TRANSFORMATION OF TEF (Eragrostis tef)

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CHAPTER 2 CONTROLLING PLANT HEIGHT AND LODGING IN TEF (Eragrostis tef, Zucc.) USING GIBBERELLIN BIOSYNTHESIS INHIBITORS

Abstract

Tef (E. tef) is a small seeded nutritious cereal and a primary food source in Ethiopia grown on over 2.56 million ha in Ethiopia. Tef productivity is low, 1.0 t per ha, due to several factors, among which lodging is the most critical causing direct losses of about 23% under natural condition. High yielding cultivars are usually tall and more susceptible to lodging and breeding effort has not yet succeeded to decouple height from yield. Inhibitors of gibberellin (GA) biosynthesis such as chlormequat chloride (CCC) are used extensively to restrict growth and improve lodging resistance in cereals. First, responsiveness of tef plants to GA3 and CCC application was determined using two tef varieties Gea Lammie (short) and DZ-01-196 (tall). At 10-2M CCC plant height was reduced by 43% and 21% in the tall and short variety, respectively, within six weeks after plant emergence. CCC at 10-1M reduced tiller number in both varieties. More detailed analysis of growth regulator application by including Paclobutrazol (PBZ) on the tall tef variety DZ-01-196 revealed that, both CCC and PBZ reduced culm length, with a much stronger reduction from paclobutrazol. Grain yield on the other hand was not affected by CCC treatment. CCC-treatment reduced culm length by affecting all internodes, with the 1st- 3rd internodes, followed by the 6th and 7th most severely affected, whereas paclobutrazol treatment strongly affected all internodes, with greatest effect on the uppermost 4 internodes. Internode diameter was unaffected by both CCC- and paclobutrazol-treatments. A steady increase in mean internode diameter until the 6th internode was found for CCC-treated and also control plants revealing a poor tapering in tef plants. Reduction of GA amount in tef might be a target for improving lodging resistance allowing uncoupling of plant height and yield.

Introduction

Tef (Eragrostis tef (Zuccagni) Trotter) is a panicle bearing, small-seeded nutritious cereal grown extensively in Ethiopia in diverse climatic and soil conditions with low risk of failure (Assefa et al., 2010). Tef is grown on about 2.6 million ha and accounts annually for about 28% of the total acreage of cereal production in Ethiopia. However, tef suffers from low productivity with average yields of only 1.0 t ha-1. Among the factors contributing to low yield, lodging is the most important (Assefa et al., 2010; Tefera et al., 2003; Yu et al., 2007). In general, lodging interferes with water and nutrient transport, reduces light interception, provides a favourable environment for disease, increases harvesting cost and losses and decreases grain yield and quality (Tripathi et al., 2003). It occurs either by buckling / bending at the basal culm internodes, or due to root lodging or failure of the anchorage system of the plant (Assefa et al., 2000; Ketema, 1983; Pinthus, 1973). Culm length and the strength of the basal part of the culm are considered major factors associated with lodging sensitivity (Rajala, 2003; Tripathi et al., 2003). In cereals, improvement of lodging resistance has been predominantly achieved by reducing plant height, in particular by chemical inhibition of gibberellin (GA) production (Rademacher, 2000) or by the use of semi-dwarf varieties with reduced GA biosynthesis or signal transduction (Hedden, 2003). Chlormequat chloride (CCC), the most commonly used plant growth retardant (PGR), blocks GA biosynthesis by inhibition of the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (CPP) by CPP synthase (Rademacher, 2000). Triazole PGRs, such as paclobutrazol (PBZ), inhibit the conversion of the GA precursor ent-kaurene to ent-kaurenoic acid (Rajala, 2003; Hedden and Graebe,1985). In general, PGRs have been extensively used in many crops to reduce lodging through shortening of the stem and to maintain a steady improvement in grain yield (Berry et al.,2004; Rajala, 2003).Reduction in plant height due to PGR treatment, is associated with reduced elongation of internodes particularly of the uppermost internodes and peduncle (Sanvicente et al., 1999;Rajala, 2003). CCC inhibits stem elongation in wheat (Humbries et al., 1965) and in oilseed rape. Foliar treatment with a combination of CCC, ethephon and imazaquin reduced main stem length in barley where shortening of the uppermost three internodes contributed significantly to the reduction (Sanvicente et al., 1999). PBZ application was found to reduce stem length and lodging in rice and increase yield by up to 15% compared to controls (French et al., 1990). In tef, cultivars bred for improved grain yield possess a tall phenotype and are highly susceptible to lodging (Assefa et al., 2010; Yu et al., 2007). Thus, lodging susceptibility has prevented the introduction of higher yielding varieties with good grain quality, and also hampered the use of input-intensive husbandry. Currently, there is no detailed information available for tef on the effect of PGR treatment on lodging and yield responses. The objective of this study was therefore to investigate morphological and yield changes in tef following GA biosynthesis inhibitor treatment under controlled environmental conditions. Results obtained demonstrate that CCC treatment of tef plants significantly reduces plant height without affecting yield.

