenex luna C-18, 5μ,

250mm×4.6 mm) with an extended guard column was used as stationary

phase and column temperature was maintained at room temperature. The

review was made on various parameters of phenolic compound investigated


59

in plant using HPLC analysis (Table -3.1). Based on reviewed literature the

following parameters were set for HPLC analysis. The mobile phase was a

gradient elution of water containing 0.2% H3PO4 (solvent A) and methanol

containing 0.2% H3PO4 (solvent B) at a flow rate of 1 ml/min. The gradient

program of solvent B in A (v/v) was as follows: 0-10 min 10% B; 10-65 min

75% B; 75-76 min 10%; 76-95 min 10% A. The column was equilibrated

prior to injection of the next sample with the mobile phase for 20 min and

comparing their retention times with those of 15 standards (Table -3.3)

identified the phenolic compounds.


60


Plants have the ability to synthesize a large number of aromatic substances,

most of which are phenols or their oxygen-substituted derivatives (Cowan

1999). Most are secondary metabolites, which may be in the form of simple

phenols and phenolic acids, quinones, flavones, flavonoids, flavonols, tannins,

terpenoids, essential oils and alkaloids. All of the phenolics have one or more

benzene rings with one or more hydroxyl groups that may be variously

elaborated with methyl, methoxyl, amino or glycosyl groups (Naczk and

Shahidi 2004). Some, such as terpenoids, give plants their odors and flavor;

many compounds, like tannins and quinones, are responsible for plant

pigment, and others, such as some alkaloids, may be toxic to the consumer.

Phenols have not been completely studied because of the complexity of their

chemical nature and the extended occurrence in plant materials. In many

instances, these substances serve as plant defense mechanisms against

predation by insects, herbivores and microorganisms (Williams and Harborne

1989, Cowan 1999, Beckman 2000). Different studies showed that there are

often large increases in phenolic synthesis in plants after attack by plant

pathogens (Matern et al. 1995, De Ascensao and Dubrey 2003). Many plant

materials and foods have not yet received much attention as sources of

antioxidant phenols due to limited popularity or lack of commercial

applications.

Phenols function as antioxidants in plants and amongst these polyphenolic

compounds are considered to be the most effective. Thus the plants that have

rich phenolics are the best source for antioxidants. Phenolics are classified

into four categories: preventive antioxidants, radical scavenging antioxidants,

repair and de novo antioxidants, and adaptation antioxidants (Noguchi et al.

2001). It was suggested that radical scavenging antioxidants have the

greatest benefits and their chemical structure is of key importance (Tannin-

Spitz et al. 2007). The efficiency of the added antioxidants depends, besides

the structural features, on many other factors such as concentration,


61

temperature, light, type of substrate, and physical state of the system (Mariutti

et al. 2008).

Generally, the source of antioxidants is edible plant parts taken as food, little

attention is paid to the foliage or other parts which are waste after harvest.

Banana plant contains many phenolic in significant amount. Leaves are rich

source of phenolic compounds and hence are studied in fully expanded

leaves of all four varieties. Changes in antioxidant potential of four Musa

varieties were shown in Figure 3.1 with standard error. Antioxidant potential

found that ranging from 28.92-41.50, 37.53-47.95, 27.54-48.18 and 28.89-

47.17 μg/mg dwt equivalent to ascorbic acid in Khasdi, Soneri, Desi and Red

variety respectively. The average value of all four varieties were 33.42, 41.08,

34.01 and 38.02 μg/mg dwt in Khasdi, Soneri, Desi and Red variety,

respectively (Table -3.1). It was observed that antioxidant potential was the

highest in Soneri variety, while the lowest was observed in Khasdi variety

(Figure 3.1). The antioxidant potential found to be fluctuating in Red variety.

The antioxidant potential of fruits and vegetables is generally positively

correlated with their content of polyphenols (Hertog et al.1995, Stoner and

Mukhtar 1995, Robards et al. 1999, Wang and Lin 2000, Benzie 2003). Musa

tissues are rich in phenolic compounds which can potentially interfere with

carotenoid extraction and/or analysis (Levizou et al. 2004). Studying seasonal

changes in contents of phenolic compounds in Rhus, Euonymus and Acer

leaves, Ishikura (1976) have found that the total phenol per leaf increased

rapidly at the early growth stages but thereafter the content was kept rather

constant. Also, Males et al. (2003) indicated that the aerial parts of Crithmum

maritimum collected before flowering and at the beginning of flowering were

the richest in total polyphenols. Nevertheless, Ayan et al. (2007) reported that

total phenol content reached the highest level at floral budding in Hypericum

hyssopifolium and Hypericum scabrum and at full-flowering in Hypericum

pruinatum.

