Establishment in vitro cultures
Shootlets cultures of globe artichoke (Green Globe cultivar obtained from Vegetable Research Department, Horticulture Research Institute, Agricultural Research Center, Egypt) were in vitro established on Murashige and Skoog [11] (MS) medium supplemented with 1 mg/l 6-benzyladenine (BA) according to method developed by Bekheet et al. [12]. Subcultures were performed every 4–5 weeks, in order to get enough mother stock cultures (Fig. 1a). Callus cultures were initiated from leaf explants as described by Bekheet et al. [13] and maintained on MS medium amended with 0.1 mg/l Naphthaleneacetic acid (NAA), 0.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D), and 0.5mg/l Kinetin (kin) by transferring onto fresh medium four weeks intervals [Fig. 1b].
Cryopreservation procedure
Shoot apices (1 cm) and callus inocula (250 mg) of globe artichoke taken from in vitro grown cultures (after the third subculture) were used as explants for cryopreservation experiments. The explants were precultured on filter-sterilized loading solution (2 M glycerol and 0.4 M sucrose dissolved in MS medium) using Petri dishes and incubated at 25 °C for 24 h. Shoot apices were directly immersed into the solutions, while callus inocula were wrapped in a small strip of sterilized cotton. After the loading treatment, the explants were transferred into 2 ml polypropylene sterile cryovials and exposed to 10% of dimethylsulfoxide (DMSO) in MS medium or Plant Vitrification Solutions 2 [PVS2; 15% (w/v) ethylene glycol, 15% (w/v) DMSO, 30% (w/v) glycerol, 0.4 M sucrose in MS medium, Sakai et al. [8] for different exposure times, i.e., 20, 40, 60, or 80 min at 0 °C. For each treatment at least ten cryotubes were employed. The explants were then suspended into cryotubes with 0.5 ml fresh of the two vitrification solutions and the tubes were directly immersed into liquid nitrogen (LN) (at − 196 °C) and kept for at least 48 h.
Thawing and recovery
After cryostorage, cryotubes were taken from liquid nitrogen container (Fig. 1c) and rapidly thawed in a beaker filled with sterile water at 40 °C for about 1.5 to 2 min until most of the ice has melted. After rewarming, the cryoprotection solution was removed from the cryotubes; the explants were rinsed with MS liquid medium for 20 min at room temperature and blotted on filter paper. Then, the explants were transferred onto recovery media [MS medium + 1 mg/l BA for shootlets regeneration (Fig. 1d) and MS medium + 0.1 mg/l NAA, 0.5 mg/l 2,4-D, and 0.5 mg/l kin for callus growth (Fig. 1e)], and then all the cultures were incubated under standard conditions of illumination and temperature.
Survival and regrowth of cryopreserved shoot apices
Assessment of survival percentages and regeneration of the cryopreserved shoot apices were carried out after four weeks of culturing on the recovery medium. The survival was recorded (after 2 weeks) based on visual observation as the formation of green tissue developing from the shoot apices (%, no of green shoot apices/total no of shoot apices in freezing × 100). Regeneration was recorded (after 4 weeks of culturing on recovery medium) when a minimum of two leaves arise from the emerging bud ((%, no of shoot apices that elongated or produced new shoots/total no of shoots × 100). Also, a number of proliferated shootlets was recorded after culturing for 12 weeks on the regeneration medium.
Survival and regrowth of cryopreserved callus cultures
Four weeks after inoculation on callus maintenance medium, survival was evaluated based on visual observation by examining the colors of callus cultures. The creamy ones (with increase of the volume) had survived; the brown ones had died. Also samples for each treatment were subjected to regrowth. Fresh weight and growth value of the survival callus cultures were determined after 8 weeks of inoculation.
$$ \mathrm{Growth}\ \mathrm{Value}=\frac{\mathrm{Final}\ \mathrm{fresh}\ \mathrm{weight}-\mathrm{Initial}\ \mathrm{fresh}\ \mathrm{weight}}{\mathrm{Initial}\ \mathrm{fresh}\ \mathrm{weight}} $$
Microscopy
To assess the damage caused by the pre-treatment and freezing process, sample of the cryopreserved callus exposed for different times (20, 40, 60, or 80 min) to PVS2 were taken and then fixed in Karnovsky’s solution [14] at room temperature before being washed in 0·05 m Sörensen phosphate buffer pH 72. Samples were progressively dehydrated through ethanol solutions to a final concentration of 100 % and then photographed under a light microscope (Nicon, Japan Labophot-2 Microscope) equipped with KL 2500 LCD light sources (Nicon, Japan) was used to analyze the cell growth.
Tissue culture medium and incubation conditions
Tissue culture media were solidified with 0.7% agar and supplemented with 30 g/l sucrose, 100 mg/l myo-inositol, 1 mg/l pyridoxine-HCl, 1 mg/l nicotinic acid and 0.2 mg/l thiamine-HCl. The pH was adjusted to 5.8 before autoclaving at 121 °C and 1.5 Ib/M2 for 25 min. In all treatments, the growth regulators were added to the culture medium prior to autoclaving. Cultures were normally maintained at 25 ± 2 °C and 16 h photoperiod provided by white fluorescent tubes (3000 lux light intensity).
Experimental design and statistical analysis
The experiments were set up as a separate completely randomized design and repeated two times using. Data were statistically analyzed using standard error (SE) according to the method described by Snedecor and Cochran [15]. The results are presented as means of 25 replicates.
