MORINGA OLEIFERA TREES USING TISSUE CULTURE TECHNIQUES

IN VITRO PROPAGATION AND PRODUCTION OF SOME

NATURAL ANTIMICROBIAL COMPOUNDS FROM

MORINGA OLEIFERA TREES USING TISSUE CULTURE

TECHNIQUES

Submitted By

Fawzia Ghareeb Abd El-Naby Sayed Ahmed

B.Sc. of Agricultural Science (General), Faculty of Agriculture, Cairo University, 1988

Diploma of Environmental Sciences, Institute of Environmental Studies & Research,

Ain Shams University, 1998

M. Sc. of Environmental Science, Institute of Environmental Studies & Research,


A Thesis Submitted in Partial Fulfillment

Of

The Requirement for the Doctor of Philosophy Degree

In

Environmental Science

Department of Environmental Agricultural Science

Institute of Environmental Studies and Research

Ain Shams University

2014

APPROVAL SHEET




TECHNIQUES

Submitted By







This Thesis Towards a Doctor of Philosophy Degree in

Environmental Science Has been Approved by: Name Signature

1-Prof. Dr. Seham Salah El-Din EL-Hawary ………………

Emeritus Prof. of Pharmacognosy

Faculty of Pharmacy - Cairo University

2-Prof. Dr. Hassan Mohamed Fadel ……………….

Prof. of Pomology - Department of Horticulture

Faculty of Agriculture - Ain Shams University

3-Prof. Dr. Nadia Mohamed Sokkar ………………

Prof. of Pharmacognosy - Faculty of Pharmacy

Cairo University

4-Dr. Ahmed Abd El-Hamid Awad ……………….

Associate Prof. of Pomology - Department of Horticulture


2014




TECHNIQUES

Submitted By







A Thesis Submitted in Partial Fulfillment

Of

The Requirement for the Doctor of Philosophy Degree

In

Environmental Science

Department of Environmental Agricultural Science

Under The Supervision of: 1-Prof. Dr. Wafaa Hasanien Wanas

Prof. of Pomology - Department of Horticulture


2-Dr. Nadia Mohamed Sokkar

Associate Prof. of Pharmacognosy

Faculty of Pharmacy - Cairo University

3-Dr. Ahmed Abd El-Hamid Awad

Lecturer of Pomolgy - Department of Horticulture


4-Dr. Essam Mohamed A. Youssef Emeritus Prof. Researcher in Department of Woody Plants

Horticultue Research Institute - Agriculture Research Center

2014

ACKNOWLEDGEMENT

First and foremost, I feel always indebted to

Allah, the beneficent and merciful

I wish to express my gratitude to Prof. Dr. Wafaa Hassanien

Wanas Prof. of Pomology, Dept. of Horticulture, Fac. of Agric., Ain Shams

Univ. for her supervision, her valuable guidance, useful suggesions,

continuous, sincere help and advice with no limits during the different

phases of this investigation.

Great thanks to Prof. Dr. Nadia Moh amed Sokkar professor of

cognize, Faculty of Pharmacy, Cairo University for here supervision, efforts

and helps.

I'm extremely grateful to Dr. Ahmed Abd El-Hamid Awad

Associated Prof. of pomology, Dept. of Horticulture, Fac. of Agric., Ain

Shams Univ. for his supervision, stimulating criticism and continuous efforts

in writing and reviewing the manuscript.

Deep thanks are offered to Prof. Dr. Essam Mohamed A. Youssef

Professor and Senior Researcher at Tissue Culture and Germplasm

Conservation Researches Lab. Horticulture Researched Institute, Agriculture

Research Center, Giza, for his supervision, suitable suggestion, following up

the stages of laboratory work.

Also I wish to express my deep thanks to Dr. Mai

Mohamed Raslan, Lecturer of Biotechnology, Faculty of Post Graduate

Studies, Bani-Suef University, for here kind help especially in HPLC

analysis.

I`m extremely grateful to the staff of Tissue Culture Research

Laboratory, Hort. Res. Institute. Agric., Res., Cent., Giza for offering all

facilities and helping.