In present study, total phenol content in leaves of four Musa varieties were

shown in Figure 3.2. Chlorogenic acid used to prepare standard calibration


62

curve for total phenol content and it shown 0.964 R2 value. The average

values of total phenol content were 641.12, 730.69, 567.00 and 725.83 μg/mg

dwt equivalent to chlorogenic acid in Khasdi, Soneri, Desi and Red variety,

respectively (Table -3.2). It was observed that highest phenolic content was

907.23 μg/mg dwt in Red variety, while lowest was 294.80 μg/mg dwt in Desi

variety. Sellami et al. (2009) had shown that during the early vegetative stage,

phenolic acids were predominant with 118.37mg/100 g of dwt, and during the

other growth stages, it was decreased. Papageorgiou et al. (2008) reported

high amounts of phenolic acids in Lamiaceae plants and in sweet marjoram. Total phenolic content was detected by Mueller and Beckman (1974) in three

Musa cultivars. The total phenol was ranged from 157-246 μg/g fresh weight.

The monophenol content in leaves of different Musa varieties were shown in

Figure 3.3. The standard calibration curve for monophenol was prepared

using hydroxybenzine. The average values of monophenols measured were

62.27, 95.81, 68.94 and 136.91 μg/mg dwt equivalent to hydroxybenzine in

Khasdi, Soneri, Desi and Red variety, respectively (Table -3.2). The highest

phenolic content was observed 195.84 μg/mg dwt in Red variety and lowest

was 43.16 μg/mg dwt in Khasdi variety (Figure 3.3).

Changes in di-hydroxyphenol content measured in leaves of four Musa

varieties are presented in Figure 3.4. Pyrocatachol was used as standard to

prepare calibration curve of di-hydroxyphenol. Average values of di-

hydroxyphenol were 27.94, 45.80, 53.88 and 89.08 μg/mg dwt equivalent to

pyrocatachol in Khasdi, Soneri, Desi and Red variety, respectively (Table -

3.2). There was similar pattern in variation among Red and Khasdi varieties

while reverse was observed in Soneri and Desi varieties with variation in leaf.

Patel and Thaker (2007) had measured the monophenol content in the

different age groups of internodes. The higher levels of monophenols in all

stages of the internode development were observed. The dihydroxy phenols

were varies from developing stages of internodes. Pansuriya et al. (2006) had

estimated phenolic contents from leaf explants and calluses of Morus alba L.

Diphenols and monophenols were higher in callus cells as compared to

explants.


63

These results indicate high variability in the content of the investigated

biologically active compounds with Musa varieties. According to Luckner

(1980), as plant maturation progresses, there appears to be an ordered

expression of the genome such that particular enzymes or group of enzymes

are synthesized or activated at a particular stage of plant development.

Several studies on the characterization of phenolic substances show that RP-

HPLC is by far the most convenient and comprehensive technique for

separating the individual components in plant extracts (Zgo´rka and Kawka,

2001). HPLC with UV-vis multiwavelength detector was used since all

phenolic compounds show intense absorption in the UV region of the

spectrum. The present method is simple, easy to use, and effective enough

for the identification and quantification of major phenolic compounds in plants.

The qualitative and quantitative analysis of phenols was performed in four

Musa varieties using HPLC technique. The amounts of phenolic compounds

detected in all samples were presented in Table -3.4. These results were

expressed in μg/g dry sample in Table -3.5. The samples were denoted as

Khasdi (K), Soneri (S), Red (R) and Desi (D). Tannic acid, quercetin,

chlorogenic acid, syringic acid and caffeic acid was detected in all four Musa

varieties, while gentisic acid detected only in Desi variety (Table -3.5).

The most abundant phenolic compound detected was mandelic acid i.e.

