iLl^t i. M. BU ICtbrarg Nortl) (Earoltna S^UU Imnerattg QK711 D2? ^-- THIS BOOK IS DUE ON THE DATE INDICATED BELOW AND IS SUB- JECT TO AN OVERDUE FINE AS POSTED AT THE CIRCULATION DESK. JAN -4 1984 Camfariiige Natural ^nenre iMamiate Biological Series. General Editor: — Arthur E. Shiplev, M.A. FELLOW AND TUTOR OF CHRIST's COLLEGE, CAMBRIDGE. »■ C. COIIEGE OTA.&Ui PRACTICAL PHYSIOLOGY OF PLANTS. aonton: C. J. CLAY and SONS, CAMBRIDGE UNIVERSITY PRESS WAREHOUSE, AVE MARIA LANE, AND H. K. LEWIS, 136, GOWEK STREET, W.C. (Slassoto: 263, ARGYLE STREET. ILcipjig: F. A. BROCKHAUS. i^cia lorfe: MACMILLAN AND CO. JSombag: GEORGE BELL AND SONS. PRACTICAL PHYSIOLOGY OF PLANTS BY FRANCIS DARWIN, M.A., F.R.S., FELLOW OF Christ's college, Cambridge, AND READEB IN BOTANY IN THE UNIVERSITY, AND THE LATE E. HAMILTON ACTON, M.A., fellow and lectubeb of ST John's college, Cambridge. WITH ILLUSTRATIONS. SECOND EDITION. CAMBRIDGE : AT THE UNIVERSITY PRESS. 1895 [All lUghts reserved.] First Edition Oct. 1894 Second Edition Oct. 1895 CAMBRIDGE : PRINTED BY J. AND C. F. CLAY, AT THE UNIVERSITY PRESS. PREFACE. Edward Hamilton Acton died in the early part of the present year. This is not the place to speak of what we, his friends, have lost by his death, nor of his promising career as a man of science. All that I can do here is to explain in what way I have dealt with that part of the book for which he was especially responsible. Part II, on the chemistry of metabolism, was entirely written by Mr Acton, and is here reprinted practically as he left it. The only changes are two or three corrections found in his interleaved copy of the book, and a few verbal and typographical alterations introduced for the sake of uniformity. The book (as explained in the preface to the 1st Edition) originated in the following way. In 1883 I began a course of instruction in the physiology of plants, of which the chief feature was the demonstration of experiments in the lecture-room. Some my 17140 VI PREFACE. years later a different arrangement was made ; the students were required to perform the experiments for themselves, and at the same time laboratory work in the chemistry of metabolism was organised by Mr Acton. To enable the students to carry out their work, written instructions were needed, and the present book is the result of an extension and elaboration of what we prepared for our classes. The book makes no pretence to completeness, it contains merely such a selection of experimental and analytical work as seems suitable for botanical students. Part I, which deals with general physiology, is necessarily of a somewhat more elementary character than Part II, which treats a particular department of physiology in a more special manner, and presupposes a greater amount of knowledge on the part of the student. A few experiments which experience has shown to be unsuitable have been omitted in the present edition. The chief additions are: — Exps. 5 and 52 (Timiriazeff's eudiometer), Exp. 83 (the importance of stomata in gaseous interchange), Exps. 118 a, 118 b, 118 c (Stahl's cobalt method), Exp. 205 A (Pfeffer and Czapek's method of localising geotropic irritability in roots), Exp. 249 A (chemotaxis in Bacteria), Exps. 249 B and c (chemotaxis in pollen-tubes). PREFACE. vil The references to the literature of Part I have been increased in number, and they now give a fuller, though still but a rough guide to the published authorities. The references to Sachs' books appear to have been a source of difficulty to some of our readers. I therefore give the full titles of those to which reference is made. Physiologie Vegetate, recherches su7^ les conditions d'existence des plantes, et sur le jeu de leurs organes. Traduit de I'Allemand avec I'autorisation de I'auteur, par Marc Micheli. Paris, V. Masson et Fils, 1868. [This is the translation of Sachs' Handhuch der Eooperimental- Physiologie der Pflanzen, being volume iv of Hofmeister's Handhuch der Physiologischen Botanik, Leipzig, 1865. Arbeiten des hotanischen Instituts in Wurzbiirg, heraus- gegeben von Prof Dr Julius Sachs. Leipzig, Engelmann. Band i. 1874, Band ii. 1882, Band ill. 1888. Text-hook of Botany, moiyhological and physiological, by Julius Sachs, Professor of Botany in the University of Wlirzburg. Edited with an Appendix by Sidney H. Vines, M.A., D.Sc, F.L.S., Fellow and Lecturer of Christ's College, Cambridge. Second Edition, Oxford, at the Clarendon Press, 1882. [This is a translation of Sachs' Lehrbuch der Botanik (Edit. 1874).] Vorlesungen uher Pflanzen-Physiologie von Julius Sachs. Leipzig, Englemann, 1882. [An English trans- D. A. b Vlll PREFACE. lation was published in 1887 by the Clarendon Press under the title Lectures on the Physiology of Plants.] Gesammelte Ahhandlungen ilber Pflanzen-Physiologie von Julius Sachs. Leipzig, Engelmann. Band I. 1892, Band ii. 1893. [This is referred to in the text as Sachs' Collected Papers^ I gladly take this opportunity of expressing my thanks to Mr F. F. Blackman, Demonstrator of Botany in the University, for much valuable help in the arrangement of the experiments in Part I. Also to the Cambridge Scientific Instrument Company for the use of the cliches for Figs. 25 and 26. FRANCIS DARWIN. Botanical Laboratory, Cambridge. September, 1895. CONTENTS, PART I. GENERAL PHYSIOLOGY. CHAPTER I. ON SOME OF THE CONDITIONS AFFECTING THE LIFE OF PLANTS. Section A. Respiration. 1. Eespiration ; production of COg by germinating seeds or buds. 2. Absorption of COo by potash. 3. Sachs' method. 4. Winkler-Hempel apparatus. 5. Timiriazeff's Eudiometer. 6, 7, 8. Intramolecular respiration. 9, 10. Kise of temperature during respiration. 11. Succulents . pp. 1 — 13. Section B. The effect of various temperatures: of certain poisons : and of electrical shock. 12. Injurious temperature demonstrated on Oxalis leaf. 13. Do., injected leaf. 14. Do., beetroot. 15. Do., dry and soaked seeds. 16. Circulation of pro- toplasm, Sachs' hot-box. 17. Velten's method. 18. Circulation of protoplasm, effect of CO.,. 19. Do., chloroform. 20. Oxalis leaf killed by chloroform. 21. Do. by phenol. 22. Do. by induced current. 23. Effect of induced current on circulating protoplasm pp. 14 — 20. b'2 CONTENTS. CHAPTER II. ASSIMILATION OF CARBON. Section A. rormation of starch. 24. Sachs' Iodine method. 25. Schimper's method. 26. Variegated leaves. 27. Disappear- ance of starch in darkness, 28. Effect of dull light. 29. Local effect. 30. Gardiner's experiment. 31. Kays of different refran- gibility. 32. Terrestrial leaves under water. 33. Stomata and gaseous exchange. 34. Excess of CO2. 35. Plants deprived of COo. 36. Gain in weight. 37. Translocation. 38. Assimilation of sugar. 39. Do., formaldehyde. 40. Leucoplasts pp. 21 — 34. Section B. Evolution of oxygen. 41. Bubbles of gas. 42. Light of varying intensity. 43. Dependence on presence of CO2. 44. Temperature and gas evolution. 45. Chloroform. 46. Co- loured lights. 47. Collection of gas evolved. 48. Engelmann's blood method. 49. Phosphorus method. 50. Gas analysis, Pfeffer's method. 51. Gas analysis, Winkler-Hempel apparatus. 52. Timiria- zeff's Eudiometer. 53. Engelmann's bacterial method. 54. Diffusion of gas through cuticle pp. 35 — 49. Section C. Reactions of chlorophyll and of some other pig- ments. 55. Separation by benzene, ether, olive oil. 56. Action of light. 57. Aeration and effect of light. 58. Action of acid. 59. Action of copper salts. 