molecular analyses of salvia africana

186 Pages·2009·4.86 MB·English

MOLECULAR ANALYSES OF SALVIA AFRICANA-
LUTEA L. TRANSGENIC ROOT CLONES FOR
SECONDARY BIOACTIVES

WATSIE PRINCESS NEO RAMOGOLA

BSc (Hons)

Submitted in fulfilment of the academic requirements for the degree
of MASTER OF SCIENCE in the Institute for Plant Biotechnology,
Faculty of AgriSciences, Department of Genetics

Stellenbosch University

DECEMBER 2009

i
DECLARATION

I, the undersigned, hereby declare that the work contained in this thesis is my own
original work and that I have not previously submitted it in its entirety or in part, at any
university for a degree.

____________________________________
Watsie Princess Neo Ramogola

I declare that the above statement is correct.

_________________________________
Doctor Nokwanda Makunga
(SUPERVISOR)

______/_____/__________
Date

ii
DEDICATION

This thesis is dedicated to my Mother;
Matshediso Ramogola
“Mama ke a leboga”

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SCIENTIFIC OUTPUTS

(i) PUBLICATIONS IN PEER-REVIEWED JOURNALS

Kamatou GPP, Makunga NP, Ramogola WPN and Viljoen AM (2008) South African
Salvia species: A review of biological activities and phytochemistry. Journal of
Ethnopharmacology 119: 664-672

(ii) CONFERENCE OUTPUTS

Ramogola WPN, Makunga NP and van Staden J (2008) Molecular analysis of Salvia
africana-lutea L. organ cultures. Joint South African Association for Botanists (34th)
and Southern African Society for Systematic Biology Annual Congress (University of
Johannesburg). South African Journal of Botany 74: 376

Makunga NP, Colling J, Horsthemke HR, Ramogola WPN and Van Staden J (2007)
Changing the chemical mosaic of South African medicinal plants through
biotechnological strategies. South African Association for Botanists 33rd Annual
Congress (University of Cape Town). South African Journal of Botany 73: 299

Makunga NP, Colling J, Horsthemke HR, Ramogola WPN and van Staden J (2007)
Adopting biotechnological strategies-plants from South Africa as targets. PSE
Congress: Plants for Human Health in the Post-Genome Era (Helsinki, Finland).