215.13 μg/g dwt in Desi variety, while in Soneri variety it was 91.0 μg/g dwt

(Table -3.5), Vanillic acid was found only in two varieties (Table -3.4); highest

amount was 8.60 μg/g dwt in Khasdi, while 0.58 μg/g dwt was found in Desi

(Table -3.5). Eugenol also found in only two varieties viz. Soneri and Desi

(Table -3.4). The eugenol was 0.90 μg/g dwt detected in Desi, while 0.82 μg/g

dwt in Soneri (Table -3.5).

Gallic acid was found in Soneri, Desi and Red varieties (Table -3.4). The

highest amount was observed in Soneri while it was lowest in Red (Table -

3.5). Anisic acid was recorded in Desi and Red varieties (Table -3.4). The

highest value was observed in Desi i.e. 6.69 μg/g dwt (Table -3.5). Ferulic


64

acid was observed in Desi, and in Red varieties (Table -3.4). The value of

ferulic acid measured in Red was 0.22 μg/g dwt (Table -3.5). Ellagic acid was

observed in Soneri, Desi and Red varieties (Table -3.4). Ellagic acid was

ranged from 0.68 to 1.26 μg/g dwt (Table -3.5).

Coumarin was recorded in Khasdi, Soneri, Desi and Red (Table -3.4).

Coumarin was ranged from 0.14 to 0.81 μg/g dwt among the measured

samples (Table -3.5). Salicylic acid, caffeic acid, syringic acid and chlorogenic

acid were determined in all four Musa varieties (Table -3.4). The highest

amount of salicylic acid was measured in Khasdi variety while caffeic acid was

measured in Desi variety i.e. 4.72 and 0.45 μg/g dwt, respectively, the highest

amount of syringic acid and chlorogenic acid were measured in Soneri variety

i.e. 2.26 and 3.34 μg/g dwt, respectively.

Quercetin and tannic acid were observed in all four varieties of Musa (Table -

3.4). Quercetin and tannic acid were ranged from 0.47-3.78 and 8.17-34.76

μg/g dwt, respectively (Table -3.5). Thus, it seems that quercetin and tannic

acid was found in all four Musa varieties studied.

All phenolic compounds were unequivocally confirmed by relative retention

value and spectra of authentic standards. C-18 stationary phase was used in

this study to separate phenolic compounds of plant samples, produced

satisfactory separation of these compounds. Proestos and Komaitis (2008)

reported the highest amount of ferulic acid (6.9 mg/100 g dry sample) in

Origanum majorana, and syringic acid had detected only in Rosmarinus

officinalis. Also, the relatively high contents of ferrulic acid (895- μg/g), caffeic

acid (983-2561 μg/g) and chlorogenic acid (1720-2076 μg/g) had observed in

Bromus family (Pare et al. 1994).

The quercetin levels of peas hull (V. uniguiculata) (55.0 mg/kg) reported by

Huang et al. (2007). Quercetin was the second most abundant flavonol

detected in the fruits analyzed by some authors (Sultana and Anwar 2008).

Highest level was detected in mulberry (359.4 mg/kg), followed by apricot

(322.1 mg/kg), and apple (119.5 mg/kg). Van Der Sluis et al. (2004) reported


65

that quercetin glycosides were the main flavonol of apple. Franke et al. (2004)

reported quercetin content of apple (M. sylvestris). Siddhuraju and Becker

(2003) reported that quercetin and kampeferol levels of M. oleifera leaves

(Sohanjana) ranged from 6340 to 27,490 mg/kg and 647 to 1050 mg/kg,

respectively. Silva et al. (2006) reported that photosynthesis reaction taking

place in leaves results in the greater concentration of the phytochemicals.

Gallic acid is recorded as the most potent antioxidants among the simple

phenolic acids (Soobrattee et al. 2005). The phenolic compounds can be

presente in special glandules into the vegetative structure, depending on the

plant organ, the cultivation edafoclimatic conditions, stress, and age of the

plant (Roda et al. 2003). The quercetin concentration changes even with

position at the same leave surface, as was reported for Nicotiana attenuata

(Roda et al. 2003). The phenolic compounds were found mainly in the leaves

and roots of Centella asiatica. The extracts of fennel seeds, rosemary leaves,

macela flowers, and turmeric roots obtained by supercritical fluid extraction

presented higher yields in phenolic compounds. Overall the phenolics were

found in the higher concentrations in the banana leaves. A detail study is

needed for the selection of best solvent and extraction procedure to make use

of this waste product.

