An X-Ray Study of Waxy Coatings from Plants. Comparative Investigations, and the Application of a New Micro-Method for X-Ray Diffraction to Structural Problems with Ancillary Observations under the Polarising and the Electron Microscope

As an introduction, in chapter I, a survey is given of the microscopic structures of wax cuticles on plants distinguished by De Bary (1871) and of the older opinions concerning their submicroscopic structure and their formation. The importance of obtaining a clear picture of their still very insufficiently known submicroscopic structure in order to gain insight into the problems concerning their formation, has been pointed out. Then some more recent investigations into the submicroscopic structure of the wax on, and in the outer wall of the epidermis have been mentioned and the desirability of carrying out X-ray investigations has been argued. In chapter Ila, the X-ray investigation of a wax cuticle by means of a standard equipment for the study of X-ray diffraction in use in the Laboratory of Technical Physics, Delft, has been described. As an object to study, the wax layer of the sugar cane stem was chosen in places where it consists of rods growing nearly perpendicularly to the cuticle. In sect. 1, this choice has been justified, a description of the object has been given as well as an account of technical details concerning the preparation, the method of taking X-ray photographs and some difficulties connected with the small size of the object. The X-ray diagrams obtained have been described in sect. 2, and it is ascertained what conclusions about the structure of the wax layer they allow to be drawn without carrying out special calculations. It appeared that the crystallites in the wax layer are preferentially orientated with one of their axes perpendicular to the cuticle, whereas they can occupy all possible positions by turning around that axis. In this orientation, however, a great angular dispersion of the positions of the crystallites occurs, which might be due equally to lack of parallism between the rods of the layer as to imperfect orientation of the crystallites in the individual rods. In addition, the diagrams show that the longitudinal direction of the chain molecules is perpendicular to the axis of orientation. This implies that the chain molecules of the wax are lying with their longitudinal direction parallel to the cuticle and perpendicular to the longitudinal direction of the rods. In sect. 3 we have tried to calculate the identity period of the axial direction of the crystallites which is orientated perpendicular to the surface of the cuticle, in order to be able to index this axis. The calculation is greatly handicapped by the considerable angular dispersion in the positions of the axis under discussion. It is found that the axial direction is probably [no]. In sect. 4 it is ascertained, by means of the reciprocal lattice, in which places on the photograph maxima of blackening must be expected if the crystallities are perfectly orientated with the abovementioned axial direction perpendicular to the cuticle. The desirability is argued of obtaining a photograph actually showing those maxima as a confirmation of the more or less doubtful conclusions from the first-mentioned photographs with regard to the indexing of the axis of orientation of the crystallites. In chapter Ila, the X-ray investigation of the wax rods by means of a newly developed micro-method has been recorded. In sect. 1 it is stated that the desired photograph mentioned above might be obtained from a small packet of perfectly parallel wax rods. The impossibility of obtaining such a diagram by means of the available standard equipment, and the necessity of a special micro-method for that purpose have been argued. Then the general importance which a micro-method might have for X-ray studies of microscopic and, especially, biological objects, has been discussed. Since no description of such a method in a design suitable for most biological objects was found in the literature, investigations were carried out in order to develop a generally applicable micro-method. These investigations, however, have not been described here. The results were briefly mentioned. The next section describes how, by means of the newly developed micro-method, a micro-diagram could be obtained from a small bundle of parallel wax rods. This diagram shows maxima in the places predicted before in sect. 4 and thus confirms that the axial direction with which the crystallites are orientated perpendicular to the cuticle, has been correctly indexed on the ground of the diagrams obtained earlier. The diagram enables the identity period of this axis to be determined with much more accuracy than the diagrams obtained first. It also confirms that in the individual wax rods, a comparatively perfect orientation of the crystallites occurs. Chapter III describes the examination of the wax rods by means of the polarising microscope and the electron-microscope in order to elucidate their submicroscopic structure further. This concerns in particular the orientation of the crystallites with respect to the directions in the wax rods normal to their longitudinal direction, those details not being attainable by means of X-ray diffraction on account of the small diameter of the rods. When using high magnifications under the polarising microscope it appeared that the rodlets were coarsely ribbed. The orientation of the crystallites in the ribs could be indicated on account of the observed birefringence phenomena combined with data from the literature about the birefringence of waxes. They are orientated with the longitudinal direction of the molecules in radial directions of the rodlet. We have disputed Weber’s claim (1942) to having demonstrated the same direction of the molecules in wax rods of Phragmites. Sect. 2 gives some considerations on the limits of the dimensions of the crystallites and on the possibilities for the structure of the rods within the range of those limits. In sect. 3 it has been described how the picture of the submicroscopic structure obtained in the preceding sections, could be confirmed and supplemented by making use of the electron-microscope. The conclusions concerning the submicroscopic structure of the wax rodlets to which this investigation has given rise, are summarised after chapt. III. Finally, in chapter IV, some general view-points on the place of origin of the cuticular plant waxes and on the formation of their submicroscopic structure have been put forward. Sect. 1 deals with the place of formation of the wax on the basis of data from the literature. In sect. 2 the factors which might possibly affect the structure of the wax coatings are briefly discussed. The composition is suggested to be the most important factor. Therefore, in sect. 3, it is ascertained whether waxes giving similar X-ray powder diagrams also tend to exhibit similar microscopic structures; this has been found, in general, to be true. In sect. 4 it is attempted to give an explanation of the structure of the wax rods. Furthermore it is discussed in particular whether it is necessary to ascribe a direct organising influence to the protoplasm in the formation of the structures of wax cuticles strongly suggesting such an influence. We have advanced possibilities of formation of those structures as an indirect effect of chemical activity of the protoplasm. Finally the conclusions from this chapter are summarised.