Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips
| ISSN: |
1432-2242
|
|---|---|
| Keywords: |
Key words Pisum sativum ; Cell-cycle synchronization ; Plant chromosome and nuclei isolation ; Flow cytometric analysis and sorting ; PRINS
|
| Source: |
Springer Online Journal Archives 1860-2000
|
| Topics: |
Biology
|
| Notes: |
Abstract A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 µM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of and respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.
|
| Type of Medium: |
Electronic Resource
|
| URL: |