Table 3.1. Review on various parameters of phenolic compounds investigated in plants using HPLC analysis.

No. Sample Compounds

investigated Mobile phase

Stationary

phase

Flow Rate

Injection volume

Detection

(UV-vis)

nm

Reference

1

Vitex agnus-castus (Verbenaceae), Origanum dictamnus (Lamiaceae), Teucrium polium (Lamiaceae), Lavandula vera (Lamiaceae), Lippia triphylla (Verbenaceae)

Phenolic compounds (gallic acid, gentisic acid, p-coumaric acid, vanillic acid, ferulic acid, catechin, quercetin, apigenin, naringenin, eriodictyol, luteolin, caffeic acid, rutin, hydroxytyrosol, tyrosol, p-hydroxybenzoic acid and butylated hydroxytoluene)

H2O 1% glacial acetic acid (A), H2O with 6% glacial acetic acid (B), and H2O-acetonitrile (65:30 v/v) with 5% glacial acetic acid (C)

5 µm ODS2

4.6x250 mm

0.5 ml

min-1 20 µl

280 nm Proestos et al.

2006

2

Rosmarinus officinalis, Origanum dictamnus, Origanum majorana, Teucrium polium (all belonging to Labiatae), Vitex agnus–cactus (Verbenaceae) and Styrax officinalis (Styracaceae)

Phenolic compounds (gallic acid, gentisic acid, p-coumaric acid, vannilic acid, ferulic acid, syringic acid, catechin, quercetin, apigenin, naringenin, eriodictyol, luteolin, caffeic acid, rutin, and p-hydroxybenzoic acid)

H2O 1% glacial acetic acid (A), H2O with 6% glacial acetic acid (B), and H2O-acetonitrile (65:30 v/v) with 5% glacial acetic acid (C)

5 µm ODS2

4.6x250 mm

0.5 ml

min-1 20 µl

280 nm for

Phenolics and 320,

370nm for flavones

and flavonoles

Proestos and Komaitis 2008

3 Capparis spinosa (Capparaceae),

Phenolic compounds (gallic acid, gentisic acid, p-coumaric

H2O 1% glacial acetic acid (A),

5 µm ODS2

0.5 ml 20 µl 280 nm

for Proestos et al.

2006

Castanea vulgaris (Cupuliferae), Cuminum cyminum, Foeniculum vulgare (Umbelliferae), Geranium purpureum (Geraniaceae), Humulus lupulus,Urtica dioica(Urticaceae), Jasminum officinalis (Oleaceae), Nepeta cataria, Origanum dictamnus (Labiateae), Phytolacca americana (Phytolaccaceae), Ruta graveolens (Rutaceae), Spartium junceum (Leguminosae), Styrax officinalis (Styracaceae)

acid, vanillic acid, ferulic acid, syringic acid, catechin, quercetin, apigenin, naringenin, eriodictyol, luteolin, caffeic acid, epicatechin, rutin, hydroxytyrosol, p-hydroxybenzoic acid and butylated hydroxytoluene)

H2O with 6% glacial acetic acid (B), and H2O- acetonitrile (65:30 v/v) with 5% glacial acetic acid (C)

4.6x250 mm

min-1 Phenolics and 320,

370nm for flavones

and flavonoles

4 Canarium album (Burseraceae)

Phenolic compounds (gallic acid, ellagic acid, ferulic acid, quercetin, syringic acid, protocatechic acid, chlorogenic acid, caffeic acid, sinapic acid, methyl gallate, ethyl gallate.

0.5% (v/v) acetic acid (A) and methanol (B)

5 µm C18

250x 4 mm

0.6 ml

min-1 - 280 nm He and Xia

2007

5 Origanum majorana (Lamiaceae)

Phenolic compounds (gallic acid, caffeic acid, dihydroxyphenolic acid, epicatechin, chlorogenic acid, syringic acid, vanillic acid, rutin, p-coumaric acid, ferulic acid, rosmarinic acid, quercetin-3-rhamnoside, trans-2-dihydroxycinnamic acid, luteolin, coumarin, quercetin, cinnamic acid, apigenin, amentoflavone and flavone)

Acetonitrile (A) and H2O with 0.2% sulphuric acid (B)

4 µm ODS2

4.6x250 mm

0.5 ml

min-1 20 µl 280 nm Sellami et al.

