for Tolerance to Aluminium Toxicity By Ermias Abate Desta

203 Pages·2016·3.29 MB·English

by  Ermias Abate Desta

Pre-breeding of Tef [Eragrostis tef (Zucc.) Trotter] for Tolerance to
Aluminium Toxicity

By

Ermias Abate Desta

B.Sc. Plant Sciences (Alemaya University, Alemaya, Ethiopia)
M.Sc. Horticulture and Crop Protection (University of Jordan, Amman, Jordan)

A thesis Submitted in Partial Fulfilment of The Requirements for the Degree of
Doctor of Philosophy (PhD) in Plant Breeding

The African Centre for Crop Improvement
School of Agricultural, Earth and Environmental Sciences
College of Agriculture, Engineering and Science
University of KwaZulu-Natal
Pietermaritzburg
Republic of South Africa
November 2015
Thesis Summary
Tef [Eragrostis tef (Zucc.) Trotter] is the most widely grown cereal crop in Ethiopia. Its
grain is used for human consumption and the straw is an important and highly valued
livestock feed. Soil acidity and Al toxicity are among the major production constraints
affecting tef in Ethiopia. Utilization of lime and other non-genetic acid soil management
options is constrained by various socio-economic factors. Development of cultivars
with tolerance to Al-toxicity is a complementary approach to liming in the production
of globally important crops such as wheat, rice, maize, barley, sorghum and rye.
However, no breeding for tolerance to Al toxicity in tef had been undertaken previously.
Hence, this research project was initiated in order to address the following objectives:
1. To assess the perceptions, challenges and coping mechanisms of farmers dealing
with soil acidity and Al-toxicity in problem areas of north western Ethiopia;
2. To characterize the reactions of released tef varieties to soil acidity and the
associated Al-toxicity;
3. To determine the extent of genetic diversity among tef germplasm collected from
areas of Ethiopia with acid soil;
4. To isolate and characterize EMS-induced mutants of tef for tolerance to Al-toxicity
and other important agronomic traits;
5. To evaluate the use of hydroponics system as a phenotyping platform to screen
for Al-tolerance in tef, using root measurement and haematoxylin assay methods.
There is no information on breeding for Al-tolerance in tef. Therefore, relational
background literature was collated on other cereals on their mechanisms of Al-toxicity,
tolerance mechanisms, genetic control, screening methods and marker assisted
breeding. The information obtained from such sources was used to develop and
undertake the subsequent breeding activities on tef.
In order to meet the set objectives, several laboratory, greenhouse, and field
experiments were conducted at the Amhara Regional Agricultural Research Institute
(ARARI), Ethiopia, from December 2012 to June 2015.
A Participatory Rural Appraisal (PRA) study was conducted in three Districts of north
western Ethiopia that are affected by acid soils, in order to assess the state of soil
acidity, and to determine its perceived causes and indicators, and to document the
coping strategies of the farmers. Semi-structured interviews, group discussions and
i
soil analyses were the main techniques used to generate data in this background
study. Farmers’ perceived the causes of soil acidity to include: soil erosion; poor
nutrient recycling; the abandoning of traditional fertility management practices; the
unbalanced and/or minimal use of external inputs; and the exclusive use of acid-
forming, inorganic fertilizers. Soil erosion, soil acidity, the high cost of mineral fertilizers
and lime, cash shortages, and a lack of acid tolerant crop varieties were ranked as the
top constraints. Species tolerance to soil acidity was found to be one of the major
factors that influenced crop choice by farmers. A decline in genetic diversity and the
rapid expansions of newly introduced, acid tolerant crops such as oat and triticale were
noticed. The pH (H O) of most of the soils in the study sites was in a strongly acidic
2
range (4.6–5.5). Gashena Akayita of Banja District was the most acidic of all and had
high levels of exchangeable Al. The limitations of the current coping strategies
suggested the need to introduce compatible technologies that would ensure the
sustainable management of the soils in the region, by the small-scale farmers there.
