i Cranfield University at Silsoe Institute of Water and Environment Ph.D Thesis 2004 Luc Joseph Gabriel Bonneau Drought resistance of willow short rotation coppice genotypes Supervisor Professor William Stephens December 16, 2004 This thesis is submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy ii ABSTRACT This thesis reports on an investigation of drought resistance of willow SRC genotypes Experiments were conducted at Silsoe, Bedfordshire, in pots and field trials in 2002 and in lysimeters in 2003 to evaluate the range of water use efficiency (WUE) of 50 willows varieties (Salix sp.) and isolate morpho-physiological traits related to WUE and drought resistance Within the genotype pool tested there was a wide range of responses The results depict the morpho-physiology of an ideal candidate that plant breeding could produce for drier area of UK, which are summarised below Its cuttings not develop calluses when stored in darkness at +4°C After planting, the candidate does not grow rapidly but has an early exponential phase of stem elongation, after a year of growth it has few stems per stool (< 5) Its long, narrow (Rl/w > 8) hairless leaves are characterised by small adaxial epidermal cells (AECS < 330µm2) The ideal candidate prioritises less biomass to its root system (root/shoot < 0.8) mainly in the top 0.2 m When grown under optimum condition, the large leaf area has high stomatal conductance and leaf temperature As water stress progresses, the leaf area decreases leaving little time for leaves to senesce and few yellow leaves remain on the stems The stomatal conductance decreases slowly and the leaf temperature is almost unaffected If water stress occurs before August the candidate is able to recover faster the initial physiological state and grow new leaves when re-watered The results indicate that the best parents to produce such candidate are S viminalis and S schwerinii or their related hybrids Water use (WU) of high yielding willow short rotation coppice hybrids is similar which indicates that the opportunity to reduce WU is limited and that productivity can be only improved by increasing WUE to produce above ground biomass and drought resistance The current willow breeding programme has great chance to produce hybrids with high WUE however the production of a progeny population from high yielding hybrids that contrast widely in resistance to water stress is recommended In theory, from such a population, valuable data on morpho-physiological traits related to drought resistance and high WUE can be collected and help genomics to develop quantitative trait loci to the condition that reference hybrids are grown along to quantify the level of water stress experienced by the planting iii ACKNOWLEDGEMENTS Many people deserve to be thanked for their support, encouragement and above all friendship during these rather intense three years To Professor William Stephens who supervised the project and had a paramount input into my work and left me plenty of liberty to conduct the research, I don’t know how I will ever be able to thank him enough To the Cranfield University staff for their professionalism; special thanks to: Ian Seymour, Tim Hess, Euan Brierley, Gabriela Lovelace, Leon Terry, Roger Swatland, Nigel Janes and last but not least Simon Medaney I would like to thank my parents, my sisters and my brother for their everlasting love, and their understanding towards my choice of career To David and Fabien my “blood brother” who shared the adventure with me and were always there for me Good luck to them in the completion of their Ph.D I am thankful to all my friends back home who trusted me in the completion of the research: Cedric Journet, Damien Baguenard, Dominique Vrignaud, Sebastien Caillaud, Dominique Juteau, Nicolas Hay, Cecile Demonchy, Melanie Lardeux, Benjamin Legras, Stephane Cornu, Janique Tourgis and Thomas Martin Over a year I was shared between my coppice and the Silsoe students, the experience as Student President was profoundly rewarding but what a busy life! Still I will miss my Silsoe experience This would not have been the same without my friends Melinda and Mark Dresser, my best wishes for their new life as parents The Silsoe experience gave me many friends for life and I would like to especially thank Sara Chaler Navarro, Sophie Bourreau, Sophie Goldenberg, Caroline Souchal for their support, Emmanuel Bekoe for sharing my daily office life and Ashish Kumar for sharing all the good times of the last months The project was funded by the Department of Trade and Industry iv TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES xiii LIST OF PLATES xviii LIST OF APPENDICES xix SYMBOLS AND ABBREVIATIONS xx CHAPTER Introduction 1.1 Background 1.2 Plant breeding 1.3 Silsoe project 10 1.4 Objectives 13 1.5 Thesis structure 14 CHAPTER The