2009

6 Plants, Fruit and Vegetables

Phenols and Acid hydrolyzed (kaempferol, triglycoside, apigenin glycosides, kaempferol diglycoside, luteolin glycoside, acylated kaempferol glycosides, chalcone, luteolin and kaempferol)

- - - - - Robards et al. 2003

7 Hippophae rhamnoides (Elaeagnanceae)

Phenolic compounds (gallic acid, protocatachuic acid, parahydroxybenzoic acid, vanillic acid, salicylic acid, cinnamic acid, p-coumaric acid, ferulic acid and caffiec acid)

2% acetic acid (A) and methanol (B)

5 µm ODS2

4.6x250 mm

1 ml min-1 20 µl 255 to

325 nm Arimboor et

al. 2008

8 Aromatic amino-acids Phenolic acids Aromatic - - - - - Waksmundzk

in Plants carboxylic acids a-Hajnos et al. 1998

9 Alpinia zerumbet (Zingiberaceae)

Phenolic compounds (p-hydroxybenzoic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid and cinnamic acid)

H2O with 1% acetic acid (v/v) (A) and methanol: acetonitrile:acetic acid (95:4:1, v/v/v) (B)

5 µm ODS

4.6x250 mm

0.8 ml

min-1 5 µl 280 nm Elzaawely et

al. 2007

10 Vitis vinifera Flavonoids and aromatic compounds (Dihydroflavonols, quercetin and kaempferol)

Acetonitrile/acetic acid/ H2O (35:2:63)(A) and 2% acetic acid (B)

5 µm C18

300x4.6 mm

0.8 ml

min-1 20 µl 240 to

460 nm

Masa and

Vilanova 2007

11 Origanum dictamnus (Labiatae)

Phenolic compounds and Flavonoids (rosmarinic acid, vanillic acid, caffeic acid, apigenin, eriodictyol, taxifolin, quercetin dehydrate, fisetin, luteolin, catecthin, diosmetin and thymol)

H2O (A), Methanol (B) and Acetonitrile (C), each contains 0.2% trifluroacetic acid

5 µm C18

250x4.6 mm

1 ml min-1 -

190 to 400 nm

Kouri et al. 2007

12 Vitex agnus castus (Verbenaceae)

Diterpenes and Flavonoids (rotundifurane and casticin)

Acetonitrile/ H2O (40/60, v/v)

10 µm C18

125x4 mm

0.8 ml

min-1 20 µl 258 nm Cossuta

et al. 2008

13 Indian medicinal plants (133 Spp., 64 Family)

Phenolic compounds, Phenolic acids, Tannins, Flavonoids, Curcuminoids, Coumarins, Lignans, Quinines and Anthocyanins

2.5% formic acid (A) and 100% Methanol (B)

5 µm C18

250x4 mm

0.8 ml

min-1 20 µl

190 to 550 nm

Surveswaran et al. 2007

14 Camellia sinensis (Theacea)

Poly phenols and Flavanols (gallic acid, caffeine, catechin, gallocatechin, epigallocatechin, epicatechin, epigallocatechin-3-gallate, gallocatechin-3-gallate and epigallocatechin-3-gallate)

M-I 0.12% Ammonium acetate (pH 5.5) (A) and methanol (B) M-II Acetoni- trile:ethyl acetate:0.1% orthophophoric acid (8.5:2:89.5,v/v/v) (A) and acetonitrile:water (1:1,v/v)(B)

4 µm C18

150x3.9 mm and

2-3 µm 100x4.6

mm

1 ml min-1 - 280 nm Baptista

et al. 1998

15 Ginkgo biloba

Phytochemicals (terpene, trilactones, ginkgolides, Bilobalide, flavonol, glycosides, quercetin, biflavones, proanthocyanides and alkylphenols) Ginkgolic acid; Phenolic acids; 6-Hydroxykynurenic acid; 4-O- Methylpyridoxine; Polyprenols)

- - - - - Beek et al. 2002

16 Alpinia zerumbet (Zingiberaceae)

Phenolic compounds and dihydro-5,6-dehydrokawain (Syringic acid, p-hydroxybenzoic acid, vanillin, p-coumaric acid, ferulic acid