Thirty three Released Varieties and selected accessions of tef were evaluated for their
tolerance to soil acidity in pot trials. Twenty eight of these were then evaluated under
field conditions. The results revealed the presence of significant genetic variability
within the test genotypes. Nearly all the test genotypes were highly sensitive to soil
acidity and Al-toxicity. However, a local landrace that is widely grown in Banja, a
District severely affect by soil acidity, consistently outperformed the other genotypes
both under pot and field conditions. There were changes in the ranking of the tef
genotypes tested under pot and field conditions, which suggested the need to consider
other edaphic and climatic factors when breeding for Al-tolerance. Overall, the grain
yield of the test genotypes and the tolerant local landrace were less than the national
mean yield of tef, identifying the need to develop varieties with better tolerance of acid
soils and the associated Al-toxicity, aiming for superior agronomic performances in
acid soils.
Twenty-seven tef accessions collected from three regions of Ethiopia that are affected
by acid soils were evaluated, together with released breeders’ varieties, and selected
breeding materials for genetic diversity, using 16 selected and highly polymorphic SSR
markers. Analysis of molecular variance (AMOVA) showed highly significant
differences (P<0.001) among and within populations. Despite the wide geographical
separation of the collection sites, 88.5% of the accessions from acid soils were
ii
grouped into two clusters (Clusters II and III) while 90% of the breeding materials and
the Released Varieties were grouped into Cluster I. A significant degree of genetic
differentiation was observed among the populations. Accessions from the north
western Ethiopia exhibited a significant level of variation for most of the genetic
diversity parameters. The number of private alleles was significantly higher for tef
plants from acid soils than the Released Varieties and the breeding materials the Pair-
wise estimates of genetic identity and gene flow showed higher values existed
between the Released Varieties and breeding materials.
About 15,000 M seeds were screened under acid soil conditions along with the M
2 0
mutagenized seeds of the parent variety Tsedey and an Al-tolerant local landrace,
Dabo banja. Twenty one M plants with root lengths of greater than the mean plus
2
standard deviation of the tolerant check were selected and their M progenies were
3
characterized for Al-tolerance and morpho-agronomic traits under greenhouse and
field conditions, respectively. There were highly significant differences for Al-tolerance
between the M mutant lines and the parent (P<0.001); and between the M mutant
3 3
lines and the sensitive check (P<0.001). However, there was no significant difference
between the M mutant lines and the tolerant check. The result of the morpho-
3
agronomic characterization revealed the presence of significant differences between
the M mutants for 16 of the 20 quantitative traits measured.
3
Five levels of AlK(SO ) .12H O were evaluated (0, 150, 250,350, 450, 550 µM) in order
4 2 2
to select the optimal concentration of Al that can most efficiently discriminate between
sensitive and tolerant tef genotypes, using a hydroponic growing facility and
measuring root lengths. The haematoxylin staining method was also assessed as a
tool for the visual evaluation of tef varieties for Al-tolerance using selected test
genotypes. There were highly significant differences (P<0.001) between the
treatments, both for dose of Al and for genotype sensitivity to Al. The maximum
differences in relative root length (RRL) (%) and root length (RL) (mm) between the
sensitive and the tolerant genotypes were observed at the Al level of 150 µM Al. This
concentration efficiently discriminated between 28 test genotypes with different levels
of sensitivity to Al-toxicity. A visual assessment of the reactions of two sensitive and
two tolerant genotypes to haematoxylin staining using 0, 150 and 250 µM of
AlK(SO ) .12H O showed differential staining reactions in their roots that were
4 2 2
consistent with their prior root growth measurements.
iii
Declaration

