A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension.
BACKGROUND
Meiotic chromosome preparation is a key step in plant meiotic research. Pollen mother cell (PMC) wall elimination is beneficial to cytogenetic experimental procedures. Without wall interference, these procedures are easier and more successful. In existing methods it is difficult to eliminate PMC walls completely and uniformly. In this paper, we present an improved method for digesting PMC walls, and one for providing massive chromosomal spreads on a slide for other cytogenetic experimental procedures.


RESULTS
Three plants were selected to exhibit the modified meiotic chromosome preparation method. PMCs were dispersed as single cells and incubated in a mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase) for 1.5-2.5 h. In total, 28.28 % cells were lost during this process. There were 800-1900 spreads on every slide and no PMC wall interference was found on any of the slides. The spreads were also evenly distributed on the slides. More spreads were obtained when PMC and protoplast densities in the suspension were increased. All three plants' spreads were successfully used to locate a 5 s rDNA conserved sequence. The Nicotiana hybrid's spreads were successfully used to identify the hybrid's parental genome.


CONCLUSIONS
This is an alternative method for meiotic chromosome preparation. Through this method, PMC walls can be completely and uniformly eliminated, and hundreds of spreads on every slide can be obtained. These spreads can be successfully used for DNA in situ hybridization.
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Jiangbo
J
Dang
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Qian
Q
Zhao
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Xing
X
Yang
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Zhi
Z
Chen
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Suqiong
S
Xiang
Southwest University, College of Horticulture and Landscape, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715 China.
Guolu
G
Liang
Meiosis; PMC wall; Pollen mother cells; Protoplast; Suspension