Abstract:

Objective: The purpose of this study is to develop a technique for high quality chromosome preparation of root tips, in order to improve the fluorescence in situ hybridization(FISH) system for 45S rDNA and 5S rDNA of Hydrangea macrophylla 'Endless Summer' with dispersed and good morphology chromosomes in the cell division phase, which would provide a basis for improving the molecular cytogenetic study of Hydrangea. Method: Root tips taken from semi-lignified branches of H. macrophylla 'Endless Summer' through hydroponic culture were pre-treated with 1 MPa nitrous oxide, 0.7 mmol·L^-1 cycloheximide and 2 mmol·L^-1 8-hydroxyquinoline respectively. Chromosome was prepared after meristem enzymolysis of the pre-treated root tips with a mixture of 4% cellulase and 2% pectinase, and the metaphase chromosome was hybridized in situ with 45S rDNA and 5S rDNA probes. Result: 1) Materials pre-treated with 1 MPa nitrous oxide for 0.5 h, their chromosomes were still insufficiently condensed and too long for analysis; those pre-treated for 1 h, the chromosomes condensation were appropriate, and the centromeres of most chromosomes could be identified; pre-treatment for 2 h, the chromosomes were over-condensed and punctate. Materials pre-treated with 0.7 mmol·L^-1 cycloheximide for 1 h and 3 h, their chromosomes were insufficiently condensed; pre-treatment for 5 h, the chromosomes were properly condensed, but the centromere positions of most chromosomes were not discernible. All materials pre-treated with 2 mmol·L^-1 8-hydroxyquinoline for 2 h, 4 h or 6 h, their chromosomes condensation were all appropriate, but the chromosome centromeres could not be identified. So 1 h treatment with 1 MPa nitrous oxide is the most suitable pre-treatment condition for H. macrophylla 'Endless Summer' root tips. 2) Three enzymatic treatment time gradients were used. Chromosomes digested for 0.5 h, their cytoplasm could be observed obviously around the metaphase and the nucleus; chromosomes digested for 1 h, their background were clean and the chromosomes well dispersed; when digestion time expanded to 2 h, the chromosomes were easily lost and many incomplete mitotic phases were observed. Therefore, the most suitable treatment for H. macrophylla 'Endless Summer' root tip was digested in the mixture of 4% cellulase and 2% pectinase at 37℃ for 1 h. 3) H. macrophylla 'Endless Summer' is diploid with 36 chromosomes (2n=2x=36). The FISH of 45S rDNA long and short probes showed similar effects, both produced strong hybridization signals which located at the end of the short arm of a pair of acrocentric chromosomes. Both the long and the short 5S rDNA probes only revealed weak FISH signals, but the detection rate of the long probes was higher than that of the short probes. The 5S rDNA located at the long arm ends of one pair of telocentric chromosomes. Conclusion: This study successfully established a high quality metaphase chromosome preparation and rDNA physical localization technical system for H. macrophylla. This system can efficiently analyze and determine the chromosome number and rDNA distribution in Hydrangea plants. This can be used in Hydrangea resources identification and provide a basis and guidance for Hydrangea hybrid breeding.

Key words: Hydrangea macrophylla, chromosome preparation, rDNA, fluorescence in situ hybridization(FISH)