Effect of blanching and freezing on the physical properties, bioactive compounds, and microstructure of garlic (Allium sativum L.).

The aim of this work was to evaluate the impact of blanching on the physical properties of frozen garlic cloves and to explore the relationship between quality changes and microstructure. A short-term blanching treatment (100 °C  for 45 s, 90 °C  for 45 s, and 80 °C  for 60 s) before freezing did not affect the total organosulfur compound content. In a preliminary research, blanching conditions were determined to be 100 °C  for 45 to 80 s. Under these conditions, peroxidase was inactivated, but organosulfur compounds were retained. Mechanical and color tests showed a damaging effect of blanching and freezing on frozen garlic blanched for 60 and 80 s at 100 °C . Compared to frozen fresh garlic, frozen garlic treated by blanching for 45 s at 100 °C  retained 2871.49 ± 200.24 µg/g of allicin, although 81.83% of peroxidase was inactivated; browning and hardness improved by 49.97 and 48.01%, respectively. According to scanning electron microscopy, significant damage to the microstructure was observed in both frozen fresh garlic and frozen garlic after 60 s and 80 s of blanching at 100 °C . Moreover, 1 H low-field nuclear magnetic resonance (LF-NMR) indicated that blanching for 60 s and 80 s induced an increase in free water in garlic tissues, resulting in further damage after freezing. As peroxidase was efficiently inactivated, the microstructure and organosulfur compounds were better preserved, and blanching treatment at 100 °C  for 45 s before freezing is a potential method for obtaining frozen garlic with high sensory and nutritional qualities. PRACTICAL APPLICATION: Freezing helps to overcome challenges associated with growing seasons and the deterioration of garlic during storage. After frozen garlic is thawed, it is prone to some undesirable changes, such as enzymatic browning and softening. Minimal blanching (45 s at 100 °C ) pretreatment can help to maintain the bioactive compounds of garlic and prevent texture and color deterioration caused by freezing directly.

Structure and morphology of cellulose fibers in garlic skin.

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.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

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."

'Laba' garlic processed by dense phase carbon dioxide: the relation between green colour generation and cellular structure, alliin consumption and alliinase activity.

BACKGROUND: 'Laba' garlic is usually processed by soaking garlic in vinegar for more than 1 week during winter. It is popular for its unique green colour and tasty flavour. Greening is desirable and required for this product as its characteristic. Dense phase carbon dioxide (DPCD) had a significant effect on the greening of intact garlic (Allium sativum L.) cloves. The relation between green colour generation and alliin consumption, alliinase activity and the cellular structure of garlic, respectively, were investigated in this work. The effects of treatment time, pressure and temperature of DPCD were also analysed and discussed. RESULTS: DPCD had a significant effect on the cellular structure of garlic cells. Garlic protoplast underwent greater morphological change after DPCD treatments at higher temperatures while the amount of precipitate increased with greater treatment time and temperature. Common trends on garlic greening and alliin consumption were observed except for DPCD treatment at 10 MPa and 65 °C. The alliinase activity decreased with increasing treatment time, pressure and temperature. It reached the lowest level at 13 MPa and 55 °C. CONCLUSION: The formation of the green colour was a comprehensive result of DPCD on changing cellular structure, alliin consumption and alliinase activity. DPCD treatment at 10 MPa and 55 °C was the optimum condition for the greening of 'Laba' garlic. This work further facilitated the application of DPCD in the industrial production of 'Laba' garlic. © 2015 Society of Chemical Industry.

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.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

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.

Flower development in garlic: the ups and downs of gaLFY expression.

