thesis...deep sense of gratitude to my major advisor dr. muneshwar prasad, a sstt. professor-cum-jr....

“Micropropagation of Banana cv. Malbhog for production of

quality planting material”

THESIS

SUBMITTED TO THE

DEPARTMENT OF HORTICULTURE (FRUIT & FRUIT TECHNOLOGY)

BIHAR AGRICULTURAL COLLEGE

BIHAR AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE (AGRICULTURE)

IN

HORTICULTURE

By

Kiran Bharati Reg. No. M/Hort./169/BAC/2014-15

DEPARTMENT OF HORTICULTURE (Fruit & Fruit Tech.)

Bihar Agricultural College

BIHAR AGRICULTURAL UNIVERSITY

Sabour – 813 210, Bhagalpur, Bihar, India

2016

BIHAR AGRICULTURAL UNIVERSITY,

SABOUR, BHAGALPUR

Dr. Muneshwar Prasad

Asstt. Prof.-cum-Jr. Scientist

Department of Horticulture

(Fruit & Fruit Technology)

Bihar Agricultural College Sabour,

Bhagalpur – 813 210 Bihar, India

Dated ______________

Certificate - I

This is to certify that the thesis entitled “Micropropagation of banana cv.

Malbhog for production of quality planting material” submitted in partial

fulfilment of the requirements for the award of the degree of Master of Science

(Agriculture) in the subject of Horticulture of the faculty of Agriculture, Bihar

Agricultural University, Sabour, Bhagalpur, Bihar, is genuine record of bonafide

research work carried out by Miss Kiran Bharati, Regd. No.

M/Hort./169/BAC/2014-15, under my guidance and supervision. No part of the

thesis has been submitted for any other degree or diploma.

It is further certified that such help or information received during the

course of this investigation and preparation of the thesis have been fully

acknowledged.

(Muneshwar Prasad)

Major advisor

(Advisory committee)

Certificate – II

We, the undersigned members of the Advisory Committee of Miss Kiran

Bharati, Reg. No. M/Hort./169/BAC/2014-15, a candidate for the degree of Master

of Science (Agriculture) in the subject of Horticulture majoring in Pomology, have

gone through the manuscript of the thesis and agree that the thesis entitled

“Micropropagation of banana cv. Malbhog for production of quality planting

material” may be submitted in partial fulfilment of the requirement for the award of

the degree.

(Muneshwar Prasad)

Major Advisor Advisory Committee

Endorsed:

Chairman

Department of Horticulture


Members

1. Dr. Hidayatullah Mir


Deptt. of Hort. (Fruit & Fruit Technology)

(Member from Major Subject)

2. Dr. Awadhesh Kumar Pal


Deptt. of Plant Breeding & Genetics

(Member from Minor Subject)

3. Dr. Basudev Kole


Deptt. of Statistics, Mathematics &

Computer Application

(Member from Supporting Subject)

4. Dr. Sanoj Kumar


Deptt. of Agricultural Engineering

(Nominee of the Dean PGS)

Certificate – III

This is to certify that the thesis entitled “Micropropagation of banana cv.

Malbhog for production of quality planting material” submitted by Miss Kiran

Bharati, Regd. No. M/Hort./169/BAC/2014-15 to the Bihar Agricultural University,

Sabour, Bhagalpur, Bihar in partial fulfilment of the requirements for the award of the

degree of Master of Science (Agriculture) in the subject of Horticulture, has been

evaluated satisfactory and approved by the Student’s Advisory Committee on

_____________

(Muneshwar Prasad)

Major advisor

(Advisory committee)

Name & Signature

of

External Examiner

Endorsed:

Chairman

Deptt. of Horticulture


1. Dr. Hidayatullah Mir


Deptt. of Hort. (Fruit & Fruit Technology)

(Member from major Subject)

2. Dr. Awadhesh Kumar Pal


Deptt. of Plant Breeding & Genetics

(Member from Minor Subject)

3. Dr. Basudev Kole


Deptt. of Statistics, Mathematics &

Computer Application

(Member from Supporting Subject)

Assoc. Dean-cum-Principal

Bihar Agricultural College, Sabour

Bhagalpur – 813 210

4. Dr. Sanoj Kumar


Deptt. of Agricultural Engineering

(Nominee of the Dean PGS)

DRI-cum-Dean, PGS

Bihar Agricultural University

Sabour, Bhagalpur – 813 210 [

Department of Horticulture (Fruit & Fruit Technology)

Bihar Agricultural College, Sabour

Bihar Agricultural University, Sabour, Bhagalpur (Bihar)-813210

Title: Micropropagation of banana cv. Malbhog for production of

quality planting material

Abstract

Banana belongs to the genus Musa and family Musaceae. It is one of the most important source of tropical fruits in the world market because it is a significant staple food as well as a major export commodity. Banana is the second major fruit crop after mango in Bihar. Two distinct tracts growing banana in Bihar are Vaishali region and North Eastern Koshi region. Important varieties grown in Bihar are Alpan, Malbhog, Kanthali, Champa, Kothia, Chinia and Chini Champa. Many of these varieties such as Malbhog and Alpan have no match for their fruit quality. These excellent varieties are on verge of extinction due to problem of diseases and non-availability of quality propagules. The conventional method of vegetative propagation in banana is by means of daughter sucker. However, the constraints of conventional propagation is the variation in age and size of sucker, non-uniform crop, prolonged harvesting difficulty in management and lack of availability of large quantities of sword suckers at any given time. In vitro propagation of bananas provide excellent advantages over traditional propagation, including a high multiplication rate, physiological uniformity, the availability of disease-free material all the year round, etc. Therefore, present study was performed with the objective to establish aseptic shoot culture of banana cv. Malbhog and hardening of rooted plantlets for field transfer using different (alone or in combination) sterilants, PGRs and potting mixtures. One of the most commonly encountered problems in in

vitro culture establishment is the contamination. The efficiency of sterilizing agents was evaluated in terms of maximum aseptic explants sprout.Maximum aseptic shoot culture establishment (45.00) was obtained by surface sterilizing the explants with ethanol (70%) for 30 sec + HgCl2

(0.1%) for 25 minutes.Addition of cytokinins alone or in combination with auxin was found essential for shoot initiation and multiplication. Minimum no. of days for shoot initiation (18.4 days), longest length of shoot (5.1 cm) and maximum no. of shoot/ explant (4.20) was obtained on PGR combination NAA 0.5 + BAP 5.0 mg/l.Half strength MS media with IBA 1.5 mg/l was found significantly higher over all other auxin treatments for various rooting parameters. ½ MS media supplemented with 1.5 mg/l IBA gave best response in terms of root formation frequency (66.67%), least no. of days for root formation (18.00), maximum no. of roots/explant (3.80) and longest length of root (4.0 cm). Maximum survival for hardening of plantlets was found in case of coco peat (89%) followed by sterile soilrite (79%). For various morphological parameters like plant height, leaf length, leaf width, leaf number, etc. coco peat gave better result over other. Thus, an attempt was made to standardize efficient and rapid micropropagation protocol which will be useful in expanding area under cultivation of Malbhog banana.

Dr. Muneshwar Prasad Kiran Bharati

CONTENTS

S.NO PARTICULAR PAGE

NO.

I INTRODUCTION

1-5

II REVIEW OF LITERATURE

6-29

III MATERIALS AND METHODS

30-42

IV EXPERIMENTAL FINDINGS

43-50

V DISCUSSION

51-57

VI SUMMARY AND CONCLUSION

58-59

VII BIBLIOGRAPHY

i-xiii

LIST OF TABLES

Table

No.

Particulars Page

No.

3.1 Composition of MS medium and stock solutions

33

3.2 Preparation of stock solutions of different

phytohormones

34

3.3 Surface sterilant treatment duration to prevent

contamination

38

3.4 MS basal medium treatments with different

concentration of

growth regulators for shoot multiplication

40

3.5 Half MS basal medium treatments with different

concentration of growth regulators for root

initiation

40

4.1 Effect of surface sterilization treatments on

establishment of shoot tip explant of banana cv.

Malbhog

44

4.2 Effect of MS basal medium treatments with

different concentration of growth regulators on

shoot multiplication

45

4.3 Effect of Half MS basal medium treatments with

different concentration of growth regulators for

root initiation

48

4.4 Effect of different potting mixtures on hardening

of banana cv. Malbhog microprogated plantlets

48

4.5 Effect of different potting mixtures on

morphological characteristics of banana cv.

Malbhog micropropagated plantlets

50

LIST OF PLATES

Sl. No.

Title of Plate

After Page

4.1 Explant inoculation into MS media

50

4.2 Cultures showing contamination in Malbhog banana explants

50

4.3 Cultures showing establishment of banana

explants

50

4.4 Cultures showing shoot initiation and shoot proliferation

50

4.5 Shoot multiplication in Malbhog banana

50

4.6 Root initiation and proliferation in Malbhog banana

50

4.7 Hardening of plantlets in different potting

mixtures

50

4.8 Plantlets in secondary gardening

50

ACKNOWLEDGEMENT

I deem it to be a rare privilege and golden opportunity to express my deep sense of gratitude to my Major Advisor Dr. Muneshwar Prasad, Asstt.

Professor-Cum-Jr. Scientist, Department of Horticulture (Fruit and Fruit Technology),Bihar Agricultural College, Sabour, Bhagalpur for his inspiring,

learned guidance, untiring help and constant encouragement in carrying out the research and preparation of this manuscript.

With the same spirit, I express my fathomless gratitude to benevolent and ever generous members of my Advisory Committee, Dr.

Hidayatullah Mir, Asstt. Professor-Cum-Jr. Scientist, Deptt. of Horticulture, (Fruit and Fruit Technology), Dr. Awadhesh Kumar Pal, Asstt. Professor-

cum-Jr.-Scientist., Deptt. of Plant Breeding and Genetics, Dr. Basudev Kole, Asstt. Professor- Cum-Jr. Scientist, Deptt. of Statistics, Mathematics and

Computer Application.

I am much obliged to my Nominee of Dean PGS Dr. Sanoj Kumar, Asstt. Professor- cum-Jr.-Sci., Deptt. of Agricultural Engineering, for

providing necessary facility to carry out the research work in an effective manner.

I am highly grateful to My Chairman Prof. (Dr.) Feza Ahmed, University Professor-cum-Chief Scientist, Deptt. of Horticulture (Fruit and

Fruit Technology) and also grateful to Dr. V. B. Patel University Professor-cum- Chief Scientist Deptt. of Horticulture (Fruit and Fruit Technology)

B.A.C., Sabour for providing adequate facilities and valuable suggestions during the course of investigation.

My sincere thanks to Dr. R. R. Singh, Sr. Scientist-cum-Assoc.

Professor, Dr. Sanjay Sahay, Sr. Scientist-cum-Assoc. Professor, Dr. Ruby Rani, Asstt. Professor-cum-Jr. Scientist, Dr. K. Karuna, Asstt. Professor-cum-

Jr. Scientist, Department of Horticulture (Fruit and Fruit Technology) for their kind help and guidance during my entire study period as well as during preparation of manuscript.

I am very much thankful to Dr. Ravindra kumar Jr- Scientist cum-

Asstt. Prof, Dr. Kalyan Barman, Asstt. Professor-cum-Jr Scientist, Dr. Samik Sen Gupta, Asstt. Professor-cum-Jr Scientist, Dr. Manoj Kundu, Asstt.

Professor-cum-Jr Scientist, Dr. Rupa Rani, Asstt. Professor-cum- Jr Scientist, Deptt. of Horticulture (Fruit and Fruit Technology), for their very valuble

suggestion, remark and co-operation during the lab work.

I am also extending my heartiest thanks to Pranay Prasad and Anil kumar, tissue culture lab, Department of Plant breeding and Genetics.

I am also grateful to Dean, (Agriculture), DRI-cum-Dean PGS, Dr. B.

C. Saha, BAU, Sabour & Assoc. Dean-cum-Principle, BAC, Sabour for providing necessary financial facilities and encouragement to perform my research work for M. Sc. (Ag.) degree programme.

I am indeed very much obliged to Dr Ajoy Kumar Singh, Hon’ble Vice-Chancellor, BAU, Sabour, Bhagalpur, Bihar for providing all facilities during

the course of my investigation.

I also pay my heartiest thanks to all the staffs of Tissue Culture Lab and Department of Fruit and Fruit Technology, College, Library and

University for their unending help during my research and study.

I am also well wishes who helped me a lot in various ways during the entire period of my investigation.

I have no word to express my deepest and heartiest gratitude to my

parents Late Dr. Ram Kripal Mahto and Mrs. Veena Devi and Brothers for their love, affection, encouragement and blessings to achieve my goal.

I would like to record my special and heartiest thanks to my seniors

Nishi Kumari, Pushpa Kumari, Kanchan Bhamini, Amrita Kumari, Ruchi Kumari, Ravindra Kumar Singh, Piyush Shrivastava, Praveen Mishra,

Sachin Tyagi, Awadh Kishor Sah, Vikash Kumar, Vishal Nirgude, Alok Kumar, Ram Yadav, my Dearest batchmates Abhilasha Sinha, jyoti Kumari, Brajesh Nandan, Nishant Kumar, Syed Rajaul Islam, Malaviya Amit, Anjli

Kumari, Pratima Kumari, Anisha Kumari, Abha Kumari, Jyoti Bharti and Reena Kumari and my lovely junior Ankita, Shweta, Aditi, Deeplata, Vinay

,Amresh, Shashank, and Shivbhavan.

I wish to convey my heartiest thanks to all those who has helped me during my study programme.

