Author: Fu Ying, Sun Meng-xiang, Yang Hong-yuan and Zhou Chang
J Integr Plant Biol 1997, 39 (8): -.
The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var.
macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10%
mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central
cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion
products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high
concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central
cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic
maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component
cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded
with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with
1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water,
and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell
and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to
develop into small cell clusters.