Genome-Wide Identification and Characterization of KNOTTED-Like Homeobox (KNOX) Homologs in Garlic (Allium sativum L.) and Their Expression Profilings Responding to Exogenous Cytokinin and Gibberellin.

Garlic (Allium sativum L.) is an important vegetable and is cultivated and consumed worldwide for its economic and medicinal values. Garlic cloves, the major reproductive and edible organs, are derived from the axillary meristems. KNOTTED-like homeobox (KNOX) proteins, such as SHOOT MERISTEM-LESS (STM), play important roles in axillary meristem formation and development. However, the KNOX proteins in garlic are still poorly known. Here, 10 AsKNOX genes, scattered on 5 of the 8 chromosomes, were genome-wide identified and characterized based on the newly released garlic genome. The typical conserved domains of KNOX proteins were owned by all these 10 AsKNOX homologs, which were divided into two Classes (Class I and Class II) based on the phylogenetic analysis. Prediction and verification of the subcellular localizations revealed the diverse subcellular localization of these 10 AsKNOX proteins. Cis-element prediction, tissue expression analysis, and expression profilings in responding to exogenous GA3 and 6-BA showed the potential involvement of AsKNOX genes in the gibberellin and cytokinin signaling pathways. Overall, the results of this work provided a better understanding of AsKNOX genes in garlic and laid an important foundation for their further functional studies.

Reduction of 3-Deoxyglucosone by Epigallocatechin Gallate Results Partially from an Addition Reaction: The Possible Mechanism of Decreased 5-Hydroxymethylfurfural in Epigallocatechin Gallate-Treated Black Garlic.

5-Hydroxymethylfurfural (5-HMF) is a harmful substance generated during the processing of black garlic. Our previous research demonstrated that impregnation of black garlic with epigallocatechin gallate (EGCG) could reduce the formation of 5-HMF. However, there is still a lack of relevant research on the mechanism and structural identification of EGCG inhibiting the production of 5-HMF. In this study, an intermediate product of 5-HMF, 3-deoxyglucosone (3-DG), was found to be decreased in black garlic during the aging process, and impregnation with EGCG for 24 h further reduced the formation of 3-DG by approximately 60% in black garlic compared with that in the untreated control. The aging-mimicking reaction system of 3-DG + EGCG was employed to determine whether the reduction of 3-DG was the underlying mechanism of decreased 5-HMF formation in EGCG-treated black garlic. The results showed that EGCG accelerated the decrease of 3-DG and further attenuated 5-HMF formation, which may be caused by an additional reaction with 3-DG, as evidenced by LC-MS/MS analysis. In conclusion, this study provides new insights regarding the role of EGCG in blocking 5-HMF formation.

Cryopreservation of Plant Shoot Tips of Potato, Mint, Garlic, and Shallot Using Plant Vitrification Solution 3.

Cryopreservation of shoot tips facilitates long-term storage of plant genetic resources which can otherwise only be propagated vegetatively. The vitrification approach using the cryoprotectant plant vitrification solution 3 (PVS3, 50% sucrose and 50% glycerol) is easy to handle, has shown to produce high regrowth percentages in a number of potato, mint, garlic, and shallot accessions, and is, thus, highly suitable for routine cryopreservation of plant genetic resources. In the current chapter, the vitrification procedure is described for potato, mint, garlic, and shallot and includes details about modifications for the different plant species. Special emphasis is given on the preparation of the different culture media, solutions, the culture conditions prior and post-cryopreservation, and the preparation of the shoot tips from different sources. Furthermore, protocols to introduce plants into in vitro culture and methods to estimate cryopreservation success are provided.

Mechanism study of the beneficial effect of sodium selenite on metabolic disorders in imidacloprid-treated garlic plants.

As an effective neonicotinoid insecticide, imidacloprid (IMI) has been widely used in crop production, but its residue affects normal plant growth. Selenium (Se) is a non-essential mineral nutrient in higher plants, that acts as the active centre of glutathione peroxidase (GSH-Px), which removes harmful peroxides. In this study, we investigated the mechanism by which selenium improves the growth status of IMI-treated garlic plants through analyses of apparent morphology and antioxidant enzyme activity as well as the dynamic changes in nutrients and metabolites in the plants. The results showed that 80 µg/kg Na2SeO3 had a strong effect on alleviating the damage in garlic plants exposed to IMI (1.2 mg/kg) by increasing the absorption of mineral elements to enhance the synthesis of chlorophyll and antioxidant enzymes. A nontarget metabolomics analysis based on gas chromatography-mass spectrometry (GC-MS) indicated that the addition of Na2SeO3 to IMI-treated garlic could reconstruct the plant metabolic distribution by enhancing the nitrogen and indole metabolism, maintaining lower concentrations of secondary metabolites and maintaining the balance of the plant energy metabolism. Our study provides novel insights into the molecular mechanisms by which garlic plants responds to IMI exposure and suggests the use of selenium with IMI-contaminated plants as a solution for the advancement of sustainable agricultural pesticide use.

