An excision and squash technique for analysis of the cell cycle in the root quiescent centre of maize

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The quiescent centre of primary roots of Zea mays L. (cvs LG 11 and Golden Bantam) consists of a population of slowly cycling diploid cells. These metabolically inactive cells may be triggered to synthesise DNA under specific conditions and constitute a good model for studying the regulation of the cell cycle. An excision and squash technique is described for the quiescent centre which, when coupled with Feulgen and fluorochrome staining, allows nuclear DNA contents to be determined by microdensitometry in less than a day. This technique was coupled with experiments in which excised quiescent centres were placed on solid culture medium into which hormones and radioactive DNA precursors were incorporated. In complementary and control experiments [methyl‐3H]thymidine was supplied to intact roots (with or without root caps) by means of fibre‐glass cubes as donors.Progression of the cell cycle was followed by microdensitometry and autoradiography. Distribution of DNA content was similar in excised and squashed quiescent centres and in histological sections. Labelling experiments showed that the quiescent centre is made up of cells that differ in their cycle time. While some labelled cells had reached mitosis after 8 h, others were still in G2 after 16 h of continuous labelling. Excision and culture of the quiescent centre resulted in a dramatic activation of the cell cycle as shown by the labelling index that increased from 15% in intact roots fed during 16 h with [methyl‐3H]thymidine to 31% in excised quiescent centres to which radioactive precursor was supplied during the same time. Supplying hormones (50 μM abscisic acid [ABA], 0. 1 μM zeatin, 1 μM zeatin riboside) to quiescent centres via the culture medium restored their inactivity (labelling indices dropped to 1% after ABA. and to 11% after zeatin and zeatin riboside treatments. respectively).