effects of water stress on the growth and biomass production of 50 varieties of Salix 15 2.1 Introduction 15 2.2 Material and methods 16 2.2.1 Varieties and cuttings selection 16 2.2.2 The field trial 19 2.2.3 The pot trial 22 2.2.4 Growth monitoring .24 2.2.5 Statistical analysis 25 2.3 Results 27 2.3.1 Weather 27 2.3.2 Stem length and biomass .30 2.3.3 Stem elongation and elongation rate 38 2.4 Discussion 46 2.4.1 Climate and weather 46 2.4.2 Trials design .46 v 2.4.3 Willows and water stress .49 CHAPTER Morpho-physiological traits linked with water stress resistance and stem biomass 53 3.1 Introduction 53 3.2 Material and methods 57 3.2.1 The field and pot trials 57 3.2.2 Willow cuttings early stages of growth .57 3.2.3 Willow leaves .59 3.2.4 Willow morphology .66 3.2.5 Statistical analysis 68 3.3 Results 70 3.3.1 Descriptive statistics 70 3.3.2 Further statistics 77 3.4 Discussion 85 CHAPTER Water use, biomass production and water use efficiency of five Salix hybrids 91 4.1 Introduction 91 4.2 Material and methods 96 4.2.1 Variety selection 96 4.2.2 Lysimeter research trial 98 4.2.3 Stem biomass .107 4.2.4 Water use efficiency (WUE) 108 4.2.5 Statistical analysis 109 4.3 Results 109 4.3.1 Seasonal and monthly water use 109 4.3.2 Monthly and seasonal water use efficiency .111 4.4 Discussion 115 CHAPTER Leaf population, leaf area and biomass partitioning of five Salix hybrids grown in lysimeters under two water regimes 122 5.1 Introduction 122 5.2 Material and methods .124 5.2.1 Experimental layout 124 vi 5.2.2 Leaf area 125 5.2.3 Biomass partitioning 127 5.2.4 Statistical analysis 128 5.3 Results 128 5.3.1 Length of leaf bearing stem .128 5.3.2 Model of Leaf Area 133 5.3.3 Biomass partitioning and WUE 135 5.3.4 Correlation between WUE and other variables 139 5.4 Discussion 141 5.5 Conclusions 146 CHAPTER Transpiration and photosynthesis of five Salix hybrids .148 6.1 Introduction 148 6.2 Material and methods .154 6.2.1 Treatments 154 6.2.2 Soil water and water use 155 6.2.3 Stomatal conductance (gs), Photosynthetic rate (A), instantaneous water use efficiency (WUEi) and leaf temperature 157 6.2.4 Chlorophyll fluorescence 160 6.2.5 Statistical analysis 165 6.3 Results 165 6.3.1 Soil water 165 6.3.2 Daily water use pattern over progressive water stress .169 6.3.3 Stomatal conductance (gs), photosynthetic rate (A) and instantaneous water use efficiency (WUEi) .173 6.3.4 6.4 Discussion 182 CHAPTER 7.1 Chlorophyll fluorescence 179 Conclusions and recommendations 190 Outcomes 190 7.1.1 Drought effects on yield and development 190 7.1.2 Morphological traits related to drought resistance 191 7.1.3 Water use and water use efficiency 192 7.1.4 Morpho-physiological changes 192 vii 7.1.5 Leaf physiology 193 7.1.6 Use of morpho-physiological traits characterising high WUEstem and drought resistance 195 7.2 Recommendations 195 7.3 Further work .197 REFERENCES 199 APPENDICES I viii LIST OF TABLES Table 2-1 List of 50 varieties used in the Silsoe trials The commercial names of the varieties, the parents’ pedigrees, regions of origin are indicated when possible * indicates pure species Bedfordshire; 2002-2003 .17 Table 2-2 Codes and number of cuttings replaced on 18/06/02 in the field trial; Silsoe, Bedfordshire; 2002 21 Table 2-3 Codes and number of cuttings replaced at harvest in January 2003 in the field trial; Silsoe, Bedfordshire 22 Table 2-4 Number of cuttings missing at harvest in the field trial; Silsoe, Bedfordshire; 2003 .30 Table 2-5 Analysis of variance of the stem biomass production of two populations of 39 hybrids and 10 pure species, grown in the field trial; Silsoe, Bedfordshire; 2002 The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 31 Table 2-6 Means and standard errors of the means (sem), of two growth variables at harvest (biomass and maximum stem length) for a population of 49 varieties of Salix (39 hybrids 10 pure species) grown in field in 2002 and 2003 and pot trial in 2002; Silsoe, Bedfordshire .31 Table 2-7 Factorial analysis of variance of stem biomass at harvest for a population of 39 hybrids of Salix grown in field trial in 2002 and in 2003 after coppicing; Silsoe, Bedfordshire The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 32 Table 2-8 Analysis of variance of the stem biomass production of 39 hybrids, grown in the field trial in 2002; Silsoe, Bedfordshire The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 33 Table 2-9 Factorial analysis of variance of 39 hybrids grown in two sites in 2002 (irrigated field and the water stressed pot trial) Silsoe, Bedfordshire The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 .34 ix Table 2-10 Key periods (identified from Figure 2-7), where a general change in behaviour was observed in the pot trial in comparison to the field trial for most 39 hybrids; Silsoe, Bedfordshire; 2002 43 Table 