Toxicity assessment of palladium oxide nanoparticles derived from metallosurfactants using multi assay techniques in Allium sativum.

In today's world, nanotechnology is reaching practically every ground and entering the human lifestyle by becoming a part of it. Thus, it is vital to check the cytotoxic and genotoxic effects of nanosubstances on plants, as they are the base constituent of ecosystem. The present work deals with the toxicity evaluation of metallosurfactant derived palladium oxide nanoparticles towards Allium sativum (Garlic cloves). The nanoparticles were prepared using microemulsion quenching method (a softer approach) using palladium metallosurfactants as precursors. The three ligands used were cetyltrimethylammonium chloride (CTAC), dodecylamine (DDA) and hexadecylamine (HEXA). Further, their characterization was done using TEM, Size Distribution curve, FESEM, EDS, XRD and Zeta potential. Garlic (Allium sativum) cloves were used to investigate the cytotoxicity and genotoxicity of fabricated PdO NPs. To check the cytotoxicity, optical microscopy was employed and for the genotoxic assessment, different parameters such as chromosomal aberrations in the mitosis, circular dichroism, and gel electrophoresis were utilized. From mitosis study, chromosomes aberrations were confirmed such as chromosomes stickiness, breakage, C-Mitosis, delay in anaphase, spindle fibre abnormality, laggard, vagrant and condensed chromosomes. Morphology of A. sativum clove, rooting and shooting pattern in the presence of PdO nanosuspension was observed. From all the experiments, it was concluded that all the three PdO nanosuspension are toxic in nature to both the cells and to genome, although, bishexadecyltrimethyl ammonium palladium tetrachloride (PdCTAC) Ns was found to be the most cytotoxic and genotoxic. Gel electrophoresis also confirmed the complete degradation of DNA in the presence of PdCTAC Ns.

A comparative study on the accumulation, translocation and transformation of selenite, selenate, and SeNPs in a hydroponic-plant system.

Plants can play important roles in overcoming selenium (Se) deficiency and Se toxicity in various regions of the world. Selenite (SeIV), selenate (SeVI), as well as Se nanoparticles (SeNPs) naturally formed through reduction of SeIV, are the three main Se species in the environment. The bioaccumulation and transformation of these Se species in plants still need more understanding. The aims of this study are to investigate the phytotoxicity, accumulation, and transformation of SeIV, SeVI and SeNPs in garlic, a relatively Se accumulative plant. The spatial distribution of Se in the roots were imaged using synchrotron radiation micro-focused X-ray fluorescence (SR-µXRF). The chemical forms of Se in different plant tissues were analyzed using synchrotron radiation X-ray absorption spectroscopy (SR-XAS). The results demonstrate that 1) SeNPs which has the lowest phytotoxicity is stable in water, but prone to be converted to organic Se species, such as C-Se-C (MeSeCys) upon uptake by root. 2) SeIV is prone to concentrate in the root and incorporated into C-Se-C (MeSeCys) and C-Se-R (SeCys) bonding forms; 3) SeVI with the lowest transformation probability to organic Se species has the highest phytotoxicity to plant, and is much easier to translocate from root to leaf than SeNPs and SeIV. The present work provides insights into potential impact of SeNPs, selenite and selenate on aquatic-plant ecosystems, and is beneficial for systematically understanding the Se accumulation and transformation in food chain.

Probing the binding effects of zinc and cadmium with garlic phytocystatin: Implication of the abiotic stress on garlic phytocystatin.

Abiotic stress induced by heavy metals retards the growth and development of plants. Therefore, it is essential to have an insight into the potential toxic effects of heavy metals. The present article investigates the effect of zinc and cadmium on the structure and function of garlic phytocystatin (GPhyCys). The cysteine proteinase inhibitory assay showed a reduction in the inhibitory activity upon binding with zinc and cadmium. UV-vis absorption spectroscopy revealed the complex formation of zinc and cadmium with garlic phytocystatin. Fluorescence quenching experiment confirmed the quenching of fluorophores upon binding of zinc and cadmium. Synchronous and 3-dimensional fluorescence spectroscopy suggest the alteration in the microenvironment around aromatic residues of garlic phytocystatin upon binding with the above metals. Circular dichroism showed a reduction in the alpha-helical content of native garlic phytocystatin. Scanning electron micrographs showed the morphological changes in the native garlic phytocystatin upon addition of zinc and cadmium. The observations confirmed the alteration in structure and conformation of garlic phytocystatin upon interaction with zinc and cadmium. It can be safely concluded that the high concentration of zinc and cadmium can alter the functioning of cysteine proteinase present in garlic and affects the growth and development of plants.