At the last but not the least I am extremely grateful to the almighty

who inspired and helped me to come up where I am today.

Sabour Dated: ......../........./2016 (Kiran Bharati)

LIST OF ABBREVIATIONS

% Percentage NAA Naphthalene Acetic Acid

°C Degree Celsius NaOH Sodium hydroxide

ANOVA

Analysis of Variance PGR Plant Growth Regulator

BA Benzyl adenine pH Power of Hydrogen

BAP 6- Benzyl amino purine psi Pounds per square inch

CD Critical Difference RH Relative Humidity

cm Centimeter SE Standard error

CRD Completely Randomized Design

std Standard

et al. et alii ('and others') UV Ultraviolet

GA3 Gibberellic acid V Volt

gm Gram v/w Volume/Weight

HCl Hydrochloric acid w/v Weight/Volume

hr Hour Fig. Figure

IAA Indole-3-acetic acid mm Milimeter

IBA Indole-3-butyric acid MT Metric ton

INTs Intensity Ha Hectare

KCl Potassium chloride cv cultivar

KIN Kinetin v/v Volume/volume

Mg Magnesium NaOCl Sodium hypochorite

MgCl2 Magnesium chloride CW Coconut Water

mg/l Milligram per litre 2,4-D 2, 4-dichlorophenoxyaceticacid

min Minute TDZ Thidiazuron

ml Millilitre 2 ip N6 - (2-isopentyl) adenine

mM Millimolar Sec Second

mm Millimeter

MS Murashige and Skoog

N Normality

Chapter-I

Introduction

Banana belongs to the genus Musa and family Musaceae. It is

known as one of the most important source of tropical fruits in the

world market as it is a significant staple food as well as a major

export commodity (Rahman et al., 2013). Bananas and Plantains

(Musa spp.) are some of the earliest crop plants which have been

domesticated by humans (Singh et al., 2011). Bananas and

Plantains (Musa spp.) are the most important and most widely

grown fruit crop in India. Banana is commonly known as "Apple of

Paradise" and botanically "Musa paradisiaca". The genus name

Musa is thought to be derived from the Arabic name for the plant

(Mouz) which, in turn, may have been applied in honour of

Antonius Musa (63-14 BC), physician to Octavius Augustus, first

emperor of Rome (Hyam and Pankhurst, 1995). The name banana

is derived from Arabic word "banan” = finger (Boning, 2006) and

was thought to be used in Guinea (West Africa) concomitant with

the introduction of fruit by the Portuguese. Oldest record of edible

bananas come from India (600 BC), known only by Hearsay in the

Mediterranean region in the third century BC (Horry et al., 1997). It

is believed that bananas were first introduced to Europe in the 10th

century. Early in the 16th century, Portuguese mariners

transported the plant from the West African coast to South

America. The wild types found in cultivation in the Pacific have

been traced to Eastern Indonesia from where they spread to the

Marquesas and gradually to Hawaii (Arvanitoyannis et al., 2008).

Bananas are very adaptable plants and geographically bananas

grows best between latitude 20°N and 20°S, where there are

predominantly tropical conditions. For growth and flower

production, optimal temperatures should be between 22°C and

INTRODUCTION

2

31°C with rainfall of 2000-2500 mm, spread evenly throughout the

year (Robinson and Sauco, 2010). Many plants are found in the

genus Musa but, those that bear edible fruits are the most

significant. Banana is a good source of carbohydrate, proteins,

vitamins and other nutrients (Kalimuthu et al., 2007) having 67-

137 calories per 100 g fruit. It is also a good source of potassium,

phosphorus, calcium and magnesium. The fruit is easy to digest

and is free from fat and cholesterol. Banana powder is used as the

first baby food. Its year round availability, affordability, varietal

range, taste, nutritive and medicinal value makes it the favourite

fruit amongst all classes of people. In addition to fruit, plantain and

banana cultivars provide medicines, beverages, fibres, edible floral

parts, dyes, fuel, stem for cooking and cordage. It help in reducing

risk of heart disease when used regularly and is recommended for

patients suffering from high blood pressure, arthritis, ulcer and

gastroenteritis. The unripe fruit can be brewed to form beer and

wine or processed into sauce, flour, chips, crisps, smoked products

and confectionary. Unripe fruit is also a source of amylase and

starch. Male floral buds can be eaten as a boiled vegetable, whereas

pseudostems are a source of fibre for the manufacture of rope,

paper and textiles. India leads the world in banana production with

an annual output of about 30.08 million tonnes (NHB, 2015). Major

banana growing states in India are Maharashtra, Tamil Nadu,

Gujarat, A.P., Karnataka, M.P., Bihar, U.P., West Bengal, Assam,

Kerala, and Orissa. Among them, Tamil Nadu is the leading state

with an area of 118 thousands ha with total production of 5650

thousand MT. Banana is the third major fruit crop after mango in

Bihar, it occupies an area of 34.31 thousands ha with an annual

production of 1526.50 thousands MT and a productivity of 44.06

MT/ha (NHB, 2015). Two distinct tracts of banana growing in Bihar

are Vaishali region and North Eastern Koshi region. Important

varieties grown in Bihar are Alpan, Malbhog, Kanthali, Champa,

INTRODUCTION

3

Kothia, Chinia and Chini Champa. Many of these varieties such as

Malbhog, Alpan and Chinia have no match for their fruit quality.

These excellent varieties are on verge of extinction due to problem

of diseases and non-availability of quality propagules.

Bananas and plantains are propagated vegetatively because

almost all cultivated banana cultivars are triploid, seedless or seed

sterile. The materials used for conventional propagation include

corms, large and small suckers, and sword suckers (Cronauer and

Krikorian, 1984; Arias, 1992). The main method of vegetative

propagation in banana is by means of daughter suckers formed at

the base of pseudostem suckers (5 to 10 in number depending on

the variety). Traditionally, sword suckers with narrow leaves,

weighing approximately 500-1000 g are the preferred planting

material for vegetative propagation. Banana propagated through

vegetative means suffer from slow multiplication, bulkiness, and

poor phytosanitary quality (Vuylsteke, 1989). The major constraint

for conventionally propagating banana is the lack of ready

availability of large quantities of sword suckers. The problem is felt

more acutely in non-availability of sword suckers consistently.

Besides, fungal, bacterial, nematode and viruse pathogens

are also transmitted through vegetative propagation. This defect

continues crop after crop, affecting the yield adversely and thus,

bulking of new varieties is also difficult through vegetative method.

No genetic variation can be expected, and hence they are incapable

of adapting to new condition. Similarly, due to the variation in age

and size of sucker, the crop is not uniform, harvesting is prolonged

and management becomes difficult. Therefore, in vitro clonal

propagation i.e. tissue culture plants (properly hardened secondary

seedlings) are recommended. Several researchers have reported the

regeneration of Musa spp. via. micropropagation (Krishnamoorthy

INTRODUCTION

4

et al., 2001; Kagera et al., 2004; Madhulatha et al., 2004 and Roels

et al., 2005). In vitro propagation of bananas provide 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 to conventional plants and faster

growth in the early growing stages compared to conventional

materials (Arias, 1992). Tissue culture also plays a vital role in the

distribution of germplasm conservation and safe exchange of

internal planting material. Mass propagation of selected genotype,

somaclonal variation techniques, genetic engineering and other

biotechnological applications can be utilized for banana

improvement and are based on reliable plant regeneration

protocols. Tissue cultured bananas are now getting commercial

acceptance among farmers owing to their uniform, fast growing

nature, pest and disease free seedling and early maturity of crops

than suckers. Tissue culture has been proven to be potential

technology to produce millions of identical plantlet, which are

disease free and true to parental type (Akbar and Roy, 2006).

Production of plants in test tubes facilitate safe movement and easy

handling of germplasm between laboratories within and across

countries. The yields and returns are expectedly higher (Hussein,

2012). Different in vitro techniques have been applied to banana

including shoot regeneration from cultured tissue by organogenesis

and somatic embryogenesis for micropropagation, embryo rescue,

somaclonal variation and gene transfer by somatic hybridization

and transformation (Sipen et al., 2011).

In Bihar “Malbhog (Musa paradisiaca AAB group)” is very

popular with high commercial value, has high demand in market

due to its sweet aroma, taste and higher postharvest life. However,

INTRODUCTION

5

shortage of planting material and synchronisation of fruit ripening

are two major bottlenecks that cause unavoidable trouble to

banana growers. So, use of tissue culture technique and

development of micropropagation protocol in the elite cultivar of

Bihar such as Malbhog will save them from extinction and will help

in their further expansion in new areas. Keeping all these factors in

mind and in order to ensure large scale production of quality

planting material of cv. Malbhog, the present study on

“Micropropagation of banana cv. Malbhog for production of quality planting material” is being planned with the following

objectives:

1. To study the establishment of aseptic shoot culture of banana

cv. Malbhog.

2. To study the success for establishment of micropropagated

plantlets during hardening.

*****

Chapter-II

Review of Literature

This chapter deals with presentation of relevant and

comprehensive review of literature on the present experiment

“Micropropagation of banana cv. Malbhog for production of quality planting material”. This is very essential aspect to provide

an insight into the previous research work helping in meaningful

interpretation of the findings.

Tissue culture technique cover a wide range of techniques

including in vitro culture of organs (shoot tips, root tips, runner

tips, stem segments, flowers, anthers, ovaries, ovules, embryos etc.)

tissues, cells and protoplasts. The beginning of plant tissue culture

took place in 1902 when the well-known German plant

physiologist, Gottlieb Haberlandt did cell culture. He developed the

concept of in vitro cell culture and regarded as the father of plant

tissue culture. Micropropagation is the practice of rapidly

multiplying stock plant material to produce a large number of

progeny plants, using modern plant tissue culture methods. It is

used to produce a sufficient number of plantlets for planting from a

stock plant which does not produce seeds or does not respond well

to vegetative reproduction. Following are the methods of

micropropagation:

1) Meristem culture

2) Callus culture

3) Suspension culture

4) Embryo culture

5) Protoplast culture

In vitro banana production technology is a superior

technology over traditional method (Sucker-propagated) of banana

Review of literature

7

production with respect to optimal yield, uniformity, disease-free

planting material and true to type plants. They are cheaper to

transport than conventional suckers coupling with virus indexing,

allows for safe movement and exchange of germplasm (Ngomuo et

al., 2014). The rapid proliferation obtained in tissue culture allows

nurserymen to meet an unexpected demand for a particular

cultivar; a million or more plants can be produced in a year from a

single meristem tip.

But, the response of banana tissues in vitro is highly

genotype specific. For instance, the response of AAB type banana is

much poorer than that of AAA (Bhagyalakshmi and Singh 1995;

Hirimburegama and Gamage 1997; Roels et al. 2005). In AAB, the

growth of axilliary buds in vivo is inhibited by a high degree of

apical dominance. Also, during sterilization, the living materials

should not lose their biological activity and only contaminants

should be eliminated; therefore explants are surface sterilized only

by treatment with disinfectant solution at suitable time and

concentrations for a specified period.

Apart from this, success of micropropagation depends upon

the following factors as:-

1. Nutrient media.

2. Concentration of different growth regulators like the effect of

cytokinins in the course of shoot induction.

3. Type of cytokinin to be used which is able to induce largest

amount of shoots.

4. Interaction of genotype and environmental effect.

So, the given below review of literature is aimed at

highlighting the factors that lead to success of micropropagation of

banana, potential of improvement in its micropropagation, the


8

problems encountered in micropropagation of banana and the ways

to solve them.

2.1 Tissue culture

Plant tissue culture is the science of growing plant cells,

tissues or organs isolated from mother plant on artificial media. It

include techniques and methods appropriate to research into many

botanical disciplines and several practical objectives. Both

organized and unorganised growth is possible in vitro (George,

1993).

Ahloowalia et al. (2004) referred plant tissue culture as

growing and multiplication of cells, tissues and organs of plants on

defined solid or liquid media under aseptic and controlled

environment. The commercial technology is primarily based on

micropropagation, in which rapid proliferation is achieved from

tiny stem cuttings, axillary buds and to a limited extent from

somatic embryos, cell clumps in suspension cultures and

bioreactors. The process of micropropagation is usually divided

into several stages i.e., pre-propagation, initiation of explants,

subculture of explants for proliferation, shooting, rooting and

hardening.

Venkatachalam et al. (2007) established for the first time that

the regeneration and rapid micropropagation protocol will be of

great use in conserving the endangered cultivars without the risk of

genetic instability.

A tissue-culture technique in which propagules are cloned

from tissue taken from a single plant is known as micro-

propagation (Perez and Hooks, 2008).


9

2.2 Tissue culture of banana

Sengar et al. (2010) concluded that micropropagation of

bananas remain as the next best alternative of natural regeneration

with tremendous potential for production of quality planting

material. Thus, the farmers are benefited by tissue-cultured plants

because of ease of multiplication of their variety of choice. He also

suggested that micropropagation is user-friendly technique which

does not require much expertise and is suitable for adoption by

small and marginal farmers and success of micropropagation

depends on method, variety and price of initiation media.

Further, Gitonga et al. (2010) evaluated a micropropagation

protocol for local banana (Musa spp., Muunju landrace) in Kenya

as an alternative to reduce the unit cost of tissue culture

micropropagation.

Ngomuo et al. (2014) reported that the planting materials of

banana obtained through conventional methods (suckers) do not

meet the increasing demand for planting and they are of poor

quality. Tissue culture is the approach which can solve these

problems.

Gray and Daniels (2015) described micropropagation as the

practice of growing plants like banana from meristematic tissue or

somatic cells of superior plants in vitro.

