Evidence for the natural occurrence of zeatin and derivatives: compounds from maize which promote cell division.

Three laboratories previously have obtained from Zea mays kernels (milk stage) one or more 6-(substituted)aminopurines capable of greatly promoting cell division in plant tissue cultures.1-3 At least two of the laboratories have worked on a single compound4 which has been identified as 6-(4-hydroxy-3-methylbut-2-enyl)aminopurine.5 The name zeatin has been applied to this particular compound.6 Only a small portion of the total cell-division activity in crude extracts from kernels seems to be due to zeatin'-3 and its actual existence in the kernel has been questioned.7 We now present evidence strongly indicating that zeatin does occur naturally in its unsubstituted form and also in nucleoside and nucleotide forms. Assay.-The active materials in the maize extracts were detected by using the soybean (Glycine max, var. Acme) tissue test.3' 8 This tissue requires a substance like zeatin for continued cell division and has proved to be very sensitive to zeatin and similar compounds. For example, in recent tests we have obtained measurable growth responses to as little as 5 X 10-11 M zeatin. Most assays were performed with small quantities of extracts which contained rather low concentrations of active materials. With such low concentrations, the soybean test shows considerable variation of fresh weights for individual pieces in a particular assay but still a high degree of reproducibility from test to test. Repeats of all experiments reported here have given consistent results. Stock cultures of the soybean tissue have been maintained on a medium containing (mg/liter): KH2PO4, 300; KNO3, 1000; NH4NO3, 1000; Ca(NO3)2 .4H20, 500; MgSO4-7H20, 71.5; KCl, 65; MnSO4 .4H20, 14; NaFe ethylenediaminetetraacetate, 13.2; ZnSO4 7H20, 3.8; H3BO3, 1.6; Cu(NO3)2.3H20, 0.35; (NH4)6 Mo7024 *4H20, 0.1; i-inositol, 100; nicotinic acid, 0.5; pyridoxine HCI, 0.1; thiamin * HCl, 0.1; a-naphthaleneacetic acid, 2; kinetin, 0.5; sucrose, 30,000; and Bacto-agar, 10,000. The pH was adjusted to 5.8 (NaOH). A preparation to be tested for cell-division activity was added to this medium with the kinetin omitted, the pH was adjusted to 5.8 with NaOH or HCl, and sterilization was achieved by autoclaving. Each of four 125-ml Erlenmeyer flasks containing 50 ml of hardened medium was planted with four pieces of the stock soybean tissue. After growth for 28 days at 27°C and constant fluorescent lighting at about 40 ft-c, the pieces were weighed individually. The fresh weight averages indicate relative amounts of cell division. Extracts.-Frozen kernels (milk stage) of Zea mays, var. Golden Cross Bantam, were extracted by macerating in either cold or boiling 95 per cent ethanol. The final concentration of ethanol was adjusted to 70 per cent (assuming the weight of kernel was due entirely to water). The extract was cooled and the filtered precipitate discarded. The filtrate was used in all of the experiments. Chromatography.-Ethanol solutions or other preparations were streaked on sheets of Whatman #1 filter paper. For development, one of the following mix-