Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer

 By using highly regenerative calluses, we developed a stable transformation system in garlic (Allium sativum L.). The temperature and number of days of co-cultivation with Agrobacterium tumefaciens was shown to be an important factor in transient expression of the uid A gene. After a culture period of 5 months in selection medium containing hygromycin, 20 shoots were induced from ca. 1000 calluses, among which 15 plants expressed β-glucuronidase activity upon staining with X-Gluc. Shoots developed into transgenic garlic after 1 month. Integration of the uid A gene was confirmed by Southern blot analysis for genomic DNA of transgenic garlic plants. Received: 25 October 1999 / Revision received: 16 February 2000 / Accepted: 22 February 2000     

Infraspecific differentiation of garlic (Allium sativum L.) by isozyme and RAPD markers

Garlic (Allium sativum L.) is a sterile species of considerable variability with respect to morphological and physiological features. The crop presumably originated in West to Middle Asia from its progenitor A. longicuspis Regel and was transported from there to the Mediterranean and other areas of cultivation. In order to clarify older classification schemes, often based on small or biased collections, we used isozyme and RAPD markers to analyze and structure a collection of 300 accessions, many of which were gathered in Middle Asia close to the assumed center of origin. All of the accessions were first investigated with isozymes, and 48 were selected for a RAPD analysis. The resulting molecular markers were used to construct neighbor-joining dendrograms to group the accessions and to indicate the genetic distances between them. Based on the dendrograms and in conjunction with some morphological features, we propose an infraspecific classification of garlic with four major groups. In agreement with the results of other workers, A. longicuspis lies within the range of the species A. sativum. Numerous forms with varying degrees of domestication are part of our longicuspis group, from which presumably the more derived cultivar groups originated. The origin and spreading of the crop are discussed with respect to the geographical distribution and the genetic distances of the accessions.     

Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis

Plants were regenerated by somatic embryogenesis from long-term callus cultures derived from five garlic (Allium sativum L.) cultivars. Thirty-five of these plants were subjected to RAPD analysis. The frequency of variation was found to be cultivar dependent: approximately 1% in the two clones Solent White and California Late and around 0.35% in another three clones, Chinese, Long Keeper and Madena. Certain band changes were found in regenerants of different cultivars, suggesting the existence of a mutation-sensitive part of the garlic genome. The karyotypes of another 75 regenerants derived from the same callus cultures of three parental garlic clones were examined. Of these plants, 9.3% were found to be tetraploids, 4% aneuploid and 2.6% showed a change in the position of the secondary constriction. No association could be shown between the rate of variation for molecular and cytological characters either by comparing cultivars or examining individual regenerants. Received: 30 July 1996 / Revision received: 28 October 1996 / Accepted: 12 November 1996     

Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoots

Hyperhydricity is a physiological disorder frequently affecting shoots propagated in vitro. Since it negatively affects shoot multiplication vigor, and impedes the successful transfer of micropropagated plants to in vivo conditions, hyperhydricity is a major problem in plant tissue culture. In commercial plant micropropagation, there are reports of up to 60% of cultured shoots or plantlets which demonstrate hyperhydricity, which reflects the pervasiveness of this problem. The phenomenon has been correlated to water availability, microelements, and/or hormonal imbalance in the tissue culture. In this study, the ultrastructure and the characteristics of reactive oxygen species between hyperhydric and normal shoots of garlic were studied. We observed that in some cells of hyperhydric tissues, the intranuclear inclusion was separated, the mitochondrion was swollen and its intracristae had splits, the organelles were compressed against the cell wall, and the chloroplasts and intergranal thylakoids were also compressed. Additionally, the content of chlorophyll and soluble protein in hyperhydric shoots decreased significantly. For instance, chlorophyll a decreased 43.61%, chlorophyll b decreased 49.29%, chlorophyll a+b decreased 48.10%, and soluble protein dropped 47.36%. In contrast, the O₂ generation rate and H₂O₂ level increased 45.36% and 63.98%, respectively, obviously higher than the normal shoots. Lipoxygenase activity and malondialdehyde content in the hyperhydric shoots increased significantly, while the electrolyte leakage rose, indicating a serious membrane lipid peroxidatic reaction. Superoxide dismutase, peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase activities in hyperhydric tissue were all significantly higher than in normal leaf tissue. The antioxidant metabolism demostrated a close connection between hyperhydricity and reactivated oxygen species.     

