118 REACTIONS INVOLVED IN [CH. 



Their evidence may be summed up under two headings : viz. evidence 

 from (1) presence of oxidising enzyme, (2) presence of chromogen. 



1. Presence of oxidising enzyme. 



In order to demonstrate the existence of oxidising enzymes in the 

 tissues, Keeble & Armstrong have adopted an excellent microchemical 

 method. The mode of procedure is to place the tissue to be examined 

 in either a 1 % solution of benzidine in dilute alcohol, or in an equally 

 dilute solution of a-naphthol and incubate at 37 C. 1 If no direct oxidase 

 reaction results, the material is removed from the tube and washed 

 with a dilute solution of hydrogen peroxide. The above method was 

 employed with great success on petals of Primula sinensis. It was 

 found that a-naphthol gave a delicate lilac-blue colour with oxidases, 

 but only detected them in the bundle sheath of the veins, whereas 

 benzidine gave a brown coloration and detected oxidases both in the 

 epidermis and in the bundle sheath. The results as regards Primula 

 sinensis may be summed up as follows : 



Flowers from all coloured varieties and recessive whites (see p. 177) 

 gave a benzidine reaction in the epidermis and an a-naphthol reaction 

 in the bundle sheath. These tissues being the chief seat of the pigment, 

 it may be said that the distribution of enzyme and pigment in coloured 

 varieties is practically coincident. Flowers of dominant white varieties 

 gave no oxidase reaction. In the case of a blue variety with inhibited 

 white patches on the corolla segments, there was a more or less corre- 

 sponding lack of oxidase reaction in the inhibited patches. It was found 

 that the inhibitor could be removed by treatment with hydrocyanic acid 

 and other methods, and the above varieties then gave the usual benzidine 

 and a-naphthol reactions. Flowers of certain flaked (magenta and 

 white) varieties showed no oxidase reaction in the white parts. 



Other genera were also investigated by the same method. In 

 Sweet William (Dianthus barbatus) flowers, it was found that the 

 oxidase reaction was entirely proportional to the amount of pigmenta- 

 tion. Of white varieties, some were found to contain oxidase; white 

 parts of other varieties were found to contain no oxidase. In Geranium 

 sanguineum the purple type contains epidermal oxidase, but the white 

 variety gives no such reaction. In the Sweet Pea (Lathyrus] and 

 Garden Pea (Pisum), no whites were found which did not contain 



1 Benzidine and naphthol are artificial acceptors. Whether the reaction obtained is 

 direct or indirect depends on the presence of a natural peroxide suitable for acceptors 

 used. Absence of direct action does not preclude a system peroxide-peroxidase in the 

 plants. 