My sincere thanks are, also, due to the member staff of the

Department of Drugs, Fac. of Pharma., Cairo Univ. for their valuable

guidance during the chemical determination of the biocompounds.

i

ABSTRACT

The experiments were carried out during the years from 2010 to 2014 at

Tissue Culture and Germplasm Conservation Research Lab. Hort. Res. Inst.,

Agric. Res. Center, Giza, Egypt, to investigate some factors that affecting

propagation and production of biocompounds such as quercetin and

kaempferol from different explant types of Moringa oleifera Lam. trees

using tissue culture techniques. The results obtained from micropropagation

of moringa experiments showed that Clorox at 10% for 15 min and mercuric

chloride (MC) at 0.1 % for 15 min significantly gave the lowest

contamination and the highest survival percentages of stem nodal explants.

The lowest contamination and survival percentages were observed with

shoot tip explants exposed to 0.1 % mercuric chloride for 7 min. MS

medium gave the highest significant number and length of proliferated

shoots/explant compared with WPM and B5 media. The highest

multiplication rates were achieved using modified MS medium

supplemented with 30 g/L sugar, 1.0 mg/L BAP. Repeating subculture up to

3 times increased average number and length of proliferated shoots. The

highest rooting percentage and average root number and length were

achieved on 3/4 strength MS medium. As for the effect of PGR on rooting

stage, using NAA at 2.0 mg/L with 3/4 MS gave the highest rooting

percentage. Rooted plantlets were successfully acclimated on mixture of peat

moss: sand: vermiculite at 1:1:1 v/v.

As for callus induction, data proved that the highest callus formation

percentage was recorded for leaf explants taken from in vitro grown

microshoots cultured on MS medium provided with 2,4-D at 2.0 mg/L. The

callus growth (accumulative fresh weight) increased with increasing period

ii

of incubation and the most favorable conditions were MS medium

supplemented with 2.0 mg/L 2,4-D plus 2.0 mg/L kin followed by4.0 mg/L

2,4-D plus 2.0 mg/L kin. Results on biocompounds produced from in vitro

callus cultures cleared that the most favorable conditions for callus growth

(fresh and dry weight) was that callus cultured on MS medium supplemented

with 2.0 mg/L 2,4-D plus 2.0 mg/L kin and incubated under 35°C compared

with the other treatments under investigation (UV, microwave and

incubation temperature). HPLC analysis indicated that quercetin was

detected in a very small percent in leaves of mother tree and increased in

callus cultured into medium containing 2.0 mg/L 2,4-D plus 2.0 mg/L kin

incubated at 35 and 25°C. While, kaempferol could not be detected in all

treatments under study or mother tree except callus culture of 4.0 mg/L 2,4-

D plus 2.0 mg/L kin exposed to 100 and 200 w microwave for 10 and 20 sec.

Also 30 w of UV-C for 30 min induced callus culture of 2.0 mg/L 2,4-D plus

2.0 mg/L kin to produce kampferol. Antimicrobial activity was studied for

all extracts of callus exposed to plant growth regulators, incubation

temperature, UV and microwave treatments as well as extracts of leaves and

seeds of mother tree, greenhouse seedlings and in vitro shoots. The extracts

were tested against two fungi and three bacterial species.

Erwinia amylovora and Staphylococcus aureus were more sensitive

to leaves and seeds extracts of mother tree, respectively. While, Salmonella

typhis was strongly affected by extract of callus cultured on medium

contained 2.0 mg/L kin plus 4.0 mg/L 2,4-D and microwaved at 200 W for

10 sec. Microwave induced callus cultured on medium contained 2.0 mg/L

2,4-D plus 2.0 mg/L kin to produce antifungal substances, using extract of

callus exposed to microwave irradiation at 100 W for 10 sec. recorded the

largest inhibition zone of Aspergillus niger. Whereas, applying the extract of

callus exposed to microwave irradiation at 200 W for 20 sec. recorded the

largest inhibition zone for Penicellium digitatum.

LIST OF CONTENTS

Page 1. Introduction 1

2. Review of literature 5

3. Material and methods 59

4. Results and discussions 78

4.1. In vitro propagation 78

4.1.1. Establishment stage 78

4.1.2. Multiplication stage 85

4.1.3. Rooting stage 120

4.1.4. Acclimatization stage 128

4.2. In vitro calls formation 133

4.2.1. Effect of plant growth regulators 133

4.2.2. Effect of plant growth regulators, temperature degree,

ultraviolet and microwave irradiation.