Utilization of hydrolytic enzymes for the extraction of cycloalliin from garlic (Allium sativum L.)

In the present study, enzyme assisted extraction of organosulfur compounds, especially cycloalliin, from garlic (Allium sativum L.) was examined using various commercial cellulases, and the changes of the cycloalliin contents in garlic extract were investigated after storage at 40 °C and 60 °C for 30 days. Among the commercial enzymes tested, Ultraflo L showed the greatest yield of cycloalliin compared to other cellulases. The conditions were optimized to include 2.5% (v/w) addition of Ultraflo L, 1 h incubation at 40 °C and a pH of 6.0. Under the optimum conditions, the contents of cycloalliin achieved 1.5-folds increase in the enzyme-assisted garlic extract compared with the non-enzymatic extraction. In addition, the cycloalliin content was also significantly increased 3.8-fold after storage at 60 °C for 15 days. The polyphenol content was also significantly increased by 3-fold after at 60 °C for 30 days. Overall, Ultraflo L proved to be an efficient method to extract cycloalliin from garlic.     

Analysis of the genetic diversity of garlic (Allium sativum L.) germplasm by SRAP

Cluster analysis and principal component analysis were used to investigate the genetic diversity of 40 garlic germplasms analyzed with 23 sequence-related amplified polymorphism (SRAP) primer combinations. A total of 130 polymorphic loci were detected among these germplasms, with an average of 5.65 polymorphic loci per SRAP primer combination. The percentage of polymorphic loci was 69.1%, whereas the mean effective number of alleles, the mean Nei's gene diversity, and the mean Shannon's information index were 1.4446, 0.2788, and 0.4365, respectively. Cluster analysis revealed that the 40 germplasms could be divided into 3 groups. The results of principal component analysis were consistent with those of unweighted pair-group method with arithmetic averages (UPGMA) clustering analysis. The Shannon-Weaver information index ranged from 0.2419 to 0.4202, indicating that the garlic germplasms had high genetic diversity.     

Evaluation of some garlic (Allium sativum L.) mutants resistant to white rot disease by RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic diversity among eight garlic mutants resistant to white rot disease (Sclerotium cepivorum). Twelve of the 13 synthetic random primers were found to identify polymorphism in amplification products. Mutants characterised with moderate resistance to white rot were closely related to the control using cluster and correlation analyses. On the other hand, highly resistant mutants were quite distant from the control with low correlation coefficients. The banding patterns produced by primer OPB‐15 (GGAGGGTGTT) with highly resistant mutants may by used as genetic markers for early selection of resistant plants.     

Generation of chlorsulfuron-resistant transgenic garlic plants (Allium sativum L.) by particle bombardment

We established an effective biolistic transformation procedure fortransferring foreign genes into garlic (Allium sativumL.),which we demonstrated by generating transgenic plants resistant tochlorsulfuron, a sulfonylurea herbicide. We subcultured callus tissue from theapical meristem of garlic cloves and repeatedly selected calli with brittle,non-mucilaginous surfaces for over six months, to increase transformationefficiency. We then constructed recombinant DNA that contained the acetolactatesynthase (ALS) gene from a chlorsulfuron-resistantArabidopsis mutant, the cauliflower mosaic virus 35Spromoter, the -glucuronidase (GUS) reporter gene, and the hygromycinphosphotransferase (HPT) selectable marker gene. The garlic calli werebombarded twice with tungsten particles coated with the DNA constructs. Transformed calliwere efficiently selected by embedding them in solid agar medium containing 50mg l^–1 hygromycin B. Selected propagules wereregenerated into 12 independent plants. We confirmed that the transgenes wereintegrated and expressed in the plants using PCR-Southern and Northern blotanalyses and by -glucuronidase expression assay forGUS. The regenerated plants survived in the presence of 3mg l^–1 chlorsulfuron, demonstrating that theirALS was insensitive to this herbicide. These results illustrate the successfultransformation of foreign genes into garlic plants. The set of proceduresdeveloped in this study is applicable to the generation of transgenic garlicplants with other agronomically beneficial traits. These authors contributed equally to this work     