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ABSTRACT 
THESIS COMPOSITION 
ACKNOWLEDGEMENT 
ABBREVIATIONS AND SYMBOLS 
LIST OF FIGURES 
LIST OF TABLES 
CHAPTER ONE: INTRODUCTION
1.1. The problem of lodging 
1.2. Plant architecture and lodging
1.3. Genetic control of lodging resistance
1.3.1. GA genes and lodging
1.3.2. Manipulation of plant height using GA genes
1.4. Brassinosteroid (BRs) genes and lodging 
1.5. Induced mutation 
1.6. Plant growth regulators for plant height control 
1.7. Lodging in E. tef
1.7.1. E. tef growth
1.7.2. Pheno-morphic features related to lodging
1.7.3. E. tef breeding for lodging resistance
1.8. Working hypothesis and aim of study
CHAPTER CHAPTER TWO: CONTROLLING PLANT HEIGHT AND LODGING IN TEF (Eragrostis tef) USING GIBBERELLIN BIOSYNTHESIS INHIBITORS
2.1. Abstract 
2.2. Introduction
2.3. Materials and methods
2.3.1. Plant material
2.3.2. Plant growth
2.3.3. Plant growth regulators (PGRs) treatment
2.3.4. Growth measurement
2.3.5. Analysis of endogenous GA content
2.3.6. Data analysis
2.4. Results 
2.4.1. Experiment I
2.4.1.1. Analysis of endogenous GA content
2.4.2. Experiment II
2.4.2.1. Culm and panicle growth
2.4.2.2. Internode growth
2.4.2.3. Tillering and above ground biomass yield
2.5. Discussion
CHAPTER THREE: TRANSFORMATION OF TEF (Eragrostis tef)
3.1 Abstract
3.2 Introduction 
3.3 Materials and methods 
3.3.1 Preparation of planting material and culture
3.3.2 Immature embryo isolation, callus induction and culture growth
3.3.3 GA20x and nptII marker gene plasmids
3.3.4 Agrobacterium culture, inoculation and co-cultivation
3.3.5 Plant regeneration
3.3.6 Preparation of plant material and culture
3.3.7 DNA isolation and PCR Screening of E. tef regenerants
3.3.8 Phenotypic measurement and characterization of T1 generation
3.3.9 Analysis of endogenous content
3.4 Results
3.4.1 Plant transformation
3.4.2 Transgene detection
3.4.3 Phenotypic characterization
3.4.3.1 Culm, internode and panicle length
3.4.4 Analysis of endogenous GA content
3.5 Discussion 
CHAPTER FOUR: ISOLATION, CHARACTERIZATION AND EXPRESSION OF GA GENES WITH PARTICULAR EMPHASIS ON GA20ox IN TEF (Eragrostis tef)
4.1 Abstract 
4.2 Introduction 
4.3 Materials and methods
4.3.1 Plant material and plant growth
4.3.2 Genomic DNA isolation
4.3.3 Gene identification and isolation
4.3.4 Isolation of complete E. tef GA20ox coding region
4.3.5 Cloning and sequencing of PCR DNA products
4.3.6 DNA sequence analysis and phylogentic analysis
4.3.7 RNA isolation and cDNA synthesis
4.3.8 Isolation of the Reduced height (Rht) and other E. tef genes
4.3.9 GA20-oxidase expression in E. tef
4.3.10 Expression of E. tef GA20-oxidase1 (EtGA20ox1) in E. coli
4.3.11 HPLC analysis
4.3.12 Southern blot analysis
4.4 Results 
4.4.1 Isolation of GA genes from E. tef
4.4.2 Putative E. tef GA20ox isolation and cloning
4.4.3 EtGA20ox copy number
4.4.4 GA20ox expression in E. tef
4.4.5 In vitro enzymatic activity of GA20ox in a heterologous system
4.4.6 Isolation of the Rht and other E. tef genes
4.5 Discussion
CHAPTER FIVE: EVALAUTION AND ANALYSIS OF MUTANT TEF(Eragrostis tef) LINES FOR DWARFISM FOR LODGING RESISTANCE
5.1 Abstract 
5.2 Introduction 
5.3 Materials and methods
5.3.1 Plant material
5.3.2 Plant growth and GA treatment
5.3.2.1 Growth measurement
5.3.2.2 Data analysis
5.4 Results 
5.4.1 Culm height, internode length and diameter
5.4.2 Panicle length, tillering, biomass and yield
5.5 Discussion
CHAPTER SIX: GENERAL DISCUSSION AND FUTURE
PERSPECTIVE
REFERENCES 
APPENDIX

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