2-11 Means, standard errors of the mean (sem) and Analysis of variance of stem elongation rates (mm d-1) recorded during seven periods for a population of 39 hybrids of Salix grown in the field (n=117) and pot (n= 156) trial; Silsoe, Bedfordshire; 2002 The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 43 Table 2-12 Factorial analyses of variance of the stem elongation rates during seven periods for a total population of 39 willow hybrids grown in field and pot trial; Silsoe, Bedfordshire; 2002 The levels of significance are represented as: ns: non significant; *: significant at p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 44 Table 3-1 After storage indices, to 5, of three morphological traits used to assess the morpho-physiological state of willow cuttings after eight weeks of storage at +4 °C; Silsoe, Bedfordshire; 2002 .58 Table 3-2 Willow SRC emergence growth index to 10, used on willow SRC to weeks after planting; Silsoe, Bedfordshire; 2002 59 Table 3-3 Hair density index of willow leaves and stem apex; Silsoe, Bedfordshire; 2002 61 Table 3-4 Branch type index of willow SRC; Silsoe, Bedfordshire; 2002 68 Table 3-5 Branch positions index of willow SRC Silsoe, Bedfordshire; 2002 68 Table 3-6 Mean, standard error of the mean (sem), analysis of variance (ANOVA), 95% confidence interval (CI) and skewness of seven variables measured on the early stages of development of cuttings of 50 willow varieties; Silsoe, Bedfordshire; 2002 The significance of ANOVA is represented as na: not applicable; ns non significant; * significant at p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n=150 in the field, n=200 in the pots 71 Table 3-7 Mean, standard error of the mean (sem), analysis of variance (ANOVA), 95% confidence interval (CI) and skewness of leaf population ratio on stem (Rleaf) on four dates measured on the leaves of 50 varieties of willows Field trial; Silsoe, Bedfordshire; 2002 The significance of ANOVA is represented as * significant at p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 n=150 72 x Table 3-8 Mean, standard error of the mean (sem), analysis of variance (ANOVA), 95% confidence interval (CI) and skewness of eight variables measured on the leaves of 50 varieties of willows Field trial; Silsoe, Bedfordshire; 2002-2003 The significance of ANOVA is represented as na: not applicable; ns non significant; * significant at p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 74 Table 3-9 Mean, standard error of the mean (sem), analysis of variance (ANOVA), 95% confidence interval (CI) and skewness of five variables measured from the willow of 50 varieties of willows Field trial; Silsoe, Bedfordshire; 2002 The significance of ANOVA is represented as na: not applicable; ns non significant; * significant at p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 76 Table 3-10 Significance and order of morpho-physiological traits most correlated with stem biomass production in the field in 2002 Data collected on 39 Salix hybrids grown in the field and the pot trials at Silsoe, Bedfordshire; 2002-200377 Table 3-11 Significance and order of morpho-physiological traits the most correlated with relative stem biomass production Data collected from 39 Salix hybrids grown in the field and in the pot trials at Silsoe, Bedfordshire; 2002-2003 78 Table 3-12 Three K-means clustering analysis results, cluster members, mean stem biomass (kg plant -1) and 95% confidence interval (CI) The variables used were extracted earlier in a set of Kendal tau analyses on ranks of the correlation of the variables with the stem biomass production Silsoe trials, Bedfordshire; 2002 Tora (50), Ashton Stott (49), Resolution (36), Endurance (37) and LA980289 (31) are highlighted as indicator hybrids The letters represent Fisher least significant differences (LSD) post hoc grouping p ≤ 0.05 80 Table 3-13 Three K-means clustering analysis results, cluster members, mean relative stem biomass production (%) and 95% confidence interval (CI) The variables used were extracted earlier in a set of Kendal tau analyses on ranks carried on the correlation of the variables with the relative stem biomass production; field trial; Silsoe, Bedfordshire; 2002 Tora (50), Ashton Stott (49), Resolution (36), Endurance (37) and LA980289 (31) are highlighted as indicator hybrids The letters represent Fisher least significant differences (LSD) post hoc grouping P ≤ 0.05 82 ...ii ABSTRACT This thesis reports on an investigation of drought resistance of willow SRC genotypes Experiments were... 1.2 Plant breeding 1.3 Silsoe project 10 1.4 Objectives 13 1.5 Thesis structure 14 CHAPTER The effects of water stress on the growth and biomass production... 5.4 Discussion 141 5.5 Conclusions 146 CHAPTER Transpiration and photosynthesis of five Salix hybrids .148 6.1 Introduction 148 6.2 Material and methods