Anti-hyperlipidemia of garlic by reducing the level of total cholesterol and low-density lipoprotein: A meta-analysis.

BACKGROUND: This study aimed to understand the impact of garlic on improving blood lipids using a meta-analysis. METHODS: A literature search of the PubMed, EMBASE, and Cochrane Library databases was performed using keywords such as "garlic" and "hypercholesterolemia," and the deadline "July 14 (th), 2017." After extracting relevant details, each selected literature was evaluated for quality according to the quality evaluation criteria of bias risk recommended by Cochrane Collaboration recommendations and heterogeneity tests were performed. Standardized mean difference (SMD) and 95% confidence interval (CI) were evaluated using R 3.12 software. The publication bias was assessed using Egger method. RESULTS: A total of 14 eligible papers published from 1981 to 2016 were included. The quality of the literatures was of moderate to high qualities. The values of TC (SMD = -1.26, 95% CI, -1.86 to -0.66), low-density lipoprotein (LDL) (SMD = -1.07, 95% CI, -1.67 to -0.47), and high-density lipoprotein (HDL) (SMD = 0.50, 95% CI, 0.06-0.94) after taking garlic in the experimental group and the control group have statistical significance, while there was no significant difference of TG in the 2 groups (SMD = -0.16, 95% CI, -0.87-0.55). However, the result of HDL was reversed when removed some of the literatures. No significant publication bias among the eligible studies with values of TC (P = .0625), LDL (P = .0770), HDL (P = .2293), and TG (P = .3436). CONCLUSION: Garlic can reduce the level of TC and LDL instead of HDL and TG, indicating the ability of anti-hyperlipidemia.

Evaluation of weed control efficacy and crop safety of the new HPPD-inhibiting herbicide-QYR301.

QYR301, 1,3-Dimethyl-1H-pyrazole-4-carboxylic acid 4-[2-chloro-3-(3,5-dimethyl-pyrazol-1-ylmethyl)-4-methanesulfonyl-benzoyl]-2,5-dimethyl-2H-pyrazol-3-yl ester, is a novel HPPD-inhibiting herbicide and was evaluated to provide a reference for post-emergence (POST) application under greenhouse and field conditions. The crop safety (180 and 360 g active ingredient (a.i.) ha-1 treatments) experiment revealed that wheat, paddy, garlic and corn were the only four crops without injury at both examined herbicide rates. The weed control efficacy (60 and 120 g a.i. ha-1) experiment showed that QYR301 exhibited high efficacy against many weeds, especially weeds infesting paddy fields. Furthermore, it is interesting that both susceptible and multiple herbicide resistant Echinochloa crus-galli (L.) Beauv. and Echinochloa phyllopogon (Stapf) Koss, two notorious weed species in paddy field, remained susceptible to QYR301. Further crop tolerance results indicated that 20 tested paddy hybrids displayed different levels of tolerance to QYR301, with the japonica paddy hybrids having more tolerance than indica paddy hybrids under greenhouse conditions. Results obtained from field experiments showed that QYR301 POST at 135 to 180 g a.i. ha-1 was recommended to provide satisfactory full-season control of E. crus-galli and Leptochloa chinensis (L.) Nees and to maximize rice yields. These findings indicate that QYR301 possesses great potential for the management of weeds in paddy fields.

Cytotoxicity and chromosomal aberrations induced by methanolic extract of Cuscuta reflexa and its pure compounds on meristematic cells of Allium species.

Cuscuta reflexa (Convolvulaceae), is commonly known as amarbel or akashbel. In Bangladesh and Nepal some of the tribes use C. reflexa against edema, body ache, cancer, skin infections and liver disorders. Despite its traditional uses there is no information regarding genotoxic effects of either the plant extract or its pure compounds. Methanolic extract of C. reflexa (MECR) and pure compounds derived from it namely, odoroside H, neritaloside, and strospeside, were evaluated in Allium cepa L. and A. sativum L. for their effects on root growth, root apical meristem mitotic index (MI) , and chromosomal aberrations (CAs). In this study, we adopted a new method of calculating percent change in root length. MECR caused a concentration- and time- dependent inhibition in root length at 100 - 10000µg/ml in A. cepa root. It was accompanied by a subsequent decline in MI which is an indicative of its cytotoxic effect. On the contrary, at low concentrations a significant rise in root length was noticeable. In A. sativum, MECR also reduced the root length having IC50 values ~8 x and 4.3 x lower than A. cepa. A variety of CAs were evident in both Allium systems after treatment with MECR, odoroside H and neritaloside. Thus in MECR, cardenolides glycosides, i.e. odoroside H and neritaloside could be accountable for its genotoxicity.

Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots.

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200µM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs.

[Influence of Four Kinds of PPCPs on Micronucleus Rate of the Root-Tip Cells of Vicia-faba and Garlic].

In order to determine the degree of biological genetic injury induced by PPCPs, the genotoxic effects of the doxycycline (DOX), ciprofloxacin (CIP), triclocarban (TCC) and carbamazepine (CBZ) in the concentration range of 12.5-100 mg · L⁻¹ were studied using micronucleus rate and micronucleus index of Vicia-fabe and garlic. The results showed that: (1) When the Vicia-faba root- tip cells were exposed to DOX, CIP, TCC and CBZ, micronucleus rates were higher than 1.67 ‰ (CK1), it was significantly different from that of the control group (P < 0.05), and the micronucleus index was even greater than 3.5; With the increasing concentrations of the PPCPs, the micronucleus rates first increased and then decreased. (2) When the garlic root tip cells were exposed to DOX, CIP, TCC and CBZ respectively, the micronucleus rates were less than those of the Vicia-faba, while in most treatments significantly higher than that of the control group (0.67‰). The micronucleus index was higher than 3.5 in the groups exposed to CIP with concentrations of 25, 50, 100 mg · L⁻¹ and TCC and CBZ with concentrations of 25 mg · L⁻¹; With the increase of exposure concentrations, the micronucleus rate showed a trend of first increasing and then decreasing as well. (3) Under the same experimental conditions, the cells micronucleus rates of the garlic cells caused by the four tested compounds were significantly lower than those of Vicia-faba. (4) The micronucleus index of the root tip cells of Vicia-faba and garlic treated with the four kinds of compounds followed the order of CIP > CBZ > TCC > DOX. These results demonstrated that the four compounds caused biological genetic injury to root-tip cells of Vicia-faba and garlic, and the genetic damage caused to garlic was significantly lower than that to Vicia-faba. The damages caused by the four kinds of different compounds were also different.

Efectos del Allium sativum, ajo, en pacientes con dislipidemia en la ciudad de Huancayo. Estudio preliminar / Effects of Allium sativum, garlic, in patients with dyslipidemia in the city of Huancayo. Preliminary Study

Objetivo: Determinar el efecto del consumo de cápsulas de Allium sativum en una dosis diaria de 1 g por 12 semanas en pacientes con dislipidemia residentes en la ciudad de Huancayo (3200 msnm). Materiales y Métodos: Estudio experimental de un solo grupo de intervención con comparación pre-post. Se evaluaron a 33 sujetos (hombres y mujeres) con diagnóstico de dislipidemia de acuerdo a los criterios ATP III; quienes recibieron cápsulas con dosis de 1g diario por doce semanas. Se analizaron los valores de colesterol total, LDL-c, HDL-c y triglicéridos antes y después de la intervención. Resultados: Después de doce semanas, se encontraron reducciones estadísticamente significativas (p<0.001) de los valores de colesterol total (Δ 62.4 mg/dl; IC 95%: 59.1-65.7), LDL-c (Δ 63.7 mg/dl; IC 95%: 60.3-67.1) y Triglicéridos (Δ 21.5 mg/dl; IC 95%: 14.3-28.7) y aumento del HDL-c (Δ 4.1 mg/dl; IC 95%: 2.9-5.3). Conclusión: La intervención por doce semanas con cápsulas de Allium savitum en pacientes con dislipidemia mostró efectos estadísticamente significativos en los niveles de colesterol total, LDL-c, HDL-c y triglicéridos. Se recomienda realizar estudios clínicos aleatorizados para poder evaluar en real magnitud las tendencias observadas en estos resultados preliminares.

[Effect of cadmium stress on physiological characteristics of garlic seedlings and the alleviation effects of exogenous calcium].

