THE BIOCHEMISTRY OF PLANT VIRUSES 37 



practicable. At present the limit is of the order of 5 to 10 mg. of dry virus 

 per kilogram of fresh leaf material, but for consistent successful purification 

 a larger quantity than this is desirable. This very factor limits the viruses 

 susceptible to biochemical study to those few which are exceptionally 

 infectious, and which indeed may not be wholly representative of plant 

 viruses as a whole. In addition to this difficulty of isolation, which may well 

 be overcome by the use of novel techniques, present-day methods of physical 

 and chemical investigation, highly developed as they are, require at least 

 2 milligrams of j)ure virus material, so that the problem of purifying many 

 of the interesting plant viruses also becomes one of dealing with relatively 

 vast volumes of material. 



A. Groiving the Virus-Infected Plants 



In addition to the requirement of a source containing a substantial 

 quantity of the virus material, there is also a requirement for certain prop- 

 erties m the plant sap as expressed from the virus-infected hosts. Because 

 of this, plants which contain large amoimts of gums, latex, or which have 

 miusually large quantities of phenolic constituents are to be avoided. In 

 fact even such conditions as the age and maturity of the host plants are 

 exceptionally important. Thus, for example, in the purification of virus 

 from tobacco plants, young, vigorously growing plants with a growth which 

 horticulturalists would term "soft" are to be preferred, although this is 

 not always true. Young plants also tend to contain less pigment, and, in 

 general, are easier to deal with than are older plants. For routine j)urification 

 work, plants are best raised from seed, when they are usually, though not 

 invariably, free from virus disease, and transplanted singly at the cotyledon 

 stage, or shortly afterward, into individual 3-inch diameter pots containing 

 a sterilized compost which has an adequacy of nitrogen, preferably in the 

 form of an insoluble organic substance like gromid horn, phosphate, and 

 potassium, as well as vermiculite or peat to ensure a sufficiency of water. 

 The plants are best grown under glass and inoculated when small: thus, 

 tobacco plants should be some 6 inches from leaf tip to leaf tip when inocu- 

 lated with a virus giving a systemic infection. For viruses such as the tobacco 

 necrosis viruses which cause only local infections, the leaves to be inoculated 

 should be about 5 to 6 iuches long and sufficiently mature to have a hard, 

 somewhat smooth surface, because the younger, soft leaves are not so 

 susceptible. The leaves are harvested after maximum virus content has been 

 reached, which wiU vary with the virus, the plant species, climatic conditions, 

 etc., and which must be determined empirically. 



The harvested leaves may then be sorted into those most likely to contain 

 large amounts of virus. This procedure, coupled with the removal of leaf 