I, Ermias Abate Desta, declare that
1. The research reported in this thesis, except where otherwise indicated, is my
original work.
2. The thesis has not been submitted for any degree or examination at any other
university.
3. This thesis does not contain other persons’ data, picture, graphs or other
information, unless specifically acknowledged as being sourced from other
persons.
4. This thesis does not contain other persons’ writing, unless specifically
acknowledged as being sourced from other researchers. Where other written
sources have been quoted, then:
a. Their words have been re-written but the general information attributed to them
has been referenced.
b. Where their exact words have been used, then their writing has been placed in
italics and inside quotation marks, and referenced.
5. This thesis does not contain text, graphics or tables copied and pasted from the
internet, unless specifically acknowledged, and the source being detailed in the
thesis and in the reference section.
Signed
…………………………………………………………………………………………………
Ermias Abate Desta
As the candidate’s supervisors, we agree to the submission of the thesis:
………………………………………………………………………………………………….
Prof. Shimelis Hussein (Supervisor)

…………………………………………………………………………………………………
Prof. Mark D. Laing (Co-Supervisor)
iv
Acknowledgments
First of all, I would like to thank God, the Almighty, for giving me the strength and the
patience to accomplish this work.
Special thanks to the Alliance for a Green Revolution in Africa (AGRA) for offering me
the study grant. I also thank the Amhara Regional Agricultural Research Institute
(ARARI) for giving me study leave, and for administrative and logistical support during
my research work in Ethiopia.
I would like to express my deepest gratitude to my principal supervisor, Professor
Shimelis Hussein, for his unwavering support, encouragement, guidance and
understanding throughout the process of the proposal write-up, field and lab execution
of the research, analysis of results, and the writing of this thesis. I am always grateful
for the faith he had in me, even at times when I was fatigued.
I also owe a special thanks to my co-supervisor, Professor Mark D. Laing, for his
thorough and refining evaluation of the proposal and the thesis document. I am
particularly thankful for his inspiring and insightful conversation on my research work.
His compassion was highly appreciated.
I am grateful to my in-country co-supervisor, Dr Fentahun Mengistu, for his valuable
comments and suggestions in the process of proposal development and thesis write-
up. I am always indebted to his encouragement and the trust he had in me.
I would like to express my heartfelt thanks to Dr Charles Higgins and Judy from USA
for their help and encouragement. Without their support and facilitation, the most
demanding part of my thesis, the hydroponics research, would not have been possible.
I am also grateful to Professor Leon Kochian and Dr Jon E. Shaff, from Cornell
University, for their technical advice on establishment of the hydroponics system. The
recipe of the modified Magnavaca’s nutrient solution was kindly provided by Dr Jon E.
Shaff.
I thank Dr Zerihun Tadele from University of Bern, Switzerland, for the provision of
seeds of mutant populations of tef used in this study. His support and encouragement
was so helpful.
I am very appreciative of the support of Dr Amelework Beyene who assisted in
statistical analysis of the molecular diversity data.
v
I am most grateful to the administrative staff of the African Centre for Crop
Improvement (ACCI) for hosting my study at the University of KwaZulu-Natal (UKZN).
Without their caring and attentive support, timely accomplishment of this research
work would have been impossible. My special thanks to Rowelda Donnelly. I am also
grateful to the academic staff of ACCI, the Plant Breeding Department and all those
who generously shared their knowledge in the training programme. I also thank the
ICT Division of UKZN.
Several people directly and indirectly helped me to undertake this research. I
acknowledge the support given by Mitiku, Gedefaw, and Atalay in tef germplasm
acquisition, seed increase and their assistance in undertaking the field experiments. I
am grateful to Yeshitila Merene, Dr Birru Yitaferu, Dr Akalu Teshome, Dr Minale
Wondie, Minilik Getaneh, Dr Tesfaye Feyisa, Dr Gizaw Desta, Dr Endale Gebre, Dr
Hailu Tefera, Dr Kibebew Assefa, Dr Solomon Chanyalew, Birhanu Agumas, Mulugeta
Alemayehu, Kemilew Muhe, Moges Mesele, Anteneh Abewa, Fikremariam Asargew,
Dr Tilahun Tadesse, Dr Yigzaw Dessalegn, Dr Tilaye Teklewold, Mekonen Getahun,
Dr Tadele Amare, Mengistu Muche, Misganaw Fente, Balew Ferede, and Abebe (from
Awi station) for their collaboration in one way or another.
I acknowledge the support and encouragement from the staff and management of
ARARI and the Adet Agricultural Research Centre. Specifically, I would like to thank
Mastewal, Beteha, Ejigitu, Birhan, Asasu, Getinet, Zinash, Worknesh, Sisay, Gashaw,
Abebe, and Hulubanchi for their help and encouragement.
My special thanks go also to Regional, Zonal, District, and Kebele level staff of the
Agriculture Development Departments, and the many farmers who collaborated in the
PRA and the field study. I am also grateful to the Ethiopian Institute of Biodiversity
Conservation (IBC) and the National Tef Improvement Programme for kindly providing
the tef germplasm used in this study.
Thanks also to my fellow postgraduates at ACCI-UKZN for sharing a friendly and
wonderful environment during the year of course-work and proposal development. I
have been privileged to have many brothers and sisters in Christ who cherish me
despite my shortfalls. Dear friends, you are too many to list here. You are all
recognized and whole-heartedly acknowledged for your invaluable support and
encouragement. You were with me when I needed you most. Thank you.
vi
Finally, my heartfelt thanks to my family, relatives and friends who gave me the support
and courage to overcome this daunting task. I am particularly grateful to my wife,
Ayalnesh Asresie, for taking full-care of our lovely children during my absence. The
unconditional love from my children, Eyerusalem, Meba and Barok, was wonderfully
nurturing. My mom, Dereje, Woinishet, Hailemariam, and Etagegn, thank you for the
support that you compassionately provided me.
vii
Dedication

