Effect of Explant ' s Size and Phytohormonal Composition of Nutritive Medium on Post-Vitification Recovery of Garlic Meristems

Investigation of the effect of explants dimensions and composition of nutritive medium on morphometric parameters of devitrified garlic meristems will enable a significant optimizing of the cryopreservation techniques intended to establish a collection of its cultivars at low temperature bank. Apexes were subjected to dehydration with plant vitrification solution (1M sucrose + 2M glycerol + 2.5 M ethylene glycol based on Murashige-Skoog salt medium) for 120 min, placed into a polypropylene container and immersed into liquid nitrogen. Thawing was performed in a water bath at 40°C, washing of cryoprotectants was carried-out by a two-fold transfer into the medium with 0.3 M sucrose. It has been shown that the solution under study was non-toxic to garlic meristems and allowed the obtaining of a high level of viability. After cryopreservation by means of vitrification of garlic meristems of ‘Manuylivskyy’ spring variety the number of regenerated plants reached (56.3 ± 11.2)%, and ‘Duchess’ winter variety did (44.8 ± 3.7)%. For the cultivation of the cryopreserved garlic meristems as the phytohormone the kinetin is advisable to be used in concentration of 0.5 mg/l. It has been shown that after cryopreservation by vitrification the garlic meristems of 2–3 mm had the highest viability (60–70%). Larger meristems (3.5–4 mm) had a high regenerative potential but low viability (below 25%). In smaller meristems (1–1.5 mm) the viability rates were reduced (30–47%) as well as a regenerative potential. In the meristems of 0.5 mm a regenerative potential was absent.

Ukrainian National Collection of garlic (Allium sativum L.) comprises over 110 samples characterized with a high quality production, adaptation to growing conditions, resistance and tolerance to diseases and pests.The National Genetic Bank is designated not only to collect, register and study the samples, but to preserve the genetic diversity of these unique forms which are necessary for the improvement of contemporary cultivars and for developing the new ones [14].Ukrainian investigators use the methods to establish different types of collections.Active banks (IVAG (the in vitro active genebank)) are intended for the samples often used in breeding practice, thus requiring a medium-term storage at low positive temperatures.In the low-temperature banks (IVBG (the in vitro base genebank)) the explants of the recovered plants are kept at the temperature of liquid nitrogen (-196°C) [4,11,13,18].Preservation at ultra-low temperatures enables to avoid the exhaustion of reserve substances, accumulation of toxins, decay and activation of enzyme complexes, lipid oxidation, degradation of functional genetic systems, as well as poorly studied biochemical or biophysical processes associated with aging of cells [4,13].
In 2002 the group of E.R.J. Keller developed the cryopreservation method for garlic meristems, which involved a two-stage dehydration with the vitrifying solution (plant vitrification solution 3 (PVS)), which consisted of glycerol and sucrose in the 50:50 ratio [12,13].But implication of this cryopreservation method for our tasks did not result in satisfactory outcomes of viability, most meristems formed callus.Our studies showed the perspectives of another vitrifying solution (PVS N) developed for cryopreservation of potato and grape meristems, which enabled to simplify significantly the dehydration procedure and to get a high percentage of regeneration [15,16].
It was shown previously [10,13,19,20] that the proportions of cytokinins, auxins and gibberellic acid in the culturing medium did not affect the viability of the meristems of Vitis, Citrus, Anigozanthos, Pyrus species at the cultivation stage, but affected the development of devitrified apexes into shoots.The size and stage of development of the meristems isolated from in vitro plants had a significant impact on the cryopreservation outcome.The sample must have an apical dome and some primordial leaves, but nevertheless its size has to be small enough [13,17].For example, the viability and growth of Cassava meristems are significantly increased if cryopreservation involved apexes of 1-2 mm.The apexes of calocassia (Calocassia esculenta) of 0.5-0.8mm, containing 1-2 pairs of primordial leaves were effectively dehydrated by the plant vitrification solutions and were better recovered after cryopreservation than the apexes of larger sizes.
There were no studies on cryopreservation of garlic meristems to establish the IVBG collections in Ukraine so far.Therefore the purpose of this study was to evaluate the effectiveness of PVS N, as well as the biotechnological techniques for clonal micropropagation of garlic in the in vitro culture, which foresee the determining of the size of explants and phytohormonal composition of regenerative medium for obtaining a high viability rate of plants of cryopreserved meristems.