60. Stability of the copper compound. 61. Spectroscopic examination. 62. Anthocyan in Kicinus, &c. 63. Floridese. 64. Brown sea-weeds . . . pp. 50— 53. Section D. Production of chlorophyll, etiolation, sun- and shade-leaves. 65. Appearance of the green colour. 66. Etiolin and light. 67. Pinus. 68. Chlorophyll formation and tempera- ture. 69, 70. Do. and oxygen. 71. Do. and iron salts. 72. Form of etiolated plants. 73. Sun- and shade-leaves. pp. 54—57. CHAPTER III. FURTHER EXPERIMENTS ON NUTRITION. Section A. Water-culture. 74. Method. 75. Potassium salts necessary. 76. Phosphoric acid necessary. 77. Experi- ments with Lemna. 78. Calcium oxalate formation. 79. Nitrate reaction pp. 58 — 67. CONTENTS. XI Section B. Nutrition of Fungi and of Drosera. 80. Method. 81. Various cultures. 82. Puccinia. 83. Hanging-drop cultures. 84. Germination of spores. 85. Drosera, digestion of white of egg. 86. Drosera, benefit from feeding .... pp. 68 — 72. Section C. Functions of roots. 87. De Saussure's experiment. 88, 89, 89 A. Eoot pressure. 90. Moll's experiment. 91. Absorption by means of dead roots . pp. 73 — 78. CHAPTER IV. TRANSPIRATION. Section A. Absorption of water. 92. Potometer. 93. Kohl's method. 94. Effect of sunshine. 95. Effect of wind. 96. Effect of light. 97 — 100. Negative pressure. 101. Permeability of mem- branes. 102. Oozing of water from wood. 103. Permeability of splint-wood. 104. Recovery of flaccid shoot. 105. Emulsion ex- periment. 106, Injection with cocoa-butter. 107. Compression. 108. Incisions. 109. Cross-cuts. 110. Do,, course shown by eosin. 111. Air-pump and potometer. 112. Strasburger's air- pump experiment pp. 79 — 96. Section B. Loss of water. 113. Loss of weight during transpi- ration, 114. Transpiration compared with evaporation from water. 115. Loss compared with absorption. 116. Spring balance. pp. 97— lOl. Section C. Stomata, bloom, lenticels. 117. Stomatal trans- piration. 118, Stipa-hygrometer. 118a. Stahl's cobalt method. 118 b. Stomata in withered leaves, 118 c. Effect of salt on the stomata, 119, Stomata and intercellular spaces, 120, Leaf injected with water. 121. Frost effects, 122, Blocking of stomata with water. 123. Movements of stomata. 124. Do, with induced current. 125. Lenticels and intercellular spaces. 126. Bloom as affecting transpiration pp. 102 — 111. CHAPTER V. PHYSICAL AND MECHANICAL PROPERTIES. Section A, Imbibition, hygroscopic movements, polariscope, osmosis. 127, Laminaria, microscopic observation, 128. Lami- naria, increase not uniform in all directions. 129. Imbibition of seeds. XU CONTENTS. temperature effect. 130. Imbibition ; salt solution. 131. Stipa, action of. 132, 133. Stipa, temperature. 134. Stipa, salt solu- tion. 135. Stipa, mechanism of movement. 136. Nobbe's ex- periment. 137. Variability in the swelling of seeds. 138. Kise of temperature accompanying imbibition. 139. Work done during imbibition. 140. Polariscope. 141, Polariscope, observations on strained glass rods. 142. Traube's artificial cell. 143. Slowness of diffusion. 144. Kelation of membrane to diffusing fluid. 145. Absorp- tion of methylene blue by living cell . . . . pp. 112 — 124. Section B. Tiirgor. 146. Plasmolysis, microscopic observations. 147. Kecovery after plasmolysis. 148. Osmotic strength of cell-sap in terms of KNOg. 149. Isotonic coefficient. 150. Do., micro- scopic method. 151. Hydrostatic pressure in turgescent tissue. 152. Pfeffer's gypsum method pp. 125—132. Section C. Tensions of tissues. 153. Longitudinal tensions. 154. Extension of pith in water. 155. Changes in transverse di- mensions of pith. 156. Tangential dimension. 157. Shortening of roots. 158. Imperfect elasticity of tissues. 159. Cyclometer. 160. Hofmeister's experiment. 161. Loss of rigidity. 162. In- crease in length. 163. Splitting turgescent tissues. 164. Split- ting a root. 165. Splitting a pulvinus . . . pp.133 — 141. CHAPTER VI. GROWTH. Section A. Experiments witliout special apparatus. 166. Method. 167. Free oxygen necessary. 168. Respiration necessary, 169. Effect of salt solution. 170. Growth at various temperatures. pp. 142—145. Section B. Distribution of growth. 171. Distribution in roots. 172. In air-roots. 173. In stems. 174. Grand period, time observation. 175. Growth and plasmolytic shrinking pp. 146 — 148. Section C. Auxanometers. 176. Methods. 177. Descent of the weight measured on a scale. 178. Micrometer screw. 179. Arc-indicator. 180. Microscope. 181. Self-recording aux- anometer. 182. Do., simple form. 183, 184. Growth and tem- perature, microscopic method. 185. Growth and respiration, micro- CONTENTS. XIU scopic method. 186. Growth and temperature, auxanometer. 187. Growth and light, auxanometer. 188. Growth and Hght, Phycomyces. 189. Growth and light, Sinapis. 190. Periodicity, auxanometer ........ pp. 149 — 162. CHAPTER VII. CURVATURES. Section A. Geotropism. 191. Eegion of growth and region of curvature, roots. 192. Do., stems. 193. Subsequent changes in curvature. 194. Grass-haulms. 195. Noll's experiment, grass- haulms. 196, 197. Geotropism and respiration. 198. Johnson's experiment. 199. Pinot's experiment. 200. Knight's experiment. 201. Sudden curvature. 202, 203. After effect . pp. 163—172. Section B. Curvatures due to injury &c. 204. Decapitated roots. 205. Decapitation prevents perception of stimulus. 205 a. Do., Pfeffer's experiment. 206. Recovery after decapitation. 207. Cur- vature due to injury. 208. Ciesielski's experiment. 209. Drooping of leaves in frost pp.173 — 179. Section C, Heliotropism. 210. Positive heliotropism. 211. After effect. 212. Light of high refrangibility most effective. 213. Nega- tive heliotropism. 214. Struggle between the effects of light and gravitation. 215. Transmitted stimulus . . pp. ISO — 183. Section D. Diaheliotropism, diageotropism &c. 216. Diahelo- tropism. 217. The movements due to specific sensitiveness ; klinostat. 217 A. Exclusion of helio- and geotropism. 218. Rectipetality. 219. Theory of klinostat, grass-haulms. 220. Do., Cucurbita. 221. Diageotropism, roots. 222. Growth of secondary roots in Hght. 223. Diageotropism, Narcissus. 224. Horizontal branches. 225. Tor- sion of internodes. 226. Buds of the yew. 227. Epinasty. 228. Epinasty and geotropism. 229. Nutation of epicotyls pp. 184—199. CHAPTER VIII. FURTHER EXPERIMENTS ON MOVEMENT. Section A. Stimulus of contact, chemical agency, moisture, changes in illumination and temperatvire. 230. Tendrils, sensi- XIV CONTENTS. tive to contact. 231. Tendrils, Pfeffer's contact experiment. 232. Mi- mosa, movements produced by stimulation, 233. Mimosa, temperature. 234. Mimosa, darkness. 235. Mimosa, continued stimulation. 236. Oxalis acetosella, sensitiveness. 237. Oxalis, Briicke's experi- ment. 238. Drosera, stimulated by meat. 239. Do., by inorganic matter. 240. Drosera stimulated by dilute solutions. 241. Drosera, inflection indirectly caused. 242. Berberis, irritable stamens. 243. Berberis, effect of chloroform. 244. Stigma of Mimulus. 245. Centaurea, irritable stamens. 246. Phycomyces, curvature towards iron. 247. Hydrotropism. 248. Movement of chloroplasts. 249. Chemotaxis, antherozoids. 249 a. Chemotaxis: bacteria. 249 b, c. Do., pollen tubes. 250. Opening and closing of tulip, temperature. 251. Tulip, sensitive to small change of temperature. 252. Crocus, mechanism of movement. 253. Light and darkness, daisy. 254. Light and darkness, Trifolium. 