Kn. Zhao et al., STUDIES OF COTYLEDON PROTOPLAST CULTURES FROM BRASSICA-NAPUS, BRASSICA-CAMPESTRIS AND BRASSICA-OLERACEA .1. CELL-WALL REGENERATION AND CELL-DIVISION, Plant cell, tissue and organ culture, 40(1), 1995, pp. 59-72
Protoplasts isolated from cotyledons of a number of cultivars of Brass
ica napus, B. campestris and B. oleracea were cultured in different me
dia to study the characteristics of cell wall regeneration and cell di
vision at early stages of culture. Time course analysis using Calcolfl
uor White staining indicated that cell wall regeneration began in some
protoplasts 2-4 h following isolation in all cultivars. 30-70% of cul
tured cotyledon protoplasts exhibited cell wall regeneration at 24 h a
nd about 60-90% at 72 h after the initiation of culture. Results also
indicated that a low percentage (0.4-5.4%) of cultured cotyledon proto
plasts entered their first cell division one day after initial culture
in all twelve cultivars. The percentage of dividing cells increased l
inearly up to 40% from 1 to 7 day, indicating that cotyledon protoplas
ts of Brassica had a high capacity for cell division. Factors that inf
luence the level of cell wall regeneration and cell division during co
tyledon protoplast culture have been investigated in this study. Cotyl
edons from seedlings germinated in a dark/dim light regime provided a
satisfactory tissue source for protoplast isolation and culture for al
l Brassica cultivars used. The percentages of protoplasts exhibiting c
ell wall regeneration and division were significantly influenced by cu
ltivar and species examined, with protoplasts from all five cultivars
of B. campestris showing much lower rates of cell wall regeneration th
an those of B. napus and B. oleracea over 24-120 h, and with the level
s of cell division in B. napus cultivars being much higher than those
in B. campestris and B. oleracea over 1-9 days. The capacity of cell w
all regeneration and cell division in cotyledon protoplast culture of
the Brassica species appears under strong genetic control. Cell wall r
egeneration in protoplast culture was not affected by the culture medi
um used. In contrast, the composition of the culture medium played an
important role in determining the level of cell division, and the inte
raction between medium type and cultivars was very significant.