GROWTH FACTOR REQUIREMENTS OF FOUR SPECIES OF ISOLATED ROOTS

ALTHOUGH THE culture of isolated root tips was reported in 1922 by Robbins and by Kotte, it was not until more than ten years later that White (1934) succeeded in maintaining excised tomato roots in vitro with undiminished growth rate over long periods of time. White found that for the successful culture of tomato roots the addition of a small amount of yeast extract to the nutrient medium was essential. Much of the further work on the cultivation of isolated roots has centered around the identification of the chemical substances responsible for this promotive effect of yeast extract on root growth. Bonner (1937) showed that vitamin B1, a component of yeast extract, is essential for the growth of the isolated pea root. A similar relation of vitamin B1 to the growth of the isolated tomato root has been demonstrated by Robbins and Bartley (1937) and later by White (1937). It has been recognized, however, that vitamin B1 is not the sole accessory root growth substance supplied by yeast extract. For the pea root nicotinic acid is a second essential factor (Addicott and Bonner, 1938). For the tomato root, on the other hand, vitamin B6 has been shown to be indispensable (Robbins and Schmidt, 1939a, 1939b). In the past, work concerning the root growth factors has centered on the roots of pea and tomato. In the present paper a comparison will be made between the growth factor requirements of the roots of four species of plants. MATERIALS AND METHODS.-In all the cultures reported here the nutrient solution reported earlier (Bonner and Addicott, 1937) as satisfactory for the growth of isolated pea roots was used. This nutrient solution contains per liter: 242 mg. of Ca(NO3)2 4H20, 42 mg. of MgSO4 7H20, 85 mg. of KNO3, 61 mg. of KCI, 20 mg. of KH2PO4, and 1.5 mg. of ferric tartrate. Sucrose was added to the medium in a final concentration of 4 per cent for roots of pea, radish and flax, and of 2 per cent for tomato roots. 'Received for publication June 30, 1939. Report of work carried out with the cooperation of the Works Progress Administration, Official Project number 665-07-3-83, Work Project number 9809. Accessory growth factors were made up in concentrated stock solutions so that they could be added, as desired, at the rate of 1.0 cc. of stock solution per liter of nutrient. The final medium was autoclaved at 15 pounds pressure for 25 minutes. All cultures were carried out in 10 cm. Petri dishes, each dish containing 20 cc. of nutrient solution and 1 to several roots, the number of roots depending inversely on the luxuriance of growth. Culturing and transferring of the roots were carried out in a culture room which could be steam sterilized. Forceps and knives were sterilized by immersion in alcohol and subsequent flaming. Contaminations by fungi or bacteria were very rare. All cultures were maintained in the dark in an air-conditioned room held at 25?0.50C. Roots were in all cases obtained from seedling plants. Seeds were sterilized in 0.1 per cent HgC12 and allowed to germinate under asceptic conditions. When the seedling roots were 10-20 mm. long, tips 4-5 mm. long were excised and used for further work. EXPERIMENTAL RESULTS.-Pea roots.-It is known (Addicott and Bonner, 1938) that isolated pea roots can be maintained in culture over periods of several months (and presumably indefinitely) provided that both vitamin B1 and nicotinic acid are supplied as growth factors in addition to the nutrient medium described above. In the presence of adequate supplies of vitamin B1 and of nicotinic acid still other factors, however, must limit the growth of the pea root. The relation of other known or possible plant growth substances to the growth of isolated pea roots in the presence of an excess of vitamin B1 and nicotinic acid was therefore investigated. In each experiment 500 freshly excised tips (variety "Perfection") were grown for one week in growth factor free medium. At the end of this week 400 roots, as nearly uniform as possible, were subcultured by the removal of 10 mm. tips and divided among the several parts of the experiment. The roots were then subcultured at regular weekly intervals. Table 1 gives the results from one experiment in which ade-