大蒜体细胞胚胎发生研究进展

大蒜生产主要靠无性繁殖 ,因此 ,进行大蒜体细胞胚发育研究具有重要意义。本文对大蒜体细胞胚发育的影响因子进行了综述 ,其中较高浓度的维生素B1 及还原态氮源可能有利于胚胎发生 ,而大蒜体细胞内含物则不利于胚胎发生。此外 ,对大蒜体细胞胚培养中存在的主要问题进行了讨论 ,并认为系统开展体细胞胚发生的细胞分子生物学机理研究、建立悬浮培养体系以及进行大蒜体细胞胚无性系变异的研究等 ,具有广阔的前景。     

Continuous callus production and regeneration of garlic (Allium sativum L.) using root segments from shoot tip-derived plantlets

Root segments from shoot tip-derived plantlets of the garlic (Allium sativum L.) clones `DDR7099', `PI383819', and `Piacenza' were utilized as an explant source for continuous, friable callus production. The best callus production occurred on root segments initially cultured on medium with 4,5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 weeks, then subcultured to medium with 4.7 μm 4-amino-3,5,6-trichloropicolinic acid (picloram) +0.49 μm 6-(γ-γ-dimethylallylamino)purine (2iP) for 8 weeks. Embryogenic, friable callus was transferred to liquid medium for 1 month and then transferred to solid regeneration medium for 14 weeks. The best shoot and root regeneration (85.3% and 35.8%, respectively) occurred on 4-month-old calli from the clone `DDR7099'. In all clones, regeneration rate decreased as callus age increased. Received: 14 October 1997 / Revision received: 26 December 1997 / Accepted: 12 January 1998     

Isolation,characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.)

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.     

Flavor formation in tissue cultures of garlic (Allium sativum L.)

Differentiated and undifferentiated cultures of garlic (Allium sativum L.) were analyzed for the study of flavor formation in cultures. Attempts were made to correlate alliin content with free and bound amino acid contents and with enzymes like phenylanine ammonialyase (E.C. 4.1.1.5) and alliin-lyase (E.C.4.4.1.4) which play important roles in formation of the flavor percursor alliin.It was observed that in differentiating cultures showing shoot formation, there is an increase in alliin content as well as in free and bound amino acid contents. Corresponding to this there was also an increase in the activity of phenylalanine ammonialyase in differentiating cultures. Alliin-lyase activity was found to be significantly different in differentiating and undifferentiated cultures. The significance of these results is discussed.     

Sister chromatid exchanges in garlic (Allium sativum L.) callus cells

A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing 100 M BrdU+0.01 M FudR+1 M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of genetic instability of plant cells cultured in vitro.Abbreviations BrdU 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FPG fluorescent-plus-Giemsa - FudR 5-fluoro-2-deoxyuridine - SCE sister chromatid exchange - SSC 0.15 M NaCl + 0.015 M Na-citrate - T thymidine-containing strand of the DNA duplex - B 5-bromo-2-deoxyuridine-containing strand of the DNA duplex - Urd uridine     

Role of garlic (Allium sativum L.) in human and plant diseases

A resurgence of interest in garlic due to recent revelations of its beneficial effects in the treatment of various human and plant diseases and also due to validation of claims made in traditional systems of medicine has resulted a plethora of publications on different aspects of garlic in recent years. Chemical constituents of garlic and their variations on the methods of isolation have been discussed in the present review. Effect of garlic and its constituents against various human and plant pathogenic and saprophytic microorganisms has also been reviewed.     

Development of morphogenic suspension cultures of garlic (Allium sativum L.)

Rapidly growing, regenerable suspension cultures were obtained from meristem-derived callus cultures of garlic (Allium sativum L.). The liquid culture medium consisted of MS salts, B5 vitamins, 3% sucrose, 1 mg l^–1 naphthalene-acetic acid (NAA) and 2 mg l^–1 6-benzyladenine (BA). The tissue in the suspension culture was yellow, smooth, organized, and proliferated as nodular clumps. Histological examination revealed that these morphogenic clumps had a well-defined epidermis. Following transfer of the morphogenic clumps to an agar-solidified medium, numerous meristems with green leaf primordia were produced.     

