The effects of monoculture and intercropping on photosynthesis performance correlated with growth of garlic and perennial ryegrass response to different heavy metals.

BACKGROUND: The potential of phytoremediation using garlic monoculture (MC) and intercropping (IC) system with perennial ryegrass to enhance the uptake of cadmium (Cd), chromium (Cr), and lead (Pb) were investigated. RESULTS: Positive correlations were found between MC and IC systems, with varying biomass. Production of perennial ryegrass was affected differently depending on the type of toxic metal present in the soil. Root growth inhibition was more affected than shoot growth inhibition. The total biomass of shoot and root in IC was higher than MC, increasing approximately 3.7 and 2.9 fold compared to MC, attributed to advantages in root IC crop systems. Photosystem II efficiency showed less sensitivity to metal toxicity compared to the control, with a decrease between 10.07-12.03%. Among gas exchange parameters, only Cr significantly affected physiological responses by reducing transpiration by 69.24%, likely due to leaf chlorosis and necrosis. CONCLUSION: This study exhibited the potential of garlic MC and IC with perennial ryegrass in phytoremediation. Although the different metals affect plant growth differently, IC showed advantages over MC in term biomass production.

Comprehensive Metabolite Profiling in Genetic Resources of Garlic (Allium sativum L.) Collected from Different Geographical Regions.

Garlic (Allium sativum) is the second most important Allium crop that has been used as a vegetable and condiment from ancient times due to its characteristic flavor and taste. Although garlic is a sterile plant that reproduces vegetatively through cloves, garlic shows high biodiversity, as well as phenotypic plasticity and environmental adaptation capacity. To determine the possible mechanism underlying this phenomenon and to provide new genetic materials for the development of a novel garlic cultivar with useful agronomic traits, the metabolic profiles in the leaf tissue of 30 garlic accessions collected from different geographical regions, with a special focus on the Asian region, were investigated using LC/MS. In addition, the total saponin and fructan contents in the roots and cloves of the investigated garlic accessions were also evaluated. Total saponin and fructan contents did not separate the garlic accessions based on their geographical origin, implying that saponin and fructan contents were clone-specific and agroclimatic changes have affected the quantitative and qualitative levels of saponins in garlic over a long history of cultivation. Principal component analysis (PCA) and dendrogram clustering of the LC/MS-based metabolite profiling showed two major clusters. Specifically, many Japanese and Central Asia accessions were grouped in cluster I and showed high accumulations of flavonol glucosides, alliin, and methiin. On the other hand, garlic accessions grouped in cluster II exhibited a high accumulation of anthocyanin glucosides and amino acids. Although most of the accessions were not separated based on country of origin, the Central Asia accessions were clustered in one group, implying that these accessions exhibited distinct metabolic profiles. The present study provides useful information that can be used for germplasm selection and the development of new garlic varieties with beneficial biotic and abiotic stress-adaptive traits.

Comparative transcriptome profiling reveals that brassinosteroid-mediated lignification plays an important role in garlic adaption to salt stress.

Garlic (Allium sativum L.) is an economically important vegetable crop which is used worldwide for culinary and medicinal purposes. Soil salinity constrains the yield components of garlic. Understanding the responsive mechanism of garlic to salinity is crucial to improve its tolerance. To address this problem, two garlic cultivars differing in salt tolerance were used to investigate the long-term adaptive responses to salt stress at phenotype and transcriptome levels. Phenotypic analysis showed four-week salt stress significantly decreased the yield components of salt-sensitive cultivar. Transcriptomes of garlics were de novo assembled and mined for transcriptional activities regulated by salt stress. The results showed that photosynthesis, energy allocation, and secondary metabolism were commonly enriched in both sensitive and tolerant genotypes. Moreover, distinct responsive patterns were also observed between the two genotypes. Compared with the salt-tolerant genotype, most transcripts encoding enzymes in the phenylpropanoid biosynthesis pathway were coordinately down regulated in the salt-sensitive genotype, resulting in alternation of the content and composition of lignin. Meanwhile, transcripts encoding the enzymes in the brassinosteroid (BR) biosynthesis pathway were also systematically down regulated in the salt-sensitive genotypes. Taken together, these results suggested that BR-mediated lignin accumulation possibly plays an important role in garlic adaption to salt stress. These findings expand the understanding of responsive mechanism of garlic to salt stress.

Influence of packaging materials on postharvest physiology and texture of garlic cloves during refrigeration storage.

