purine (2iP), and 0.3 mg/L indole-3-acetic acid (IAA) at
pH 5.8 (liquid or solid).
5. Loading solution: MS medium with vitamin, 13.7% (w/v)
sucrose, and 18.4% (w/v) glycerol (liquid).
6. Plant vitrification solution 3 (PVS3): MS medium with vitamin,
50% (w/v) sucrose, and 50% (w/v) glycerol [18] (liquid).
7. Washing solution: MS with vitamin and 41% (w/v) sucrose in
medium (liquid).
8. Washing solution for inflorescences: MS with vitamin, 27%
(w/v) sucrose, 2.0 mg/L 2iP, and 0.3 mg/L IAA at pH 5.8.
9. Regeneration medium for potato: MS medium with vitamin,
0.5 mg/L trans-zeatin riboside, 0.5 mg/L IAA, 0.2 mg/L
gibberellic acid (GA3), 3% sucrose, and 1% agar at pH 5.8 (see
Note 2).
10. Regeneration medium for mint, garlic, and shallot: MS
medium with vitamin, 0.1 mg/L 1-naphthaleneacetic acid
(NAA), 0.5 mg/L (2iP), 3% sucrose, and 1% agar at pH 5.8
(see Note 3).
3 Methods
3.1 Preparation
and Sterilization
of the Culture Media
1. Prepare the MS basal medium according to the instructions of
the manufacturer.
2. Add other components, such as sucrose and growth regulators,
as required. Use stock solutions for adding the growth regulators to reach the required final concentrations.
3. Adjust the pH of the media to 5.8 using 1 M HCl or
1 M KOH.
4. Add 1% (w/v) agar.
5. Dispense the media into suitable containers. Use culture tubes
to initiate plant propagation (10 mL culture medium) and jars
for the maintenance of shoots (50–90 mL culture medium).
6. Sterilize the media by autoclaving at 121
C for 20 min
(118 kPa steam pressure). When sterile plastic boxes are used,
autoclave the media and fill the boxes under sterile conditions.
7. Store the autoclaved media at 10
C for at maximum of
2 months. Before using, the media must be warmed up to
room temperature.
8. Store the stock solutions between À20 and 4
C.
Cryopreservation of Shoot Tips
651
pH 5.8 (liquid or solid).
5. Loading solution: MS medium with vitamin, 13.7% (w/v)
sucrose, and 18.4% (w/v) glycerol (liquid).
6. Plant vitrification solution 3 (PVS3): MS medium with vitamin,
50% (w/v) sucrose, and 50% (w/v) glycerol [18] (liquid).
7. Washing solution: MS with vitamin and 41% (w/v) sucrose in
medium (liquid).
8. Washing solution for inflorescences: MS with vitamin, 27%
(w/v) sucrose, 2.0 mg/L 2iP, and 0.3 mg/L IAA at pH 5.8.
9. Regeneration medium for potato: MS medium with vitamin,
0.5 mg/L trans-zeatin riboside, 0.5 mg/L IAA, 0.2 mg/L
gibberellic acid (GA3), 3% sucrose, and 1% agar at pH 5.8 (see
Note 2).
10. Regeneration medium for mint, garlic, and shallot: MS
medium with vitamin, 0.1 mg/L 1-naphthaleneacetic acid
(NAA), 0.5 mg/L (2iP), 3% sucrose, and 1% agar at pH 5.8
(see Note 3).
3 Methods
3.1 Preparation
and Sterilization
of the Culture Media
1. Prepare the MS basal medium according to the instructions of
the manufacturer.
2. Add other components, such as sucrose and growth regulators,
as required. Use stock solutions for adding the growth regulators to reach the required final concentrations.
3. Adjust the pH of the media to 5.8 using 1 M HCl or
1 M KOH.
4. Add 1% (w/v) agar.
5. Dispense the media into suitable containers. Use culture tubes
to initiate plant propagation (10 mL culture medium) and jars
for the maintenance of shoots (50–90 mL culture medium).
6. Sterilize the media by autoclaving at 121
C for 20 min
(118 kPa steam pressure). When sterile plastic boxes are used,
autoclave the media and fill the boxes under sterile conditions.
7. Store the autoclaved media at 10
C for at maximum of
2 months. Before using, the media must be warmed up to
room temperature.
8. Store the stock solutions between À20 and 4
C.
Cryopreservation of Shoot Tips
651