A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures.
A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase - contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.
Division of Cellular Biology, Biotechnology Institute, Akita Prefectural College of Agriculture, Minami 2-2, Ohgata, 010-04, Akita, Japan.
K
K
Masuda
S
S
Takahashi
K
K
Nomura
M
M
Inoue