Supplementary Figure 1
Fig. S1: Total viable protoplast yield from 50mL enzyme digest as a function of incubation period. The optimal
yield was obtained earliest at 12 hours of incubation under conditions used. Error bars represent standard
deviation from the mean, n= 3.
1
Supplementary Figure 2
A
B
Fig. S2: (A) FDA staining of protoplasts to examine viability via confocal microscopy, (B) differential
interference contrast field view of protoplasts from A. Bar = 100µm
2
Supplementary Data Appendix 1
Salt Medium (Modified KM Medium)
This protoplast-sourced callus regeneration medium is adapted from that described by Kao
and Michayluk (1975)
(1) KM Mineral salts (mg/l) – from Duchefa, Belgium (dry powder)
(2) Vitamins (mg/l) – from Duchefa, Belgium (1000X stock added after autoclaving)
(3) Glucose (6.8% w/v) & Mannitol (4% w/v)
(4) Sugars and sugar alcohols
Sucrose
Fructose
Ribose
Xylose
Mannose
1X
250
250
250
250
250
100 X Stock
50mL (mg)
1250
1250
1250
1250
1250
(5) Organic acids & Addenda
(pH adjusted to 5.5 with NH4OH)
Sodium pyruvate
Citric acid
Casein hydrolysate
(Enzymatic)
D-Calcium
Pantothenate
(6)
2,4-D
BAP
NAA
1X
20
40
250
1
0.2 mg/L
0.5 mg/L
1.0 mg/L
1X
Rhamnose
Cellobiose
Sorbitol
Mannitol
250
250
250
250
50mL (mg)
1250
1250
1250
1250
100 X Stock
50mL (mg)
100
Malic acid
200
Fumaric acid
1250
Folic acid
Ascorbic acid
5
Choline Cl
Riboflavin
1X
40
40
0.4
2.0
1.00
0.20
50mL (mg)
200
200
2
10
5
1
(stock prepared earlier)
(stock prepared earlier)
(stock prepared earlier)
To make 1000mL of KM (KOM) Medium: To dry weighed KM basal salts, glucose, mannitol
and vitamin powders add 10mL of Sol 4, and 10mL of Sol 5. Add 980mL dH20.
The pH of the medium is set to 5.6 (using NaOH). The medium is autoclaved before use.
3