2.2.1 Culture medium

Success of plant tissue culture depends largely on the choice

of nutrient medium, including its chemical composition and

physical form (Murashige, 1974). Several media formulations have

been reported for banana shoot tip culture but, nearly half of them

are modified MS media (Brown et al., 1995). Other popular media

include B5 (Gamborg et al., 1968), SH (Schenk and Hildebrant,


10

1972), N6 (Chu et al., 1975) and LS (Linsmaier and Skoog, 1975).

The culture medium vary in both type and concentration of

components but all have similar basic component of growth

regulators viz. nitrogen, carbohydrates, organic, inorganic, micro

and macro nutrients and vitamins.

The added phytohormones and natural extracts in the basal

medium were important for tissue culture responses. In banana,

besides carbon sources and gelling agent, some phytohormones

were also used. By changing the amount and type of growth

regulators in the medium, the cells can be stimulated to develop in

to shoots and/or roots. The most widespread used technique for

vegetative propagation in banana is in vitro micropropagation by

culturing actively growing pieces of plants under varying

concentrations of different auxins and cytokinins (Ortiz and

Vuylsteke, 1994; Vuylsteke, 1998; Mendes et al., 1999; Arinaitwe et

al., 2000; Wojtania and Gabryszweska, 2001).

Generally, the cultures are established on a separate

initiation medium, which has a lower concentration of cytokinin

than the multiplication medium, to which cultures are

subsequently transferred (Jarret et al., 1985; Novak et al., 1989).

Akbar and Roy (2006) cultured banana explants on MS

medium supplemented with 0.5 mg/l of BA, Kn and NAA and found

that addition of 10% coconut water to the medium resulted in

increased number of differentiated shoots per culture. It was also

found that acclimatization and transplantation performances of

plants was superior for those rooted in liquid medium compared to

those rooted in solid medium.

Madhulata et al. (2006) studied the effect of carbon sources

(sucrose, glucose, fructose and mannitol) on in vitro propagation of

banana and found the highest frequency of shoot proliferation on


11

the medium containing sucrose compared to all other carbon

sources. It was also reported that MS medium supplemented with

sucrose and glucose combination (1:1) at the concentration of 30

gm/l showed the optimum shoot proliferation. Further, Feng et al.

(2007) optimized medium for in vitro proliferation of banana and

found that 40 gm/l sugar not only facilitated proliferation of buds

but also controlled proportion of buds with leaf sheaths and

increased the available bud index. The highest available rooted bud

index was obtained on medium with 60 gm/l sugar.

The effect of nutrient medium constituents on growth and

development of banana plantlets produced in vitro was evaluated by

Hussein (2012). He found that the medium with sucrose at 30%

supplemented with 0.4mg/l BA was the most optimum for banana

shoot tip culture as expressed by better growth vigour, plantlet

height, fresh weight as well as stronger shoot and root system.

Ahmed et al. (2014 a) investigated the effect of different

carbohydrate source, pH and supporting media on in vitro rooting

of banana plantlets using MS medium with 0.1 mg/l IBA and

activated charcoal. Sucrose in the medium remarkably influences

the rooting of plantlets. In the absence of sucrose, culture could

not survive after 3 weeks of incubation. In the sucrose containing

media, 30 g/l gave the best result. Out of different pH levels tested,

minimum time for root initiation with longest length of root was

obtained on pH 5.5. The reduction of agar concentration from 0.8

to 0.4% in the medium improve the in vitro root and shoot

characters as compare to other supporting structures viz.

Whatman No. 1 filter paper, ordinary filter paper and brown paper.

2.2.2 Explant

Sharrock (1992) reported that in vitro cultures of both

pineapple and plantain can be initiated from any growing point of


12

the plant. Thus, dormant buds and shoots that are unsuitable for

conventional propagation, can be utilised in micropropagation.

Rates of multiplication in vitro are much higher than those

achievable in conventional propagation systems.

Jyothi et al. (1993) cultured shoot tips, eye bud or floral apex

explants of banana cv. Red banana on semi-solid MS medium

supplemented with various growth regulators. For all 3 explant

types, the shortest time taken for culture establishment (8, 9 and

14 days for shoot tip, eye bud and floral apex explants,

respectively) was obtained with 0.5 ppm NAA+3 ppm BA.

Amiri (2001) used shoot tips from small suckers of banana to

see the growth and rate of multiplication of Musa acuminata cv.

Dwarf Cavendish in vitro. Muhammad et al. (2004) reported that

banana shoot tips derived from different suckers can be used to

study multiplication rate in banana. Further, Perez and Hooks

(2008) reported that micropropagating banana through shoot tip is

the main method used for fast propagation of banana plants.

Matsumoto et al. (2010) reported that the protoplast culture

and somatic hybridization are a feasible technique of

micropropagation and to support the genetic improvement of

banana.

Ahirwar et al. (2012) used male inflorescence tip explants and

shoot tip of banana for studying micropropagation in banana.

Goswami and Handique (2013) studied the effect of three

different sizes of explants (5, 10 and 20 mm) on the establishment

of banana in micropropagation. Three cultivars (Amritsagar,

Malbhog and Chenichampa) were used for the study. They found

that larger explants (20 mm) responded well with regard to survival

of explants, days to swelling and greening of explants, emergence of


13

leaf and days to multiple bud initiation under in vitro condition as

compared to smaller explants.

2.2.3 Establishment of aseptic culture condition

Explants should be free from surface contaminants to

accomplish growth and development under aseptic conditions and

this is achieved by surface sterilization. The sterilization treatment

should be so selected that it kill the microbes without affecting the

plant tissues adversely. Contamination in tissue culture may be

caused by endogenous bacteria that escape initial disinfection or by

microorganisms introduced during tissue culture manipulations.

Both types of contaminants may survive in the plant material for

several sub-culture cycles without expressing symptoms in the

medium. There are several antibiotics which are successful in

controlling bacterial contaminants in banana tissue culture.

Oliveira et al. (2000) evaluated two methods of banana

explant disinfection and the use of 3 indicator culture media for

banana micropropagation. Shoot tips of the cultivar Pioneira were

disinfected by (1) immersion in 80% alcohol for 2 min and in (2)

sodium hypochlorite (2% active chlorine) with 4 drops of Tween-20

for 10 min, with shaking. In both treatments, explants were rinsed

3 times in sterilized distilled water, immersed individually in 90%

alcohol for 3 seconds flamed and cut to reduce their size to 0.6×0.4

cm. The explants were established on 3 indicator media: 1) 1.3 gm

meat extract/l, 5 gm peptone/l and 15 gm agar/ l, 2) pH 5.6 or MS

medium with 30 gm sucrose/l, 3) 1.8 gm phytagel/l, pH 5.7. Bud

multiplication was carried out in MS medium with 3 subcultures

(30 days each). The efficiency of TI and TII disinfection treatments

were similar. The contamination observed was only of bacterial

nature. The multiplication rate was highest when TI treatment was


14

used. MS was the best contamination indicator media and it had

the advantage of being suitable for multiplication phase.

Lima and Moraes (2006) evaluated the bacterial

contamination control methods in banana explant cv. Caipira using

NaOCl, rifampicin antibiotic and their combinations. The best

treatment for explants was the immersion in 1% (v/v) NaOCl for 10

minutes, followed by immersion in 300 mg/l rifampicin for 20

minutes. After contamination, the best treatment was the

immersion in 1% NaOCl for 10 minutes, followed by immersion in

300 mg/l rifampicin for 24 hours in the dark.

The disinfectants widely used are sodium hypochlorite (which

dates back to the mid-18th century), calcium hypochlorite, ethanol

(or isopropyl alcohol), mercuric chloride, hydrogen peroxide, silver

nitrate and bromine water to enhance effectiveness in sterilization

procedure, a surfactant like Tween-20 is frequently added to the

sterilizing solution (and in some laboratories a mild vacuum is

applied during the procedure); in general, the sterilizing solutions

containing the explants are continuously stirred during the

sterilization period (Oyebanji, et al., 2009).

Jing-Yan et al. (2011) studied the effects of different

disinfectant on explants. Mercuric chloride and 0.2% sodium

hypochlorite solutions were used to sterilize the sucker buds of

Longxuan banana which were used as explants. Sodium

hypochlorite showed better effects than mercuric-chloride on the

disinfection of explants, the sterilization rate was 90.47% and the

explants grew well without any intoxication.

Goswami and Handique (2013) did the sterilization of

explants by firstly treating with savlon for 15 minutes followed by

sterilizing with a mixture of 2% NaOCl+1 gm/l Captan or Dithane

M-45 and rifampicin (0.1%) for 45 minutes with Tween-20.


15

Thereafter, quick dipping of explants (15 sec) in 70% alcohol. On

laminar air flow cabinet explants treated with NaOCl (0.5-1%) for

15 minutes followed by treatment with 0.1% HgCl2 for 7 minutes

proved better than the other treatments.

Bohra et al. (2014) studied the aseptic culture establishment

using antibiotics with reference to their efficiency and phytotoxicity

in difficult-to-establish native Ney Poovan banana (Musa AB). They

standardize antibiotic supplement for obtaining aseptic cultures in

native banana variety Elakki Bale. Of the different antibiotics and

combinations tried, chloramphenicol was found to give 100%

aseptic cultures followed by rifampicin + chloramphenicol,

rifampicin and chloramphenicol + streptocycline combination.

Rifampicin was found to cause least phytotoxicity. The plantlets

grew normally in subsequent cycles and 96.3 % plantlets could

survive upon transfer to ex vitro conditions.

Anbazhagan et al. (2014) did surface sterilization of the

explants primarily by rinsing in tap water for 30 minutes followed

by gently rinsing with 70% ethanol for 60 seconds and with 5%

sodium hypochlorite solution for 10 minutes. Further, sterilization

procedures were carried out in laminar air flow chamber by using

0.1% HgCl2 for 5 minutes.

Helaly et al. (2014) studied the effect of nanoparticles on

biological contamination of in vitro culture of banana. Nine strains

of bacterial contaminants (Cellulomonas uda, Cellulomonas

flarigena, Corynebacterium panrometabolum, Bacillus megaterium,

Staphylococcus spp., Klebsiella spp., Erwinia cypripedii,

Pseudomonas spp. and Proteus spp.) and four fungal (Fusarium

spp., Aspergillus spp., Penicillium spp. and Candida spp.)

contaminants were identified in nanoparticles-free media of banana

in vitro cultures. They eventually led the explants death. The


16

contamination-free cultures of banana in vitro cultures were

obtained as a result of application of nano Zn and ZnO particles to

the culture MS media, with no negative effect on regeneration.

Ahmed et al. (2014) found considerable reduction in

contamination of explant by treating with sodium hypochlorite 5%

for 10 minutes after rapid rinsing in 70% ethanol for 30 sec. In the

same study he concluded that sterilization of explants with 5%

sodium hypochlorite alone was ineffective resulting in very high

contamination.

Chalak et al. (2015) studied the removal of viruses from

Lebanese fig varieties using tissue culture and thermotherapy.

They found that the shoot tip culture was reliable for elimination of

60 to 100% of fig viruses. However, stem cutting culture coupled

with thermotherapy was the most effective for shoot regeneration.

Shashikumar et al. (2015) treated the explants of Musa spp.

Karibale Monthan with 70% absolute alcohol for 6 min, 0.1 %

mercuric chloride for 10 min and again 0.2 % for 10 min, 1 %

Sodium hypochlorite for 15 min, 0.1 % cefotaxime for 5 min and

0.05 % gentamicin for 5 min. to minimize the bacterial

contamination and to promote healthy growth.

Gray and Daniels (2015) tested the effectiveness of two

different sterilization agents, 70% ethanol and 3% sodium

hypochlorite in relation to sterilization time and size of explants.

From the experiments it was concluded that 3% sodium

hypochlorite for 30 minutes on larger explants was the most

effective sterilizing agent for banana explants.

2.2.4 Condition of culture room

Light, temperature and humidity conditions provided inside

the culture room plays a significant role in success of an in vitro


17

technique. The light intensity, quality and duration are the major

factors affecting the growth of in vitro culture (Murashige, 1974,

1977). Murashige (1977) found a 16 h photoperiod satisfactory for

a wide range of plant species. Relative humidity of the culture room

is adjusted to 55±5%. Banana shoot tip cultures are incubated at

an optimal growth temperature of 28± 2ºC in a light cycle of 12-16

h with a photosynthetic photon flux (PPF) of about 60µE/m2/s.

2.2.5 Effect of different type and concentration of growth

hormone on shoot multiplication

Srangsam and Kanchanapoom (2003) observed yellow friable

calluses of banana (Musa spp.) ‘Gros Michel’, cultured on

Murashige and Skoog (MS) solid medium supplemented with 2, 4-

dichlorophenoxyaceticacid (2, 4-D) and coconut water (CW). Small

spherical, compact calluses were formed. Friable calluses were

transferred to half-MS liquid media supplemented with 1.5 mg/l 2,

4-D or 1.5 mg/l 2, 4-D in combination with 5% CW or without 2, 4-

D and CW. No shoots were produced from these media.

Embryogenic calluses were induced followed by subculture the

spherical, compact calluses to half-MS solid medium in the

presence of thidiazuron (TDZ). These embryogenic calluses gave

rise to shoots on MS germination medium containing 2.0 mg/l α-

naphthalene acetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA).

Muhammad et al. (2004) cultured the shoot tips of banana

cv. Basrai on Murashige & Skoog basal medium supplemented with

5.0 mg/l BAP and he recorded on the average, 124 plants produced

from each shoot tip after five sub culturing.

