70 BIOCHEMICAL SYSTEMATICS 



dialyzable cleavage products. Some other rather uncommon reactions 

 have been utihzed but so infrequently as to render them insignificant 

 for present purposes. 



As in any other biochemical approach, the validity of serologi- 

 cal data depends directly upon the reliability of the techniques 

 utilized. From the earliest investigations strong support for particular 

 innovations of technique has been the rule, and often the attitude has 

 been taken that other techniques, usually equally vigorously sup- 

 ported by their adherents, are, nevertheless, almost completely worth- 

 less. Controversy over technical procedure was particularly rife be- 

 tween the Konigsberg and Berlin investigators, and it seems that they 

 hardly agreed on anything. Subsequent improvements in technique, 

 to be discussed later, indicate that the controversy could only disclose 

 which group's technique represented the greater imperfection. Since 

 the question of technique in serology is exceptionally relevant to 

 a reasonably objective appraisal of the method itself, some details will 

 be included below. 



In botanical serology, seeds are most frequently used as a source 

 of antigen. These may be ground in a mortar and pre-extracted 

 with some non-polar solvent such as petroleum ether to remove lipids. 

 The ground material may also be extracted with ethanol. The protein 

 is finally extracted, most often with physiological saline, in proportions 

 of about 100 ml per 10 gms of tissue. Extraction time is controlled, of 

 course, and may represent several hours, or overnight. Sometimes 

 expressed sap is used directly. 



Considerable disagreement arose among earlier investigators 

 as to whether individual plants were serologically homogeneous or 

 whether different organs or even tissues from the same plant had 

 different antigenic complements. Mez believed that plants were homo- 

 geneous, but the Berlin group disagreed. Chester noted, however, that 

 comparisons between seed proteins and other plant parts were particu- 

 larly distinctive and supported the Berlin viewpoint. Quite recently, 

 strong evidence for antigenic heterogeneity has been adduced by 

 Kloz, et al. (1960); this evidence will be presented in detail later, 

 following some additional discussion of present methodology. If there 

 is significant adaptive enzyme formation during development of higher 

 plants, as a priori considerations and precedent from microorganisms 

 suggest, then antigenic heterogeneity may be expected. Furthermore, 

 present work on multiple enzymes, discussed earlier (Chapter 4), 

 suggests strongly that large differences in antigenic composition may 

 be expected within an organism. 



Another important question of technique relates to the pro- 

 tein concentration of a particular plant extract. For example, if a 