143

4.3. Secondary metabolites production of phenolic

compounds

159

4.3.1. Effect of plant growth regulators, temperature degrees,

ultraviolet and microwave irradiation

159

4.4. Antimicrobial activity 179

4.4.1. Antibacterial activity 179

4.4.2. Antifungal activity 184

Summary 200

References 208

Arabic summary

LIST OF TABLES

Page Table (1):Culture medium composition and strength of Murashige

and Skoog, 1962 medium (MS) used for

micropropagation of Moringa oleifera Lam.

63

Table (2): McCown WPM medium (Lloyd and McCown 1980) and

B5 medium (Gamborg et. al, 1968)

64

Table (3): Weights (g) of the dry residues of 70 % methanol

extracts obtained from 0.5 g powdered samples of the in

vitro grown calli and microshoots, leaves and seeds of

the mother tree and leaves of the greenhouse seedlings.

71

Table (4): Quercetin standard curve data 73

Table (5): Kaempferol standard curve data. 74

Table (6): Conditions for HPLC analysis of flavonoidal compounds. 75

Table (7): Effect of different sterilization treatments on disinfection

and survival percentages of Moringa oleifera Lam. shoot

tips and stem node explants

84

Table: (8-a): Effect of culture media ingredients on shoot

multiplication of Moringa oleifera Lam. stem node

explants.

91

Table (8-b): Effect of plant growth regulators on shoot


explants.

98

Table (8-c): Effect of number of subcultures on shoot


explants.

101

Table (8-d): Effect of culture media ingredients and number of

subcultures on shoot multiplication of Moringa

oleifera Lam. stem node explants.

105

Table (8-e): Effect of plant growth regulators and number of

subcultures on shoot multiplication of Moringa

oleifera Lam. stem node explants.

109

Table (8-f): Effect of culture media ingredients and plant growth

regulators on shoot multiplication of Moringa oleifera

Lam. stem node explants.

112

Table (8-g): Effect of culture media ingredients, plant growth

regulators and number of subcultures on shoot


explants.

119

Table (9): Effect of MS medium strength on in vitro rooting ability

of Moringa oleifera Lam. microshoots.

124

Table (10): Effect of different auxin (type and concentration) and

charcoal concentrations on in vitro rooting ability of

Moringa oleifera Lam. microshoots.

129

Table (11): Effect of different culture media substrates on ex vitro

acclimatization of Moringa oleifera Lam. plantlets.

132

Table (12): Effect of plant growth regulators treatments on callus

formation % and callus fresh weight of Moringa

oleifera Lam. cultures.

143

Table (13): Effect of plant growth regulators, temperature degrees,

ultraviolet and microwave irradiation treatments on

Moringa olifera Lam. callus fresh & dry weights (gm)

and moisture content (%).

156

Table (14): Effect of plant growth regulators, incubation

temperature degrees, ultraviolet and microwave

irradiation treatments on endogenous total phenols and

flavonoids content (g/100 g D.W.) of Moringa oleifera

Lam. callus cultures.

163

Table (15): Effect of plant growth regulators, temperature degrees, 171

UV and microwave irradiation treatments on

kaempferol and quercetin content (g/100 g D.W.) of

Moringa oleifera Lam. callus cultures.



irradiation treatments on antibacterial activity of

Moringa oleifera Lam. callus culture extracts.

186



irradiation treatments on Antifungal activity of Moringa

oleifera Lam. callus culture extracts.

192

LIST OF PLATES

Page

Plate 1

a, b and c Tree, flowers and fruits of Moringa oleifera Lam.

60

Plate 2

Effect of culture media and plant growth regulators on multiplication

stage of M. oleifera Lam.

88

Plate 3

Effect of MS-strength on rooting stage of M. oleifera Lam.

125

Plate 4

Moringa oleifera Lam. plantlets after 45 days from acclimatization

132

Plate 5

Effect of plant growth regulators on callus growth

145

Plate 6

Growth inhibition zone of Erwinia amylovora

195

Plate 7

Growth inhibition zone of Salmonella typhi

196

Plate 8

Growth inhibition zone of Staphylococcus aureus

197

Plate 9

Growth inhibition zone of Penecillium digitatum

198

Plate 10

Growth inhibition zone of Aspergillus niger

199

Documents

MORINGA OLEIFERA TREES USING TISSUE CULTURE TECHNIQUES