Cryopreservation and Acclimatization of Lycopersicon esculentum ( Mill . ) Genotypes

Romanian tomato (Lycopersicon esculentum Mill.) cultivars have been cryopreserved by encapsulation-dehydration and successfully acclimatized to ex vitro growth conditions. Shoot tips were excised from in vitro grown plants then precultured for 24 h in various sucrose concentrations, dehydrated up to 6 h in laminar air flow prior to direct immersion in liquid nitrogen (−196 °C) for 24 h. Different parameters have been studied: the effects of osmoprotection and desiccation duration on the regrowth of cryopreserved shoot tips, the effects of various IBA concentrations on rooting and the ex vitro acclimatization of plants recovered from liquid nitrogen. The highest frequency of regrowth (72% cv. ‘Pontica’) was obtained when encapsulated explants were precultured in 0.5 M sucrose and the moisture content (fresh weight basis) of alginate beads was 23%. The highest rooting rates (58% to 77%) for all cultivars were observed for shoots grown on MS medium supplemented with 1.0 mg/l IBA. The rooted plants could be easily acclimatized ex vitro with up to 100% survival.


Introduction
Lycopersicon esculentum (Mill.) is one of the most extensively studied species not only for their importance as crop species but also as model system for genetic, molecular or physiological analyses (Gubiš et al., 2004).Various aspects of in vitro grown tomato cultures have been studied including micropropagation, selection of cell lines resistant to biotic / abiotic stress and production of haploids (Bhatia et al., 2004) or cryopreservation (Zevallos et al., 2013).Breeding efforts have raised the need for the conservation of valuable tomato germplasm (Grout and Crisp, 1995).Advances in the characterization of its genetic diversity led to ex situ plant conservation (Bauchet and Causse, 2012).Although tomato seeds show orthodox storage behaviour and can be maintained at −20 °C (Ellis and Roberts, 1981), loss of viability over time is inevitable due to biochemical activity, which is not completely stopped at this temperature (Roberts and Ellis, 1984;Stanwood, 1985).For the long-term conservation of genetic resources the availability or development of reliable and cost effective strategies and the subsequent regeneration of plants following storage are basic requirements (Kaviani, 2010(Kaviani, , 2011)).Cryopreservation techniques optimized for differentiated plant material contributed significantly to the conservation of various plant species (Lambardi et al., 2000;Sakai and Engelmann, 2007;Ozudogru et al., 2011).Methods involving alginate encapsulation followed by desiccation have been applied to numerous plant species (González-Arnáo and Engelmann, 2006;Harding et al., 2009;Engelmann, 2011;Cruz-Cruz et al., 2013) since their development by Fabre and Dereuddre (1990).
Cryopreservation protocols can be used as such or adjusted for the purpose of cryotherapy which is emerging as a powerful technique for virus elimination and was successfully used to eradicate severe pathogens in different plant species (Wang et al., 2009).Therefore, the establishment of an efficient cryopreservation protocol could be a prerequisite for cryotherapy and virus eradication in tomato.The aim of our work was (1) to establish a protocol for cryopreservation of five elite Romanian tomato cultivars by encapsulationdehydration and (2) to acclimatize plants regenerated from low temperature storage to ex vitro growth conditions.

Plant material and growth conditions
For initiation of in vitro cultures, tomato (Lycopersicon esculentum Mill., cvs. 'Capriciu', 'Darsirius', 'Kristin', 'Pontica' and 'Siriana') seeds were surface sterilized with 75% Clorox for 15 min and rinsed three times with sterile distilled water.The seeds were then germinated on half-strength (1/2) Murashige and Skoog (1962) (MS) medium supplemented with 30 g/l sucrose and solidified with 7.6 g/l agar.The pH of the medium was adjusted to 5.7 before autoclaving.The cultures were maintained in the dark for four days and then transferred to 16 h light/8 h dark photoperiod (36 μmol/m 2 /s photosynthetic removed for 1 hour, followed by one additional hour every day and after 10 days the plants were transferred to soil in pots and exposed to normal ambient light with daytime illumination at 22±1 °C.

Assessment of recovery and statistical analyses
Survival and regrowth following cryopreservation was assessed 15 and respectively 30 days after rewarming and transfer to regeneration medium.For each treatment of the cryopreservation protocol three replicates of 10-12 explants were used and the experiments were performed twice.Encapsulated explants treated with sucrose and desiccated but not exposed to liquid nitrogen were used as controls.The frequency of survival (shoot tips which developed green tissue) and regrowth (shoot growth and development, expansion of leaves from the original shoot tip) were calculated according to the formulas: Survival (%) = (∑ surviving shoot tips / ∑ number of total shoot tips) * 100 Regrowth (%) = (∑ regrowing shoot tips / ∑ number of total shoot tips) * 100 Data for rooting of shoots recovered from liquid nitrogen storage were recorded six weeks after transfer of node segments to rooting medium and was expressed as percentage of nodal explants forming roots from the total number of inoculated nodal segments.Acclimatization was expressed as the percentage of plants which survived and showed growth under ex vitro conditions from the total total number of plants transferred to pots.
The results were expressed as the mean ± standard deviation (SD).Data expressed in percentages were subjected to arcsine transformation before statistical analysis and were analyzed by ANOVA using the Tukey's test for data comparison.