Al-Amin et al. (2009) studied the effect of different

concentrations of BAP and NAA on plant regeneration and shoot

multiplication of banana cv. BARI banana-I. Highest shoot

proliferation, longest shoot production, maximum no. of leaves and


18

longest leaves were found at concentration of 7.5 mg/l BAP + 0.5

mg/l NAA.

Jing-Yan et al. (2011) added different hormonal combinations

(0-6.0 mg/l 6-BA and 0.1-0.2 mg/l NAA) in MS medium to induce

the adventitious buds in banana cv. Longxuan and to allow its

multiplication. The best induction and multiplication medium for

adventitious buds was found at MS + 3.0 mg/l 6-BA and MS + 4.0

mg/l 6-BA + 0.2 mg/l NAA, respectively.

Aremu et al. (2012) studied the effects of five topolins

(metaTopolin= mT; metaTopolinriboside =mTR; metaMethoxytopolin

= MemT; metaMethoxytopolinriboside = MemTR and

metaMethoxytopolin 9tetrahydropyranyl = MemTTHP) on shoot

regeneration of micropropagated ‘Williams’ bananas and compared

to benzyladenine (BA). 30 µM mT resulted in the highest number of

shoots (7.3±1.0). Unlike other CK treatments requiring higher

concentrations, optimum mean shoot number per explant was

attained at the lowest concentration in MemT and MemTTHP (10

μM) treatments. In terms of abnormality index, mTR regenerated

plantlets were of the best quality across all the CKs tested.

Mondal et al. (2012) studied the effect of coconut water and

ascorbic acid on shoot regeneration in banana variety Dwarf

Cavendish. They inoculated the shoot tips on MS medium

containing BAP (Benzyl Amino Purine) 5.0 mg/l supplemented with

coconut water in various concentration (0, 50, 100, 150 and 200

ml/l) and with various concentrations (0, 25, 50, 75 and 100 mg/l)

of ascorbic acid, respectively. As the concentration of coconut water

and ascorbic acid was increased upto 100 mg/l and 50 mg/l

respectively greater frequency of explant in shoot regeneration, no.

of shoots regenerated per explant and shoot length was observed.


19

Sipen and Davey (2012) studied the different concentrations

of N6-benzylaminopurine (BAP) and Indole Acetic Acid (IAA) for

their effect on shoot multiplication and plant regeneration of the

Malaysian banana cultivars Pisang Mas, Pisang Nangka, Pisang

Berangan and Pisang Awak. Maximum shoot was produced on

medium supplemented with BAP at 5 mg/l (Pisang Nangka), 6 mg/l

(Pisang Mas and Pisang Berangan) and 7 mg/l (Pisang Awak) with

0.2 mg/l IAA. Further, Ahirwar et al. (2012) took different

concentration of BAP (0-10 mg/l), Kinetin (0-10 mg/l), NAA (0.3-0.5

mg/l) and different combination of BAP (0-10 mg/l) and NAA (0.3-

0.5 mg/l). Highest frequency of shoot regeneration (52.25), number

of shoots regenerated per explant (3.25) and shoot length (4.69)

was found at BAP concentration of 5 mg/l, Kinetin concentration of

5 mg/l and combination of 7.5 mg/l BAP + 0.3 mg/l NAA. The

addition of 5 mg/l BAP was found better than Kinetin for shoot

development from shoot tip or male inflorescence tip explants.

Similar studies were conducted by Gawad et al. (2012) in coffee tree

(Coffea arabica L.) to study the response of shooting hormone

Benzyl Adenine and Kinetin at different concentrations viz. 2.0, 4.0

and 6.0 mg/l. Full strength of MS medium supplemented with 6

mg/l BA or 6 mg/l kinetin gave the best significant result for

survival percentage of explants and multiplication rate shoot

length (4.56 cm), no. of shoots (2.88) and leaves (6.67) in 6 mg/l BA

while shoot length (2.56 cm), no. of shoots (1.64) and leaves (4.67)

in 6 mg/l KIN. While Rai et al. (2012) cultured explant of Grand

Naine on MS media supplemented with different concentrations of

BAP (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l) and NAA (0.25 and 0.5

mg/l) and found that BAP at 2.0 mg/l along with NAA at 0.5 mg/l

to be the best combination and showed optimum shoot growth.

Rahman et al. (2013) investigated the best plant growth

regulators for shoot proliferation and multiplication for cultivar


20

Agnishwar. Among different types and concentration of cytokinins

viz. 6-benzylaminopurine (BAP), kinetin (KIN), N6 - (2-isopentyl)

adenine (2iP) tested for multiplication of shoot; maximum

multiplication (95%) was obtained in MS medium containing 4.0

mg/1 BAP. The highest average number of shoots for each explant

(5.9) was found in MS medium fortified with 4.0 mg/l BAP while

maximum elongation of shoot (4.9 cm) was observed in MS medium

containing 5.0 mg/l BAP.

Ramachandran and Amutha (2013) carried out research work

on Cavendish Dwarf variety of banana. Murashige and Skoog’s

basal medium supplemented with 4 mg/l BAP and 0.2 mg/l NAA

was found to be most suitable combination for shooting. Further,

for multiplication combination of BAP 5 mg/l + NAA 0.3 mg/l was

found best while Ahmed et al. (2014) found MS medium

supplemented with BAP 4.00 mg/l + IAA 2 mg/l best for explant

establishment and shoot multiplication of banana cv. Grand Naine.

Shiv Shankar et al. (2014) did mass propagation of banana

(Musa spp.) cv. Grand Naine through direct organogenesis by using

PGRs Benzyl Adenine Purine and Kinetin. Benzyl Adenine Purine

(BAP) in five different concentrations (control, 2.0, 4.0, 6.0, 8.0 and

10.0 mg/l) were used for shoot proliferation and differentiation and

shoot multiplication rate. The study revealed that medium

supplemented with BAP 4.0 mg/l produced greater number of

shoots (55) and longer shoot (3.0±0.012 cm) when compared with

other treatments.

Reddy et al. (2014) studied the effect of diverse concentration

of 6-benzylamino purine (6-BAP) on shoots induction of Grand

naine plantlets (Musa spp). Modified MS (1962) medium

supplemented with seven different concentrations of 6-

Benzylaminopurine (BAP) (2 mg/l, 0.5 mg/l, 1.5 mg/l, 3.7 mg/l,


21

6.8 mg/l, 8.7 mg/l, 9.4 mg/l) were investigated. Of all, the best

shoot induction were obtained on medium supplemented with 2.0

mg/l of 6-BAP. Similarly, Anbazhagan et al. (2014) cultured shoot

tips of Musa spp. on Murashige and Skoog (MS) medium

supplemented with different concentrations of BAP, KIN and IAA

both in individual and in combined form and the best results were

obtained from MS medium supplemented with BAP + IAA at the

concentration of 3.0 mg/l and 0.5mg/l respectively.

Jamir and Maiti (2014) studied the effect of various levels of

cytokinin and auxin for in vitro regeneration of banana cultivars

Grand Naine and Jahaji. They tested various concentration of BAP

(0-6.5 mg/l). 4.5 mg/l BAP was found as the best concentration in

induction of highest no. of buds (an average of 7.05 and 7.2) with

highest mean length of 0.65 cm and 0.7 cm of shoots. But, the

shoot elongation was maximum at lower concentration of BAP (1.5

mg/l).

Helaly et al. (2014) studied the effect of nanoparticles on in

vitro cultures and organogenic regeneration of banana. The highest

percent of somatic embryogenesis was observed in MS media

supplemented with 100 mg/l nano Zn followed by nano ZnO.

Excellent shooting, rooting and regenerated plantlets were also

observed in MS + 100 mg/l nano Zn and ZnO.

Qamar et al. (2015) optimized micropropagation protocol

supplemented with different concentrations and combinations of

benzyl amino purine (BAP) (0, 2, 4, 6 mg/l) and Indole Acetic Acid

(IAA) (0.5 and 1.0 mg/l) for banana (Musa spp.) genotypes GCTCV-

215 (AAA), ‘Yangambi’ Yangambi Km-5 (AAA) and FHIA-23 (AAAA).

Out of various treatments, best concentration for multiple shoot in

short period of time, maximum fresh mass of shoot for GCTCV-215

and Yangambi Km-5 was found at 4.0 mg /l BAP + 0.5 mg/l IAA


22

but, for shoot length; combination of 4.0 mg/l BAP with 1.0 mg/l

IAA was found to be most suitable.

Hasan and Khasim (2015) evaluated different cytokinins such

as 2 ip, Kinetin, Zeatin and BAP for in vitro multiplication of

banana var. Robusta. Among different cytokinins tested, BAP was

found to be best at an optimum concentration of 5 mg/l. Similar

research work were conducted by Shashikumar et al. (2015) with

BAP, TDZ and coconut water at various concentrations. He

recorded high frequency of shoot initiation (93.33) at 5 mg/l BAP.

The synergetic effect of BAP (4 to 6 mg/l), TDZ (0.1 to 1.2 mg/l) and

coconut water (0.1 to 0.9 ml/l) induced multiple shoot buds and

this was optimum at the concentration of 5 mg/l BAP, 0.5 mg/l

TDZ and 0.5 ml/l coconut water with 15.90 ± 1.66 frequency of

shoots per propagule.

Suman and Kumar (2015) did the micropropagation of

banana cv. Malbhog on Murashige and Skoog (MS) medium

supplemented with different concentrations and combinations of

Indole Acetic Acid (IAA) and Benzyl Amino Purine (BAP). This

combination resulted in differentiation of adventitious shoots. The

maximum differentiation of shoots (92.05 %) was observed on MS

medium with 0.57 μM IAA + 17.74 µM BAP. The number of shoots

per culture was 16.75. The subculture of differentiated shoots on

the same medium resulted in further differentiation (91.97 %) of

more than 15 shoots per culture.

2.2.6 Study of different concentration of growth hormone on

rooting

Al-Amin et al. (2009) used half strength MS medium

supplemented with different levels of IBA (0, 0.5, 1.0 and 1.50

mg/l) and IAA (0, 0.5 and 1.0 mg/l) for root initiation in banana cv.


23

BARI-1. The highest number of roots (6.50) and longest length of

root (5.88 cm) was obtained on 0.5 mg/l IAA + 0.5 mg/l IBA.

Sipen and Davey (2012) found that half strength MS medium

fortified with 1 mg/l NAA was suitable for root regeneration from

scalps in Malaysian banana cultivars Pisang Mas, Pisang Nangka,

Pisang Berangan and Pisang Awak. They found maximum of 7

roots from cv. Pisang Nangka and Pisang Berangan and mean root

length was also maximal (4.5 cm) in the latter two cultivars.

Rai et al. (2012) inoculated multiplicated shoots of banana cv.

Grand Naine (G-9) on rooting media incorporated with either IBA or

NAA (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and Charcoal (2 g/l)

for root induction. IBA (2 mg/l) and Charcoal (2 g/l) produced

maximum number of roots (8.5) with a lot of root hairs.

Rahman et al. (2013) investigated the best plant growth

regulator for induction of roots in banana cv. Agnishwar. Among

different types and concentration of auxin viz. Indole-3-Butyric

Acid (IBA) and α-naphthalene acetic acid (NAA), IBA at 1.0 mg/l

was found most suitable for rooting of shoot (96% rooting).

Ramachandran and Amutha (2013) transferred cultured

plants of banana cv. Cavendish Dwarf into media containing

activated charcoal and hormone (NAA-1.5 mg/l).

Ahmed et al. (2014) obtained rooting on MS (half strength)

medium fortified with IBA 1.00 mg/l and activated charcoal 200

mg/l.

Aremu et al. (2014) conducted rooting experiments involving

the use of indole-3-acetic acid, indole-3-butyric acid, naphthalene

acetic acid, smoke–water (SW) and karrikinolide (KAR1).

Significantly increased number and length of roots were obtained in

SW and KAR1 compared to the control. Overall, when compared to


24

BA, the use of topolin demonstrated higher mean shoot number per

explant (MemT and MemTTHP) at lowest CK concentrations and the

ease of rooting during the shoot proliferation phase (MemTTHP).

Shiv Shankar et al. (2014) assessed the development of roots

in regenerated shoots of Musa spp. cv. Grand Naine by treating the

shoot with five levels of kinetin (control, 0.5, 1.0, 1.5, 2.0 and 2.5

mg/l) supplemented in MS medium. Root development was not

observed in the medium devoid of hormone. Whereas, increased

number of roots (4.0), length of roots (6.0 cm) and length of shoots

(6.5 cm) was observed in medium with 1.0 mg/l kinetin.

Anbazhagan et al. (2014) found that best root formation

(96%) and root number / explant (11.80) of in vitro developed

shoots of Musa spp. could be achieved on half strength MS medium

supplemented with IBA at 1.0 mg/l.

Jamir and Maiti (2014) studied the effect of IBA and NAA on

rooting of banana cultivars Grand Naine and Jahaji. For rooting,

NAA and IBA were used individually at the concentration of 0, 0.1,

0.2 and 1 mg/l. Cent percent rooting and also highest no. of

functional roots (6.33 and 5.2) with moderate root length (2-4 cm)

were achieved on medium with 1 mg/l NAA.

Shashikumar et al. (2015) found that MS medium

supplemented with 1.0 mg/l IBA induced 5.33 ± 1.21 number of

roots with a mean root length of 7.50 ± 1.87 cm in banana cv.

Karibale Monthan.