H2O with 1% acetic acid (v/v) (A) and methanol:acetonitrile:acetic acid

5 µm ODS C18 250x 4.6

mm

0.8 ml

min-1 5 µl 280 nm Elzaawely

et al. 2007

and cinnamic acid) (95:4:1,/v/v) (B)

17 Food anthocyanins, isoflavones and flavanols

Polyphenols, Anthocyanidins, Isoflavones and Flavanols - - - - - Valls

et al. 2009

18 Cordia Americana (Boraginaceae)

Rosmarinic acid, 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid, rutin and quercitrin

Water-acetonitrile-formic acid (90:10:0.1) (A) and acetonitrile-formic acid (0.1%) (B)

5 µm C18

5x100 mm

0.5 ml

min-1 20 µl 330 nm

Geller et al. 2010

19 Melissa officinalis Polyphenolics And terpenoids (rosmarinic acid and luteolin-7-O-glucoside)

H2O -H3PO4, 85% (100:0.3) (A) and MeCN-H2O-H3PO4, 85% (80:20:0.3) (B)

4 µm Lichrocart

125 RP

2 ml min-1 25 µl 330 nm Carnat,

et al. 1998

20 Vasconcellea pubescens (Caricaceae)

Phenolic compounds (α- rhamnose, coumaric acid, caffeic acid and rutin)

1% formic acid (A) and MeOH (B)

5 µm RP- C18 250x4.6

mm

0.5 ml

min-1 20 µl 254 and

320 nm Simirgiotis et al. 2009

21

Vernonia amygdalina (Compositae) Corchorus olitorius (Tiliaceae) Ocimum gratissimum (Labiateae) Manihot utilissima (Euphorbiaceae)

Flavonoids and Cynnamoil derivatives (caffeoyl quinic acid, chlorogenic acid, rutin, luteolin, 1,5-Dicaffeoyl-quinic acid, apigenin 6-O or 7-O-glucur, Flavonoid, Kaempferol, Ferulic acid, Amentoflavone, caffeoyl quinic derivative, hyperoside, isoquercitrin,

H2O acidified to pH 3.2 by formic acid (A) and acetonitrile (B)

4 µm C12

150x4 mm

0.4 ml

min-1 -

200-400 nm

Ola et al. 2009

vicenin, caffeic acid, rosmarinic acid, cichoric acid, caffeoyl derivatives, cirsiliol, cirsimaritind and nevadensine)

22

Vernonia amygdalina (Compositae) Corchorus olitorius (Tiliaceae) Ocimum gratissimum (Labiateae) Manihot utilissima (Euphorbiaceae)

Flavonoids and Cynnamoil derivatives (caffeoyl quinic acid, chlorogenic acid, rutin, luteolin, 1,5-Dicaffeoyl-quinic acid, apigenin 6-O or 7-O-glucur, Flavonoid, Kaempferol, Ferulic acid, Amentoflavone, caffeoyl quinic derivative, hyperoside, isoquercitrin, vicenin, caffeic acid, rosmarinic acid, cichoric acid, caffeoyl derivatives, cirsiliol, cirsimaritind and nevadensine)

H2O acidified to pH 3.2 by formic acid (A) and acetonitrile (B)

5 µm Polaris-

ether (Varian) 250x4.6

mm

0.8 ml

min-1 - 200-400

nm Ola

et al. 2009

23 Lactuca sativa (Asteraceae)

Polyphenols (caffeic acid derivative (2, 5-caffeoylquinic acid, isochlorogenic acid, quercetin 3-O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-(6-malonylglucoside), chicoric acid and caffeic acid derivative)

Acetonitrile and Water (adjusted to pH 3.2 by H3PO4)

5 µm C18

150x3 mm

0.6 ml

min-1 - 190-450

nm Romania

et al. 2002

24 Platycladus orientalis Flavonoids (rutin, quercitrin, quercetin and amentoflavone)

Methanol (A), acetonitrile (B) and

5 µm XDB

0.8 ml 10 µl

190-800 nm

Lu et al. 2006

18mM sodium acetate buffer adjusted to pH 3.5 with glacial acetic acid (C)