Effects of osmoprotection on survival and regrowth of shoot tips
In preliminary experiments the regrowth ability of nonencapsulated and encapsulated tomato shoot tips was assessed.The non-encapsulated shoot tips started to grow after 3-4 days in culture and within 10 days the shoot formation was 100%.Encapsulation of explants did not significantly affect regeneration although shoot growth was slower and shoot formation ranged between 96% (cvs.'Capriciu' and 'Siriana') and 100% (cvs.'Pontica', 'Kristin' and 'Darsirius').In our current study a significant decrease in survival and regrowth of non-cryopreserved shoot tips was observed with increasing concentration of sucrose (Tab.1).Altough, results obtained for survival can offer information about the efficiency of osmoprotection in early stages of recovery for establishment of a cryopreservation protocol essential is the recovey of shoot tips, therefore, only this parameter will be further considered.It was shown that preculture of shoot tips in sucrose plays an important role in the acquisition of resistance to desiccation and liquid nitrogen exposure (Suzuki et al., 2006;Barraco et al., 2011) increasing the efficiency of cryopreservation in many species (Padrò et al., 2012;Kim et al., 2012;Feng et al., 2013;Le Bras et al., 2014).
active radiation) at 24±1 °C.Shoot tips (2-3 mm in length) consisted of the apical meristem covered by 2-3 leaf primordia were excised from 1 month old in vitro plants under a stereo microscope in sterile conditions and were further used in cryopreservation studies.

Encapsulation and osmoprotection
For encapsulation of shoot tips two solutions were prepared, a 3% (w/v) Na -alginate (low viscosity 250 centipoise -cps) solution in Ca 2+ -free liquid MS medium containing 30 g/l sucrose and a 100 mM CaCl2 solution in MS medium.The pH of the encapsulation matrix was adjusted to 5.7 after the addition of sodium alginate.Both solutions were sterilized by autoclaving.For encapsulation shoot tips were transferred to the Na-alginate solution and then to the sterile CaCl2 solution and beads (approximately 4-5 mm in diameter) were formed instantaneously (Fig. 1a).After 30 min of polymerization the CaCl2 solution was drained off and the beads were washed using liquid MS medium without sucrose (pH 5.7).Further the beads were osmoprotected in liquid MS medium containing sucrose in various concentrations (0.25, 0.5, 0.75 and 1.0 M) for 24 h on a rotary shaker (98 rpm) at 24±1 °C and 16 h light/8 h dark photoperiod.

Desiccation, cryopreservation and regrowth
Osmoprotected beads were blot dried using sterile filter paper, transferred to open Petri dishes (10 cm diameter) and desiccated in sterile laminar air-flow up to 6 h.During air desiccation the environmental conditions of the room were monitored for temperature (24±1 °C) and relative humidity (RH 65±4%).At 1-h intervals desiccated beads were placed in 1.8 ml cryovials (5 beads/cryovial) and immersed into liquid nitrogen contained in a 25 l Dewar flask for 24 h.Rewarming was performed in water bath at 38 °C for 2 min and than shoot tips were transferred in Petri dishes on semisolid (5.5 g/l agar) MS medium for regrowth under the same light and temperature conditions as mentioned above.

Moisture content of the alginate beads
To determine the optimum desiccation time for high regrowth percentages following storage in liquid nitrogen the amount of water loss in the beads was monitored.For dry weight determination, ten beads for three replications were weighed and dried at 60 °C until constant weight was attained.The percentage of moisture content was expressed on fresh weight basis using the formula: MC (%) = [(fresh weight -dry weight) / fresh weight] x 100.

Rooting and acclimatization
For rooting, one node shoot segments (2-2.5 cm in length) from shoots regenerated following cryopreservation were transferred to MS medium supplemented with 1.5 mg/l N 6benzyladenine (BA) and various concentrations (0, 0.5, 1.0, 1.5 mg/l) of indole-3-butyric acid (IBA).For each auxin concentration 10 nodal explants were used.After six weeks roots were formed and plants were removed from in vitro growth conditions, washed (to remove agar) and planted in sterile perlite in 150 ml plastic cups stored in glass boxes (50 x 50 x 30 cm, length/width/height) covered with glass lids to ensure high humidity in the initial stages of the acclimatization period.First day under ex vitro growth conditions the lids were

