Development and application of an optimized drop-slide technique for metaphase chromosome spreads in maize

Abstract

ABSTRACTChromosome spreads are important for complex molecular cytogenetic studies. An adequate chromosome spreading method for identification and isolation of the maize B chromosome, however, has not been reported. We used the maize inbred lines, B73 and Mo17, the hybrid YD08 line and the landrace DP76 to develop an optimized chromosome spreading method. We investigated the effects of four treatment factors on the quality of metaphase chromosome spreads using a factorial analysis of variance. Optimal conditions for metaphase spreading were identified using regression analysis based on multifactor orthogonal design of four treatment factors with five levels for each factor. We developed optimal conditions for metaphase spreading as follows: nitrous oxide treatment for 2 h, glacial acetic acid fixation for 2 h, enzyme hydrolysis for 6.0 h, and a drop height of 35 cm for cell suspension. We obtained high quality metaphase chromosome spreads with large metaphase areas, large numbers of chromosomes, few chromosome overlaps and high frequency of intact metaphases. Our optimized drop-slide procedure was markedly better than the traditional flame smear technique. We identified 487 B chromosomes in three forms from maize landraces from Southwest China. We found no relation between the C-band number and B chromosome. Single B chromosomes also were isolated directly from a metaphase chromosome drop-slide using a micromanipulator. Our optimized method provides a simple, efficient and reproducible procedure for preparing high quality plant chromosome spreads.