C-18 150x4.6

mm

min-1

25 Cymbopogon citratus (Gramineae)

Flavonoids (caffeic acid derivatives, caffeic acid, p-coumaric acid derivative, luteolin derivatives and apigenin derivatives)

5% aqueous formic acid v/v (A) and methanol (B)

5 µm ODS-2 250x4.6

mm

1 ml

min-1 - 200-300

nm Figueirinha et al. 2008

26

Petroselinum sativum and celery (Apium graveolens) Umbelliferae (Apiaceae) and Urtica dioica (Urticaceae)

Plant phenolics (Ascorbic acid, gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, naringin, rosmarinic acid, hesperidin, rutin, isoquercitrin, Myricetin, quercetin, luteolin, kaempferoland apigenin)

Methanol (A) and 0.2% of o-H3PO4 in distilled water (B)

5 µm C18

250×4.6 mm

1ml min-1 - 280 nm Yildiz

et al. 2008

27

Rosmarinus officinalis,Origanum dictamnus, Origanum majorana, Teucrium polium (Labiatae), Vitex agnus–cactus (Verbenaceae)and Styrax officinalis (Styracaceae)

Plant phenolic (Gallic acid, gentisic acid, p-coumaric acid, vannilic acid, ferulic acid, syringic acid, catechin, quercetin, apigenin, naringenin, eriodictyol, Caffeic acid, Rutinand and p-Hydroxybenzoic acid)

Water with 1% glacial acetic acid (A), water with 6% glacial acetic acid (B), and water-acetonitrile (65:30,v/v) with 5% glacial acetic acid (C)

5 µm ODS2

4.6x250 mm

0.5 ml

min-1 20 µl 320 and

370 nm

Proestos and

Komaitis 2008

28 Borago officinalis L. Plant phenolic Rosmarinic acid

Acetic acid in water as (A) and acetonitrile as (B)

5 µm ODS-80TS 2mm IDx25

cm

0.3 ml

min-1 - 280 nm

Bandoniene and

Murkovic 2002

29

Dactylis glomerata; Festuca rubra L.; Festuca ovina L.; Bromus marginatus; Tilia cordata; Uncaria tomentosa

Phenolic compounds; (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, m-coumaric acid, o-coumaric acid, benzoic acid, ferulic acid, sinapic acid, rosmarinic acid, cinnamic acid, 3,4-dihydroxybenzaldehyde, p- hydroxybenzaldehyde, syringaldehyde and vanillin)

0.3% (v/v) acetic acid in water (A) and methanol (B)

3 µm BDS C18 4.6×100

mm

0.6 ml

min-1 20 µl 190-400

nm Štˇerbová et al. 2004

30 Vegetables Fruits and Medicinal plants

Kaempeferol, Quercetin and Myricetin

3% trifluoroacetic acid (A) and acetonitrile and methanol (80:20 v/v) (B)

5 µm ODS C18 250 x4.6

mm

1 ml min-1 20 µl 360 nm

Sultana and

Anwar 2008

31 Oxalis tuberosa Mol.

Phenolic compounds p-coumaric, o-coumaric, protocatechuic, ferulic, gallic, caffeic, chlorogenic and p-hydroxybenzoic), flavonols (quercetin, rutin, myricetin and kaempherol), and flavanones

Water: acetic acid (94:6, v/v, pH 2.27) (A) and acetonitrile (B)

5 µm RP18

250x4.6 mm

0.5 ml

min-1 -

200-700 nm

Chirinos et al. 2009

32 Hamamelis virginiana L.

Phenolic compounds (Catechins; Gallic acid)

0.1% (v/v) orthophosphoric acid in water (A) and 0.1% (v/v) orthophosphoric acid in methanol (B)

5 µm C18

150x4.6 mm

1 ml min-1

10 µl 210 nm Wang et al. 2003

Table 3.2. Average value of antioxidant potential, total phenol content, monophenol and diphenol of four Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red, respectively].

μg/mg dwt K S D R

Antioxidant activity 33.42 41.8 34.01 38.02

Total phenol content 641.12 730.69 567.00 725.83

Monophenols 62.27 95.81 68.94 136.91

Dihydroxyphenols 27.94 45.8 53.88 89.08

Table 3.3. The standard phenolic compound used in HPLC analysis with their properties.