Tolerance of encapsulated shoot tips to desiccation and cryopreservation
The highest regrowth in all cultivars was obtained for shoot tips treated either with 0.5 M or 0.75 M sucrose in combination with 3 or 4 h desiccation time (Fig. 2a-e).Nondehydrated cryopreserved shoot tips did not survive regardless of genotype (Fig. 2a-e).The extent of sucrose treatment varied from 24 h (González-Arnáo et al., 1993) to several days (Reed et al., 2005).The initial water content of the osmoprotected beads was up to 84%, declined to 25-35% within the first 3 h of air drying and achieved 13-19% following 6 h of desiccation with consequences on shoot regrowth following liquid nitrogen storage in all cultivars.Our findings showed that the moisture content of the alginate beads is critical to ensure regrowth of cryopreserved tomato shoot tips (Fig. 2a-e).Combining osmoprotection of the beads in sucrose with their desiccation, a reduction of the water content was achieved to a level which allowed shoot regrowth following cryopreservation.To ensure high recovery rates after liquid nitrogen storage the moisture content of the beads had to be between 21-26% with a maximum regrowth (72% cv.'Pontica') at 23% moisture content of the alginate beads (Fig. 2d).Although, the alginate bead moisture content that ensures high regeneration after cryopreservation is approximatelly 20% 468 (Engelmann et al., 2008), high regrowth was obtained at lower or higher values of beads moisture content (Padrò et al., 2012).The decline in regrowth of cryopreserved shoot tips with increasing desiccation is may be due to the osmotic shock at higher sucrose concentrations leading to decreased viability of encapsulated shoot tips after liquid nitrogen exposure (Rabba'a et al., 2012).Following osmotic dehydration an extensive evaporative desiccation is necessary to achieve moisture content compatible with cryopreservation (Dumet et al., 2002).Due to desiccation of explants most or all freezable water is removed from cells and vitrification of internal solutes takes place during rapid exposure to liquid nitrogen avoiding lethal intracellular ice crystallization (Engelmann, 2000).Sucrose treatment and the moisture content of alginate beads are key factors for successful application of encapsulation-dehydration (Gonzalez-Benito et al., 1998).Cryopreserved shoot tips developed directly shoots and no callus formation was observed (Fig. 1b, d).Cryopreservation of tomato has been reported for wild tomato seeds (60% regrowth) (Zevallos et al., 2013(Zevallos et al., , 2014)), shoot meristems (52% regeneration) (Grout et al., 1978), and pollen (Karipidis and Douma, 2011).19.4 ± 2.3 e 1.6 ± 0.5 d 1.7 ± 0.4 c 0.5 47.2 ± 1.5 d 5.6 ± 0.5 bc 2.0 ± 0.5 c 1.0 69.4 ± 2.5 a 5.3 ± 1.1 c 5.5 ± 1.0 a 'Darsirius' 1.5 61.1 ± 2.8 b 2.6 ± 0.5 d 2.5 ± 1.0 bc 0 25.0 ± 1.7 e 2.6 ± 0.5 d 2.3 ± 0.7 c 0.5 41.6 ± 1.7 d 7.0 ± 2.6 ab 3.

Rooting and ex vitro acclimatization
Rooting occurred also on medium without IBA but was significantly reduced in all cultivars compared to the medium with auxin (Tab.2).The highest rooting rates for all cultivars were obtained for shoots grown on MS medium supplemented with 1.0 mg/l IBA, whereas the highest number of roots was noted on medium with 0.5 mg/l IBA with significant differences among cultivars (Tab.2).The acclimatization percentages for plants derived from cryopreserved shoot tips were high, ranging between 90 and 100% (cvs.'Kristin' and 'Capriciu') with no significant differences between cultivars.The recovered plants showed growth in length, developed new leaves, were morphologically similar to the control plants and no visible malformations were observed in any of the tested cultivars.Rooting of in vitro grown shoots prior their transfer to soil in pots was a prerequisite for successful ex vitro acclimatization (Fig. 1e, f).

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The conditions for acclimatization of tissue cultured plants have been reviewed (Pospíšilová et al., 1999;Chandra et al., 2010).Important issues during acclimatization include, the exposure of plants to light (which enables them to establish photosynthesis), appropriate temperatures without major air fluctuations (Lesar et al., 2012) or the control of relative humidity (Jang et al., 2011).Plant acclimatization following in vitro culture can be improved by application of abscisic acid to the last subculture (Pospíšilová et al., 2009), using an alternate substrate (Deb and Imchen, 2010).

Conclusions
A simple and efficient protocol for shoot tips cryopreservation of five Lycopersicon cultivars using encapsulation-dehydration was developed in the present study.Shoots recovered from liquid nitrogen storage were rooted and successfully acclimatized to ex vitro growth conditions.Further cryopreservation techniques must be evaluated to increase regrowth percentages.

Fig. 1 .Fig. 2 .
Fig. 1.Cryopreservation of tomato shoot tips: (a) alginate coated shoot tips; (b) regrowth of shoot tip (4 weeks after liquid nitrogen treatment), cst -cryopreserved shoot tip, rsregrowing shoot; (c, d) shoots 6 weeks after liquid nitrogen treatment ('Darsirius' and 'Capriciu'), circle -dead shoot tip; (e) rooted shoots ('Kristin'); (f) acclimatized tomato plant ('Kristin'); Bar 0.5 cm Tab. 1.Effect of preculture with different sucrose concentrations on survival and regrowth of non-cryopreserved tomato shoot tips Data represent mean values ± SD.Values followed by the same letter within a column are not significantly different (P ≤ 0.05).Tab.2. Effect of various concentrations of IBA on rooting of shoots regenerated from encapsulated and cryopreserved shoot tips after six weeks of culture *