**4.4 Apple tree**

338 Current Frontiers in Cryopreservation

Potato explants (Fig. 1b.) were multiplied by nodal cuttings in plastic boxes (Vitro Vent container, Duchefa) on 100 ml modified MS medium (Grospietsch *et al*., 1999) with 7 g L-1 agar and 30 g L-1 sucrose, without myo-inositol and phytohormones, with a decreased amount of nitrogen at pH 5,5. Nodal cuttings were cultivated at 22 ± 1 °C, 80 µmol m-2 s-1 and photoperiod 16/8 h light/dark (L/D) ( Fig. 1b). Subculture interval was 3-4 weeks.

Nodal cuttings were planted in the same conditions as the pre-cultured plants but only 50 ml medium was used per one box. After 4 day pre-culture, lateral buds elongated to at least 1 mm (Fig. 2b). Subsequently, 25 ml 2 M sucrose was added into each container and

> onto aluminum foils (20 x 6 x 0.05 mm) Dehydration above silicagel for 1,75 – 2 h.

laboratory temperature.

0,2 mg L-1 GA3)

cryopreservation.

Plunging aluminum foils into liquid nitrogen

Stored in cryovials (two foils with 20 shoot tips per vial).

Alluminum foils plunged rapidly into the water bath at a

recovery as a new explant development (Fig. 12b.) Plant regeneration was evaluated 2 and 8 weeks after

Maternal plants were cultivated on a multiplication medium without phytohormones at 22 ± 1 °C, 80 µmol m-2 s-1, photoperiod 16/8 h (L/D); subculture interval was 8 weeks. Modified solid medium (Murashige & Skoog, 1962) without casein and myoinositol, with decreased amount of nitrogen (25 % (w/v) of NH4NO3 and 50 % (w/v) of KNO3 of the original Murashige and Skoog medium), with 40 g L-1 glucose, pH 5,5 without phytohormones was

Transfer immediately onto the recovery medium (Grospietsch *et al*., 1999) with the same composition as the pre-culture medium but with phytohormones (0,5 mg L-1 IAA + 0,.5 mg L-1 Kinetin +

Survival was defined by shoot tip growth and by green color and

Shoot tips (Fig. 2b.) (1-2 mm) were transferred onto a filter paper moistened with 14 ml 0,7 M sucrose and phytohormones of the same composition as in a recovery medium (0,5 mg L-1 IAA + 0,5 mg L-1 Kinetin + 0.2 mg L-1 GA3) at 22 ± 1 °C and photoperiod 16/8 h (L/D) and shielded with a leaf of paper for overnight. The second day the shoot tips were transferred (20 tips per foil)

explants were cultivated at the same conditions for the next 5-6 days.

Cryoprotocol Steps The Procedure

Survival and regeneration evaluation

Table 2. Cryopreservation steps of potato

used as the multiplication medium (MSH).

Shoot tips dehydration

Cryopreservation

Thawing

**4.3 Hop** 

**4.2 Potato** 

*In vitro* plants (Fig. 1d) were cultivated in 100 ml Ehrlenmeyer flasks with 20 ml of MS medium, 3% (w/v) sucrose, 6 g L-1 agar, supplemented with GA3 1 mg L-1, BAP 1 mg L-1, IBA 1 mg L-1, at 20 ± 1 °C, 8/16 (L/D) photoperiod of light intensity 100 µmol m-2 s-1.


Table 3. Cryopreservation steps of apple tree