This study aims to investigate the postharvest physiology and texture of garlic cloves packaged in polyethylene terephthalate (PET), polyethylene (PE), aluminized kraft paper (AKP), single kraft paper (SKP), and mesh bag. Germination rate, electrical conductivity, respiration intensity, water content, and texture were determined during 180 d storage at -2 °C. Results showed that the germination of garlic cloves packaged in PET, PE, and AKP was effectively inhibited during storage. PE effectively reduced the degree of damage to the cell membranes of the garlic cloves. PE and SKP significantly inhibited respiratory intensity during storage. Garlic cloves water content did not change significantly in 90 d storage which packaged in PE and SKP. PE exhibited better effect on the texture and freshness of garlic cloves than the other materials. In conclusion, PE is the best packaging material for maintaining the quality attributes and extending the shelf lives of garlic cloves.

Resistance and clonal selection among Allium sativum L. germplasm resources to Delia antiqua M. and its correlation with allicin content.

BACKGROUND: Garlic is the second largest allium crop after onion and is grown all over the world. The onion maggot (Delia antiqua M.) is a pest that seriously affects the yield and quality of garlic. Cultural controls and insecticides have several potential problems, including pesticide residue and development of resistance. Screening resistant varieties is an ideal alternative method. RESULTS: The resistance of 213 accessions of garlic clones against onion maggot was identified. The results showed that the pest index was between 5.56% and 91.11%, with classification into six groups by cluster analysis: HR (highly resistant), R (resistant), MR (moderately resistant), MS (moderately susceptible), S (susceptible) and HS (highly susceptible). Among these accessions, 9 and 30 were HR and R to onion maggot, respectively. Comparing the resistances of seven pairs of accessions between the original accessions and their progenies showed that single bulb clonal selection could be an effective way to improve allicin content, onion maggot resistance and most morphological traits. The relationship between allicin content and resistance was investigated, and a significant positive relationship was found. Accessions with a high content of allicin have great potential as resistant accessions. CONCLUSION: This study showed significant differences among garlic germplasm in their response to Delia antiqua M. Some accessions were highly resistant and tolerant. Utilization of these accessions will help minimize environmental pollution, preserve agro-ecosystems and biodiversity, and make management processes more economical. Furthermore, these accessions could be used in breeding programs to develop new maggot-resistant onion cultivars. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Transcript profiling reveals an important role of cell wall remodeling and hormone signaling under salt stress in garlic.

Salt stress is one of the environmental factors that evidently limit plant growth and yield. Despite the fact that understanding plant response to salt stress is important to agricultural practice, the molecular mechanisms underlying salt tolerance in garlic remain unclear. In this study, garlic seedlings were exposed to 200 mM NaCl stress for 0, 1, 4, and 12 h, respectively. RNA-seq was applied to analyze the transcriptional response under salinity conditions. A total of 13,114 out of 25,530 differentially expressed unigenes were identified to have pathway annotation, which were mainly involved in purine metabolism, starch and sucrose metabolism, plant hormone signal transduction, flavone and flavonol biosynthesis, isoflavonoid biosynthesis, MAPK signaling pathway, and circadian rhythm. In addition, 272 and 295 differentially expressed genes were identified to be cell wall and hormone signaling-related, respectively, and their interactions under salinity stress were extensively discussed. The results from the current work would provide new resources for the breeding aimed at improving salt tolerance in garlic.

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.

Induction of reactive oxygen species and the potential role of NADPH oxidase in hyperhydricity of garlic plantlets in vitro.

Hyperhydricity is a physiological disorder associated with oxidative stress. Reactive oxygen species (ROS) generation in plants is initiated by various enzymatic sources, including plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cell wall-bound peroxidase (POD), and apoplastic polyamine oxidase (PAO). The origin of the oxidative burst associated with hyperhydricity remains unknown. To investigate the role of NADPH oxidases, POD, and PAO in ROS production and hyperhydricity, exogenous hydrogen peroxide (H2O2) and inhibitors of each ROS-producing enzyme were applied to explore the mechanism of oxidative stress induction in garlic plantlets in vitro. A concentration of 1.5 mM H2O2 increased endogenous ROS production and hyperhydricity occurrence and enhanced the activities of NADPH oxidases, POD, and PAO. During the entire treatment period, NADPH oxidase activity increased continuously, whereas POD and PAO activities exhibited a transient increase and subsequently declined. Histochemical and cytochemical visualization demonstrated that specific inhibitors of each enzyme effectively suppressed ROS accumulation. Moreover, superoxide anion generation, H2O2 content, and hyperhydric shoot frequency in H2O2-stressed plantlets decreased significantly. The NADPH oxidase inhibitor was the most effective at suppressing superoxide anion production. The results suggested that NADPH oxidases, POD, and PAO were responsible for endogenous ROS induction. NADPH oxidase activation might play a pivotal role in the oxidative burst in garlic plantlets in vitro during hyperhydricity.