The lack of sexual processes prohibits genetic studies and conventional breeding in commercial cultivars of garlic. Recent restoration of garlic flowering ability by environmental manipulations has opened new avenues for physiological and genetic studies. The LEAFY homologue gaLFY has been shown to be involved in the floral development, while two alternatively spliced gaLFY transcripts are expressed in flowering genotypes. In the present work, quantitative real-time PCR and two techniques of RNA in situ hybridization were employed to analyze spatiotemporal expression patterns of the gaLFY during consequent stages of the garlic reproductive process. Temporal accumulation of gaLFY is strongly associated with reproductive organs, significantly increased during florogenesis and gametogenesis, and is down-regulated in the vegetative meristems and topsets in the inflorescence. The two alternative transcripts of the gene show different expression patterns: a high level of the long gaLFY transcript coincided only with floral transition, while further up-regulation of this gene in the reproductive organs is associated mainly with the short gaLFY transcript. It is concluded that gaLFY is involved at different stages of the sexual reproduction of garlic. These new insights broaden our basic understanding of flower biology of garlic and help to establish conventional and molecular breeding systems for this important crop.

In situ comparative studies on subnucleolar distribution and configuration of plant rDNA.

The distribution and configurations of nucleolar DNA in Pisum sativum L., Allium sativum L., Triticum aestivum L. were analyzed by specific cytochemical staining using NAMA-Ur. It has been observed that in the nucleoli of different plant species, the DNA occupied different positions in different areas, which may imply a different status and strategy of rDNA transcription. Our results showed irregular clumps of rDNA surrounding FCs in semi-circular formations in P. sativum and T. aestivum, indicating a regular pattern of rDNA distribution and supporting the helix model of rDNA configuration. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, which suggests a possible origin for nucleolar DNA.

Unlocking variability: inherent variation and developmental traits of garlic plants originated from sexual reproduction.

Recent collections of fertile garlic (Allium sativum) accessions from Central Asia allow a detailed study of seedling developments and the evaluation of inherent variations. We hereby provide a comprehensive account of the ontogenesis of a population of garlic seedlings and their vegetative and reproductive traits. A nucleotide binding site profiling marker technology was applied to provide conclusive evidence for the cross-pollination nature of garlic, and to compare the levels of polymorphism between progeny derived from a single mother clone fertilized by several pollinators. The seedlings' population demonstrates a large variation in vegetative and reproductive characters, including bulbing ability, bulb color and size, clove number, and response to environmental conditions, similar to that of the genepool of vegetatively propagated garlic clones. In addition, a large variation in flowering and seed production ability was recorded. The understanding of garlic physiology, the availability of the large variability unleashed by sexual reproduction, and the possible utilization of sexual hybridization opens the way for genetic studies and breeding work.

Reproductive development and phenotypic differences in garlic are associated with expression and splicing of LEAFY homologue gaLFY.

Modern garlic (Allium sativum L.) cultivars are sterile and propagated only vegetatively. The recent discovery of fertile genotypes in Central Asia and the restoration of flowering and fertility by environmental manipulations open the way for in-depth florogenetic, genetic, and molecular research in garlic. In the present work, two bolting garlic accessions were employed: #3026, developing normal flowers and seeds, and #2509, in which flowers abort at the early stages of development. Morphological studies showed transition of the apical meristems from the vegetative to the reproductive stage and inflorescence initiation in both genotypes. Low temperatures promote transition of the apex and stem elongation, but have no effect on the phenotypic expression of the inflorescence development. The initial stages of reproductive development in non-flowering #2509 plants were followed by abortion of floral primordia at the differentiation stage. A search for genes involved in the control of flowering in garlic resulted in identification of the garlic LEAFY/FLO homologue, gaLFY. Further comparative analyses of gene expression revealed two gaLFY transcripts, differing in 64 nucleotides, with clear splicing borders. The short variant transcript was identified in both genotypes throughout all development stages, whereas the long variant appears in the flowering genotype #3026 only during reproductive development. The phenotypic differences in garlic, with regard to flowering, may be associated with the efficacy of the splicing process.

Increase in the permeability of tonoplast of garlic (Allium sativum) by monocarboxylic acids.

