EXPERIMENTAL STUDIES IN THE GENUS Salicornia L. (CHENOPODIACEAE) A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Science in the University of London and for the Diploma of Imperial College by SABAH MANAF NASSERULLA M.S.c. University of London December, 1979 Imperial College of Science and Technology Department of Botany and Plant Technology Field Station, Silwood Park Ascot, Berkshire. 2. ABSTRACT The main study has involved experimental work on phenotypic variation in the annual species of Salicornia, employing a sand culture with subirrigation technique. Biometrical studies of the progeny grown in culture showed - that descendents of different parent plants remained significantly distinct, while anther length, pollen grain diameter and certain mor- phological characters (associated with the degree of compression of the fertile segments) showed a correlation with chromosome number (diploid v. tetraploid). Chromatography of the phenolic glycosides in plants 'of known chromosome number, grown in culture, showed two groups when subjected to Principal Components Analysis with one being diploid (the S. europaea group), the other tetraploid (the S. procurnbens group). The only separate recognisable chromatographically, S. pusilla, fell as might be expected in the diploid group. Analysis of field populations showed a good separation into upper and lower marsh groups, presumably because of the preference of diploids for the former level and tetra- ploids for the latter. Cultivation at different nitrogen levels produced interesting changes in the balance between dominance by the apical characters (low nitrogen levels) and by the lateral branches (higher nitrogen levels).. Low nitrogen levels also favoured increase in seed size and caused an advancement of flowering time. Variation in phosphorus levels produced much less marked effects, but elevated sodium chloride 3. levels induced stunting of growth. Doubling the daylight ambient light intensity caused a . significant increase in branch numbers per plant. Analysis of variance also revealed that samples of differ- ing parentage had differing responses to variation in nutrient levels. In addition, attention is drawn to certain characters (such as fertile segment number and angle of branching) which are little or not at all affected by the nutrient treatments. Analysis of soils from sites of Salicornia populations showed gradients with maximum nitrogen and sodium chloride levels in the upper marsh, and also showed the nitrogen level to be low overall in comparison with normal agricultural soils. Taken in conjunction with the culture experiments, these results indicate that Salicornia has adapted to low nitrogen levels, but not to low phosphorus (pre- sumably because phosphorus is adequate for the plant need). 4. CONTENTS PAGE TITLE PAGE • .. 1 ABSTRACT 2 CONTENTS LIST OF FIGURES 8 LIST OF TABLES ... 13 LIST OF PHOTOGRAPHS 19 LIST OF APENDIX TABLES 21 CHAPTER I INTRODUCTION ... 26 1.1 General ... 26 1.1.1. The morphological characters ... 26 1.1.2. The life history ..: 29 1.1.3. Pollination 32 1.1.4. Geographical distribution of Salicornia.. 35 1.1.5. Economic uses of Salicornia spp. 35 1.2 The species problem in Salicornia 36 1.3 Taxonomic treatment of Salicornia in North—West Europe 45 CHAPTER II CULTURING OF SALICORNIA 50 2.1 Choice of the culture technique . 51 2.2 Description of the apparatus 000 51 2.3 Operation of the apparatus 58 2.4 Selection of the culture solution .. • 60 2.5 Source and treatment of the sand before use 63 2.6 Cleaning of the glassware and the culture equipments used in the growth experiments. ... 64 CHAPTER III MORPHOLOGICAL BEHAVIOUR OF DIFFERENT TYPES OF SALICORNIA UNDER UNIFORM GROWTH CONDITIONS... 66 3.1 Experimental procedures 66 3.1.1. Plant collection 66 3.1.2. The growth conditions 69 5. CONTENTS (Contined) PAGE 3.1.3. Cytological technique ... 70 3.1.3.1. Seed germination ... 70 3.1.3.2. Staining technique and the 72 preparation of permanent slides: 3.2 General observation on Salicornia in culture ... 73 3.3 The statistical analysis of the morphological. characters 78 3.3.1. Vegetative morphological characters 78 3.3.2. Microscopical characters ...- 94 3.3.3. Fertile segment ratios 96 3.4 General conclusion ••• 104 CHAPTER IV CHROMATOGRAPHIC STUDY OF THE PHENOLICS OF SALICORNIA SPP. ... 106 4.1 Plant source ... 106 4.2 Preparation of the plants for analysis ... 107 4.3 Experimental procedures ... 107 4.3.1. One-dimensional chromatographic techniques. ... 110 4.3.2. Two-dimensional chromatographic techniques. 4.3.2.1. Chromatography of the phenolic glycosides 4.3.2.2. Chromatography of the simple phenols and phenolic acids 4.4 Results 4.4.1. One-dimensional chromatography of the direct acid hydrolysates 4.4.2. Two-dimensional' chromatography for the phenolic glycosides 118 4.4.3. Phenolic acids and simple phenols in selected individuals of Salicornia ... 