**4.1 Garlic**

The unripe topsets were surface sterilized by chloramines and 75% ethanol and from this point, all preparations were performed under sterile conditions using sterile instruments and culture media in a laminar flow box. Opening the surface sterilized topset in sterile condition, all the inside structures were sterile (Fig. 1a). The sterile unripe bulblets were removed and cuts were made to the clusters or clumps of 3-8 bulbils. The bulbils varied in the thickness (approximately 2 mm) and in the length (3-5 mm) depending on the genotype and stage of ripening. Inside the unripe bulbils shoot tips were (Fig. 2a) with meristematic tissue.

Pre-culture of unripe clusters of bulbils was done on the MS culture medium (Murashige & Skoog, 1962) with 0,2 mg L-1 BAP and 0,02 NAA mg L-1 with 10 % sucrose for 20-24 h at 22 °C and 16 h light in Petri dishes sealed with Parafilm.

Fig. 1. Plants grown in *in vitro* conditions ready for dissection of shoot tips: (a) Garlic. Scale bar, 5 mm; (b) Potato. Scale bar, 1 mm; (c) Hop. Scale bar, 5 mm and (d) Apple tree. Scale bar, 10 mm.

*al*., 1999; Hirai & Sakai, 1999). The encapsulation of shoot tips prolongs the dehydration period up to seven hours at a low relative humidity. The alginate beads without shoot tips had approximately the same dehydration-time curve. On the contrary no encapsulated shoot tips were completely dehydrated up to 1hour. The static dehydration of shoot tips was

The unripe topsets were surface sterilized by chloramines and 75% ethanol and from this point, all preparations were performed under sterile conditions using sterile instruments and culture media in a laminar flow box. Opening the surface sterilized topset in sterile condition, all the inside structures were sterile (Fig. 1a). The sterile unripe bulblets were removed and cuts were made to the clusters or clumps of 3-8 bulbils. The bulbils varied in the thickness (approximately 2 mm) and in the length (3-5 mm) depending on the genotype and stage of

Pre-culture of unripe clusters of bulbils was done on the MS culture medium (Murashige & Skoog, 1962) with 0,2 mg L-1 BAP and 0,02 NAA mg L-1 with 10 % sucrose for 20-24 h at

Fig. 1. Plants grown in *in vitro* conditions ready for dissection of shoot tips: (a) Garlic. Scale bar, 5 mm; (b) Potato. Scale bar, 1 mm; (c) Hop. Scale bar, 5 mm and (d) Apple tree. Scale

ripening. Inside the unripe bulbils shoot tips were (Fig. 2a) with meristematic tissue.

done over the various saturated salt solutions.

22 °C and 16 h light in Petri dishes sealed with Parafilm.

a b

c d

**4. Cryoprotocols** 

**4.1 Garlic** 

bar, 10 mm.

Fig. 2. The size and shape of shoot tips used for cryopreservation. (a) Garlic. Scale bar, 1 mm; (b) Potato. Scale bar, 0,25mm; (c) Hop. Scale bar, 0,25 mm and (d) Apple tree. Scale bar, 1 mm.


Table 1. Cryopreservation steps of garlic

Comparison of Cryopreservation Methods of

Cryoprotocol Steps The Procedure

**4.4 Apple tree** 

Shoot tips dehydration

Cryopreservation

**5. Thermal analysis** 

Thawing

Vegetatively Propagated Crops Based on Thermal Analysis 339

Nodal cuttings were planted in the same conditions as during explant multiplication but only 50 ml medium was used per one box (Fig 1c). After 7-10 d pre-culture, lateral buds elongated to 1-2 mm. Then the explants were transferred into cold acclimation conditions at 4 °C for 7-10 days. Subsequently 25 ml 0,7 M sucrose was added into each container and

*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,

One to two week subcultivation on fresh MS medium,

(8/16, L/D) of light intensity 25 µmol m-2 s-1

Beads are gently dried on a sterile filter paper

Differential scanning calorimetry (DSC) belongs to thermal methods that can be used for measurement and determination of phase and glass transitions for cryopreservation. In principle, the DSC measures the temperatures and heat flows associated with transitions in

Cold hardening for 4-6 weeks at +4± 1 °C, short photoperiod

Petri dish with 16 ml of MS medium and poured with 16 ml of

Encapsulation of shoot tips (Fig. 2d. ) with meristematic cells in 3% w/v dipped into alginate in 0,75M sucrose for 10 min and then drop into 0,1 M CaCl2 in 0,6 M sucrose for 10 min to net

Additional dehydration in the laminar flow box at laboratory temperature and dehydrated for different time up to 4 -5 hours, Placed in 2 ml cryovials and plunged in liquid nitrogen.

Shoot tips with beads were warmed up by plunging cryovials in

Survival was defined by shoot tip growth and by green color and regenerated as a new explant development (Fig. 12d.)

IBA 1 mg L-1, at 20 ± 1 °C, 8/16 (L/D) photoperiod of light intensity 100 µmol m-2 s-1.

2M sucrose solution for 48 hours

the alginate and form a bead

40 °C water

Survival and regeneration evaluation

Table 3. Cryopreservation steps of apple tree

explants (Fig. 1c.) were cultivated at the same conditions for the next 7-10 days. Cryopreservation steps for hop shoot tips were the same as for potato (see Tab. 2).

### **4.2 Potato**

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


Table 2. Cryopreservation steps of potato

#### **4.3 Hop**

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 used as the multiplication medium (MSH).

Nodal cuttings were planted in the same conditions as during explant multiplication but only 50 ml medium was used per one box (Fig 1c). After 7-10 d pre-culture, lateral buds elongated to 1-2 mm. Then the explants were transferred into cold acclimation conditions at 4 °C for 7-10 days. Subsequently 25 ml 0,7 M sucrose was added into each container and explants (Fig. 1c.) were cultivated at the same conditions for the next 7-10 days.

Cryopreservation steps for hop shoot tips were the same as for potato (see Tab. 2).
