Garlic Potyviruses Are Translocated to the True Seeds through the Vegetative and Reproductive Systems of the Mother Plant.

Garlic lost its ability to produce true seeds millennia ago, and today non-fertile commercial cultivars are propagated only vegetatively. Garlic viruses are commonly carried over from one generation of vegetative propagules to the other, while nematodes and arthropods further transmit the pathogens from infected to healthy plants. A recent breakthrough in the production of true (botanical) garlic seeds resulted in rapid scientific progress, but the question of whether viruses are transmitted via seeds remains open and is important for the further development of commercial seed production. We combined morpho-physiological analysis, fluorescence in situ hybridization (FISH), and PCR analysis to follow potyvirus localization and translocation within garlic fertile plants and seeds. Spatial distribution was recorded in both vegetative and reproductive organs. We conclude that garlic potyviruses are translocated to the seeds from the infected mother plant during flower development and post-fertilization, while pollen remains virus-free and does not contribute to seed infection. Therefore, the main practical goal for virus-clean seed production in garlic is the careful maintenance of virus-free mother plants. Although garlic pollen is free of potyviral infection, the male parents' plants also need to be protected from contamination, since viral infection weakens plants, reducing flowering ability and pollen production.

Crosstalk in the darkness: bulb vernalization activates meristem transition via circadian rhythm and photoperiodic pathway.

BACKGROUND: Geophytes possess specialized storage organs - bulbs, tubers, corms or rhizomes, which allow their survival during unfovarable periods and provide energy support for sprouting and sexual and vegetative reproduction. Bulbing and flowering of the geophyte depend on the combined effects of the internal and external factors, especially temperature and photoperiod. Many geophytes are extensively used in agriculture, but mechanisms of regulation of their flowering and bulbing are still unclear. RESULTS: Comparative morpho-physiological and transcriptome analyses and quantitative validation of gene expression shed light on the molecular regulation of the responses to vernalization in garlic, a typical bulbous plant. Long dark cold exposure of bulbs is a major cue for flowering and bulbing, and its interactions with the genetic makeup of the individual plant dictate the phenotypic expression during growth stage. Photoperiod signal is not involved in the initial nuclear and metabolic processes, but might play role in the later stages of development, flower stem elongation and bulbing. Vernalization for 12 weeks at 4 °C and planting in November resulted in flower initiation under short photoperiod in December-January, and early blooming and bulbing. In contrast, non-vernalized plants did not undergo meristem transition. Comparisons between vernalized and non-vernalized bulbs revealed ~ 14,000 differentially expressed genes. CONCLUSIONS: Low temperatures stimulate a large cascades of molecular mechanisms in garlic, and a variety of flowering pathways operate together for the benefit of meristem transition, annual life cycle and viable reproduction results.The circadian clock appears to play a central role in the transition of the meristem from vegetative to reproductive stage in bulbous plant, serving as integrator of the low-temperature signals and the expression of the genes associated with vernalization, photoperiod and meristem transition. The reserved photoperiodic pathway is integrated at an upstream point, possibly by the same receptors. Therefore, in bulb, low temperatures stimulate cascades of developmental mechanisms, and several genetic flowering pathways intermix to achieve successful sexual and vegetative reproduction.

Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.).

BACKGROUND: Garlic is cultivated and consumed worldwide as a popular condiment and green vegetable with medicinal and neutraceutical properties. Garlic cultivars do not produce seeds, and therefore, this plant has not been the subject of either classical breeding or genetic studies. However, recent achievements in fertility restoration in a number of genotypes have led to flowering and seed production, thus enabling genetic studies and breeding in garlic. RESULTS: A transcriptome catalogue of fertile garlic was produced from multiplexed gene libraries, using RNA collected from various plant organs, including inflorescences and flowers. Over 32 million 250-bp paired-end reads were assembled into an extensive transcriptome of 240,000 contigs. An abundant transcriptome assembled separately from 102,000 highly expressed contigs was annotated and analyzed for gene ontology and metabolic pathways. Organ-specific analysis showed significant variation of gene expression between plant organs, with the highest number of specific reads in inflorescences and flowers. Analysis of the enriched biological processes and molecular functions revealed characteristic patterns for stress response, flower development and photosynthetic activity. Orthologues of key flowering genes were differentially expressed, not only in reproductive tissues, but also in leaves and bulbs, suggesting their role in flower-signal transduction and the bulbing process. More than 100 variants and isoforms of enzymes involved in organosulfur metabolism were differentially expressed and had organ-specific patterns. In addition to plant genes, viral RNA of at least four garlic viruses was detected, mostly in the roots and cloves, whereas only 1-4% of the reads were found in the foliage leaves. CONCLUSIONS: The de novo transcriptome of fertile garlic represents a new resource for research and breeding of this important crop, as well as for the development of effective molecular markers for useful traits, including fertility and seed production, resistance to pests and neutraceutical characteristics.

Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production.

Commercial cultivars of garlic (Allium sativum) do not produce flowers and seed; hence, information on microgametogenesis and genetic knowledge of this important crop is unavailable. Recently, physiological studies enabled flowering and fertility restoration in garlic bolting genotypes by environmental manipulations, thus broadening of the genetic variation and facilitating genetic studies. The present report provides first detailed description of the development of male gametophytes in 11 garlic genotypes varying in their fertility traits. Morphological and anatomical studies revealed completely fertile genotypes, as well as variation in anther and pollen development and disruption of the male organs and gametes at different developmental stages. Three types of plant sterility were observed, including complete sterility, male sterility and environmentally induced male sterility. The ITS1 and ITS2 regions of rRNA of the studied genotypes proved to be strongly conservative and thus did not correspond with the phenotypic expression of fertility or sterility in garlic. On the other hand, two-dimensional protein separation maps revealed significant differences between fertile and sterile genotypes, as well as between developmental stages of microsporogenesis. Further research is needed to investigate the internal mechanisms and environmental component of garlic sterility, as well as the possible molecular markers of these traits.

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.

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.

Differential effects of tomato (Lycopersicon esculentum mill) matrix on the volatility of important aroma compounds.

Significant tomato matrix effects on the volatility of certain fresh tomato odorants were found. The concentrations of odorants such as (E,E)-2,4-decadienal, beta-damascenone, and beta-ionone, in crushed fresh tomato fruit obtained by solid-phase microextraction (SPME), resulting from a tomato matrix calibration curve were 5.5-, 2-, and 12-fold higher, respectively, than those calculated by calibration based on buffer solutions. Static headspace analyses indicated that, in most cases, the tomato matrix significantly retains the odorants relative to the buffer solution. Thus, the concentration of odorants in the headspace of tomato is lower than expected compared to a simple matrix such as buffer. CaCl(2), although needed in crushed fruit tissue to block enzymatic activity, was found to interact specifically with 2-isobutylthiazole, reducing its content in the headspace by at least 6-fold. If a matrix effect is found, analysis of the odorant molecule contents in the headspace rather than in the food is recommended in order to better evaluate their access to the olfactory receptors.