127 4.5 The effect of nitrogen deficiency and phosphorus deficiency on the chromatographic pattern of the phenolic glycosides..,. ..• 131 4.6 Chromosome number and the chromatographic pattern of the phenolic glycosides 135 4.7 Taxonomic considerations of the chromatographic data 138 6. CONTENTS (Continued) PAGE CHAPTER V PHENOTYPIC VARIABILITY IN SALICORNIA SPP. 145 5.1 Scope and purpose of the experiments 146 5.2 The effect of nitrogen deficiency and phosphorus deficiency on the morphology of Salicornia ... 146 5.2.1. Experimental procedures • .. 147 5.3 The effect of increased light intensity on the morphology of SaZicornia 151 5.3.1. Experimental procedures 151 5.4 Results and discussion 153 CHAPTER VI MORPHOLOGICAL BEHAVIOUR OF SALICORNIA SPP. IN RELATION TO DIFFERENT LEVELS OF NITROGEN, PHOSPHORUS AND SODIUM CHLORIDE ... 167. 6.1 Material for the experiment ... 168 6.2 The effect of different levels of nitrogen and phosphorus on the morphology of SaZicornia.. 168 6.2.1. Experimental procedures ... 168 6.2.2. Results ... 172 6.2.2.1. The effect of nitrogen treat- ments on the morphology 173 6.2.2.2. The effect of phosphorus treat- ments on the morphology 190 6.2.3. Discussion 203 6.3 The effect of different levels of sodium chloride on the morphology of Salicornia 217 6.3.1. Experimental procedures 218 6.3.2. Results • .. 220 6.3.3. Discussion 235 6.4 Development of betacyanin in SaZicornia 244 6.5 Assessment of value of certain taxonomic 249 characters 6.5.1 Results 250 6.5.1.1 Vegetative morphological characters 251 6.5.1.2. External measurements of the fertile segments 254 7. CONTENTS (Continued) PAGE 6.5.1.3. Fertile segment indices ... 257 6.5.2. General conclusions ... 261 6.6 Taxametric studies ... 262 CHAPTER VII FIELD STUIES ... 289 7.1 Soil analysis of Salicornia populations ... 289 7.1.1. Collection of the soil samples ... 289 7.1.2. The method adopted for the soil analysis ... 291 7.1.3 Results ... 294 7.1.4 Discussion 294 7.2 Comparison of the results of the soil analyses with the growth behaviour of Salicornia in the greenhouse and in the field ... 300 7.3 Chromatographic analysis of natural populations of Salicornia ... 307 7.3.1. Collection of the plant material ... 307 7.3.2. Experimental procedures ... 307 7.3.3. Results ... 308 7.3.4. Discussion ... 313 SUMMARY 321 ACKNOWLEDGEMENTS 329 REFERENCES 330 APPENDIX TABLES 344 8. LIST OF FIGURES (Numbered consecutively within each Chapter) PAGE 1.1 General morphology of annual Salicornia. ... 27 2.1 General sketch of the automatic subirrigation system which had been used for culturing Salicornia plants. ••• . 53 3.1 Shows the level of significance for the vegetative characters measured, between the cultured SaZicornia groups. ... 80 3.2- Comparison the mean values of shoot length, total number of 3.12 primary branches from the main axis,total. number of sterile segments in the main axis, terminal spike length, number of fertile segments in the terminal spike, length of the upper- most branch from the main axis, length of the lowermost branch from the main axis, length of sterile segment in the lower part of the plant, length of the sterile segment below the. terminal spike, angle of the uppermost branch with the main axis and angle of the lowermost branch with the main axis respectively for the 25 cultured types of Salicornia of known chromosome number (2X = diploid, 4X = tetraploid), with + 2 standard deviation as an estimate of the range. ... 85 3.13 Stomatal guard cell length, pollen grain dimension and anther length means and standard error of the mean of 25 cultured types of Salicornia. ... 95 3.14 Fertile segment components which are measured in the present investigation. ... 98 and for 3.15 Mean values of the fertile segment ratios; B, B F the 25 cultured types of Salicornia. ... 99 E 3.16 Mean values of the fertile segment ratios; D,and D for the 25 cultured types of Salicornia. 100 9. LIST OF FIGURES (Continued) PAGE , 3.17 Mean values of the fertile segment ratios. C+D A+B , and 2B 2C EEF for the 25 cultured _ types of SaZicornia. ... 101. D-F 3.18 Mean values of the fertile segment ratios; and E+(D-F) E-F -7E4(15-:-.-,- for the 25 cultured types of Salicornia. 102 3.19 Scatter diagrams of pollen grain size, fertile segment ratios T3- nd —C+D2B with the anther length for the 25 cultured types of Salicornia (A= diploid, , = tetraploid). ,103 4.1 Patterns of one-dimensional chromatograms from different types of Salicornia in Forstal and in BAW (Butanol-acetic acid-water). ... 114 4.2 Patterns of one-dimensional chromatograms from different types of SaZicornia in PhoH (water-saturated phenol) and in water. ... 115 4.3 Master chromatogram of the phenolic glycosides spots detected from the 29 types of Salicornia.