Cytological and biophysical comparative analysis of cell structures at the microsporogenesis stage in sterile and fertile Allium species.

MAIN CONCLUSION: Using a live-cell-imaging approach and autofluorescence-spectral imaging, we showed quantitative/qualitative fluctuations of chemical compounds within the meiocyte callose wall, providing insight into the molecular basis of male sterility in plants from the genus Allium. Allium sativum (garlic) is one of the plant species exhibiting male sterility, and the molecular background of this phenomenon has never been thoroughly described. This study presents comparative analyses of meiotically dividing cells, which revealed inhibition at the different microsporogenesis stages in male-sterile A. sativum plants (cultivars Harnas and Arkus) and sterile A. ampeloprasum var. ampeloprasum (GHG-L), which is phylogenetically related to garlic. Fertile species A. ampeloprasum (leek) was used as the control material, because leek is closely related to both garlic and GHG-L. To shed more light on the molecular basis of these disturbances, autofluorescence-spectral imaging of live cells was used for the assessment of the biophysical/biochemical differences in the callose wall, pollen grain sporoderm, and the tapetum in the sterile species, in comparison with the fertile leek. The use of techniques for live-cell imaging (autofluorescence-spectral imaging) allowed the observation of quantitative/qualitative fluctuations of autofluorescent chemical compounds within the meiocyte callose wall. The biophysical characterisation of the metabolic disturbances in the callose wall provides insight into the molecular basis of male sterility in A. sativum. In addition, using this method, it was possible for the first time, to determine precisely (on the basis of fluctuations of autofluorescence compounds) the meiosis stage in which normal microsporogenesis is disturbed, which was not visible using light microscopy.

Optimization of Water Content for the Cryopreservation Of Allium sativum In Vitro Cultures by Encapsulation-Dehydration.

BACKGROUND: There is a general requirement to determine and correlate water content to viability for the standardization of conservation protocols to facilitate effective cryostorage of plant germplasm. OBJECTIVE: This study examined water content as a critical factor to optimize the cryostorage of Allium sativum. MATERIALS AND METHODS: Stem discs were excised from post-harvest, stored bulbs prior to cryopreservation by encapsulation-dehydration and water content was determined gravimetrically. RESULTS: Survival of cryopreserved stem discs was 42.5 %, with 22.5 % exhibiting shoot regrowth following 6 h desiccation. Gravimetric data demonstrated a correlation between water content corresponding with survival / regrowth from desiccated, cryopreserved stem discs. For encapsulated stem discs a 25 % residual moisture and corresponding water content of 0.36 g H2O g-1 d.wt correlated with maximal survival following ~6.5 h of desiccation. CONCLUSION: The data concurs with the literature suggesting the formation of a stable vitrified state and a 'window' for optimal survival and regrowth that is between 6 - 10 h desiccation. Further studies using differential scanning calorimetry (DSC) are suggested to substantiate these findings.

In Vitro and Cryopreservation Techniques for Conservation of Snow Mountain Garlic.

Garlic is an important medicinal herb of culinary value by imparting its flavors and odors to the food. Allicin, a notable flavonoid in garlic, is a powerful antibiotic and antifungal compound. Due to poor bioavailability, garlic is of limited use for oral human consumption. Being sexually sterile, propagation of garlic is done by individual cloves from a bulb which increases the chances of transfer of viral diseases. In this chapter, an efficient and improved regeneration protocol for explant establishment and shoot multiplication under in vitro conditions is described. A high rate of shoot multiplication is obtained on MS medium supplemented with 0.5 mg/l BAP, 1.0 mg/l KN, and 2.0 mg/l GA3. Addition of 1.0 mg/l NAA to MS medium resulted in rooting at the shoot bases. A detailed method for encapsulation of explant in sodium alginate beads and their cryopreservation using encapsulation-dehydration is also described.

Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation.

KEY MESSAGE: This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Tubulin cytoskeleton during microsporogenesis in the male-sterile genotype of Allium sativum and fertile Allium ampeloprasum L.

KEY MESSAGE: Microsporogenesis in garlic. The male-sterile Allium sativum (garlic) reproduces exclusively in the vegetative mode, and anthropogenic factors seem to be the cause of the loss of sexual reproduction capability. There are many different hypotheses concerning the causes of male sterility in A.sativum; however, the mechanisms underlying this phenomenon have not been comprehensively elucidated.Numerous attempts have been undertaken to understand the causes of male sterility, but the tubulin cytoskeleton in meiotically dividing cells during microsporogenesis has never been investigated in this species. Using sterile A.sativum genotype L13 and its fertile close relative A. ampeloprasum (leek), we have analysed the distribution of the tubulin cytoskeleton during microsporogenesis. We observed that during karyokinesis and cytokinesis, in both meiotic divisions I and II, the microtubular cytoskeleton in garlic L13 formed configurations that resembled tubulin arrangement typical of monocots. However, the tubulin cytoskeleton in garlic was distinctly poorer (composed of a few MT filaments) compared with that found in meiotically dividing cells in A. ampeloprasum. These differences did not affect the course of karyogenesis, chondriokinesis, and cytokinesis, which contributed to completion of microsporogenesis, but there was no further development of the male gametophyte. At the very beginning of the successive stage of development of fertile pollen grains, i.e. gametogenesis, there were disorders involving the absence of a normal cortical cytoskeleton and dramatically progressive degeneration of the cytoplasm in garlic. Therefore,we suggest that, due to disturbances in cortical cytoskeleton formation at the very beginning of gametogenesis, the intracellular transport governed by the cytoskeleton might be perturbed, leading to microspore decay in the male-sterile garlic genotype.

[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).

Micropropagation and cryopreservation of garlic (Allium sativum L.).

Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

Analysis of interferograms of multi-layered biological samples obtained from full field optical coherence tomography systems.

Several methods were developed in the past to analyze interferograms produced by optical coherence tomography, and successfully applied to simulated or animated samples. However, these techniques do not cope with noisy and distorted interferograms from biological tissues. In this paper, known techniques, including the fast Fourier transform and several variations of the continuous wavelet transform, were employed to analyze the interferogram data. However, to cope with the difficulties in biological data, pre- and post-processing procedures and adaptive thresholding were developed to provide stability and robustness. Additionally, three-dimensional structural models of the biological samples were constructed, and revealed information like the number and locations of interfaces, the layer thickness and pattern, and abnormalities.

'Personalisation' of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects.

Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5 percent glycerol + 17.5 percent sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90 percent (37.5 percent glycerol + 15% DMSO + 15 percent EG + 22.5 percent sucrose) for 10 to 30 min or B1-100 percent (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40 degree C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.

Regression of preestablished cholesterol gallstones by dietary garlic and onion in experimental mice.

We have recently reported the health beneficial potential of dietary garlic and onion in reducing the incidence and severity of cholesterol gallstone (CGS) during its experimental induction in mice. In the current study, the efficacy of dietary garlic and onion in regressing preestablished CGS was investigated in experimental mice. After inducing CGS in mice with a lithogenic diet for 10 weeks, they were maintained on basal diets containing 0.6% dehydrated garlic or 2% dehydrated onion for a further 10 weeks. Dietary garlic and onion, either raw or heat processed, regressed preformed CGS in mice up to 53% to 59%, whereas the regression in the basal control diet group was only 10%. The antilithogenic potency of garlic was decreased by its heat processing, but not in the case of onion. Biliary cholesterol was significantly decreased in garlic- and onion-fed animals. Biliary cholesterol saturation index and hydrophobicity index were significantly lowered by dietary garlic and onion. Serum and liver cholesterol levels were decreased by feeding these spices during post-CGS induction period. Hepatic hydroxymethylglutaryl-coenzyme A reductase activity was increased after feeding garlic and onion, whereas activities of the cholesterol-degrading enzymes cholesterol-7α-hydroxylase and sterol-27-hydroxylase were increased in spice-fed groups. These results indicate that feeding garlic and onion effectively accelerates the regression of preformed CGS by promoting cholesterol desaturation in bile. This observation is significant in the context of evolving dietary intervention strategy to address regression of existing CGS and stopping the possible recurrence.

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.