255. Nyctitropic movements, Trifolium. 256. Do., Mimosa, self-recorded. 257. Para- heliotropism, Averrhoa pp. 200 — 226. Section B. Autonomous movements. Periodicity. 258. Cir- cumnutation. 259. Do., twining plants. 260. Autonomous movements, Trifolium. 261. Do., Averrhoa. 262. Do., Desmo- dium. 263. Periodicity, light and darkness, daisy. 264. Perio- dicity, temperature, daisy. 265. Contrast, daisy pp. 227 — 234. PART II. CHEMISTRY OF METABOLISM. CHAPTER IX. INTRODUCTION. SOLVENTS. METHODS OF EXTRACTION. GENERAL NOTES ON APPARATUS AND MANIPULATION. Introductory. Preparation of material to be examined. Preparation of extracts : non-nitrogenous plastic substances. Preparation of ex- tracts: nitrogenous plastic substances. Filtration. Evaporation of solutions. Changes occurring in solutions on keeping pp. 237 — 248. CONTENTS. XV CHAPTER X. PROTEIDS. AMIDES. AMMONIA. NITRATES, &C. Literature. Practical classification of nitrogenous plastic sub- stances. Qualitative examination for proteids insoluble in water, soluble in dilute alkali — for proteids soluble in water — for peptones and albu- moses — for amides — for ammonia, nitrates, nitrites. Estimation of proteids — of peptones and albumoses. Estimation of amides. Estima- tion of ammonia, nitrates and nitrites. Experiments on nitrogenous metabolism. Qualitative examination of Onobrychis sativa for proteids &G. Comparison of amounts of proteids, peptones, amides in seeds of Onobrychis and in shoots of the same grown under various conditions. Comparison of amounts of ammonia, nitrates, nitrites in shoots of Onobrychis from plants variously treated . . pp. 249 — 260. CHAPTER XI. OILS AND FATS. GLYCERINE. Literature. Extraction of oils and fats. Qualitative examination of benzene extract. Reactions of glycerin. Quantitative examination. Determination of total oils and fats — of free fatty acids — of glycerin. Experiments. Determination of oils and fats in seeds of Lepidium — in seedlings of Lepidium, young and old . . . pp. 261 — 265. CHAPTER XII. TANNINS AND GLUCOSIDES. Literature, Extraction of tannins and glucosides. Many different bodies included under heading tannin and glucoside. Qualitative tests for tannins. Qualitative tests for phloroglucin. Removal of tannins before examining for sugars. Determination of whether a tannin is a glucoside or not, Glucosides. Identification of salicin. Examination for certain sugars. Experiments. Testing extract of willow-bark for tannin, salicin, sugars. Estimation of certain sugars in young and old fruits of Musa sapientum pp. 266 — 275. XVI CONTENTS. CHAPTER XIII. DEXTRINS AND SUGARS, GLUCOSES, CANE-SUGAR, MALTOSE, &C. Literature. Soluble carbohydrates. Qualitative test for fermentable sugars. Estimation of fermentable sugars. Kemoval of dextrins. Tests for glucoses, cane-sugar, maltose, mannite, pentoses. Estimation of glucoses, cane-sugar, maltose. Calculation of results. Experiments on sugars. Testing leaves of Tropaeolum majus for various sugars. Estima- tion of fermentable sugars in leaves and roots of Beta vulgaris. Estima- tion of various sugars in leaves of Beta vulgaris under different conditions. pp. 276 — 289. CHAPTER XIV. STARCH. CELLULOSE. Literature. Estimation of starch and cellulose. Experiments on starch. Estimation of starch in the potato by different processes — in leaves of Acer pseudo-platanus under different conditions. In grains of wheat before and after germination . . . pp. 290 — 294. CHAPTER XV. ORGANIC ACIDS AND SALTS. Literature of organic acids. Qualitative examination for organic acids. Determination of 'acidity' of extracts. Literature of inorganic salts. Preparation of ash of tissues. The constituents of the ash. Estimation of chlorine — phosphoric acid — alkalies — in ash. Estimation of calcium oxalate in tissues. Experiments on organic acids. Compari- son of acidity of juice from old and young rhubarb petioles — of acidity and amounts of sugars in juice from ripe and unripe apples. Experi- ments on inorganic salts. Weights of ash from normal and etiolated leaves. Estimation of phosphoric acid and alkalies in leaves and grains of barley — of calcium oxalate in young and old leaves of Sempervivum tectorum pp. 295— 302. CONTENTS. XVU CHAPTER XVI. UNORGANISED FERMENTS. (ENZYMES.) Literature. Extraction of enzymes. Comparison of activity of extracts. Experiments on diastatic ferments. Preparation of solid diastase. Influence of filtration on diastatic power of extracts. Com- parison of diastatic power of malt and ungerminated barley — of leaves of Pisum sativum and Trifolium pratense. Experiments on invertase and glycase. Decomposition of a glucoside (salicin) by an enzyme (synaptase) from another plant .... pp. 303 — 311. CHAPTER XYII. GENERAL EXPERIMENTS. The increase in weight of growing Spirogyra. The influence of in- organic salts on the formation of starch. The changes in the reserve materials of an oily seed during germination under different conditions. pp. 312—313. APPENDIX I. Notes on the results likely to be obtained in experiments on metabo- lism pp. 314 — 322. APPENDIX II. List of reagents and material required for experiments on meta- bolism pp. 323 — 326. LIST OF ILLUSTRATIONS. FIG. PAGE 1 Apparatus for demonstrating respiration .... 2 2 Apparatus for estimating COo produced during respiration . 4 3, 4, 5 Winkler-Hempel apparatus for gas-analysis . . . 7, 8 6 Foil clamp for holding cover-slips together under water, for use with Velten's hot-stage 18 7 Apparatus for the preparation of water free from CO., hut not free from oxygen 28 8 Arrangement for the culture of plants in an atmosphere free from CO., 30 9 Apparatus for gas-analysis for use in experiments on assimi- lation 42 10 Timiriazeff's eudiometer 46 11, 12 Lemna cultivated in various nutrient solutions . . 64, 65 13 Apparatus for demonstrating root pressure .... 75 14 Another apparatus for the same purpose .... 77 15 The potometer, for estimating the absorption of water by a cut branch 80 16 A modification of Kohl's apparatus for the same purpose . 88 17 A method of preventing evaporation from the surface of a flower-pot 97 18 Apparatus for comparing loss by transpiration with the water absorbed . . • 99 19 A spring-balance for use in transpiration experiments . 101 20 A hygrometer made of the awn of Stipa for performing Garreau's experiment 103 21 Devaux's gelatine method of making an air-tight junction with the j)etiole of a leaf 108 LIST OF ILLUSTRATIONS. XIX FIG. PAGE 22 Apparatus for demonstrating the hygroscopic properties of the awn of Stipa 114 23 Diagram illustrating the use of turgescent tissue in De Vries' experiments on isotonic coefficients 126 24 Tracings from split portions of the hypocotyl of Ricinus used in experiments on isotonic coefficients .... 127 25 Pfeffer's gypsum method 132 26 Arrangement for demonstrating that a turgescent shoot loses rigidity when bent 139 27 Micrometer-screw used in growth experiments . . . 151 28 Recording auxanometer 154 29 Method of using the auxanometer-lever .... 158 30 Tracing illustrating the effect of an increase of temperature on growth 160 31 Hanging writer for recording geotropic or other movements on a revolving drum . . 172 32 Illustrating the curvature of a root which has recovered from the effects of decapitation 176 33 Curvature of roots produced by small pieces of card attached to the tips 177 34 Drooping of laurel leaves in a frost 179 35 A twig of Veronica salicifolia exposed to oblique illumination 184 36 The Minostat 186 37 Section showing part of the mechanism of the klinostat . 