Immersion of intact aged garlic (Allium sativum) cloves in a series of 5% weak organic monocarboxylate solutions (pH 2.0) resulted in green color formation. No color was formed upon treatment with other weak organic acids, such as citric and malic acids, and the inorganic hydrochloric acid under the same conditions. To understand the significance of monocarboxylic acids and their differing function from that of other acids, acetic acid was compared with organic acids citric and malic and the inorganic hydrochloric acid. The effects of these acids on the permeability of plasma and intracellular membrane of garlic cells were measured by conductivity, light microscopy, and transmission electron microscopy. Except for hydrochloric acid, treatment of garlic with all three organic acids greatly increased the relative conductivity of their respective pickling solutions, indicating that all tested organic acids increased the permeability of plasma membrane. Moreover, a pickling solution containing acetic acid exhibited 1.5-fold higher relative conductivity (approximately 90%) as compared to those (approximately 60%) of both citric and malic acids, implying that exposure of garlic cloves to acetic acid not only changed the permeability of the plasma membrane but also increased the permeability of intracellular membrane. Exposure of garlic to acetic acid led to the production of precipitate along the tonoplast, but no precipitate was formed by citric and malic acids. This indicates that the structure of the tonoplast was damaged by this treatment. Further support for this conclusion comes from results showing that the concentration of thiosulfinates [which are produced only by catalytic conversion of S-alk(en)yl-l-cysteine sulfoxides in cytosol by alliinase located in the vacuole] in the acetic acid pickling solution is 1.3 mg/mL, but almost no thiosulfinates were detected in the pickling solution of citric and malic acids. Thus, all present results suggest that damage of tonoplast by treatment with monocarboxylates such as acetic acid may be the main reason for the greening of garlic.

[Ultracytochemical localization of enzymes and DNA analysis during the development of garlic].

It is believed that during storage, the parenchyma cells of garlic (Allium sativum L.) bulb would wither and fade gradually, and nutrients released inside the cells become available for the germination and growth of the young bud. In this study, The distributions of acid phosphatase (APase) and Adenosine Triphosphatase (ATPase) during germination were analyzed based on the method of lead precipitation at the electron microscopic level. It was found that their activities presenting in plasma membrane, cell wall and plasmodesma increased along the different developmental periods during storage. The fact that the most intensive enzymatic activity of APase and ATPase appeared at germination indicates that degradation, transformation and exportation of cell matrix are helpful in complete translocation of nutrient to new bud. The DNA in the degrading parenchyma cells was analyzed using the agarose electrophoresis. Results clearly showed a typical DNA ladder on the gel, indicating that gene-controlled, programmed cell death may contribute to the degradation of garlic parenchyma cells.

Changes in sucrose and glycerol content in garlic shoot tips during freezing using PVS3 solution.

Changes in moisture content (MC), sucrose and glycerol concentration in garlic shoot tips were monitored during loading and unloading with PVS3 solution. Upon PVS3 treatment, shoot tip MC decreased rapidly and sucrose and glycerol concentrations increased rapidly during the first 30 min. Sucrose and glycerol concentrations increased more slowly thereafter. Shoot tip MC in after PVS3 treatment was affected by their size, but not by sucrose concentration of the preculture medium. As the size of shoot tips increased, so their MC increased after PVS3 treatment. However, sucrose and glycerol concentrations decreased after PVS3 incubation, and concentrations in dehydrated shoot tips were much lower than those measured in non-air dried controls. During unloading with 1.2 M sucrose medium, shoot tip MC increased rapidly during the first 10 min, whereas glycerol concentration decreased steadily over 90 min. Loading and unloading of PVS3 solution in garlic shoot tips follows the principle of solute bulk flow.

[Studies on the change of intercellular connection and the appearance and function of ectodesmata-like structure in garlic clove during the dormancy development].

The characteristics of intercellular connection have been observed during the dormancy development of garlic by the transmission electron microscope (TEM). The results indicated that the appearances of the different states of intercellular connection were physiological adaptation to the development of garlic clove during storage. Ectodesmata-like existed at the wall boundary between the declining tissue and the living cells in the garlic during germination period. By Confocal Laser Scanning Microscopy (CLSM) combined with fluorescence probe, 457Da Lucifer Yellow (LYCH) which was impermeable to the membrane, could enter the living parenchyma cell through symplastic route. The result proved that ectodesmata-like structure, which may be recognized as the modified plasmodesata, still retained physiological activity during a certain time and function as the channels of the symplastic transport of nutrient.

Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb.

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.