Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L) root tips for flow cytometric analysis and sorting

被引:44
作者
Lee, JH
Arumuganathan, K
Kaeppler, SM
Kaeppler, HF
Papa, CM
机构
[1] UNIV NEBRASKA,CTR BIOTECHNOL,LINCOLN,NE 68588
[2] UNIV NEBRASKA,DEPT AGRON,LINCOLN,NE 68583
[3] UNIV NEBRASKA,USDA ARS,WHEAT SORGHUM & FORAGE RES UNIT,LINCOLN,NE 68583
关键词
flow karyotype; hydroxyurea; plant chromosome sorting; trifluralin;
D O I
10.1139/g96-088
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The goal of this research was to optimize treatment parameters to synchronize the cell cycle of maize root tip meristem cells. Levels of hydroxyurea, a DNA synthesis inhibitor, were assessed for their utility in accumulating cells at the G1 phase of the cell cycle. Trifluralin, amiprophos-methyl, and colchicine were used to accumulate cells containing metaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxyurea for 18 h, incubating for 1 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 1 mu M trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection of seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a simple slicing procedure. The chromosome preparations were found to be suitable for flow cytometric characterization and sorting. Chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated on the basis of maize chromosome size.
引用
收藏
页码:697 / 703
页数:7
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