2 Materials
2.1 In Vitro Culture
Establishment,
Maintenance,
and Propagation
2.1.1 General Equipment
1. Laminar flow bench (Scanlab “Fortuna IVF,” MTG, Bruckberg, Germany).
2. Incubators (CLF Plant Climatics GmbH, Wertingen, Germany) to adjust light and temperatures between À5 and 25
C.
3. Dissection binocular (Stemi 305; Carl Zeiss Microscopy
GmbH, Jena, Germany).
4. Heat sterilizer (250
C).
5. Autoclave.
6. UV light.
7. Gas safety burner to flame culture tubes.
8. Glass bead sterilizer to sterilize instruments.
9. Magnetic stirrer with heating.
10. pH meter.
11. Balance.
12. Benchtop open air shaker.
13. Vacuum pump and desiccator.
14. Sterile instruments: scalpel, forceps, pipette, counter, syringe,
and needle.
2.1.2 Surface
Sterilization of Plant
Material Derived from
the Field or Greenhouse
1. Tap water.
2. Ethanol 70% (v/v).
3. Hand disinfection solution.
4. Sodium hypochlorite solution [NaClO] 12% Cl, diluted 1:4 (v:
v) with deionized water.
5. Surface detergent Tween 20.
6. Autoclaved deionized water.
7. Autoclaved round filter papers, grade 598, 90 mm for preparation (Whatman GmbH, Dassel, Germany).
8. Erlenmeyer flasks, 100 mL.
2.1.3 In Vitro
Maintenance
and Propagation
1. In vitro cultures of the former subculture as sources of explants.
2. Autoclaved round filter paper, grade 595, 125 mm (Whatman
GmbH, Dassel, Germany).
3. Glass Petri dishes (150 mm).
4. Culture tubes 3 cm, 10 cm long, glass vials (750 mL), oval
culture boxes with “breathing” hermetic cover (Duchefa Biochemie BV, Haarlem, the Netherlands).
5. Aluminum foil for caps, thickness 50 μm.
Cryopreservation of Shoot Tips
649
2.1 In Vitro Culture
Establishment,
Maintenance,
and Propagation
2.1.1 General Equipment
1. Laminar flow bench (Scanlab “Fortuna IVF,” MTG, Bruckberg, Germany).
2. Incubators (CLF Plant Climatics GmbH, Wertingen, Germany) to adjust light and temperatures between À5 and 25
C.
3. Dissection binocular (Stemi 305; Carl Zeiss Microscopy
GmbH, Jena, Germany).
4. Heat sterilizer (250
C).
5. Autoclave.
6. UV light.
7. Gas safety burner to flame culture tubes.
8. Glass bead sterilizer to sterilize instruments.
9. Magnetic stirrer with heating.
10. pH meter.
11. Balance.
12. Benchtop open air shaker.
13. Vacuum pump and desiccator.
14. Sterile instruments: scalpel, forceps, pipette, counter, syringe,
and needle.
2.1.2 Surface
Sterilization of Plant
Material Derived from
the Field or Greenhouse
1. Tap water.
2. Ethanol 70% (v/v).
3. Hand disinfection solution.
4. Sodium hypochlorite solution [NaClO] 12% Cl, diluted 1:4 (v:
v) with deionized water.
5. Surface detergent Tween 20.
6. Autoclaved deionized water.
7. Autoclaved round filter papers, grade 598, 90 mm for preparation (Whatman GmbH, Dassel, Germany).
8. Erlenmeyer flasks, 100 mL.
2.1.3 In Vitro
Maintenance
and Propagation
1. In vitro cultures of the former subculture as sources of explants.
2. Autoclaved round filter paper, grade 595, 125 mm (Whatman
GmbH, Dassel, Germany).
3. Glass Petri dishes (150 mm).
4. Culture tubes 3 cm, 10 cm long, glass vials (750 mL), oval
culture boxes with “breathing” hermetic cover (Duchefa Biochemie BV, Haarlem, the Netherlands).
5. Aluminum foil for caps, thickness 50 μm.
Cryopreservation of Shoot Tips
649