大蒜种质产量和品质性状主成分聚类分析与综合评价

以40个大蒜品种为供试材料,依据数值分类学的性状选择原则,分别于大蒜生长期和采收后进行农艺性状指标的采集。估算40个大蒜品种16个农艺性状及4个品质指标的主成分,并以前3个主成分和遗传相似性系数为基础,分别作二维散点图和系统聚类分析。40份大蒜品种前7个主成分累计贡献率达85%。根据品种性状主成分表现,评选出性状优良的大蒜品种共10个。在聚类图中,在0.14的遗传相似性水平上可以把40份品种分成4类,即由5份种质组成的类群Ⅰ;由28份种质聚成的类群Ⅱ;由改良蒜等4份种质组成的类群Ⅲ,及苏联蒜等3份种质组成的类群Ⅳ。全部种质的遗传相似性系数在0.07～0.64之间,很好地揭示了品种类群间存在的亲缘关系。     

Genetic variability in callus formation and regeneration of garlic (Allium sativum L.)

Twenty garlic Allium sativum (L.) genotypes were analysed for genetic variation in their ability to form callus (in one medium) and regenerate shoots (in four different media). Genotypes showed significant differences in the analysis of variance of all the traits tested. The accession Printanor showed the best general behaviour, with 83% callus-producing explants, 44.7% organogenic explants, and 15.35 shoots/g of callus. The best regeneration medium was MBO, without growth regulators. Shoot production capacity was examined with the additive main effects and multiplicative interaction model that proved to be a powerful tool for analysis and easy comprehension of the strong genotype×medium interactions frequently observed in in vitro culture systems. Received: 5 February 1998 / Revision received: 11 May 1998 / Accepted: 1 June 1998     

Relationships between peroxidases and in vitro bulbification in garlic (Allium sativum L.)

Summary The relationship between in vitro bulbification and peroxidase activities of garlic (Allium sativum L.) was studied. Two stages could be distinguished during in vitro bulb formation characterized by the peroxidase activity, isoenzymatic patterns especially of the soluble fractions, dry weight, and bulbification index (BI). The first stage, called the morphogenic stage, started after planting until 30d of culture with a maximum soluble peroxidase activity, BI=1–0.5 and low dry weight. At that time axillary buds preformed at the base of the leaves grew and the in vitro bulb was generated. The second stage (filling in and bulb maturation) started when the BI reached 0.5 at 30 d of the ontogenic cycle, as a result of the bulb assimilate accumulation phenomenon. During the morphogenic stage the soluble peroxidase activity was maximum and the zymograms showed higher intensity bands. The second stage presented anodic ionic peroxidases and substantial increase in staining of the anodic covalent peroxidase fraction. The putative role of the different isoforms of peroxidases in relation to the bulbification process is discussed.     

Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.)

A restricted part of the undeveloped stem of the garlic clove, called the “stem disc”, which is just under the basement of the immature foliage leaves, proved to be a very potent explant for the micropropagation of garlic. Twenty to thirty tissue-cultured shoots consistently were differentiated from a single clove during 1 month of culture on phytohormone-free Linsmaier and Skoog medium. In addition, more than 90% of the shoots formed bulblets in vitro during an additional 1 month of culture. Pretreatment of the garlic bulbs at 4 °C for approximately 8 weeks before preparing the stem discs enhanced both shoot development and bulblet formation. This novel method for culturing garlic was designated the stem-disc culture method. Shoot development in this type of in vitro culture apparently is divided into four stages: expansion of tissue zones surrounded by the basal parts of the immature foliage leaves, formation of dome-shaped structures, bud differentiation directly from each dome, and development into shoots and bulblets. The dome-shaped structures appeared within 5 days of the onset of culture and had developed independently into shoots approximately 1 cm high 3 weeks later. Histological observations showed that both the internal cell organization and formation process of the dome-shaped structures were similar to those in the meristem. In addition, events leading to the formation of these dome-shaped structures appeared to be initiated by vigorous cell division in the epidermis of concentric tissue zones surrounded by the basements of immature foliage leaves. The results of several field trials showed that the stem-disc culture method is useful for the production of garlic seed plants, including virus-free plantlets. Furthermore, it is a novel field cultivation system for garlic in that the seedlings produced by in vitro-induced bulblets are used as seed instead of the usual cloves. Received: 25 November 1997 / Revision received: 4 February 1998 / Accepted: 21 February 1998