This PhD study is dedicated
to my late father, Abate Desta Mazengia,
and to my lovely children, Eyerusalem, Meba and Barok
viii
Table of contents

Thesis Summary……………………………………………………………………… i
Declaration …………………………………………………………………………... iv
Acknowledgments…………………………………………………………………... v
Dedication…………………………………………………………………………….. viii
Table of contents……………………………………………………………………. ix
Thesis Introduction…………………………………………………………………. 1
Background ……..……………………………………………………………………… 1
Constraints to tef production..………………………………………………………. 2
Problem statement…………………………………………………………………… 5
Objectives……………………………………………………………………………… 8
Outline of thesis……………………………………………………………………… 8
References …………………………………………………………………………… 9

CHAPTER 1…………………………………………………………………………… 14
A review of the Literature…………………………………………………………………… 14

1.1 Introduction…………………………………………………………………………….. 15
1.2 Development of aluminium toxicity: An overview……………………………….. 17
1.3 Mechanisms of aluminium toxicity………………………………………………… 18
1.4 Symptoms and effects of aluminium toxicity……………………………………. 18
1.5 Breeding for tolerance to Al toxicity................................................................. 19
1.6 Mechanisms of aluminium tolerance in cereals………………………………… 22
1.6.1 Exclusion mechanism…………………………………………………………….. 22
1.6.2 Internal detoxification……………………………………………………………. 23
1.7 Genetic control of Al-tolerance in cereals………………………………………… 23
1.8 Breeding for Al-tolerance in cereals……………………………………………….. 26
1.9 Screening methods for Al-tolerance………………………………………………... 28
1.9.1 Nutrient solution culture…………………………………………………………… 28
1.9.2 In vitro (tissue culture) screening method………………………………………. 30
1.9.3 Soil based screening………………………………………………………………. 31
1.9.4 Field evaluation…………………………………………………………………...... 31
1.10 Molecular marker assisted breeding for al-tolerance in cereals……………. 31
1.11 Conclusion……………………………………………………………………………. 32
References…………………………………………………………………………………… 32

ix