Crafts et al. — 196— Water in Plants 



the relation of structure to position of leaves, found that small apical leaves, having 

 numerous stomata and thick cuticle, were more xerophytic than broad basal ones. 

 Smith (1941) found a similar condition in bean. Stomatal frequency increased from 

 base to apex of the lamina, and from base to top of the plant. Salisbury (1927) found 

 that variations in stomatal number on an area basis depended upon the size of the 

 epidermal cells according to the formula : 



S 

 I r= E ■ s X 100 where (7) 



I = the stomatal index that is constant for any species. 



S =: number of stomata per unit area. 



E =: number of epidermal cells in the same unit area. 



Leaves or portions of leaves which, because of position on the plant or because of 

 xerophytic condition, are reduced in size, show high stomatal frequencies (number of 

 stomata per unit area). A similar condition is shown by exposed upper leaves, sun 

 leaves, apices and margins of leaves, and by leaves of woody as compared with herba- 

 ceous plants, and leaves of marginal vegetation as compared with shade species. All of 

 these differences, however, are due chiefly to variations in growth of the epidermal 

 cells, that is, to the spacing of the stomata and not the number developed. Under con- 

 ditions of high humidity there does, however, appear to be a reduction in the proportion 

 of stomata to epidermal cells formed. Aquatics have lower stomatal indices than land 

 plants. Poor nutritional conditions appear in some instances to reduce the stomatal 

 index. 



Methods of Measuring Stomatal Openings : — Both direct and indirect methods 

 have been utilized in studying the degree of stomatal opening. These methods have 

 been discussed by Stalfelt (1929) and Nadel (1938). The latter author critically 

 examined the alcohol fixation method of Lloyd which has been used extensively and at 

 times without due caution. 



It is obvious that where the conditions of the experiment and the nature of the 

 leaf permit, the direct microscopical observation of intact, untreated leaves is the most 

 reliable means of determining the degree of stomatal opening. Often, however, condi- 

 tions are such that this is not possible and resort must be taken to the microscopical 

 examination of treated material or to one of the more indirect methods such as porome- 

 try, infiltration, or measurement of transpiration. 



Of the various treatments which have been used to prepare the epidermis for 

 microscopical observation (drying, placing in water, and chemical treatment to pre- 

 vent stomatal change) only the alcohol fixation method proposed by Lloyd (1908) has 

 been used extensively. This method consists of stripping the epidermis from the leaf 

 and quickly immersing it in absolute alcohol. There is an immediate, rapid dehydra- 

 tion and hardening of the cellulose walls before the alcohol penetrates the cells appre- 

 ciably. Obviously, any turgor changes which might occur during the stripping and 

 immersion would tend to invalidate the method. Loftfield (1921) used the alcohol 

 method in studying some sixty dififerent species of plants. He checked the method 

 against direct observation of undisturbed leaves of alfalfa and several other species, 

 finding good agreement between the two methods. Rapid stripping and transfer of the 

 epidermis into the alcohol was found, however, to be essential to prevent dimensional 

 changes. 



A more critical analysis of the method (Nadel, 1938) showed it to be valid only 

 for plants with easily detachable epidermis while plants with adhering or partially ad- 

 hering epidermis were not found suitable. Even in the first group, if mesophyll cells 

 are left attached to the epidermis, changes in stomatal opening may take place in alcohol. 



AsHBY (1931) has compared the porometer method with Lloyd's alcohol fixation 

 method with good agreement in Geranium and Verbena. The well known porometer 

 method first introduced by Darwin and Pertz (1911) measures the rate at which air 

 can be drawn through the leaf. Thus it gives an average or statistical value for the 

 diffusion capacity rather than an absolute measure of pore size. This measure is a 

 function of mesophyll resistance, viscosity of the air, pressure difference employed, as 

 well as being subject to the mechanical difficulties of attaching the porometer cup to 

 the leaf by lightly clamping the leaf between a glass plate and a greased (beeswax- 

 vaseline) washer. Nevertheless the method has been used with apparent success (Op- 

 penheimer, 1926; Hartsuijker, 1935). 



Few attempts have been made to calculate the actual aperture of the stomata from 

 figures obtained by use of the porometer. These have usually been of an empirical 