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TABLE OF CONTENTS

DECLARATION ............................................................................................................... i
DEDICATION ................................................................................................................. iii
SCIENTIFIC OUTPUTS ................................................................................................. iv
TABLE OF CONTENTS .................................................................................................. v
LIST OF TABLES .......................................................................................................... ix
LIST OF FIGURES .......................................................................................................... x
ABBREVIATIONS......................................................................................................... xii
ACKNOWLEDGEMENTS ............................................................................................. xx
ABSTRAK .................................................................................................................. xxii
ABSTRACT ................................................................................................................ xxv
CHAPTER 1 INTRODUCTION ........................................................................................ 1
1.1 REFERENCES ...................................................................................................... 8
CHAPTER 2 LITERATURE REVIEW ............................................................................ 12
2.1 BACKGROUND INFORMATION ON GENUS SALVIA ....................................... 12
2.1.1 Botany and geographical distribution of S. africana-lutea ....................... 12
2.1.2 Biological activity of Salvia ........................................................................ 14
2.1.3 Phytochemistry of Salvia ............................................................................ 20
2.1.4 Biosynthesis of terpenes in Salvia ............................................................. 21
2.2 BIOTECHNOLOGICAL APPLICATIONS ON SALVIA SPECIES ........................ 28
2.2.1 Plant tissue culture ..................................................................................... 29
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2.2.2 Plant tissue cultures of Salvia species ...................................................... 30
2.2.3 Recombinant DNA technology ................................................................... 42
2.2.4 Agrobacterium-mediated genetic modification ......................................... 43
2.2.5 Transgenic plants with rol genes ............................................................... 47
2.2.6 Secondary metabolism in rol-transgenic plants ....................................... 49
2.2.7 Transformation of Salvia species ............................................................... 50
2.3 CURRENT BIOTECHNOLOGICAL CHALLENGES AND FUTURE PROSPECTS
.................................................................................................................................. 53
2.4 AIMS AND OBJECTIVES .................................................................................... 54
2.5 REFERENCES .................................................................................................... 55
CHAPTER 3 MICROPLANT PROPAGATION AND HAIRY ROOT CULTURE OF
SALVIA AFRICANA-LUTEA ........................................................................................ 71
3.1 INTRODUCTION ................................................................................................. 71
3.1.1 In vitro plantlet culture ................................................................................... 71
3.1.2 In vitro hairy root culture ................................................................................ 72
3.2 MATERIALS AND METHODS ............................................................................ 73
3.2.1 Micropropagation of plantlets .................................................................... 73
3.2.2. Rooting of plantlets .................................................................................... 73
3.2.3. Acclimatisation of plantlets ....................................................................... 74
3.2.4 Liquid hairy root culture ............................................................................. 74
3.2.5 Effect of different basal media on hairy root growth ................................ 75
3.2.6. Data collection and statistical analyses ................................................... 75
3.3 RESULTS AND DISCUSSION ............................................................................ 76
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3.3.1. Micropropagation of plantlets ................................................................... 76
3.3.2. Rooting of plantlets .................................................................................... 77
3.3.3 Acclimatisation of plantlets ........................................................................ 79
3.3.4 Liquid hairy root culture ............................................................................. 80
3.3.5 Effect of different basal media on hairy root growth ................................ 81
3.4 REFERENCES .................................................................................................... 85
CHAPTER 4 MOLECULAR ANALYSIS OF S. AFRICANA-LUTEA HAIRY ROOTS ... 89
4.1 INTRODUCTION ................................................................................................. 89
4.2 MOLECULAR ANALYSIS OF HAIRY ROOTS .................................................... 90
4.2.1 Polymerase chain reaction ......................................................................... 91
4.2.2 Southern hybridisation ............................................................................... 92
4.3 MATERIALS AND METHODS ............................................................................ 93
4.3.1 Genomic DNA extraction from hairy roots ................................................ 93
4.3.2 PCR amplification of rol and ags genes in the root DNA .......................... 96
4.3.3 Probe preparation and labelling ................................................................. 99
4.3.4 Southern hybridisation analysis .............................................................. 100
4.4 RESULTS AND DISCUSSION .......................................................................... 102
4.4.1 Genomic DNA isolation ............................................................................. 102
4.4.2 Optimisation of the PCR amplification ..................................................... 106
4.4.3 Validation of trangenesis of the hairy roots ............................................ 109
4.4.4 Probe preparation ..................................................................................... 110
4.4.5 Southern hybridisation ............................................................................. 111
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4.5 REFERENCES .................................................................................................. 112
CHAPTER 5 PHYTOCHEMISTRY AND PHARMACOLOGY OF S. AFRICANA-LUTEA
CLONES ..................................................................................................................... 116
5.1 STRATEGIES FOR THE DISCOVERY OF BIOACTIVE PHYTOCHEMICALS . 116
5.2 MATERIALS AND METHODS .......................................................................... 119
5.2.1 Preparation of plant extracts (Non-volatile compounds) ........................ 119
5.2.2 Extraction of essential oil (Volatile compounds) .................................... 120
5.2.3 TLC analysis .............................................................................................. 120
5.2.4 Antibacterial bioassays ............................................................................ 121
5.2.5 Antifungal bioassays ................................................................................ 122
5.2.6 Bioautographic assay ............................................................................... 123
5.2.7 GC-MS analysis ......................................................................................... 124
5.2.8 NMR analysis ............................................................................................. 126
5.3 RESULTS AND DISCUSSION .......................................................................... 126
5.3.1 TLC analysis .............................................................................................. 126
5.3.2 Antibacterial bioassays ............................................................................ 127
5.3.3 Antifungal bioassays ................................................................................ 129
5.3.4 Secondary metabolite profiles ................................................................. 130
5.4 REFERENCES .................................................................................................. 146
CHAPTER 6 GENERAL CONCLUSIONS AND RECOMMENDATIONS .................... 155
APPENDIX .................................................................................................................. 158

