ROOT-NODULES OF LEGUMINOSAE. IOI 
Did they obtain it from combined nitrogen present in the air? To settle this question, peas 
were grown under bell-jars in washed air, 7.e., in air from which all the nitric acid and ammonia had 
been removed, and the growth was just as good as in the unwashed air. Growth was also good on 
nitrogen-free soil in a closed space in a limited volume of air which could have offered to the plants 
only a trace of combined nitrogen (fig. 32). The conclusion, therefore, appeared to be irresistible 
that the peas were in some way able to assimilate free nitrogen. 
Boussingault having already shown that legumes can not directly assimilate free nitrogen, the 
only hypothesis open was some indirect assimilation through the assistance of other organisms. 
They were led to the conclusion that the root-nodule organism was the factor sought, by having 
observed that after peas had used up the stored food in the seed there often followed a period of 
nitrogen-hunger during which growth stopped and the leaves became pale or yellow, but that after 
a time the green color returned and growth was resumed. In certain plants, however, this resump- 
tion of vigorous growth never took place and the roots of such plants were observed to be nearly or 
quite destitute of root-nodules, whereas the roots of the other plants bore nodules, and the more 
abundant and better developed these were, the better the growth of the plants appeared to be. 
Fig. 33.* 
The next step, therefore, was to add and exclude nodules or nodule products, and so determine 
the results experimentally. This was done by taking 40 experimental pots containing nitrogen-free 
soil, holding 30 for checks, and to the other 10 adding 25 cc. of an extract of fertile soil, containing 
only 1 mg. of nitrogen per pot, all being planted to peas. All passed through a period of nitrogen- 
hunger, but the plants in the pots inoculated with the soil-extract all regained their green color and 
grew freely and uniformly. In only 2 of the 30 check pots did the plants grow freely. All the rest 
continued to show nitrogen-hunger, and some became quite yellow. ‘This difference in color and 
amount of growth was found to be correlated with the presence or absence of root-nodules. 
*Fic. 33. Mee 8 to 11. Stages in development of root-nodules on common garden lupin (Lupinus mutabilis). 
No. 12. Same in tranverse section as seen under a hand-lens. 
No. 13. Same as 12 but in longitudinal section; ap, external parenchyma; 7p, internal parenchyma; g6, 
vascular bundles. 
No. 14. Cross-section. x 120. 
No. 15. Mature cells of internal parenchyma containing rod-shaped corpuscles. x 320. 
No. 16. Cell showing escape of rod-shaped bodies by resorption of membrane. x 320. 
No. 17. Mass of bacteria surrounding nucleus which has survived cell membrane. x 620. 
No. 18. Isolated bacteria which have become modified and have ceased to be motile. x 620. 
After Woronine. Reduced one-eighth. 