Paulos et al. (2015) undertook to study the effect of various

concentration of growth regulators IAA (indole-3-acetic acid) (0.0,

0.5 and 1.0 mg/l) and IBA (indole-3- butyric acid) (0.0, 0.5, 1.0 and

1.5 mg/l) and their interaction on rooting in banana (Musa

paradisiaca) cv. Grand Naine. The short duration were recorded at


25

0.5 mg/l IAA – 8.9 days and 1.5 mg/l IBA – 9.4 days and

interaction of both 0.5 mg/l IAA + 1.5 mg/l IBA has given almost a

week root induction (7.33) days. The highest numbers of roots were

produced by treatment of 0.5 mg/l IAA which was 6.2 and 7.8 at 15

and 30 DAI, respectively. 1.5 mg/l IBA produced 5.1 and 7.1 roots

at 15 and 30 DAI respectively. In 0.5 mg/l IAA + 1.5 mg/l IBA

combination gave 7.0 and 8.0 numbers of roots at 15 and 30 DAI

respectively. In length of roots, it was observed that 0.5 mg/l IAA

produced longest root size 5.7 and 6.7 cm at 15 and 30 DAI,

respectively. The same concentration of IBA produced 4.5 and 5.9

cm root length at 15 and 30 DAI, respectively. In interaction, 0.5

mg/l IAA + 0.5 mg/l IBA produced 6.33 and 7.33 cm length in 15

and 30 DAI respectively.

Suman and Kumar (2015) studied the effect of IBA on rooting

of banana cv. Malbhog. They observed that the in vitro developed

shoots showed 100% rooting on MS medium supplemented with

4.92 μM Indole butyric acid (IBA).

2.2.7 Acclimatization

Acclimatization is necessary in the case of in vitro produced

plantlets because in vitro produced plant material is not adopted to

natural environmental conditions (Brainerd and Fuchigami, 1981).

They are very poorely adopted to resist the low humidity, higher

light levels and more variable temperature prevailing outside

(Wainwright, 1988). Thus, light, temperature and relative humidity

are the three major factors to be controlled during acclimatization

to natural environment.

Physical, chemical and biological properties of potting

mixture are also important factors in establishment of in vitro

raised plantlets. Thorough washing of plantlets to remove the

traces of agar and nutrient medium, dipping in 0.05% sarbendazim


26

and sterilizing the potting mixture eliminate problem of fungal

infection (Anderson, 1980 and Muniswamy et al. 1994).

Greenhouse potting mixture used for growing out banana

plantlets include 2 parts of a commercial growing media mixture

(Sunshine Professional), 1 part perlite, and 3 parts vermiculite

(medium to coarse grade). Plants are generally allowed to

acclimatize in the greenhouse for approximately 2 months and to

reach a height of about 20 cm (8 inches) before they are

transplanted to the field. (Perez and Hooks, 2008).

Rai et al. (2012) hardened rooted plantlets of banana variety

Grand Naine (G9) in portrays containing different potting mixtures

viz.soil, sand and cocopeat (1:1:1), soil sand and farmyard manure

(1:1:1) and mixture of cocopeat and sand (2:1) of which, the

mixture of Cocopeat and sand (2:1) showed maximum (96%)

survival of plantlets.

Elisama et al. (2013) studied the effect of fertigation and

Indole Butryic Acid (IBA) application in nutritive solution on growth

of Musa cavendischii plantlets during acclimatization process in

greenhouse. The experimental unit consisted of 1 transplanted

plant and daily application of 10 ml of Steiner’s nutritive solution

at 10, 25, 50, 75 and 100% without and with 1mg/l of auxin (IBA).

After 11 weeks of acclimatization, they found higher plants with

respect to plant fresh weight, dry weight, height and leaf width

which corresponded to treatment from 75 to 100% of Steiner’s

solution. The IBA application had no significant effects on the

growth of the M. cavendischii plants. There was no significant

interaction between fertigation and IBA applications.

Ahmed et al. (2014) hardened and acclimatized in vitro rooted

plantlets by using different treatments. Plants transplanted at the

age of 4 weeks after root initiation gave maximum survival (100%)


27

during transplanting. These plants were hardened in glass beaker

and polythene bags singly or in cluster. The maximum survival

during hardening (100%) was observed by covering the plantlets

with glass beaker individually and kept in culture room. Out of

various potting mixture tried, the potting mixture containing soil:

sand and FYM (2:1:1 v/v/v) gave maximum height and survival of

plantlets. The results also showed that out of different potting

mixtures tried for hardening (FYM, soil, sand and vermiculite); soil:

sand and FYM (2:1:1 v/v/v) showed cent percent survival.

Shiv Shankar et al. (2014) found the survival rate of the

plantlets in coconut coir pith to be 84.44% during primary

hardening. All the plantlets were subjected to the secondary

hardening with garden soil, sand and red soil in the ratio of 1:1:1

in polybags and all the plantlets showed 100% survivability.

Jamir and Maiti (2014) recorded that ex-vitro survival of

cultured banana var. Robusta plantlets was 98% when they were

subjected to secondary hardening in media mixture of pre-

sterilized topsoil: FYM: sand: vermicompost: cocopeat in ratio of

1:1:1/2:1/2:1 (V/V)filled in black polybags under 75% shade net

condition.

Helaly et al. (2014) studied the effect of nanoparticles (nano

Zn at 100 mg/l and 150 mg/l and nano ZnO at 100 mg/l and 150

mg/l on acclimatization of banana. Regenerated plantlets were

successfully acclimatized with about 98% efficiency for the

experimental period (one month) when treated with nano Zn 100

mg/l and nano ZnO 100 mg/l.

Anbazhagan et al. (2014) transplanted cultured plantlets of

Musa spp. into 10 cm diameter plastic pots containing a mixture of

sand, soil and vermicompost (1:1:1) and placed in the greenhouse

for hardening.


28

Qamar et al. (2015) transferred in vitro developed rooted

plantlets of Musa varieties GCTCV-215 (AAA), Yangambi (AAA) and

FHIA-23 (AAAA) from growth room to green house in polythene

bags containing garden soil and humus mixture in ratio 1:1.

2.2.8 Morphological character of hardened plants

Ahmed et al. (2014) obtained maximum height of tissue

cultured plantlets of Grand Naine (15.70 cm) in potting mixture

containing soil, sand and FYM (2: 1: 1 v/v/v) which was closely

followed by mixture containing soil : FYM (2: 1 v/v) (15.00 cm) as

well as soil: sand: vermiculite (12.80 cm). The potting mixture

containing soil and sand (2: 1 v/v) was significantly inferior to

other potting mixtures.

Bohra et al. (2014) noted significant differences for vegetative

growth parameters among propagules obtained from different

methods. They found plant height to be higher in tissue culture

derived plantlets throughout the vegetative propagation. Though,

the differences between tissue culture and sucker derived plantlets

were non-significant for initial 75 days, they became significant

thereafter. At the end of 210 days, tissue culture derived plantlets

achieved an average height of 138.83 cm, whereas those obtained

through sucker were 117.71 cm high. Shashikumar et al. (2015)

studied the morphoagronomical characters of in vitro raised plants

of Karibale Monthan at standing crop stage in comparison with the

in vivo plants. The results of the study revealed that during

vegetative growth, the height of tissue cultured plants is higher

(13.80±2.17 feet) when compared to the sucker derived plants

(12.84±1.85 feet). Tissue cultured plants showed significantly

higher values in the leaf growth such as the leaf length, leaf width

and number of leaves when compared to the in vivo plants at

standing crop stage.


29

Uzibara et al. (2015) did propagation studies on primary

hardening media (cocopeat, vermicompost, sand and vermiculite)

and secondary hardening mixtures [(red soil +sand + cocopeat

(1:1:1 v/v), red soil + sand + FYM (1:1:1; v/v) and red soil + sand +

vermicompost (1:1:1; v/v)] of in vitro propagated Red banana

plantlets. Plantlets showed cocopeat was the best medium for

primary hardening in terms of percentage survival of plantlets

(95.00 %), plantlet height (5.58 cm), number of leaves (3.20),

plantlet diameter (4.59 mm), number of primary roots per plantlet

(5.20), length of primary roots (5.18 cm) and number of secondary

roots per plantlet (25.50), whereas red soil + sand + cocopeat

(1:1:1; v/v) recorded best results in terms of plantlet height (20.50

cm), plantlet diameter (11.60cm) length of leaves (15.43 cm), width

of leaves (6.47cm), number of primary roots per plantlet (12.30)

and number of secondary roots per plantlet (331.20) followed by

red soil + sand + FYM (1:1:1; v/v) mixture were good for secondary

hardening. Finally concluded that combination of red soil + sand +

cocopeat (1:1:1 v/v) was best medium for primary and secondary

hardening of in vitro propagated Red banana.

*****

Chapter-III

Materials and Methods

The present investigation was carried out to study the

“Micropropagation of banana cv. Malbhog for production of quality planting material” during year 2015-16 in the Tissue

culture Laboratory, Bihar Agricultural College, Sabour, campus of

Bihar Agricultural University, Sabour, Bhagalpur. The details of the

experiment in respect of materials used and techniques employed

for studies have been described in this chapter.

3.1.1 Source of explants

Banana suckers cv. Malbhog were procured from Germplasm

Block, Department of Horticulture, Rajendra Agricultural

University, Pusa, Bihar and were used as explant for the

experiment. Sword suckers ranging between 1-3 feet and weighing

approximately 500 g-1 kg were dug up and excised from the mother

plants. They were washed with tap water and cut down to an

approximate size with a machete. Explants were then transported

in plastic bags to the Tissue Culture Laboratory, BAC, Sabour,

Bhagalpur, Bihar where experiments conducted.

3.1.2 Selection of explant

Shoot tips from selected sword suckers of healthy banana

plants were used as explant. These explants were very responsive

for tissue culture studies (Fig. 3.1).

Fig. 3.1: Explants used in present study a. selected sucker b. shoot tip

a b


31

3.1.3 Chemicals

Throughout the course of investigation, chemicals were used

which were only of high quality. The chemicals viz. major salts,

minor salts, growth hormones, vitamins, myo-inositol, glycine,

chelating agents (EDTA sodium salt), NAA, IAA, etc. were procured

from Sigma (USA) and Hi- Media Chemical Company, India.

However, other chemicals like sucrose, agar-agar etc. were

procured from Sisco Research Laboratories (Pvt.) Ltd., BDH and Hi-

Media Companies.

3.1.4 Culture vessels and instruments

Glasswares used for the present study were of borosilicate

quality and were procured from Borosil India Limited and Corning

Glass Company. Conical flasks and culture bottles were used for

culturing explants. Other glasswares used in the experiment

included beakers, graduated cylinders, large boiling flasks,

volumetric flasks, Erlenmeyer flasks, pipettes, etc. A refrigerator

and freezer were also used which were necessary for the storage of

stock solutions prepared media and some chemicals. Other

essential equipments included a pH meter, a top-loading balance

(preferably with a precision of 1mg or less and a weighing range of

0-200 g), a hotplate/stirrer and heating mantles. An autoclave was

used for sterilizing media, culture containers and dissecting

instruments. Equipments needed at the time of culturing include

an assortment of dissecting instruments (scalpels with removable

blades, and forceps), an ethanol dip, a spirit lamp or Bunsen

burner and a spray bottle of ethanol.

3.1.5 Washing of glasswares

Hot and cold water supplies and one or more sinks were required

for washing used glassware.

Glasswares were first washed with detergent (Teepol,

Himedia) followed by thorough washing with excess tap water. The


32

glasswares and culture vessels were finally rinsed with double

distilled water and dried in an oven at 70oC. They were then stored

in dust free cabinet till use.

3.1.6 Sterilization of culture vessels and instruments

The glassware viz. pipettes, beakers were dry heat sterilized

at a temperature of 180ºC for 2 hours in a hot bed oven whereas

the plastic wares viz. beakers, measuring cylinders were wrapped

in aluminium foil and sterilized in autoclave at 121ºC for 20 min

(15 lbs/inch²). UV light of laminar flow hood was switched on 20-30

minutes prior to operation and was turned off during the operation.

The laminar air flow was switched on prior to use and the working

floor was surface sterilized by thorough cleaning with spirit. The

forceps, scalpels, scissors were kept dipped in spirit under the

laminar flow chamber and were frequently sterilized on the flame

during the operation.

3.2 Media

3.2.1 Selection of media

Murashige and Skoog (1962) medium was selected as the basal

medium throughout the experiment as it was the most favourable

medium particularly for callusing and plant regeneration. MS

medium is a widely accepted medium for the tissue culture of

banana. For further studies an array of media was generated using

MS basal medium supplemented with different combinations and

concentrations of phytohormones. Besides MS basal medium, ½

MS medium was also used.

3.2.2 Composition of media

MS basal medium consisted of organic and inorganic salts. The

inorganic salts were salts of major and minor elements including

iron. The organic salts contained nutrients like amino acids,

vitamins and sucrose as a carbon source. Agar was used as a


33

gelling agent. The medium was further supplemented with different

combination and concentration of phytohormones.