188 38 A seedling Cucurbita which has germinated on the klinostat ; showing the frill-like growth of the heel or peg . . 193 39 Diageotropism of the flower of Narcissus poeticus . . 195 40 Leaves of clover in the day and night position . . . 222 41 The sleep-movements of Mimosa recorded on a drum by means of a hanging writer 223 42 Paraheliotropic movement in Averrhoa bilimbi . . . 225 43 Diagram representing the circumnutation of a cabbage- seedling 228 44 Leaf of Desmodium gyrans 232 45 Apparatus for distillation under reduced pressure . . 246 ERRATA. Page 67, note 1,/or " Zimmerman" read " Zimmermann." „ 70, line 5 from foot, for " developement " read " development." „ 102, last line, /or "fig. 18" read "fig. 20". „ 135, note 1, for " 1889 " read " 1888." „ 136, line 2, for " rasor " read " razor." PAET I. GENERAL PHYSIOLOGY. CHAPTER I. ON SOME OF THE CONDITIONS AFFECTING THE LIFE OF PLANTS. Section A. Respiration. Section B. Temperature — Poisons — Electricity . Section A. Respiration. The presence of free oxygen is a necessary condition of the life of all the higher plants. This fact will be more conveniently demonstrated in the chapters on growth and growth-curvatures. The present section is intended as an introduction to the study of the facts without special reference to the importance of respiration. (1) Production of CO. 2. Take a stoppered jar of about 500 c.c. capacity, fill it to one-third of its height with (in spring) horse-chestnut buds or (in winter) with beans which have been soaked in water for 12 hours and have been afterwards placed in damp cocoa-fibre for 12 hours. Place the jar in a warm room, and after 12 — 24 hours cautiously open the jar and lower a lighted taper which will be extinguished as it enters the COo produced. D. A. 1 nOtEXn LIBRARY U. C. State College RESPIRATION. [CH. I (2) AbsorjJtion of COo hy Potash, Take a filtering flask of 400 or 500 c.c. capacity, having a lateral opening as shown in fig. 1, to which a Fig. 1. Exp, 2. glass tube, A (4 or 5 mm. bore), is attached by thick rubber tubing and wire ties. The end of A dips into the €H. l] RESPIRATION. 3 mercury' in the beaker Hg. The flask contains enough germinating barley to cover a piece of wet filter-paper at the bottom of the flask. Barley germinates well in winter : it should be soaked in water for 24 hours and kept in damp air for 24 hours before use. A test-tube T half full of strong KHO is introduced into the flask, which is then closed by a sound tightly fitting rubber cork. As the COo, produced by respiration, is absorbed by the KHO, the mercury in the beaker Hg is sucked up the tube A. In starting the experiment it is necessary to warm the air in the flask before the end of A is forced into the mercury, so that as the air cools again the mer- cury may be sucked a little way up the tube to a point which will then serve as zero for subsequent observations. The warming may be done by immersing the flask in water at 40° for a few minutes ; or it may be warmed by the hands. If the mercury fails to rise within 6 hours the reason is probably to be found in the cork fitting badly. For this reason it is perhaps advisable to run melted ivax-mixture'^ round the line of contact between the cork and glass. (3) Sachs' method^. Place 100 germinating peas in a jar, A, fig. 2, closed by an india-rubber cork pierced by two holes and fitted 1 Or the beaker Hg may be filled with water. - Wax-mixture consists of resin 15 parts, bees-wax 35 parts, vaseline 50 parts. The wax and the vaseline are melted together, the resin is powdered, gradually added and stirred, ^ Physiologie (French Translation), 1868, p. 295, fig. 35. Also Pfeffer's Physiologie, i. p. 349, fig. 38. 1—2 RESPIRATION. [CH. I with glass tubes. One tube is connected with an aspi- rator so that a current of air is drawn through the vessel and keeps up continuous normal respiration. The other tube serves to admit to the flask air free from CO2; for this purpose it is connected with a filtering bottle F containing a few sticks of KHO\ The air is admitted to F through a tube T filled with soda-lime. To make sure that no extraneous CO2 enters the flask, another washing bottle B containing baryta-water is fitted between F and the experimental flask, A. The drop-aspirator figured by Fig. 2. Exp. 3. Detmer ^ answers well ; it is made from a distillation tube and is attached to a tap through which a current of water 1 We now use a washing bottle containing KHO instead of the arrangement shown in the figure [1895]. - Praktikum, p. 179, fig. 76. CH. l] RESPIRATION. 5 in detached drops passes, and produces a correspondingly slow suction-current of air at the side tube (c in Detmer's figure). The aspirator should be hung about 70 cm. above the table so as to allow the use of an outflow tube of about 60 cm. in length, which insures sufficient suction. The tube which admits air into F should be fitted with a screw clamp so as to regulate the inflow. If the sink in the laboratory is inconveniently placed the air-suction may be carried to any part of the room by means of fine lead-tubing. Between the flask and the aspirator two washing bottles P, C, containing baryta-water are fitted in which the CO2 produced by respiration of the plants is caught as BaCOg. With 100 peas the amount of BaCOg may be estimated at intervals of 20 minutes or half-an-hour. The estimation is made by titration, for which see Sutton, Volumetric Analysis, 5th Ed. pp. 80 — 89. Rough quantitative determinations may be readily made as follows. Shake up about 21 grams crystallized Barium hydrate with 1 liter of distilled water and allow it to stand for 12 hrs. in a closed flask, or till dissolved. Filter it into a stoppered bottle. Before an experiment introduce, with a pipette, 50 c.c. of this solution into the bottle P, and a further quantity into G, At the end of the experiment, when a considerable quantity of Barium carbonate has been precipitated in P, draw off 20 c.c. of the solution ^ with a pipette and titrate it quickly against ^ The liquid need not be clear, as extremely dilute HCl has no action on barium carbonate. For an accurate method of estimating the COj of respiration by titration, see Blackman in the Phil. Trans. 1895, also a full account of his work in Science-Progress, 1895. 