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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.

The knowledge of the texture and morphology of cellulose is essential for reliable modelling of cell growth and mechanical resistance of vegetal systems. Microscopic observations on thin layers of the skin of Allium sativum have shown elongated structures (i.e. cellulose fibers) imbedded in a matrix of more or less rounded cells. Examination by an optical polarizing microscope (OPM) has shown an intermittent high and low birefringence along fibers. Transversal regions with a reduced brightness along fibers are expected to contain a higher amount of amorphous lignin, hemicelluloses and waxes, some of which might also be birefringent, but at a much lower degree than cellulose. Scanning electron microscopy (SEM) has also evidenced an alternating growth of the fibers. Moreover, the negative sign of birefringence suggests a parallel orientation of cellulose nanofibrils transversally to the fiber axis. The characteristic modulation of intensity along lignocellulosic fibers can be due to variation of the cellulose concentration or orientation, perhaps caused by circadian cycles of temperature and light during growth. Indeed, imperfect orthogonal light can be totally reflected at the interface between regions with different values of the refractive index, contributing to the optical effect of banding.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2 weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5 mg l -1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25 mg l -1 2, 4-D and 1.0 mg l -1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5 mg l -1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0 mg l -1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45 pg and 33.56 pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."

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.

In this study, the antimutagenic function of the polysaccharide from Enteromorpha linza with the micronucleus test of Allium sativum root cells induced by sulfur dioxide and ultraviolet was studied. The concentration-effect relation of the two inducers was firstly evaluated. The results showed that an increase of genotoxicity damage was demonstrated and micronuclei frequency induced by sulfur dioxide and ultraviolet displayed dose dependent increases. All the doses of polysaccharide did affect the micronuclei frequency formation compared with the negative control. And also, the significant increase in inhibition rate of micronuclei frequency was observed with the increase of the dose of polysaccharide. It was showed maximum inhibition of micronuclei frequency cells (71.74% and 66.70%) at a concentration of 200g/mL in three experiments. The low molecular weight polysaccharide showed higher inhibition rate than raw polysaccharide at the higher concentration (50g/mL) in the absence of sulfur dioxide and ultraviolet. It was confirmed to be a good mutant inhibitor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Because of their unique spectral properties, quantum dots (QDs) have recently proved useful as fluorescent labels for biosensing probes. We developed a versatile QD label by modifying dsDNA with biotin and thiol groups at opposite ends and attaching it to quantum dots via a metal-thiol bond. These dsDNA-coated QDs fluorescently label their targets through biotin-streptavidin binding and show excellent histological results when used to detect biotin-labeled chromosome probes. The dsDNA coating also circumvented the common problems of aggregation and steric hindrance that occur with other QD probes.

Direct somatic embryo formation (without intervening callus) from garlic clove basal tissue was induced in which the influence of plant growth regulators (PGRs) on various explants was examined. Medium added with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were the most effective PGR combination for somatic embryo induction. It induced embryos directly in 85.5% of the basal clove explant. Callus induction was also obtained from other parts of explant and 2.0 mg/l 2,4-D induced callusing in 86.5% of the inoculated explants. Protein, amino acid and alliin content were measured in callus and in embryos. Somatic embryos had more soluble protein and free amino acid compared to callus. HPTLC analysis revealed that alliin was significantly high in somatic embryos compared to undifferentiated callus tissue; the content was even more in older embryos. The present study of Allium indicates that the event of morphogenetic development including in vitro embryogeny can effectively be analysed by monitoring the changes of biochemical profiles.

Using fluorescence and electron microscopy, the ultrastructure and symplastic transport function of Ectodesmata-like (ED-like) were studied in the declining parenchyma cells of garlic during germination period. The results showed that plasmodesmata (PD) were gradually stretched and broke into ED-like. The diameter of PD and the diameter of appressed endoplasmic reticulum (AER) in PD underwent a series of changes. F-actin and myosin might take part in the regulation of the symplastic transport of the ED-like.

The garlic cultivars grown in Brazil evolved from somatic mutations and clone selection by breeding programs and by the introduction of germplasm from other countries. Morphological characters have been used to differentiate these cultivars. Two hundred and six random amplified polymorphic DNA markers were utilized for a diversity analysis of the 17 most planted garlic cultivars in Brazil. Bootstrap analysis showed that the number of markers was efficient and sufficient to obtain a coefficient of variation of 10%. Similarity varied between 16 and 98% and cluster analysis showed that, in general, genetic similarities correlate with morphological characters of the cultivars and production cycle variation. High bootstrap values at most of the nodes supported the dendrogram stability. The grouping of most varieties agreed well with previous reports based on morphological characters. As a vegetative-propagated species, viral diseases are a key problem regarding production and quality of the bulbs, causing gradual loss of yield and decrease in storage capacity. To improve the health quality of garlic seed, a virus-free stock of garlic cloves of the Amarante cultivar was obtained. The ability to distinguish garlic cultivars to detect varietal mixing after in vitro multiplication is extremely important, since correct identification is not possible until bulbs are produced. Random amplified polymorphic DNA markers were also used to differentiate cultivars while they are in vitro and not amenable to morphological discrimination. No difference was identified between the fingerprints of the virus-free or of the infected bulks of Amarante, showing that there was no clove mixing in the handling of material in the clonal multiplication phase.