No. Compound name

Retantion time min. IUPAC name Molecular

formula Molar mass

g\mol CAS

number

1 Vanillic acid 6.1 4-Hydroxy-3-methoxybenzoic acid C8H8O4 168.14 121-34-6

2 Gallic acid 14.1 3,4,5-trihydroxybenzoic acid C7H6O5 170.12 149-91-7

3 Gentisic acid 23.1 2,5-dihydroxybenzoic acid C7H6O4 154.12 490-79-9

4 Caffeic acid 26.6 3-(3,4-Dihydroxyphenyl 2-propenoic acid C9H8O4 180.16 331-39-5

5 Anisic acid 33 2-Methoxybenzoic acid C8H8O3 152.15 579-75-9

6 Ferulic acid 37.8 (E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid C10H10O4 194.18 1135-24-6

7 Syringic acid 40.1 4-hydroxy-3,5-dimethoxybenzoic acid C9H10O5 198.17 530-57-4

8 Chlorogenic acid 51.8

(1S,3R,4R,5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid

C16H18O9 354.31 327-97-9

9 Coumarin 59.5 2H-chromen-2-one C9H6O2 146.14 91-64-5

10 Salicylic acid 64.7 2-Hydroxybenzoic acid C7H6O3 138.12 69-72-7

11 Quercetin 77.6 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one C15H10O7 302.236 117-39-5

12 Tannic acid 81.3 3,5-dihydroxy-2-(3,4,5-trihydroxybenzoyl)oxy-6-[(3,4,5-trihydroxybenzoyl)oxymethyl]oxan-4-yl] 3,4,5-trihydroxybenzoate

C76H52O46 1701.2 1401-55-4

13 Ellagic acid 82.6 2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione C14H6O8 302.197 476-66-4

14 Eugenol 84.6 4-Allyl-2-methoxyphenol C10H12O2 164.2 97-53-0

15 Mandelic acid 87.6 2-Hydroxy-2-phenylacetic acid C8H8O3 152.15 90-64-2

Table 3.4. Detection of phenols in four Musa varieties based on HPLC analysis [K-Khasdi, S-Soneri, D-Desi and R-Red, + (yellow) and – (red) indicates presence and absence of phenols, respectively].

Variety Phenols K S D R

Vanillic acid + - + -

Gallic acid - + + +

Gentisic acid - - + -

Caffeic acid + + + +

Anisic acid - - + +

Ferulic acid - - + +

Syringic acid + + + +

Chlorogenic acid + + + +

Coumarin + + + +

Salicylic acid + + + +

Quercetin + + + +

Tannic acid + + + +

Ellagic acid - + + +

Eugenol - + + -

Mandelic acid - + + -

Table 3.5. Qualitative and quantitative HPLC based analysis of phenols in four Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red].

Variety

Phenols μg/g dwt K S D R

Vanillic acid 8.60 - 0.58 -

Gallic acid - 1.21 0.99 0.63

Gentisic acid - - 0.77 -

Caffeic acid 0.17 0.32 0.45 0.15

Anisic acid - - 6.69 0.74

Ferulic acid - - 0.43 0.22

Syringic acid 1.26 2.26 2.11 1.13

Chlorogenic acid 2.35 3.34 0.82 0.91

Coumarin 0.14 0.24 0.81 0.15

Salicylic acid 4.72 4.09 4.10 2.06

Quercetin 3.78 0.68 0.47 0.55

Tannic acid 34.76 32.94 8.17 12.03

Ellagic acid - 0.99 1.26 0.68

Eugenol - 0.82 0.90 -

Mandelic acid - 91.00 215.13 -

Figure 3.1. Changes in antioxidant potential equivalent to ascorbic acid in

fully expanded leaves of Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red] vertical bars represent SE.

Figure 3.2. Changes in total phenol equivalent to chlorogenic acid in fully

expanded leaves of Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red] vertical bars represent SE.

Figure 3.3. Changes in monophenol content equivalent to hydroxybenzine

in fully expanded leaves of Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red] vertical bars represent SE.

Figure 3.4. Changes in dihydroxyphenol content equivalent pyrocatachol in

fully expanded leaves of Musa varieties [K-Khasdi, S-Soneri, D-Desi and R-Red] vertical bars represent SE.

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