Table 3.1 Composition of MS medium and stock solution

S. No. Stocks Constituent Amount (mg/l)

Inorganic

1. Major elements

Ammonium nitrate (NH4NO3) 1650

Potassium nitrate (KNO3) 1900

Calcium chloride (CaCl2.2H2O) 440

Magnesium sulphate

(MgSO4.7H2O)

370

Potassium dihyro orthophosphate (KH2 PO4)

170

2. Minor elements

Boric acid (H3BO3) 6.20

Manganese sulphate

(MnSO4.H2O)

22.3

Zinc sulphate (ZnSO4.7H2O)

8.6

Potassium iodide (KI)

0.83

Sodium molybdate

(Na2MoO4)

0.25

Cupric sulphate (CuSO4.5H2O)

0.025

Cobalt chloride (CoCl2.6H2O)

0.025

3. Iron sources

EDTA- di sodium salt (Na2EDTA.7H2O)

37.2

Ferrous sulphate

(FeSO4.7H2O)

27.8

Organic

4. Amino acids & vitamins

Glycine (C2H5NO2) 2.0

Inositol (C6H12O6) 100

Nicotinic acid (C6H5NO2) 0.5

Pyridoxine hydrochloride (C8H11NO3HCl)

0.5

Thiamine hydrochloride (C12H17ClN4 O5.HCl)

0.1

5. Carbon Source

Sucrose (C12H22O11) 30,000

6. Gelating

Agent Agar 8000


34

Table 3.2 Preparation of stock solutions of different phytoho-

rmones

Sl.

No.

Phyto-

hormones

Weight

(mg)

Initially

dissolved in

Made up

with

distilled

water

Strength

1. IAA 25 2-3 ml

ethanol 250 ml

1mg/10

ml

2. IBA 25 2-3 ml

ethanol 250 ml

1mg/10

ml

3 BAP 25

5-10 ml

slightly heated

N/10 NaOH

solution

250 ml 1mg/10

ml

4. NAA 25 2-3 ml ethanol 250 ml 1 mg/10

ml

3.2.3 Preparation of stock solutions

Stock solutions of major, minor, iron and vitamins were

prepared separately by dissolving the required amount of

component constituents in distilled water. In case of the

preparation of stock solution of major salts, CaCl2 was added at the

end and in case of stock solution of iron, the distilled water was

boiled for half an hour to make it oxygen free (Table 3.1). Stock

solution of mineral salts were kept in dark bottles and stored in a

refrigerator. Similarly, separate stock solutions of auxin (NAA, IAA

and IBA) and cytokinins (BAP) were also prepared. The stock

solution of auxin was prepared by dissolving the required amount

of constituent first in 2-3 ml of ethanol and then in distilled water

to make the desired volume. Similarly, the stock solution of

cytokinin was prepared by dissolving it in 5-10 ml N/10 NaOH and

distilled water was added to make the required volume (Table 3.2).


35

The stock solutions were stored in 50 or l00 ml volumetric flasks at

4°C in refrigerator and brought to room temperature before use.

3.2.4 Preparation of 1 litre media

It was generally prepared by dissolving the appropriate

chemicals in distilled water, adjusting the pH of the solution,

adding agar (for semi-solid media) and autoclaving. For preparation

of 1 lit medium at first a volumetric flask of 1 litre was taken. Then

the required amount of different stock solutions of major, minor,

iron, amino acids and vitamins were added into it. Phytohormones

were added from their stock solutions as per their requirement and

30 g of sucrose was also added into it. Sucrose was dissolved

gradually by adding distilled water. Further, distilled water was

added and volume was made slightly less than 1 litre. The pH of

the medium was checked on pH meter and adjusted to 5.8 by

adding drop wise dilute NaOH or dilute HCl as needed. The

medium was then brought to its precise volume i.e. 1.0 litre. This

medium was poured into 1.0 litre conical flask and 8.0 gm agar

was added as gelling agent. The whole contents of conical flask was

agitated on shaker to mix the gelling agent properly for 10-15

minutes or till the medium became transparent. 50 ml of the

medium was poured into each of culture bottles. Then the culture

bottles were capped.

3.2.5 Sterilization of prepared medium

The culture medium was sterilized in an autoclave. The

capped culture bottles containing homogenized medium were put

in perforated baskets. Date and precise medium number was

marked on each cap of the bottle. Thereafter, the basket containing

culture bottles were kept inside autoclave for 20 minutes at 121°C

at 15 psi (pounds per square inch) or 1.0 kg/cm2 pressure. After

autoclaving the culture bottles were taken out and cooled at room

temperature. The medium were left for a day to set and inoculation


36

was done the next day. The autoclaved media in flasks were stored

at 25±2 ºC for a maximum period of 10 days.

3.2.6 Transfer area and aseptic manipulations

All the aseptic manipulations such as surface sterilization of

explants, preparation and inoculation of explants and subsequent

sub culturing were carried out under aseptic conditions in the hood

of clean laminar airflow chamber. The working table of the laminar

air flow chamber was first surface sterilized with absolute alcohol

and then switching on the UV light for 20-30 minutes before work

started. The instruments used for inoculation were earlier steam

sterilized in an autoclave at 15 psi pressure and 121ºC for 20

minutes. The forceps, scalpels, scissors were kept dipped in spirit

and were flame sterilized before each inoculation. Hands were also

swabbed in 70% alcohol before inoculation. Care was taken to

avoid any obstruction of laminar air flow by placing nothing

between the work area and the source of air flow. Further, crossing

over the hands and arms were avoided.

3.2.7 Culture Incubation Room

Tissue cultured plants were incubated in an air conditioned

culture room with controlled temperature of 25±2ºC and light

intensity of 2000-3000 lux for a photoperiod of 16 h of light by cool

white fluorescent tubes. The photoperiod was controlled by means

of time switches installed in each culture room or even on each set

of shelves. Most micro propagation work were carried out under

artificial lighting provided by cool-white, fluorescent tubes.

3.2.8 Nursery area

Tissue cultured plantlets were hardened after transferring to

greenhouse. The hardening of tissue-cultured plantlets were done

gradually from low light intensity to high light intensity conditions.

To achieve this, nursery area were equipped with some facilities for

humidity control and partial shading. Plantlets were watered by

using a hosepipe or sprinkler. Plantlets were transferred into


37

different media like cocopeat, cocopeat: soil (1:1 v/v), sterile soilrite

and sterile soilrite: soil (1:1 v/v) and were gradually hardened by

progressively reducing the shade to expose the plantlets to full

sunlight just before transplanting into the field.

3.3 Culture techniques: shoot (sucker) tip culture

3.3.1 Preparation of shoot tip explants

Superfluous tissues were removed by trimming away the

outer leaf sheaths, leaf bases and corm tissues until a 6.5 to 8.5

cm3 enclosing the shoot apex is obtained. Obtained shoot apex were

carefully and thoroughly washed in running tap water to remove

soil particles and debris. Again after washing, extraneous tissues of

rhizomes were carefully removed with stainless steel knife until

rhizome tissue measuring about 4.5 to 6.5 cm3 were obtained.

3.3.2 Washing of explants

The trimmed suckers were then washed with Dettol and

Tween-20 for 20 minutes. Traces of detergent were removed by

repeated washing with running tap water for 5-10 minutes.

3.3.3 Pre-treatment of explants

The washed suckers were treated with a solution of 0.2%

Bavistin and Tween-20 for ½ hours. All the traces of chemicals

were removed by 3-4 times washing in tap water. This was followed

by dipping in streptocycline (0.05%) for 3 hours, washed in tap

water for 3-4 times. Again, dipping of explants in solution of

rifampicin (0.1%) for 1 hour, washing the explants with tap water

3-4 times and putting in solution of ascorbic acid (0.2%) for 20

minutes. After that, thorough rinsing of explants by tap water were

done for 3-4 times.Further sterilization procedure was carried out

under aseptic conditions in laminar air flow cabinet.

3.3.4 Sterilization of laminar air flow

All inner sides of laminar air flow, the working chamber,

including floor was properly wiped with ethyl alcohol using cotton

before use. Then, the inoculating instruments like forceps, needles,


38

scalpels, sterilized culture bottles, spirit lamps and media were

kept inside the chamber. The chamber was closed and the UV light

was put on for 25-30 min. and the ozone was flown for 20 minutes

to sterilize the chamber.

3.3.5 Surface sterilization of explants

The washed explants were surface sterilized under laminar

air flow firstly by rinsing in ethanol (70%) for 30 seconds and

thoroughly washing by sterile distilled water for 4-5 times to

remove traces of ethanol, peeling off of one layer of sucker using

sterilized forceps and scalpel. Then, treatment of HgCl2 was given to

explants for respective time duration as mentioned in the table 3.3.

From treatment T3 to T7 HgCl2 (0.1%) treatment was repeated twice

after removing one layer of sucker. In T3, HgCl2 treatment was of 5

min twice, in T4 of 10 min and 5 min, in T5 10 min twice, in T6 15

min and 10 min and in T7 15 min. and 15 min. After each

sterilization process explants were rinsed thoroughly with sterile

distilled water 4-5 times, each time one layer of sucker was peeled

off.

Table 3.3: Surface sterilants treatment duration to prevent

contamination.

Treatment Treatment duration

T1 Control

T2 Ethanol (70%) for 30 sec + HgCl2 (0.1%) for 5 min







39

3.3.6 Inoculation of explants

Inoculation of explants was done under sterilized laminar air

flow. At first, hands were wiped with alcohol and the instruments

like forceps, scalpels were soaked in alcohol followed by flaming on

a burner in the laminar air flow hood. The process was carried out

repeatedly during aseptic inoculation work. Tube light and air flow

were on during inoculation period. Thereafter, surface sterilized

explants were carefully inoculated by forceps into medium over the

flame of the sprit lamp. During inoculation the explants were

properly positioned on the media and were gently pressed with

forceps to secure their firm contact with the media.

3.3.7 Establishment of primary cultures

3.3.7.1 Shoot multiplication

The regenerated explants were cut aseptically and cultured

on multiplication media (Table 3.4). The initial explants sub

culturing was done and after few days explants produced well

developed shoots from the sucker. Sub culturing was done for 3-4

times on the same media at an interval of 6 weeks to get multiple

shoots.

3.3.7.2 Sub culturing

For sub culturing, in vitro grown initial explant shoots were

cut into small pieces so that each piece would contain about one

shoot. Leaf and blackish or browned basal tissues were removed to

expose the meristem. Each piece was inoculated into a similar fresh

MS medium. Through series of sub culturing, the numbers of in

vitro shoots were increased. In vitro proliferated micro shoots were

separated with the help of sterilized scalpel and trimmed the lower

base, photoactive shoots into define size and each of the micro

shoot was placed on culture medium, supplemented with different

concentrations of NAA and BAP (Table 3.4) for shoot

differentiation.


40

Table 3.4: MS basal medium treatments with different

concentration of growth regulators for shoot multiplication

Treatments Plant growth regulators (mg/l)

NAA BAP

T1 0.0 0.0

T2 0.0 1.0

T3 0.0 2.5

T4 0.0 5.0

T5 0.5 1.0

T6 0.5 2.5

T7 0.5 5.0

T8 1.0 1.0

T9 1.0 2.5

T10 1.0 5.0

3.3.8 Root initiation

Well-developed shoots with leaves were rescued aseptically

from the culture vessels and separated from each other and again

cultured on freshly prepared half MS medium containing different

concentration of IAA or IBA for root induction. Observations were

recorded for percent root formation in regenerated shoots, number

of days taken for root formation, number of roots per shoot and

root length (cm). Three different levels of IAA and IBA were used as

treatment for root induction in experiment 3 (Table 3.5).

Table 3.5: Half MS basal medium treatments with different

concentration of growth regulator for root initiation

Treatments

Plant growth regulators (mg/l)

IAA IBA

T1 0.0 0.0

T2 0.0 0.5

T3 0.0 1.5

T4 0.0 2.5

T5 0.5 0.0

T6 1.5 0.0

T7 2.5 0.0


41

3.4 Acclimatization

Rooted shoots that were 4-5 cm high and had several well

ramified roots were ready to transplant. Plantlets were removed

from culture container and agar was gently washed from roots

under running tap water. Then, the banana plantlets were dipped

in a fungicide solution (Bavistin, 2g/l) prior to transplantation to

potting media to reduce the risk of damage by fungal attack.

Individual plantlets were transplanted into following different

potting media as listed below:

Coco peat

Coco peat: Soil (1:1 v/v)

Sterile soilrite

Sterile soilrite: Soil (1:1 v/v)

At the time of transplanting care were taken not to damage the

fragile roots. Different potting mixtures covered the upper roots by

1-2 cm. After transplanting plantlets were immediately watered.

High humidity was maintained by intermittent misting.

3.5 Morphological characteristics of hardened plants

3.5.1 Height of the plant

A measuring scale was used to measure the average height of

plant.

3.5.2 Girth of the plant

Girth of the plant was measured by using a slide calliper (Mitutoyo

absolute, CD-6’CSX).

3.5.3 Leaf length

A measuring scale was used to measure the average length of leaf.

3.5.4 Leaf width

Width of leaf was also measured by a measuring scale.


42

3.5.5 Number of leaves

It was calculated by counting leaves.

3.6 Observations recorded

1. Per cent establishment of aseptic cultures from different

explants in MS media supplemented with different hormonal

combinations.

2. Number of days required for shoot initiation.

3. Length of shoots (cm).

4. Number of shoots per explant.

5. Percent root formation in regenerated shoots.

6. Number of days taken for root formation.

7. Number of roots per shoot.

8. Root length (cm).

9. Percent survival of rooted plantlets in different potting

mixtures.

10. Morphological characters of hardened plants.

3.7 Photography

Photographs of cultured tubes were taken by using Nikkor 7X wide

optical zoom ED VR camera (Model No. S3500).

3.8 Statistical Analysis

The experiments were set up in a completely randomized design

(CRD) with a minimum of 5 cultures per treatment. All the data

were analyzed by running one way analysis of variance (ANOVA)

using OP Stat. The means were compared using critical difference

to find the difference at 5% (P<0.05) level. The results are expressed

as a mean ± SE of five replications.