In the experiment, the effects of exogenous cadmium (Cd2+) and calcium (Ca2+) in nutrient solution on growth, photosynthetic characteristics, enzymes activities, main mineral elements absorption of garlic seedlings were studied. The results showed that cadmium could obviously inhibit the growth of garlic seedlings, decrease the pigment contents and photosynthetic parameters (P(n), E, g(s)) of leaves, reduced the enzymes (SOD, POD, CAT) activities and increase the MDA content of leaves, and also could reduce the N, P, K, Ca, Mg contents and increase the Cd content of roots. The growth was promoted after adding exogenous calcium to garlic seedlings under cadmium stress, which reflected that the morphological indexes were increased at first and then decreased with the increase of exogenous calcium concentrations, and were maximized when the exogenous calcium was 2 or 3 mmol x L(-1). At the same time, the pigment contents and photosynthetic parameters (P(n), E, g(s)) of leaves showed a similar tendency with the morphological indexes, and they were the highest when the exogenous calcium was 2 or 3 mmol x L(-1). In addition, adding exogenous calcium to garlic seedlings under cadmium stress enhanced the enzymes (SOD, POD, CAT) activities and decreased the MDA content of leaves, also added the N, P, K, Ca, Mg contents and reduced the Cd content of roots, and the effect was best when the exogenous calcium concentration was 2 or 3 mmol x L(-1).

Graphene quantum dots as enhanced plant growth regulators: effects on coriander and garlic plants.

BACKGROUND: We report investigations on the use of graphene quantum dots for growth enhancement in coriander (Coriandrum sativam L.) and garlic (Allium sativum) plants. The as-received seeds of coriander and garlic were treated with 0.2 mg mL(-1) of graphene quantum dots for 3 h before planting. RESULTS: Graphene quantum dots enhanced the growth rate in coriander and garlic plants, including leaves, roots, shoots, flowers and fruits, when the seeds were treated with graphene quantum dots. CONCLUSION: Our investigations open up the opportunity to use graphene quantum dots as plant growth regulators that can be used in a variety of other food plants for high yield.

Accumulation and phytoavailability of hexachlorocyclohexane isomers and cadmium in Allium sativum L. under the stress of hexachlorocyclohexane and cadmium.

The effects of Cd and HCHs with single and combined forms on Cd and HCHs phytoavailability of Allium sativum L. were investigated. The results indicated that the coexistence of Cd and HCHs presented antagonistic interactions mostly, which might be partly due to the formation of Cd-HCHs complex, compared with single stress. The bioaccumulation of Cd and HCHs in plants depended largely on their concentrations applied in pot soils, and the phytoavailability of HCH isomers was in the sequence: δ- > γ- ≥ ß- > α-HCH.

In vitro induction of tetraploid garlic with trifluralin.

Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 µM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphology of guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.

Phytotoxic and genotoxic effects of ZnO nanoparticles on garlic (Allium sativum L.): a morphological study.

The effects of zinc oxide nanoparticles (ZnO NPs) on the root growth, root apical meristem mitosis and mitotic aberrations of garlic (Allium sativum L.) were investigated. ZnO NPs caused a concentration-dependent inhibition of root length. When treated with 50 mg/L ZnO NPs for 24 h, the root growth of garlic was completely blocked. The 50% inhibitory concentration (IC(50)) was estimated to be 15 mg/L. The mitosis index was also decreased in a concentration- and time-dependent manner. ZnO NPs also induced several kinds of mitotic aberrations, mainly consisted of chromosome stickiness, bridges, breakages and laggings. The total percentage of abnormal cells increased with the increase of ZnO NPs concentration and the prolongation of treatment time. The investigation provided new information for the possible genotoxic effects of ZnO NPs on plants.

Effect of selenium treatment on mineral nutrition, bulb size, and antioxidant properties of garlic (Allium sativum L.).

Foliar selenium (Se) treatment of garlic at concentrations of 10, 50, and 100 µg of Se/mL was carried out in open field conditions in 2008 and 2009 in Estonia. Bulb weight and yield structure, content of total Se, S, N, P, K, Ca, and Mg, ascorbic acid content (AAC), pungency, total phenolics, and total antioxidant capacity (TAC) were determined. The highest level of Se decreased total S, K, and Ca in both years; no negative impact on bulb weight was observed. In 2009 Se10 treatment had significantly more bulbs with the largest diameter compared to the other treatments. In 2008, the AAC was decreased by Se50 and the content of total phenolics by all Se treatments; however, TAC was increased. Foliar Se fertilization of garlic at rates of 10-50 µg of Se/mL can be recommended to increase the number of large bulbs and increase bulb antioxidant capacity.

Development of alternative plant vitrification solutions in droplet-vitrification procedures.

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. Chrysanthemum shoot tips were very sensitive to both chemical toxicity and osmotic stress and therefore, induction of cytotoxity tolerance during preconditioning was required for successful cryopreservation. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.