April - June 2007 - Kasetsart University

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were then gently filtered through a 60 and 40 µm
nylon mesh to remove undigested tissue and
debris. The filtrate was centrifuged for 5 min at
750 rpm. The same process was repeated once
more. The protoplast pellets were purified by
floating on 20 % sucrose solution and centrifuged
at 800 rpm for 10 min. The purified protoplasts
were further washed twice with washing solution.
Protoplast yield was estimated by a
hemocytometer (Gleddie, 1995). Viability of
protoplasts was assessed using 0.01% (w/v)
fluorescein diacetate staining (FDA) (Sigma,
USA) followed by observation with a UV
fluorescence microscope (Widholm, 1972).
2. Concentration of osmoticum
solution
The best result of experiment 1 was used
in experiment 2. One gram of four-week-old in
vitro leaves was incubated in 5 ml of filtersterilized
enzyme mixture, 2% (w/v) Cellulase
Onozuka R-10 (Yacult Honsha, Japan), 0.2% (w/
v) Pectolyase Y-23 (Kyowa Chemical, Japan) in
washing solution of four varied mannitol
concentrations; 0.4, 0.5, 0.6 or 0.7 M. The
protoplasts were isolated and purified as
previously described. Protoplast yield and
viability were determined.
3. Incubation period
The best result of experiment 2 was used
in experiment 3. One gram of four-week-old in
vitro leaves was incubated in 5 ml of enzyme
mixture, 2% Cellulase Onozuka R-10, 0.2%
Pectolyase Y-23, 0.5 M mannitol, 2.5 mM
CaCl 2.2H 2O and 5 mM MES. The digestion was
performed for 3, 4, 5 or 6 hr in the dark. The
protoplasts were then harvested and purified as
previously described. Protoplast yield and
viability were determined.
4. Age of leaves
One gram of four-, six-, eight- and ten-
Kasetsart J. (Nat. Sci.) 41(2)
week-old leaves was isolated using enzyme
mixture, 2% Cellulase Onozuka R-10, 0.2%
Pectolyase Y-23, 0.5 M mannitol, 2.5 mM
CaCl 2.2H 2O and 5 mM MES, and incubated in
the dark on a gyratory shaker (40 rpm) at 25°C for
4 hr. The protoplasts were then harvested and
purified as previously described. Protoplast yield
and viability were determined.
5. Sucrose concentrations for
purification
One gram of four-week-old in vitro
leaves was incubated in 5 ml of enzyme mixture,
2% Cellulase Onozuka R-10, 0.2% Pectolyase Y-
23, 0.5 M mannitol, 2.5 mM CaCl 2.2H 2O and 5
mM MES. The protoplasts were harvested and
purified with varying levels of sucrose solution;
16, 18, 20 and 22 % and centrifuged at 800 rpm
for 10 min. Protoplast yield and viability were
determined.
Factors affecting the protoplast culture
1. Culture medium
The purified protoplasts at the density of
5 × 10 5 protoplasts/ml were cultured in two kinds
of liquid culture media; MS (Murashige and
Skoog, 1962) and KM8P (Kao and Michayluk,
1975) containing 0.2 mg/l 2,4-D, 1 mg/l NAA,
0.5 mg/l zeatin, 0.15 M sucrose and 0.3 M
mannitol incubated at 25°C in the dark. The cell
division was observed periodically with an
inverted microscope. The plating efficiency (% of
plated protoplasts which were under cell division)
and the survival rate of protoplasts were
determined after 10 days of culture.
2. Plant growth regulators
The protoplasts were cultured in liquid
MS medium containing various combinations of
growth regulators. Three culture media tested for
protoplast culture were M1 (1.5 mg/l NAA and
0.4 mg/l BA), M2 (0.2 mg/l 2,4-D, 1 mg/l NAA
and 0.5 mg/l zeatin) and M3 (0.2 mg/l 2,4-D, 2