*****

Chapter-IV

Experimental findings

The results obtained in the present investigation entitled

“Micropropagation of banana cv. Malbhog for production of quality

planting material” are presented in this chapter with the help of

heading and sub-heading and table.

4.1 Surface sterilization and establishment of shoot tip

explants

Surface sterilization of explant is of prime importance in

tissue culture to achieve high degree of success. The data regarding

the effect of different treatment duration of sterilizing agents on

banana cultivar Malbhog is furnished in Table 4.1. The efficiency of

sterilizing agents was evaluated in terms of number of aseptic

explants sprout. All the cultured explants were contaminated when

no any sterilant was used. The contamination of shoot tip explants

significantly decreased with increasing time of exposure of

sterilants. The per cent survival of explants was recorded highest

(45.00±3.33) with T6 Ethanol (70%) for 30 sec + HgCl2 (0.1%) for 25

min (15 min and 10 min each) treatment. The mortality of explants

was also increased with the increasing time of exposure of sterilant.

Although the minimum mortality observed (3.33±2.04) was with T2

(Ethanol (70%) for 30 sec + HgCl2 (0.1%) for 5 min) treatment but

percent contamination was recorded high (86.67±2.04) in this

particular treatment. Overall, Ethanol (70%) ( T6 treatment) for 30

sec + HgCl2 (0.1%) for 25 min was found the most effective and

showed maximum percent survival (45.00±3.33), less mortality

percent (28.33±2.04) and percent contamination was also recorded

low (26.6±1.67).


44

Table 4.1: Effect of surface sterilization treatments on establis-

hment of shoot tip explant of banana cv. Malbhog

Treatment Treatment duration

Contamination (%)

Mortality due to

sterilant toxicity (%)

Culture establishment (%)

T1 Control 100±0.00 0.00±0.00 0.00±0.00

T2

Ethanol (70%) for 30 sec + HgCl2 (0.1%) for 5 min

86.67±2.04 3.33±2.04 6.67±3.12

T3


73.33±1.67 8.33±2.64 18.33±1.67

T4


56.67±3.12 18.33±1.67 25.00±3.73

T5


43.33±3.12 25.00±2.64 31.67±1.67

T6

Ethanol (70%) for 30 sec + HgCl2

(0.1%) for 25 min

26.66±1.67 28.33±2.04 45.00±3.33

T7

Ethanol (70%) for 30 sec + HgCl2

(0.1%) for 30 min

20.00±2.04 50.00±2.64 30.00±4.25

CD at 0.05 4.11 7.03 7.26

4.2 Shoot tip culture

In tissue culture, establishment include all steps right from

culturing till the explants are able to produce multiple shoots. In

order to standardize a suitable shoot establishment and


45

proliferation medium, detailed experiment were conducted with

cytokinins (BAP) alone or in combination with auxin (NAA) using

MS medium. The results are presented in table 4.2.

Table 4.2: Effect of MS basal medium treatments with different

concentration of growth regulators on shoot multiplication

Treatment Composition

(mg/l)

No. of days

required for

shoot

initiation

No. of

Shoots/

Explant

Length of

shoots

(cm)

T1 Control 41.40±0.98 1.00±0.00

0.98±0.06

T2 BAP 1.0 38.00±1.38 1.40±0.24

1.80±0.37

T3 BAP 2.5 29.00±1.18 2.20±0.20

2.80±0.37

T4 BAP 5.0 22.60±1.03 3.40±0.24

4.06±0.15

T5 NAA 0.5+ BAP

1.0

28.60±1.06 1.80±0.37

2.40±0.24

T6 NAA 0.5+ BAP

2.5

21.80±0.80 3.20±0.37

4.66±0.17

T7 NAA 0.5+BAP

5.0

18.40±1.03 4.00±0.32

5.10±0.07

T8 NAA 1.0+BAP

1.0

33.00±1.79 1.40±0.24

2.00±0.31

T9 NAA 1.0+BAP

2.5

27.20±0.86 3.00±0.45

3.68±0.22

T10

NAA 1.0+BAP

5.0

25.20±0.66 3.60±0.24

4.00±0.23

CD at 0.05 3.57 0.83

0.71

Medium: MS Incubation period: 6 weeks

4.2.1 Effect of different treatments on number of days taken

for shoot initiation

The data shows that the number of days required for shoot

multiplication was significantly affected with combination of

cytokinin and auxin. The minimum number of days for shoot

initiation was taken by treatment T7 (NAA 0.5 mg/l + BAP 5mg/l)


46

i.e. 18.40±1.03 days which was at par with treatment T6 which took

21.80±0.80 days (Table 4.2) followed by treatment T4 which took

22.60±1.03 days. Treatment T1 (Control) took maximum days for

shoot initiation i.e. 41.40±0.98 days followed by treatment T2 (BAP

1.0mg/l) which took 38.00±1.38 days.

4.2.2 Effect of different treatments on number of shoots per

explant

Data of Table 4.2 shows that maximum number of shoots per

explant i.e. 4.00±0.32 was recorded highest in treatment T7 (NAA

0.5 mg/l + BAP 5 mg/l) which was found statistically at par with

treatment T10 (NAA 1.0 mg/l + BAP 5.0mg/l) and treatment T4 (BAP

5.0mg/l).

4.2.3 Effect of different treatments on length of shoots

The length of shoot was recorded maximum (5.10±0.07cm) in

treatment T7 (NAA 0.5 mg/l + BAP 5mg/l) which was found at par

with treatment T6 (NAA0.5mg/l + BAP 2.5mg/l) i.e. 4.66±0.17 cm

followed by T4 (BAP 5.0 mg/l) i.e. 4.66±0.17cm and T10 (NAA 1.0

mg/l + BAP 5.0 mg/l) treatments. i.e. 4.00±0.23 cm (Table 4.2).

4.3 Root initiation& culture

In order to standardize a suitable root initiation and

proliferation medium, detailed experiment were conducted with

different concentration of either IBA (auxin) or IAA (auxin) using

half MS basal medium. The results are presented in table 4.3.

4.3.1 Effect of different treatments on percent root formation

in regenerated shoots

Seven different treatments were carried out to find out the

effect of IBA and IAA on percent root formation. The data is

presented in table 4.3. The data in the table reveals that when IBA

added in concentration of 1.5 mg/l (treatment T3) gave significantly

best rooting response i.e. 66.67±2.64 over all other treatments


47

followed by treatment T7 (2.5 mg/l IAA) and T2 (0.5 mg/l IBA) in

which rooting responses were 58.33±2.64 and 53.33±2.04 percent

respectively.

4.3.2 Effect of different treatments on number of days taken

for root initiation

Significantly minimum number of days (18.00±0.95) taken

for root initiation was observed in treatment T3 (1.5 mg/l IBA)

followed by treatment T2 (0.5 mg/l IBA) which took 21.60±0.51

days. Maximum number of days for root initiation (32.20±0.86) was

observed in treatment T1 (control).

4.3.3 Effect of different treatments on number of roots per

shoot

It is evident from Table 4.3 that auxins exerted significant

effects on number of roots induced in shoots when sub-cultured on

half strength MS media. IBA treatment @1.5 mg/l (Treatment T3)

resulted in maximum number of roots (4.20±0.37) which was found

at par with treatment T2 (0.5 mg/l IBA), T6 (1.5 mg/l IAA) and T4

(2.5 mg/l IBA) which produced 3.40±0.24, 3.80±0.20 and

3.40±0.24 roots per shoot respectively. However, minimum

numbers of roots (1.80±0.37) were recorded in half MS media

without any auxin (control).

4.3.4 Effect of different treatments on length of root

Significant differences were found on the length of roots when

auxin were added to the half strength MS media. Maximum root

length (4.00±0.07 cm) was obtained when IBA 1.5 mg/l (Treatment

T3) was added to half strength MS media. The next best response

(3.44±0.13 cm) was obtained in treatment T2 (0.5 mg/l IBA). The

length of root was found minimum (1±0.00 cm) in control.


48

Table 4.3: Effect of Half MS basal medium treatments with

different concentration of growth regulators for root initiation

Treatment Compo-sition (mg/l)

Percent (%) root

formation

Duration (days) for

root formation

Number of roots/ Explant

Length of root (cm)

T1 Control 21.60±2.04 32.20±0.86 1.80±0.3

7 1.00±0.00

T2 IBA 0.5 53.33±2.04 21.60±0.51 3.40±0.2

4 3.44±0.13

T3 IBA 1.5 66.67±2.64 18.00±0.95 4.20±0.37

4.00±0.07

T4 IBA 2.5 45.00±2.04 23.80±0.37 3.40±0.2

4 2.56±0.04

T5 IAA 0.5 43.33±3.12 26.60±0.40 2.00±0.3

2 1.12±0.07

T6 IAA 1.5 50.00±2.64 24.20±0.58 3.80±0.2

0 3.04±0.05

T7 IAA 2.5 58.33±2.64 26.80±0.51 3.40±0.2

4 2.74±0.05

C. D. at 0.05 7.18 1.83 0.84 0.20

Medium: Half MS Incubation period: 6 weeks

4.4 Acclimatization

For acclimatization, potting mixtures like coco peat and

sterile soilrite either alone or in combination were tried. Data of

table 4.4 reveals that maximum survival percentage (89.00±2.45)

was obtained in treatment T1 (coco peat) and the next best survival

(79.00±1.87) was recorded in treatment T3 (Sterile soilrite).

Table 4.4: Effect of different potting mixtures on hardening of

banana cv. Malbhog micropropagated plantlets

Treatments Potting mixture Response (%)

T1 Coco peat 89.00±2.45

T2 Coco peat: Soil (1:1 v/v) 69.00±3.67

T3 Sterile soilrite 79.00±1.87

T4 Sterile soilrite: Soil(1:1 v/v) 66.00±2.45

C.D at 0.05 5.66


49

4.5 Morphological characteristics of hardened plants

4.5.1 Height of the plant

The data regarding the height of plant is presented in table

4.5 below. Maximum height of plant was found in coco peat (T1)

(7.98±0.19 cm) followed by T3 treatment in which sterile soilrite

were used as potting media (7.20±0.19).

4.5.2. Girth of the plant

All potting media gave almost same girth of plant and they

were statically non-significant to each other being 2.20±0.11,

1.93±0.19, 2.04±0.17 and 2.00±0.11 in coco peat, coco peat: soil

(1:1 v/v), sterile soilrite and sterile soilrite: soil (1:1 v/v) as

presented in table 4.5.

4.5.3 Leaf length

Exceptionally longest length of leaf (6.10±0.23 cm) was found

in potting media containing coco peat (T1). The second largest leaf

length (5.16±0.08) cm) was found in T3 treatment containing sterile

soilrite as potting mixture. The data is presented in table 4.5.

4.5.4 Leaf width

Leaf width also followed the same trend as in case of height of

the plant. Maximum leaf width (4.12± 0.27 cm) was found in T1

treatment containing coco peat as potting material. Non-significant

difference was found in leaf width of T3 (3.88±0.10 cm) treatment

containing sterile soilrite as potting mixture with T4 treatment

(Sterile soilrite: soil) as can be seen from the table 4.5.

4.5.5 Number of leaves

The data was found non-significant with each other for all the

four treatments T1 (Coco peat), T2 (coco peat: soil), T3 (sterile

soilrite) and T4 (Sterile soilrite: soil). The number of leaves were

found 4.20±0.31, 4.20±0.37 and 3.80±0.20 in T1, T2, T3 and T4

treatments respectively (Table 4.5).

50

Table 4.5: Effect of different potting mixtures on morphological characteristics of banana cv. Malbhog

micropropagated plantlets

Treatments Potting

mixtures Height (cm)

Girth (cm) Leaf length

(cm) Leaf width (cm) No. of leaves

T1

Coco peat 7.98±0.19 2.20±0.11 6.10±0.23 4.12±0.27 4.20±0.37

T2

Coco peat: soil (1:1 v/v)

6.12±0.19 1.93±0.19 4.98±0.13 3.72±0.14 4.00±0.31

T3

Sterile soilrite

7.20±0.19 2.04±0.17 5.16±0.08 3.88±0.10 4.20±0.37

T4

Sterile soilrite: soil (1: 1 v/v)

5.36±0.18 2.00±0.11 4.92±0.07 4.92±0.07 3.80±0.2

C. D. at 0.05 0.55 NS 0.44 0.16 NS

*****

Culture showing shoot proliferation on MS +0.5 mg/l NAA +5.0 mg/l BAP

Plate No. 4.4. Cultures showing shoot proliferation

Culture showing shoot multiplication on MS +0.5 mg/l NAA + 5.0 mg/l

BAP

Plate No. 4.5. Shoot multiplication in Malbhog banana

Chapter-V

Discussion

The potentiality of plant tissue culture in rapid propagation

and crop improvement have provided a substantial impetus for bio-

technological research. Tissue culture techniques ensure an

extremely rapid rate of multiplication which is not season

dependent and requires only a limited quantity of plant tissues as

the initial explant. The commercialization of micropropagated

banana plants is highly useful owing to its many advantages such

as early and uniform maturity, higher yield potential, etc. In the

present chapter, an attempt has been made to discuss the findings

of the present experiment in the light of present knowledge and

work done in the past with probable reasons. In order to explore

the in vitro regeneration potential of banana cv. Malbhog

experiments were conducted and observations were recorded with

respect to establishment of aseptic culture, shoot regeneration and

multiplication, rooting, acclimatization and morphological

character of hardened plants. Through the experiment, it was also

possible to find out the optimum concentration of different plant

growth regulators (PGRs) for various parameters like shoot

regeneration, shoot multiplication, rooting of regenerated shoots

and acclimatization of plantlets.

