Some Botanical Applications of the Carbon Replica Technique 



D. E. Bradley 



Research Lahoratory, Associated Electrical Industries Ltd., Aldermastoii, Berkshire 



It is likely that a knowledge of the sub-microstruc- 

 ture of the sporoderm would be of some value in a 

 study of post-glacial flora. 



The electron microscopy of the surface structure 

 of pollen grains has been made possible by the use 

 of carbon replicas: the technique was first applied 

 in this field by Muhlethaler (4). In the present work, 

 over sixty different species were studied in order 

 to ascertain firstly, the reliability of the replica 

 method developed for the purpose, and secondly, 

 the range of structures which might be encountered 

 in a wide study of pollen. 



The replica method was identical with that described 

 elsewhere (1), save for one modification. It was found 

 more satisfactory to dust pollen onto the initial Formvar 

 substrate and then disperse it with acetone or alcohol, 

 rather than to suspend the grains in water and dry down 

 a drop of the suspension. It was found that the efficiency 

 of the technique depended on the size of the pollen grain. 

 In the case of large grains, the method was only 10 % 

 efficient, i.e. only 10 out of 100 grains were replicated, 

 the remainder breaking up and leaving a hole in the 

 replica film. It is, of course, only necessary to take 

 micrographs from a few grains so that this 10% efficiency 

 was found to be quite adequate. In the case of small 

 grains, the efficiency was at least 50 ';'o. 



It was found that the use of acetone or alcohol to 

 disperse the grains caused the absorption of suffi- 

 cient moisture during the evaporation of the solvent 

 to cause the expansion of some grains. Many pollen 

 grains are deeply divided by grooves, and the swell- 

 ing causes the grooves to be pushed outwards so 

 that a grain in the form of an ovoid becomes a 

 sphere. This is a well known phenomenon. It was 

 found that the fine detail of the grain was not dis- 

 torted and that it was possible to study the structure 

 within the groove. An example is shown in figure I, 

 which is a micrograph of part of a grain of Lawium 

 alhiim. The wedge shape represents the groove. 



Structures were found to vary very greatly. In 

 some cases, there was much fine detail such as that 

 shown in figure 2 which is the surface of a grain of 



Rhododendron; most of this detail is of the order of 

 a quarter of a micron in size, and not properly re- 

 solved by the optical microscope. In many cases, 

 however, the structure was much coarser as in 

 figure 3 (Bryonia dioica) . Most of this detail which is 

 two to three microns in size, could be easily resolved 

 optically, but the electron microscope provides a 

 different viewpoint, and of course, a very much 

 clearer picture. It is certainly likely that electron 

 micrographs such as these will be valuable in a 

 study of pollen. 



The examination of fungus spores. — Figure 4 shows 

 a shadowed carbon replica of a spore of Russiila 

 venosa. This spore is believed to be covered with a 

 thin surface film known as the amyloid layer. The 

 evidence for its presence is that the spore gives a 

 very marked iodine reaction, but this reaction does 

 not occur if the spore is first treated with dilute so- 

 dium hydroxide. Some optical evidence of this film 

 has also been described. The matter is still contro- 

 versial. In the replica shown, there is certainly a 

 surface film present as it has been broken at A and 

 B. This film appears to be from 200 to 500 A in 

 thickness, according to the position on the spore. 

 The film does not extend over the plage (the rather 

 rough region immediately behind the apicle). This 



Fig. 1. Shadowed carbon replica of pollen grain of Laniiinii 

 album showing detail within the groove. Fig. 2. Shadowed 

 carbon replica of part of pollen grain of Rhododcudron. 



Fig. 3. Shadowed carbon replica of part of pollen grain of 

 Bryonia dioica. 



Fig. 4. Carbon replica of a spore of Russula venosa. {A) and 

 (B) mark places where the suspected amyloid layer has 

 broken. Shadowed. 