6 RESPIRATION. [CH. I standard decinormal hydrochloric acid, using phenol- phthalein (colourless with acids, pink with alkalis) as an indicator. Compare 20 c.c. of the original baryta solution with the same acid. The difference between the amounts of HCl required to neutralize the two samples gives a measure of the amount of baryta that has been removed from each 20 c.c. of the liquid by combining with carbon dioxide. The whole amount of CO2 produced in the experiment is of course 2^ times as great, and it may be readily estimated from the following data: 1 c.c. ^ HCl = 0-0022 gr. CO2 = 1'19 c.c. COo at 15° C. (4) Winkler- Hempel apparatus. By this apparatus the volume of CO2 given off by respiration in a kno\Mi time may be fairly well deter- mined. To get a good result it is well to use a considerable quantity of material, say 200 peas just beginning to germinate. They are placed in a conical flask of 400 c.c. or 500 c.c. capacity and closed by a good rubber cork. After 1 or 2 hours a sample of gas is drawn off for analysis. This may be managed by an arrangement similar to that shown in fig. 3, in which water flows from to i as the gas is withdrawn. The arrangement figured was meant for experiments on assimilation when several determina- tions of the CO2 in the jar J are made : in the present experiment the test-tube i is omitted and the water flows into the bottom of the flask. Thus only a single sample of gas can be analysed with accuracy since the water ;h. i] RESPIRATION. introduced into the flask absorbs the CO2 produced. A second sample of the gas may however be used if it is taken within a few minutes of the first. When the test- tube i is omitted the flask can be shaken before analysis so as to insure that there is no accumulation of CO2 at the bottom. The analysis is made in the following manner: — A strong KHO solution (1 in 2) is introduced into B (fig. 4) 1-..] o ^^^ until its level reaches A, and then by blowing down B the KHO is forced up the fine tube E and into a thick- walled india-rubber tube connected w^ith it. As soon as the solution appears at the open end of the tube, the clamp G is closed. The tubes G and F (fig. 5) of the measuring burette are then a little over half filled with 8 RESPIRATION. [CH. I distilled water, care being taken that no air bubbles remain in the connecting india-rubber tube. F is then raised till water flows out of H ; then the stop-cock L is closed and H is connected by tubing with the vessel J in fig. 3 containing the gas to be analysed. F, now nearly empty, is lowered and L opened, so that a sample of gas is drawn into the burette. L is closed and H disconnect- ed. The volume drawn in is then measured by means of U =C:^^---a Fig. 5. Exp. 4. CH. l] RESPIRATION. 9 the graduations on (r, after bringing the water in the two tubes to one level. To absorb the CO..,, H is connected with the india-rubber tubing C of the absorption pipette (fig. 4). F is raised, and L and the clamp C opened. The gas is thus forced over into D, where it is retained for a minute or so and gently shaken in contact with the KHO, the clamp C and stop-cock L being closed meanwhile. When absorption is believed to be complete the gas is sucked back into G (fig. 5) by lowering F, C and L being open. L is then closed and G and F brought to a level so that the diminished volume of gas can be again read off. The difference gives the amount of CO2 originally present. To make sure of complete absorption the gas may be again passed into D, shaken and returned, when it should show no further reduction in volume. When any potash is sucked back into G along with the gas the tubes must be carefully washed clean before being used for another sample of gas. (5) Timiriazeff's Eudiometer. The modification of TimiriazefF's eudiometer which we employ for the analysis of gas given off by assimilating water plants (see exp. 52, p. 45) may be conveniently employed for respiration experiments. Five or six germi- nating peas are placed in a test-tube (15 c.c. capacity) which is tightly closed by means of a rubber cork. It may be inverted in water or mercury, and the contained gas analysed after two hours. For this purpose it is un- corked while the mouth of the test-tube is under water and about 0'5 c.c. poured up into the funnel of the eudiometer. 10 RESPIRATION. [CH. I In one of our experiments we found the end of the inflorescence of Reseda luteola convenient material, yield- ing a good percentage of COo. (6) Intramolecular respiration. To demonstrate the fact, the following simple form of experiment may be tried. Soak 6 peas in water for 12 hours, when the seed- coats can easily be removed without injury to the embryo; the removal of the testa is necessary to avoid introducing air with the peas, the object of the experiment being to show that COo is produced in the absence of free oxygen. Fill a test-tube with mercury and invert it in a mercury trough, which should stand in a strong wooden tray. This precaution is advisable in all experiments involving the use of mercury, so that if any accident occurs the mercury may not escape and get into the cracks of the floor. Pass the peeled peas one at a time under the rim of the test-tube so that they float up into the mercury, and occupy the upper end of the test-tube. On the following day it will be found that the test-tube is half full of gas, and the peas are therefore clearly visible, instead of being partly hidden by mercury. A few drops of water are now passed in under the test- tube rim with a bent pipette, and a fragment of caustic potash added from below, in this way a strong solution of KHO is supplied, by which the COo is absorbed. (7) Another method. The Torricellian vacuum was used by Wortmann in CH. l] RESPIRATION. 11 his work on intramolecular respiration ^ A tube closed at one end, and of greater length than the height of the barometric column, is filled with, and inverted over mercury. Three or four peas are floated into the vacuum at the top of the tube. After 24 hours a depression of several cm. will be observed in the height of the column, which will, on the addition of KHO, rise to about its original level, allowance being made for any change in the barometer. (8) Pfeffers method To get accurate results another method must be followed ; the following is taken from Pfeffer's paper in his Tiibingen Untersuckungen, Vol. i. p. 637. The principle is that described in exp. 3 for estimating ordinary respiration, but instead of air, a current of hydrogen is drawn through the vessel in which the plants are contained. It is necessary to prevent the entrance of extraneous CO2 and to make sure that the hydrogen has no admixture of oxygen. (9) Rise of tempeixitin^e^. The following is Sachs' arrangement for showing the rise of temperature in germinating peas =^. We have found flowers, such as those of the dandelion (Taraxacum), 1 Sachs' Arbeiten, 11. p. 500. - The spadix of Arum is the classical material for demonstrating the heat produced by the respiration of plants. The rise of temperature only occurs during such a limited time that the experiment constantly fails and is not to be recommended for class-work. 3 Sachs' Text-book, Ed. n. p. 724. Fig. 472. 12 RESPIRATION. [CH. I treated in the same way to answer well. Gather a large handful of dandelion flowers \ cutting the stalks just below the head, place them in a large funnel supported in a beaker half filled with KHO. Hang a thermometer so that the bulb is covered by the flowers, and let the control thermometer be supported in a funnel containing coarse sawdust slightly moistened and loosely packed. This ar- rangement is meant to equalise the conditions of the two thermometers, and to prevent the thermometer among the flowers acting as a wet-bulb. We find, with the control thermometer hanging simply in the air, that the flowers keep about 2^ C. above the control temperature. As before, the whole must be covered with a bell-jar. Sachs uses a tubulated bell of which the opening is plugged with cotton-wool -. (10) Oxygen necessary. Several of the earlier observers have shown that when the air is replaced by indifferent gas the tempera- ture falls. Pfeffer^ recommends that the germinating seeds or other material should be placed in a glass balloon having three apertures — one of which serves for a thermo- meter. When the temperature of the respiring material ^ Or in winter of young flowers and buds of a small-flowered Chrysan- themum. - To demonstrate the heat of respiration, Professor Errera of Brussels (as he is good enough to inform us) uses a Leslie's Differential Thermo- meter. One of the air-bulbs is plunged in a funnel filled with germinating barley and covered loosely with damp paper. The other bulb is in a similar funnel containing killed barley. •^ See Pfeffer, Physiologic, ii. p. 403. Fig. 40. CH. l] SUCCULENTS. 