Gibberellic acid (GA(3)) is a very potent hormone whose natural occurrence in plants controls their development. Cadmium is a particularly dangerous pollutant due to its high toxicity and great solubility in water. In this study, the effect of GA(3) on Allium sativum root tip cells was investigated in the presence of cadmium. A. sativum root tip cells were exposed to CdNO(3) (50, 100, 200 microM), GA(3) (10-3 M), both CdNO(3) and GA(3). Cytogenetic analyses were performed as micronucleus (MN) assay and mitotic index (MI). Lipid peroxidation analysis was also performed in A. sativum root tip cells for determination of membrane damage. MN exhibited a dose-dependent increase in Cd treatments in A. sativum. GA(3) significantly reduced the effect of Cd on the MN frequency. MN was observed in GA(3) and GA(3) + 50 mum Cd treatments at very low frequency. MI slightly decreased in GA(3) and GA(3) + Cd treatments. MI decreased more in high concentrations of Cd than combined GA(3) + Cd treatments. The high concentrations of cadmium induce MN, lipid peroxidation and lead to genotoxicity in A. sativum. Current work reveals that the effect of Cd on genotoxicity can be partially restored with GA(3) application.

To validate the use of Allium sativum as a sensitive test model for genotoxicity, the cytogenetic effects of a commercial formulation of the pyrethroid insecticide, cypermethrin, were evaluated in the root meristem cells of A. sativum. Ultraviolet (UV) and Fourier transform infrared (FTIR) spectral measurements were also carried out to understand the interaction of cypermethrin with DNA. In a preliminary toxicity assay, the EC50 for Allium root growth was estimated to be 8 ppm. For the cytogenetic assay, root meristem cells were exposed to 1, 2, 4, 8 and 16 ppm of the test compound for 24 h, and either processed immediately for analysis or incubated in water for 24 h of recovery and then processed. Cells analyzed immediately after the exposure had a significant, dose-dependent inhibition of mitotic index (MI) and induction of mitotic and chromosomal aberrations (MAs and CAs). The 24 h recovery period reduced the effect of the test compound on the MI and percent aberrations; however, cells exposed to 8 and 16 ppm showed a significant frequency of aberrations despite the recovery period. One part per million cypermethrin was consistently negative in the assay. The data indicate that higher doses of cypermethrin produce toxicity, CAs and MAs in A. sativum. The present study indicates that A. sativum is a sensitive and reliable test system. A bathochromic shift observed in UV absorption spectra reveals that cypermethrin binds with DNA. Role of vibrational modes of the active site in the recognition and reaction of cypermethrin with DNA has been discussed. Based on spectroscopic data and structural properties, a possible mechanism has been proposed for the interaction of cypermethrin with DNA resulting in chromosomal aberrations.

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.

We used a DNA-specific staining technique to show the two states of DNA component distributed in the nucleolar region of Allium sativum cells. One state is the extended DNA fiber, and the other is the condensed DNA clump. In situ hybridization demonstrated that the extended DNA fiber was an rRNA gene. Anti-fibrillarin antibody immunolabeling revealed that these rRNA genes were located in the dense fibrillar component near the fibrillar center, including at the periphery of the fibrillar center. None was in the dense fibrillar component far away from the fibrillar center. The condensed DNA clump was located in the fibrillar center. Further observations showed that the rRNA genes in the nucleolus were all arranged around the fibrillar center and associated with the DNA clumps in the fibrillar center. Results of statistical analysis showed that the distribution region of rRNA genes occupied about one-third of the total dense fibrillar component region. Ag-NOR protein showed a similar distribution pattern to that of rDNA. Immunolabeling of an anti-RNA/DNA hybrid antibody demonstrated that the transcription sites of rRNA were located at the periphery of the fibrillar center and in the dense fibrillar component near the fibrillar center, and these sites were consistent with the location and arrangement of rDNA shown in situ. These results demonstrated that transcription of rRNA takes place around the fibrillar center and at the periphery, whereas the dense fibrillar component that was far away from fibrillar center was the non-transcription region. The DNA clumps within the fibrillar center were probably the anchoring sites for rDNA arrangement.

The aim of this work was to study the physiological mechanisms of dormancy and sprouting during post-harvest of garlic (Allium sativum L.) microbulblets produced by meristem culture of garlic seed cloves. The morphological changes occurring in garlic microbulblets were assessed from harvest till sprouting in relation with peroxidase activity and levels of gibberellins. Also the effect of a cold treatment (30 days at 4 degrees C) given 30 days after harvest was studied. The results showed that during the state of dormancy in garlic microbulblets formation of the leaf primordia and vascular differentiation of the storage leaf occurred, while increases of peroxidase activity and low levels of GA3 (the only active gibberellin identified) were found. At the end of dormancy the sprouting channel was formed, vascular differentiation established, and peaks of soluble peroxidase activity as well as of GA3 were observed. At day 90 post-harvest, garlic microbulblets showed physiologically mature and able to sprout. Further on, bud expansion and decrease of GA3 levels characterized sprouting of the microbulblets. The cold treatment enhanced GA3 levels and anticipated the sprouting process.

An efficient novel method of direct somatic embryogenesis from basal tissue of garlic clove was developed. The influence of plant growth regulators, basal medium and explant type on somatic embryo induction was examined. The best plant growth regulator combination was, 2,4-D and kinetin at 1.0 mg/L and 0.5 mg/L respectively, inducing direct somatic embryogenesis in 60% of explants. White's medium was used as basal medium and somatic embryos developed on explants after six weeks. The technique has potential applicability for problems associated with plant regeneration and virus elimination in garlic.

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.

The G1, S and G2 phases of the cell cycle in Allium cepa root tips were determined in Acriflavin--Feulgen--stained material. Pigeon erythrocyte nuclei were employed as standard for DNA dry mass determinations. Micro-interferometric and fluorometric measurements gave the average of 77,1. 10(-12) g for the G1 nuclei from the root tips.