Materials and methods
The investigations for developing the storage methods of garlic in liquid nitrogen conditions were performed in the meristems of different developmental types: 'Duchess' (winter) and 'Manuylivskyy' (spring) cultivars, which were isolated from the stems of cloves.The material was sterilized in clean room 'BAVnp Laminar-01-C' (Russia) as follows: a clove was immersed into a solution of 70% ethanol for 5-10 seconds, then was put into 2.6% solution of sodium hypochlorite and incubated for 20 min, and thereafter washed at least 5 times with a sterile distilled water [6,7].
The meristems of 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0 mm were isolated from sterilized garlic cloves using MBS-9 binocular (USSR) at 16-fold magnification, thereafter they were pre-cultivated in 10 ml glass penicillin bottles (Alhim, Ukraine) at 22°C in the dark on the surface of the Murasihe-Skoog (MS) solid nutritive medium [12] supplemented with 4.5% sucrose and by 1 mg/l of thiamine, pyridoxine and ascorbic acid.Each single experimental version comprised 30 planted explants.To prepare the solutions of cryoprotectants the MS saline was used.
(42 ± 15) deg/s.To examine the influence of cryopreservation preparation steps per se on the explants the part of meristems was treated excluding low-temperature exposure.The cryoprotectants were washedout by two consequent transfers of the meristems on filter papers saturated with 0.3 M sucrose solution.Non-frozen apical meristems not treated with the plant vitrification solution were assumed as the control in the experiments.
After re-cultivation of meristems performed to determine their viability and morphometric parameters following cryopreservation and/or treatment with plant vitrification solution they were placed to the nutritive MS medium with 0.5 mg/l 6-benzylaminopurine (BAP) + 0.1 mg/l indoleacetic acid (IAA).This medium enables the obtaining of higher regeneration of the explants during reproduction of garlic culture in vitro [6].To determine the effect of phytohormones on growth of meristems after the storage in liquid nitrogen three options of solid nutritive media for re-culturing were investigated, which differed by the content and concentration of phytohormones of cytokinins type (BAP, kinetin).The effect of meristems dimensions on their recovery after cryopreservation was investigated using MS saline supplemented with 0.5 mg/l kinetin + 0.1 mg/l IAA + 100 mg/l proline L + 100 mg/l casein hydrolyzate.The plant material was recultivated in glass penicillin bottles at 22...26°C with 16-hr-long photoperiod.The light intensity corresponded to the donor explant type: 2-3 klx for meristems during the first month of cultivation and 5 klx during the following months of the cultivation of adventive shoots.
Survival rate of the explants was determined to the 5 th day of cultivation by counting the number of meristems, which were green coloured, the viability was assessed by the number of apexes, formed the leaves and roots to the 50 th and 90 th days of cultivation and expressed as the percentage ratio to a total number of the studied meristems.The number of leaves and roots formed in regenerated plants was also counted.
The samples were registered and recorded in the laboratory journals according to the Allium spp descriptor [3].