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LIST OF TABLES

Table 2-1 Summary of pharmacological activities of some South African Salvia species .............................17
Table 2-2 Summary of biotechnological applications on some Salvia species worldwide ............................32
Table 2-3 Phenotypic characteristics produced by single effect of rol genes on transgenic tobacco plants ....
.......................................................................................................................................................................48
Table 3-1 Rooting of S. africana-lutea plantlets in PGR-free MS media .......................................................78
Table 3-2 Improvement in the acclimation process of the S. africana-lutea plantlets .................................79
Table 4-1 Primers used for the PCR amplification of the rol and ags genes incorporated in the S. africana-
lutea hairy root DNA ......................................................................................................................................98
Table 4-2 An optimised PCR reaction for the amplification of the rol and ags genes ...................................99
Table 4-3 The radioactive PCR-labelling reaction for the rol gene probe .................................................. 100
Table 4-4 Quality and concentration of the genomic DNA from S. africana-lutea root clones with different
extraction protocols (Sections 4.3.1.2 and 4.3.1.3 respectively) ............................................................... 103
Table 4-5 The fidelity of DNA samples extracted with different protocols to downstream molecular
analyses ...................................................................................................................................................... 105
Table 4-6 The effect of salt/EtOH precipitation on the quality and the concentration of the hairy root DNA
eluted with Qiagen Kit ................................................................................................................................ 106
Table 5-1 Authentic standards derivatised with MSTFA for GC-MS analysis ............................................. 125
Table 5-2 Antibacterial MIC (mg ml-1) of S. africana-lutea extracts (after 24 hours) ................................. 128
Table 5-3 Antifungal MIC (mg ml-1) of S. africana-lutea extracts (after 96 hours) ..................................... 129
Table 5-4 GC-MS library identification of some chemical compounds found in S. africana-lutea (XCaliburTM)
.................................................................................................................................................................... 137

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LIST OF FIGURES

Figure 2-1 Geographical distribution of Salvia africana-lutea in South Africa (reproduced with kind
permission of SANBI, PRECIS)……………………………………………………………………………………………………………………13
Figure 2-2 A) A bush of S. africana-lutea flowering during Spring (30th August 2006) at Stellenbosch
University, SOUTH AFRICA B) Inflorescence after the petals have dropped ……………………………………………..14
Figure 2-3 Summarised illustration of the cytoplasmic mevalonate (MVA) pathway and plastidial non-
mevalonate (non-MVA) pathway for the biosynthesis of terpenoids in plant cells (Ge and Wu 2005a)……. 26
Figure 2-4 Shikimic acid pathway adapted from Taiz and Zeiger (2006) and Wildman and Kelley (2007)….27
Figure 2-5 Schematic map of ORFs in the T-DNA of pRiA4b of A. rhizogenes (Aoki and Syōno 2000)………. 45
L
Figure 3-1 Micropropagation of S. africana-lutea plantlets……………………………………………………………………… 77
Figure 3-2 An example of transgenic liquid-shake root culture of S. africana-lutea L. in ½MS after four
weeks of sub-culture……………………………………………………………………………………………………………………………….. 82
Figure 3-3 Growth pattern of S. africana-lutea hairy root clones on solid ½MS medium…………………………. 82
Figure 3-4 Growth curves of different S. africana lutea hairy root clones in liquid PGR-free ½MS medium
over 30-day period………………………………………………………………………………………………………………………………….. 83
Figure 4-1 Troubleshooting the PCR amplification of rol and ags genes in the S. africana-lutea hairy root
DNA…………………………………………………………………………………………………………………………………………………………. 97
Figure 4-2 Genomic DNA isolated from S. africana-lutea hairy root clones with aurea buffer and bBuffers A
and B only…………………………………………………………………………………………………………………………………………… 103
Figure 4-3 Optimisation of the PCR amplification of the rol genes in the S. africana-lutea root DNA A)
Unspecific PCR amplification. B) MgCl optimisation C) Determination of the optimal hairy root DNA
2
concentration………………………………………………………………………………………………………………………………………… 108
Figure 4-4 PCR amplifications of the rol and ags genes in S. africana-lutea root and Ri plasmid DNA. A) ags
amplification (1600 bp) B) rol A amplification (300 bp) C) rol C amplification (600 bp) D) rol B amplification
(300 bp)…………………………………………………………………………………………………………………………………………………. 110
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