13 has been proved to be steadily above that of the surround- ing air, the atmosphere in the balloon is replaced by hydrogen, for which purpose the two lateral apertures will serve. The readings of the two thermometers should now become practically equal, and according to Pfeffer it is possible to re-establish the difference by readmitting air. The experiment is a difficult one and should only be attempted by a student of some experience. (11) Succulents \ In certain succulents an increase of the acidity of the cell sap is accompanied by a fixation of oxygen. A leaf of Rochea falcata is taken from the plant at the close of a hot summer-day, cut into pieces and introduced into a graduated gas- tube of simple test-tube form : it may be kept in place by a plug of glass-wool. It is not necessary to stand the tube in mercury, water will serve quite well. We have also employed an arrangement like that given in fig. 1, the KHO beiog omitted and water replacing the mercury in the beaker Hg. The apparatus is kept in the dark until the following morning, when a considerable rise in the water column is visible. As a control a similar graduated tube is fitted up with non- succulents, such as pieces of young sunflower ^. 1 See De Saussure, Recherches Chimiques, (An. xii = 1804), p. 65; also Detmer, Praktikum, p. 224, whose arrangement of the experiment we have adopted. - To complete the experiment, the relative acidity of the Rochea in the evening and next morning should be compared. See Part ii. 14 INJURIOUS TEMPERATURES. [CH. I Section B. The effect of various temperatures: of certain poisons : and of electrical shock. (12) Temperature ^ To get a rough idea of the upper limit of temperature which ordinary plants can endure, it is well to make a few simple experiments with plants in which the moment of death is marked by some obvious change, e.g. in colour. Oxalis acetosella is useful for this purpose, because death is indicated by a dingy yellow colour due to the action of the acid cell sap on the chlorophyll. Fill a beaker with water at 25^ C, and suspend in it a thermometer, to the bulb of which a leaf of Oxalis is attached. Heat the water by means of a gas flame, and note the temperature at which the leaf loses its fresh green tint. The colour begins to change at about 52° C. (13) Temperature. If the Oxalis leaf is previously injected wdth water under the air-j)ump, it changes colour at a temperature several degrees lower than in exp. 12. This is a simple way of demonstrating the fact given by Sachs (Physiologie, p. 71) that plants in air endure a temperature which they cannot bear in water. The cells of the injected Oxalis leaf acquire the tem- perature of the water more quickly than those of the uniujected leaf, and this is probably the explanation of the difference. ^ See Chapter iii., on the general conditions of plant life, in Sachs' Text-Book of Botany, Edition ii., also his Physiologie (French Trans- lation), p. 56. CH. l] INJURIOUS TEMPERATURES. 15 (14) Temperature. When a turgid cell is killed, the cell sap escapes through the dead protoplasmic wall, and if the cell sap is coloured, the escape will be a marked occurrence. The Beet-root {Beta) may be used in this way as a rough indicator of the temperature at which the protoplasm is killed. Cut a slice of beet-root, 3 or 4 mm. in thickness, w^ash it to free it from any cell sap adhering to the cut surfaces, and suspend it with a thermometer in a beaker of water at about 25° C, which is to be heated as in experiment 12, but the temperature should be allow^ed to rise very slowly. A temperature of 55^ or even 57° will be required. A similar experiment may be more accuratel}^ made under the microscope, using one of the methods described below, by which a microscopic object can be subjected to a given temperature. (15) Dry and soaked seeds'^. The effect of a high temperature depends, among other things, on the condition of the subject of the experiment. Thus, dry seeds can endure a temperature which is fatal to seeds which have been soaked. Take 20 peas, half of which {a) are to be left in water for 12 hours, or until they are thoroughly soaked, while the other 10 (6) are reserved for comparison. The dry seeds {h) are placed in a dry test-tube, w^hile the imbibed seeds (a) are placed in a test-tube half full of water : both 1 Sachs' Physiolooie (French Tr.), p. 72. Fig. 8. 16 PROTOPLASMIC CIRCULATION. [CH. I test-tubes are corked and are immersed in a beaker of water kept by means of thermostat at 60° C. for 2 hours. Both sets of seeds should now be sown in damp sawdust, — the lot (h) having been previously soaked in cold water for twelve hours : it will be found that lot (6) germinate, while (a) do not do so, and show^ other obvious signs of being dead. (16) Circulation of Protoplasm — Sachs Hot-box. Any parts of plants, in which circulating protoplasm can be observed, serve as material for studying the effects of temperature. The staminal hairs of Tradescantia or other plant-hairs are convenient, or the tentacles oi Drosera may be used. But the leaves of Elodea are perhaps most easily obtainable throughout the year. Mount a leaf of Elodea upside down in a drop of water under a large cover-glass ; look for circulating protoplasm near the mid-rib \ and subject it to gradually increasing temperature by means of any of the recognised " hot- stages," e.g. with Sachs' Hot-box. The arrangement is described and figured in Sachs' Text-Book, English Trans- lation, p. 736. It consists of a hollow-walled metal box, into which the microscope is placed so that by filling the walls with warm water the object under observation can be subjected to the desired temperature. A window admits light, and a hole in the moveable lid allows the microscope-tube and fine adjustment to project. The 1 The leaves should be cut off an hour before they are wanted, because, in winter at any rate, circulation is not visible until some time after the leaves have been cut. CH. l] EFFECT OF HEAT. 17 felt lining of the lid should be wetted and a little water should be spilt on the floor of the box, so that the atmo- sphere surrounding the object may be damp. A thermo- meter passes through a hole in the lid or, as we find more convenient, through a cork fitting one of the lateral openings. The glass slip on which the object is mounted should be separated from the stage of the microscope by a perforated plate of cork, so that the object may assume the temperature of the air, rather than that of the micro- scope, — although these two temperatures will after a time be nearly identical. The hot-box may be conveniently supported on wooden blocks and heated by a gas flame. As the warming of a considerable mass of water is a slow process it is advisable to fill the box with water 10° C. above the room tempera- ture. Notice the accelerating effect of warmth, and record the temperature at which the circulation (1) becomes