Results and discussion
Use of vitrification solutions is considered as one of the possible ways for successful low temperature preservation of biological objects with keeping a high level of viability.High concentrations of penetrating cryoprotectants (glycerol, 1,2-propane diol, ethylene glycol, etc.) affect the hardening processes during the temperature lowering, and result usually in achieving a vitrified state.Cryobiological applications are now often involving high concentrations of cryoprotectants
Since most cryoprotectants are xenobiotics, they can render cytotoxic influence on biological systems, and demonstrate various osmotic effects.Therefore, we have investigated the influence of plant vitrification solution on the recovery of garlic apical meristems after dehydration (without vitrification-warming stage).
Survival of meristems of both garlic varieties after dehydration in PVS N following 5 days of cultivation was of 80-90%.During further cultivation some meristems which were green coloured before had only primordial leaves alive, their apical dome was damaged and following cultivation did not result in development of the sprout.In other words, these meristems were non-viable.Thus, it was found that under these conditions of dehydration, the investigated by us solution had no pronounced toxic effect, therefore it can be used for cryopreservation of garlic meristems by vitrification.
An intensive formation of shoots from the meristems, stored in liquid nitrogen, started 7-10 days later, and in non-frozen ones (subjected only to dehydration stage) did to the 3 rd day of cultivation.The shoots were developed by a direct organogenesis characteristic for the meristems, cryopreserved by vitrification method [4].
According to the results presented in Table 1 the dehydration in PVS N reduced the viability of meristems of winter cultivar down to 71% and did not change it in spring one (93%), the cryopreservation significantly reduced this index down to 45% for the cultivar 'Duchess' and to 56% for the 'Manuylivskyy' one.In terms of development rate of a shoot the meristems, recovered after cryopreservation and dehydration in cryoprotective solution, were far beyond the controls.In particular, to the 90 th day of cultivation the shoot length of 'Duchess' winter variety was 135 mm in the control, 93 mm for dehydrated meristems and 81 mm in cryopreserved ones.There was significantly decreased the root length down to 45 mm in both dehydrated and cryopreserved meristems vs. 67 mm in the control, the number of leaves was unchanged.
The findings indicate that the growth and development of the apical meristems are negatively affected not only by ultra-low temperature, but also by the treatment with the solution of cryoprotectants (Figure).
Organogenesis of explants is very dependent on the appropriate balance of phytohormones.Induction of the meristems to growth and formation of shoots is achieved by adding to the medium of cytokinins (either kinetin or BAP in concentration of 10 -5 -10 -7 M) or cytokinins together with auxins.Formation of roots is promoted by supplementing of naphtyl acetic acid, IAA or indoleacetic acid to the composition of media [2].Unbalanced cytokinins composition is known to inhibit the growth of leaves and can cause morphological changes [2].
Previous studies have demonstrated that the best regeneration of shoots from apical meristems was found in the nutrient medium with added 0.5 mg/l BAP and 0.1 mg/l IAA [6], so we tried to use it for recultivation of the meristems.But the outcomes of recovering of cryopreserved meristems showed that this phitohormone in the applied concentration was less effective than kinetin in the same concentration.After introduction of 0.5 mg/l kinetin into the culture medium the growth rate and the number of leaves were significantly increased in the meristems of 'Duchess' garlic varieties, recovered after dehydration in PVS N and cryopreservation, as well there was found a tendency to increase these parameters for 'Manuylivskyy' Отже, для створення колекції сортів часнику у низькотемпературному банку необхідно виділяти cultivar.This is likely due to the very high activity of BAP because the meristems after treatment with cryoprotective solution and cryopreservation require some time to recover their functions, the need of which is confirmed by 7-10 day-long delay in development observed in dehydrated or devitrified meristems if compared with control ones.H.J. Baek et al. [1] reported that the viability of cryopreserved meristems after dehydration in PVS 3 depended on their size, but the development rate of sprouts has not been investigated by the authors.In our experiment we examined the viability and length of shoots of the sprouts regenerated from different by sizes cryopreserved meristems of 'Duchess' garlic winter varieties using PVS N.
The largest amount of regenerated plants following cryopreservation was obtained in case of using apical meristems of 2-3 mm, the viability was 60-67%.These explants formed the garlic regenerated plants of 16.8-29.0mm length during further culturing.Apical meristems of 4 mm gave in vitro the samples with the highest growth force (40 mm), but quite low viability, up to 20%.In case if the cryopreserved meristems were of 0.5 mm, the regenerative potential was completely absent (Table 3).
We believe, that larger meristems have more hydrated tissues, the penetration of cryoprotectants into the centre of the apex and water release from it during treatment with plant vitrification solution are slower than in smaller meristems, and due to this they are poorly saturated with this solution and die during freezing in liquid nitrogen.As shown in our experiments the usage of meristems of less than 2 mm was the least effective: because of the small sizes the samples suffered from a severe cytotoxicity of cryoprotectant, evidently resulting in reduced developmental indices, the tissues appeared like glassy and had such morphological abnormalities as immature roots and leaves.
culture medium for devitrified apexes should contain 0.5 mg/l kinetin, that would result finally in obtaining a high viability and a satisfactory regenerative potential.

Conclusions
Collectively, this experimental study has been shown that a plant vitrification solution (1M sucrose + 2 M glycerol + 2.5 M ethylene glycol), is non-toxic to garlic meristems under used dehydration conditions and enables the obtaining of a high viability.In particular, cryopreser-vation by means of vitrification of garlic meristems of 'Manuylivskyy' spring varieties the number of regene-rated plants reached (56.3 ± 11.2)%, and for the 'Duchess' winter varieties it was (44.8 ± 3.7)%.It is expedient to use phytohormone kinetin of 0.5 mg/l concentration for culturing of cryopreserved garlic meristems, because this allows the obtaining of better morphometric parameters (length of shoots and roots) in regenerated plants.It has been shown that after cryopreservation by means of vitrification the highest viability was inherent to garlic meristems of 2-3 mm (60-70%).Larger meristems (3.5-4 mm) have a high regenerative potential but low vitality (less than 25%).In smaller meristems (1-1.5 mm) the viability rates and regenerative potential were reduced (30-47%).In meristems of 0.5 mm under these experimental conditions the regenerative potential is absent.

Table 1 .
Effect of dehydration in plant vitrification solution (PVS N) and cryopreservation on growth of garlic apical meristems in 90 days of culturing

Table 3 .
Effect of apical mersitem size of 'Duchess' garlic clove winter cultivar on viability and growth force after cryopreservation (in 50 culturing days)