5.1 Pre- treatment and surface sterilization of explants

The plants grown under field conditions often harbours fungi

and bacteria in addition to a lot of soil and dust particles and

therefore, it becomes necessary to carry out through and effective

sterilization procedure of explants before culturing. This is achieved

by surface sterilization. The selection of sterilization treatments

should be such that it kills the microbes without adversely affecting

the plant tissues.

Discussion

52

The best result was obtained by pre- treating the explants

with bavistin (0.2%) for ½ hours followed by dipping in

streptocycline (0.05%) for 3 hours followed by dipping in rifampicin

(0.1%) for 1 hour and finally putting in solution of Ascorbic acid

(0.1%) for 20 minutes outside the laminar air flow cabinet and

then, surface sterilizing the pre-treated explants with ethanol (70%)

for 30 sec and HgCl2 for 25 minutes (for 15 minutes and 10

minutes each) inside the laminar air flow cabinet. The

contamination of explants significantly got decreased with increase

in time of exposure of sterilants. The percent survival of explants

was recorded highest (45.00±3.33) in T6 treatment. Exposure to

lesser time duration of sterilants resulted in increased

contamination whereas exposure to longer time duration reduced

the contamination but the mortality of explants increased

considerably. Similarly, significant reduction in contamination in

Cavendish dwarf variety of banana were noted by Ramchandran

and Amutha (2012) when explant were dipped in NaOCl and HgCl2

for 10 minutes each while Shashikumar et al. (2015) found

significant reduction in bacterial contamination of Musa spp. cv.

Karibale Monthan when surface sterilized with 70% alcohol for 6

minutes, 0.1% HgCl2 for 10 minutes and again 0.2% for 10

minutes, sodium hypochlorite for 15 min, 0.1 % cefotaxime for 5

min and 0.05 % gentamicin for 5 minutes. Bavistin is an antifungal

agent and rifampicin are antibacterial chemicals. Bavistin kill

pathogens by inhibiting DNA, RNA and protein synthesis (Nene and

Thapliyal, 1993). Ethanol works by denaturing proteins and

solubilizing lipid membrane. HgCl2 acts by inactivating proteins;

reacting with sulphide group of pathogens and thus killing them.

Thus, the combination of bavistin, tetracycline and rifampicin were

found very effective in controlling contamination. De Fossard (1985)

suggested a combination of physical methods (aimed at reducing

the size of microbial population) and chemical methods (killing

Discussion

53

microbes) leading to aseptic culture is most effective as compared

to single treatment. Vann Den Houwe and Swennen (2000)

demonstrated the significance of antibiotics in controlling bacterial

contamination in banana tissue culture. The antibiotic kills

bacteria by causing the cell wall to disintegrate.

5.2 Multiplication stage

In our experiment, minimum number of days for shoot

initiation, longest length of shoot and maximum number of shoot

per explant was found at NAA 0.5 + BAP 5.0 mg/l concentration.

Minimum number of days required for shoot initiation was

18.40±1.03 days, longest length of shoot recorded was 5.10±0.07

cm and maximum number of shoots per explant was found

4.00±0.32. Similar results were obtained by Ahirwar et al. (2012)

who found highest frequency of shoot regeneration (52.25), number

of shoots regenerated per explant (3.25) and shoot length (4.69 cm)

at BAP concentration of 5 mg/l or combination of 7.5 mg/l BAP +

0.3 mg/l NAA. Strosse et al. (2008) also reported NAA and BAP (5

mg/l) for highest multiplication rate and highest length of shoots in

banana. Analogical results obtained by Sipen and Davey (2012) in

Musa spp. Pisang Nangka on medium supplemented with BAP at 5

mg/l and IAA 0.2 mg/l. Previous researchers (Vuylsteke and De

Langhe, 1985; Bairu et al., 2008) also indicated that 5 mg/l (22.2

μM) BAP was the optimum concentration for most banana

cultivars. While Rahman et al. (2013) obtained maximum

multiplication (95%) and highest average number of shoots each

explants (5.9) in MS medium containing 4.0mg/l BAP. But, Rai et

al. (2012) obtained best shoot multiplication in the medium MS +

BAP (2 mg/l) + NAA (0.5 mg/l) with average of 7.5 ± 0.45 shootlets

with a mean shoot length of 6.2 ± 0.37 cm/explants. However,

Arinaitwe et al., (2000) reported that in vitro bud initiation from

banana was cultivar dependent. The different results obtained by

Discussion

54

different authors might be due to differences of genotypes and

explants used. So, the multiplication rate was decreased with

decreasing the concentration of BAP in MS medium because less

bud formation (cease of the cell division) and also the multiplication

rate decreasing with increasing the concentration of BAP in MS

medium due to abnormality development of the buds. The

application of high BAP concentration to initiate bud formation

from explants were reported by Zaffari et al. (2000) and

Subramaniam et al. (2008) in Cavendish banana cultivar Brasilian

(AAA). The explants cultured on MS medium without growth

regulator produced shortest shoot length (0.98±0.07 cm) and only

single shoot besides being kept after normal incubation period of 6

weeks. The result of the present experiment agree with the findings

of Al-Amin et al. (2009) who obtained only single shoot and shorter

shoot length (1.05 cm) at 20 and 30 DAI when explants cultured on

MS medium without growth regulator.

5.3 Rooting stage

The presence of auxins in the medium is necessary for

induction and development of roots from shoots regenerated from

shoot tip explant in tissue culture. The concentration of auxin

required is often critical to provide sufficient stimulus to initiate

roots while preventing the excessive formation of callus (Yoeman,

1986). Maximum root formation percentage, minimum number of

days for root formation, maximum number of roots per shoot and

maximum root length was found on IBA 1.5 mg/l. Maximum root

formation percentage was 66.67±2.46, minimum number of days

for root formation was18±0.95, maximum number of roots per

shoot was 4.20±0.37 and maximum root length was 4.00±0.07cm.

Some percentage of root formation (21.60±2.04) and minimum

length (1.00±0.00 cm) of root was obtained on ½ MS basal medium

without auxin. Similar results were obtained by Al-Amin et al.

(2009) who obtained 2.00 cm root length with control treatment.

Discussion

55

Rooting can be stimulated when individual shoots are transferred

to ½ MS basal medium alone (Cronauer and Kriokrian, 1984;

Jarret et al., 1985). However, auxin may induce further root

initiation (Vuylsteke, 1989). But the result of current investigation

do not agree with findings of Rai et al. (2012). He observed no

rooting in auxin free basal medium (control). Similarly, at lower

level of IBA (0.25 mg/l) treatments, he noted hardly any root

emergence. But, on all the higher concentrations of IBA so tested

(1.5 and 2.0 mg/l) good rooting response was noted. They reported

IBA to be more effective than NAA in root induction. Rahman et al.

(2013) also reported IBA was better than NAA in rooting of shoots

in banana cv. Agnishwar. IBA was the most potent root inducer.

Many workers have found indole butyric acid (IBA) 1 mg/l for

inducing rooting within one to four week in banana (Vasane et al.,

2010; Roy et al., 2010; Rahman et al., 2013). Babylatha (1993)

observed maximum rooting in half strength MS media

supplemented with 5.0 mg/l IBA. Efficiency of IBA in root induction

was also observed in grape (Chakravorty et al., 1986). However, the

maximum percent of adventitious root formation was observed in

half strength MS medium supplemented with IBA 1.5 mg/l and

NAA 1.0 mg/l (Govindaraju et al., 2012). But, Ahirwar et al. (2012)

reported significantly increased frequency of root regeneration

(75.75%), number of root regenerated per shoot (5.0) and shoot

length (4.7) in the medium containing 1.0 mg/l NAA.

5.4 Hardening

Hardening of in vitro raised plantlets is done so as to make

them adaptable to the natural environment. It is a critical process

due to anatomical and physiological peculiarities. On

transplanting, excessive water loss from the plantlets has been

recorded which was attributed to the improper development of

cuticle and slowness of stomatal response to water stress.

Therefore, a period of humidity acclimatization was considered

Discussion

56

necessary for the newly transferred plantlets to adapt to the

natural environment. Physical, chemical and biological properties

of potting mixtures are important in the establishment and growth

of in vitro produced plantlets. Maximum survival percentage of

rooted shoots were obtained on coco peat (89.00±2.45) followed by

sterile soilrite (79.00±1.87). Coco pit: soil and sterile soilrite: soil

(1:1 v/v) gave survival rate of 69 and 66 percent respectively. The

results of present experiment agreed with the finding of Shankar et

al. (2014) who found the survival rate of the plantlets in coconut

coir pith to be 84.44% during primary hardening. Similar results

were found by Rai et al. (2012) who recorded survival rate of 96 %

on medium containing coco peat and sand in the ratio of 2:1 among

different growth media viz., soil, sand and cocopeat (1:1:1), soil

sand and farmyard manure (1:1:1) and mixture of cocopeat and

sand (2:1). Coco peat is an ingredient that can keep the soil loose,

which in turn enable roots to spread out easily and thus, giving

more breathing space and aeration and consequently better plant

growth is achieved. Anbazhagan et al. (2014) transplanted cultured

plantlets into 10 cm diameter plastic pots containing a mixture of

sand, soil and vermicompost (1:1:1) and placed in the greenhouse

for hardening. Mixing soil, sand and FYM might have helped in

giving better grip for roots, ample aeration and sufficient organic

matter.

5.5 Morphological characteristics

Maximum height of plant was found in coco peat (T1)

(7.98±0.19 cm). Girth of plants were statically non-significant to

each other; being maximum in sterile soilrite (2.20±0.11). Leaf

length, leaf width and number of leaves followed the same trend.

Maximum value for leaf character was found in coco peat media

being 6.10±0.23 cm, 4.12±0.27 cm and 4.20±0.37 for leaf length,

leaf width and number of leaves, respectively. The current finding is

in accordance with Uzibara et al. (2015) who also found coco speat

Discussion

57

to be far superior to other potting media in terms of percentage

survival of plantlets (95.00 %), plantlet height (5.58 cm), number of

leaves (3.20), plantlet diameter (4.59 mm), number of primary roots

per plantlet (5.20), length of primary roots (5.18 cm) and number of

secondary roots per plantlet (25.50). He also found vermicompost to

be unsatisfactory. This may be due to better aeration, water

holding and nutrient supplying capacity of coco peat as compared

to vermiculite, sand and vermicompost. The poor result obtained

with vermicompost may be explained by its structure which

became muddy and compact (Dewir et al., 2005). But, contrary to

this; Ahmed et al. (2014) obtained maximum height of tissue

cultured plantlets of Grand Naine (15.70 cm) in potting mixture

containing soil, sand and FYM (2: 1: 1 v/v/v) in secondary

hardening.

*****

Chapter-V

Summary and Conclusion

The present investigation entitled “Micropropagation of

banana cv. Malbhog for production of quality planting material”

was carried out at the plant tissue culture laboratory, Bihar

Agricultural College, Sabour. The cultivar chosen for

micropropagation of banana was Malbhog. Shoot tip were being

used as explant. The results obtained during the course of

investigation are summarized below:-

1. The per cent survival of explants was recorded highest (45.00)

with T6 treatment i.e. when explants were surface sterilized

with Ethanol (70%) for 30 sec and HgCl2 (0.1%) for 25

minutes. The mortality of explants was also increased with

the increase in time of exposure of sterilants but, the

contamination was significantly reduced. At lesser time

duration of treatments, contamination rate was high.

2. Treatment with HgCl2 (0.1%) for longer time duration was

found effective in controlling contamination.

3. Addition of cytokinins alone or in combination with auxin

was found essential for initiation and multiplication phase.

4. MS media containing NAA 0.5 + BAP 5.0 mg/l proved best for

getting highest number of shoots per explant (4.00), longest

length of shoot (5.10 cm) and least duration for shoot

initiation (18.40 days).

5. The addition of auxin in the medium along with cytokinin

increased the length of shoot and number of shoots per

explant upto certain level indicating that a certain optimum

concentration is required for good growth and multiplication.

6. Half MS basal media supplemented with IBA 1.5 mg/l gave

maximum rooting response percentage (66.67), minimum

number of days for root formation (18.00), maximum number

Summery and conclusion

59

of roots per explant (4.20) and maximum length of root (4.00

cm).

7. Addition of auxin at optimum concentration was essential for

getting good rooting response, longest length of root and

number of roots per explant because addition of less or

excess auxin both influence rooting and other rooting

parameters.

8. Maximum survival percentage of plantlets was obtained with

coco peat (89.00). Survival in other potting mixture was

comparatively low.

9. Overall, Coco peat again proved best potting media for

maximum plant height (7.98 cm), girth (2.20 cm), leaf length

(6.10 cm), leaf width (4.12 cm) and number of leaves (4.20).

So, it can be concluded that an attempt was made for

micropropagation protocol for banana cv. Malbhog. Our results

described various factors that influence the establishment, shoot

multiplication, root induction and acclimatization of Malbhog

banana. The response of AAB type (Malbhog banana) is much

poorer than that of AAA group. In AAB group the growth of

auxiliary buds in vivo is inhibited by a high degree of apical

dominance. Furthermore, getting quality propagules (to be used

as explant) is also a major concern because Malbhog banana

plantations are very less. Therefore, there is need of further

research work to be conducted for establishing a rapid and an

efficient protocol for achieving mass scale micropropagation of

Malbhog banana so as to save them from extinction, spreading

area under cultivation, getting reliable and disease free planting

material.

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*****

thesis...deep sense of gratitude to my major advisor dr. muneshwar prasad, a sstt. professor-cum-jr....

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