slower (42°— 46°C.); (2) stops altogether (about 46'— 50° C). (17) Veltens methocV. A simpler and quicker plan is that of Velten, which, however, should not be used with a valuable microscope. The objective and the preparation are immersed in water contained in a glass dish standing on the stage of the microscope. A siphon, provided with a tap, allows warm water to run into the dish, while a second siphon and tap ^ Flora, 1876, p. 177, also F. Darwin, Q. Journal Microscopical Science, N.S. Vol. xvii, p. 245. Cf. Pfeft'er, Zeitschr. fur loiss. Mikro- skopie, 1890. D. A. 2 18 EFFECT OF COo. [CH. I provides for the overflow. The object is mounted between two cover-glasses, which are gently clamped together by a Fig. 6. Exp. 17. bit of tinfoil of the form shown in fig. 6, the flaps being bent up at 45° along the dotted lines. Unless some such plan is adopted, the upper cover-glass is liable to be washed off by currents in the water. The same method may be used to subject circulating protoplasm to a low temperature. (18) Effect of GO,. To observe the effect of gases on circulating proto- plasm, the Elodea leaf is mounted in a small drop of water, on the under surface of a cover-glass forming the roof of a gas chamber: if the cover-glass projects fairly well beyond the edges of the hole on which it lies, the apparatus can be made sufficiently gas-tight by painting the edges of the cover-glass with olive oil ; or the glass may be fixed with putty. Having under observation a circulating cell, attach the tube of the gas chamber to the COo-generating apparatus ^ and observe that the proto- The CO2 must be made to bubble through water before it reaches the gas chamber. CH. l] CHLOROFORM. 19 plasm comes to rest : by disconnecting and allowing air to pass, the circulation can be renewed, In this experiment the CO2 acts, not by preventing access of oxygen, but as a narcotic. This can be shown by connecting with a hydrogen-generator; the rapid retardation previously observed Avill be absent. (19) Chloroform. The same apparatus serves to demonstrate the effect of chloroform and other hurtful vapours. Shake up one per cent, of chloroform in a bottle of water, through which (by means of an aspirator) a current of air is made to bubble. The air, thus charged with chloroform is allowed to pass through the gas-chamber. The circulation may be stopped without killing the leaf (20) Chloroform. The effects of poisons may also be conveniently de- monstrated on the leaf of Oxalis acetosella, using the colour test already described. Shake up 1 c.c. chloroform in 200 c.c. water in a stoppered bottle and add an Oxalis leaf cut into small pieces. Note the time required for the discoloration to occur. (21) Carbolic acid. (Phenol.) Make the same experiment, substituting 0*5 per cent, carbolic acid for chloroform-water. (22) Tetanising current. If an Oxalis leaf is impaled on a pair of needles (in an insulated handle) connected with the induction coil, 2—2 20 ELECTRIC SHOCK. [CH. I the region between the punctures is killed and becomes discoloured when the current passes : the needle points should not be more than 2 — 3 mm. apart. (23) Tetanising current Two triangles of platinum foil are sealing- waxed on to a glass-slip, the points being about 1 mm. apart. To make the platinum adhere well it is necessary to heat the glass over a flame until the wax between the glass and the metal is thoroughly soft, and then to apply pressure. An Elodea leaf is mounted in water so that a cell, showing circulation, lies between the points, and by connecting the foil triangles with an induction coil, the effect of the tetanising current can be observed. The wires from the coil are most conveniently connected by means of the insulated screw-binders, obtainable from instrument makers ; in the absence of screw-binders the following arrangement will be found to answer quite well. A cork ring is sealing-waxed on to each foil-triangle near its base, and into the little vessels so made, mercury is poured, into which the connecting wires are placed. To get a rough idea of the current needed, it is advisable to note the position of the coil when the current is just bearable on the tongue, and compare it with the position of the coil when the protoplasmic circulation has been stopped. CHAPTER II. ASSIMILATION OF CARBON. Section A. Formation of Starch. Section B. Evolution of Oxygen. Section C. Reactions of Chlorophyll. Section D. Conditions of Chlorophyll formation : Etio- lation : sun and shade leaves. Section A. Formation of Starch. (24) Sachs Iodine-method^ (lod-Probe). This is a macroscopic method well adapted for many experiments. Almost any leaves will serve as material for the demonstration of the method, but since in research it is of importance to employ material which allows of rapid work, the choice of plants is a point to be con- sidered. Submerged water-plants are useful, and among land plants, Tropceolum and clover {Trifolium) are especially valuable. The leaves to be tested are to be boiled for about one minute in water'', when they should be flaccid 1 Sachs' Arbeiten, iii. p. 1. ~ Sachs allows a longer period, viz. 10 minutes ; he states also that the addition of a few drops of strong KHO to the boiling water hastens the process. 22 ASSIMILATIOX. [CH. II and free from intercellular air. They are then placed in alcohol warmed to 50° — 60° C. : cold alcohol will remove the chlorophyll equally well but not so quickly : if the specimens are not wanted at once the best results will be obtained by putting them in the sun for a few hours. The preliminary boiling in water must on no account be omitted, it shortens the process of decolorising in the most remarkable manner; of this it is easy to convince oneself by trying, for instance, to decolorise an Enteromorpha without the hot-water treatment. To produce the iodine reaction, place the decolorised leaves in alcoholic tincture of iodine diluted with water ^ to the colour of dark beer. In a few minutes they will be stained, and after washing in fresh water, they should be spread out on a white plate so that their tint — by which the amount of starch is roughly gauged — may be well seen. When full of starch they are almost black, and with less amounts of starch the colour sinks through purple, grey, and greenish grey to the yellow tint of starchless leaves. (25) Schimpers method^. In some cases it is necessary to use the microscope, this is especially necessary when the amount of starch present is small, or where, as in Schimpers researches, the distribution of starch in the leaf is minutely studied. Prepare a strong solution of chloral hydrate by dis- 1 It is not necessary to use distilled water. 2 Bot. Zeitung, 1885. CH. Il] IODINE METHOD. 23 solving the crystals in as much distilled water as will just cover them ^ The solution is now coloured by the ad- dition of a little tincture of iodine, and is ready for use. Delicate leaves, such as those of submerged water-plants, when placed in Schimper's solution, are rendered so trans- parent that every detail of starch-distribution can be studied in the leaf examined as a transparent object under the microscope. (26) Variegated leaves. Test Sachs' method on a variegated leaf such as that of the ivy (Hedera) or of Arundo donax. In the case of the ivy a rough plan of the green and white parts of the leaf must be traced on paper placed under the leaf, which may best be done by tracing a broken line with a blunt instru- ment dotted along the lines separating the chlorotic from the green parts of the leaf The iodine-stained leaf is then compared with the plan. With Arundo no such process is necessary, the chlorotic regions are in longi- tudinal stripes, and it is only necessary to cut out of the leaf a short piece, which, after staining in iodine, can be replaced between the base and apex of the leaf to wdiich it belonged : the colourless stripes in the fresh parts cor- respond to yellow stripes in the stained part, and the purple to the green. Both the extraction of the chloro- phyll and the staining with iodine are slow processes in the case of Arundo. (27) Disappearance of starch in darkness. Either of the methods may be tried on submerged ^ Chloral hydrate 8 parts, water 5 parts. 24 ASSIMILATION. [CH. II water-plants (e.g. Elodea, Potamogeton) which have been placed in the dark room for about four days. The control-plants must be grown either out of doors or in a greenhouse. (28) Effect of dull light Sachs' method may be used to demonstrate a fact, the knowledge of which is of practical value to the physiologist^, namely, that plants in a laboratory suffer from want of light far more than would be readily supposed — and that accordingly experimental plants can- not be too carefully kept in the best light available. Choose two equally vigorous pots of clover, let one remain in bright diffused light out of doors, and place the other on a table in the middle of the laboratory. The plant in the laboratory must be under a bell- jar on account of the dryness of the air, and therefore to make the control experiment fair the plant out of doors should also be under a bell. After two days compare the amounts of starch in the two plants. (29) Local eff^ect Various means may be used to convince oneself that assimilation is confined to the illuminated regions of a leaf. Part of a leaf may be darkened, while still attached to the plant, by bending it down and burying the apical half in a flower-pot of finely sifted dry earth. The leaf should be buried one day and examined in the afternoon of the following day, taking care before the leaf is un- covered to mark on it the depth to which it was buried. 1 See Detlefsen. Sachs' Arbeiten, iii. p. 88. nOPERTY LIBRARY N! C. State College CH. Il] PHOTOGRAPHIC METHOD. 25 (30) Gardiner's experiment'^. A plant growing in a flower-pot (for convenience of moving) is placed in the dark for 24 hours, or until the leaves are found to be free from starch. One of the leaves is now covered with a photographic negative and left exposed to bright light out of doors, or in a green- house, until the evening, when the leaf is tested for starch. It will be found that an accurate copy of the photograph has been printed in starch. (31) Effect of rays of different refrangihility. The effect of the different parts of the spectrum may be demonstrated by a method similar to that described in Exp. 29, as has been done by Timiriazefif^ In the ab- sence of the necessary apparatus we may compare the. effects of light transmitted through coloured fluids. Fill a couple of double-walled bell-jars, (1) with potassium bichromate solution, (2) with ammoniacal CuSO^ solution. Under each bell place a young TrojKeolum or clover plant in a small pot, or a seedling plant of any kind dug up and placed with its roots in a bottle of water. The bell-jars should stand in saucers of dry earth or sawdust, so as to ensure the exclusion of colourless light. They must be exposed to diffused light— in sunshine the temperatures are not the same in the two bell-jars. The exposure should be for 1^ or 2 days. The plants in the blue light will be almost starchless. 1 W. Gardiner, Annals of Botany, iv. p. 163. — /V97 2 Timiriazefif, Comptes rendus, T. ex. p. 1346. b-VT) cesses '^ )o VckVAi-C 26 ASSIMILATION. [CH. II (32) Terrestrial leaves under water. To show that the leaves of land-plants do not form starch as those of aquatic plants do under waiter \ it is only necessary to tie a leaf so that it is partly immersed in a beaker of water. The experiment may be started in the morning and concluded on the afternoon of the following day. (33) The imi:)ortance of the stomata in supplying the path for gaseous exchange I For this experiment leaves should be employed in which the stomata are all on the lower surface. Stahl uses Primus padus; we find Sparmannia africana gives good results, and no doubt many other plants would answer the purpose. The lower surface of one half of a leaf is carefully painted with vaseline, or as Stahl re- commends with melted cocoa-fat and beeswax. The plant having been exposed to a good light for two days, the leaf is subjected to the iodine test. The painted half (in which the stomata are blocked) will be either quite or nearly starchless, while the control half shows a normal amount of starch. (34) Effect of excess of CO,. To show that excess of CO.2 diminishes assimilation =^ floating water-plants are convenient. We use Callitriche, and possibly Lenma might be used, but these must be 1 Nagamatz (Sachs' Arheiten, iii.) shows that leaves covered with bloom can assimilate under water. 2 Stahl, Botan. Zeitung, 1894. See also F. F. Blackman, Phil. Trans. 1895, and in Science Progress, 1895. 3 Godlewski. Sachs' Arheiten, i. p. 34.3. CH. Il] EXCESS OF COo. 27 kept a long time in the dark before they are destarched. Two graduated jars of 200 c.c. capacity are filled with and inverted over water, and plants of Gallitriche, which have been previously deprived of starch, are passed under the edge and allowed to float up. Into one jar equal quantities of air and COg, while into the other 12 volumes of air to one of COo are passed. The propor- tion of COo in the atmospheres so prepared does not of course remain constant, since the water absorbs the gas. But if the experiment is started in the evening and concluded in the evening of the next day, one jar will certainly contain far more than the optimum of COo, while the other will not fall much below the optimum. A still simpler plan is to use beakers of about 800 c.c. capacity inverted in saucers of water. The beakers are graduated as follows : into one 550 c.c. of water is poured and the level marked with a diamond, a second mark being made after the addition of 50 c.c. The other beaker is marked at 300 and 600 c.c. The beakers are filled with water and inverted in saucers, and the rosettes of Callitriche floated up under the rims of the beaker. Three hundred c.c. of air are now introduced into one beaker and 550 c.c. into the other, using a finger bellows for the purpose; afterwards COg is added until each beaker contains 600 c.c. of mixed gas, one containing 50 p.c, the other 8 p.c. of CO.2. In our experiments the Callitriche exposed to 50 c.c. COg showed hardly any starch, while the control-plants were black with it. The experiment may be more accurately performed with a pair of graduated tubes inverted over mercury 28 ASSIMILATION. [CH. II (covered with a few drops of water) and containing leaves of land-plants. (35) Plants deprived of CO^. To show that the formation of starch depends on the presence of COo it is necessary to cultivate plants in such a way that they have access to oxygen but not to Water-plants. Water which has been boiled and allowed to cool in a /' P 1 1 A \ Tf^Pr^ iiyi