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.     

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     

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.)

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.     

Regeneration of whole plants from Gracilaria asiatica Chang et Xia protoplasts (Gracilariaceae,Rhodophyta)

A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.     

An in vitro method for the conservation and storage of garlic (Allium sativum) germplasm

Shoot cultures of three garlic (Allium sativum) cultivars were kept in various temperatures and media in order to maintain their viability without subculture. A high level of viability was recorded after 16 months of culture at 4°C with 100 gl^-1 sucrose in B-5 medium.     

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     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

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.     

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Cadmium accumulation and oxidative burst in garlic (Allium sativum)

To investigate the temporal sequence of physiological reactions of garlic (Allium sativum) to cadmium (Cd) treatment, seedlings developed from cloves were grown in increasing concentrations of CdCl2, ranging from 1-10 mM, for up to 8 days in sand. Analysis of Cd uptake indicated that most Cd accumulated in roots, but some was also translocated and accumulated in leaves at longer exposure time (after 12h) and higher concentrations (5 and 10mM) of CdCl2. Changes in activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), were characterized in leaves of garlic seedlings. Cd (5 and 10 mM) initially inhibited the activities of SOD and CAT but thereafter recovered or even increased compared with control plants. POD activities at 5 and 10 mM of Cd increased more than 3-4 times over control plants within 12 h and then dropped, but were still higher than controls at the end of the experiment. Otherwise lipid peroxidation enhanced with the increasing of incubation time and concentrations of external Cd. Leaves exposed to 1 mM CdCl2 showed a less pronounced response and only a small reduction in shoot growth. These results suggested that in leaves of garlic seedlings challenged by CdCl2 at higher concentrations, induction of these various enzymes is part of a general defense strategy to cope with overproduction of reactive oxygen. The possible mechanism of antioxidative enzymes changing before Cd accumulation in leaves of garlic seedlings is discussed.     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

大蒜分生组织培养脱病毒和快速繁殖技术

采用茎尖分生组织培养技术,获得了大蒜（Ａｌｌｉｕｍ ｓａｔｉｖｕｍ Ｌ．）的无病毒试管苗。通过基本培养基和激素配比试验,筛选出最佳的培养基组成,进行脱病毒苗的快速繁殖。结果表明：诱导愈伤组织的最适培养基为：ＭＳ＋ＢＡ０．２ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ,月生长率达１２．７０倍;诱导丛生芽的最适培养基为：Ｂ５＋ＢＡ０．５ｍｇ／Ｌ＋ＩＡＡ０．２ｍｇ／Ｌ,丛生芽繁殖系数高达２５．５倍,技术上达到了快速繁     

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.)

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic;Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of thein vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.     

Plant regeneration from protoplasts of wild barley (Hordeum murinum L.)

Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - L1, L2 medium according to Lazzeri et al. 1991 - L3 medium medium according to Jähne et al. 1991a     

大蒜花序轴离体培养器官发生途径的解剖学研究

以大蒜品种‘三月黄’(Allium sativum L.cv. Sanyuehuang)花序轴为外植体进行离体培养,对其器官发生过程进行了形态学和解剖学观察。结果显示:大蒜花序轴离体培养不经过愈伤组织,通过器官直接发生途径形成不定芽,其不定芽起源于大蒜花序轴维管组织韧皮部一侧周围的皮层薄壁细胞,属于外起源;皮层薄壁细胞经脱分化后,由最先形成的拟分生组织发育为茎尖分生组织,然后环绕其形成叶原基,茎尖和叶共同构成一个完整的不定芽;大蒜花序轴离体培养发生的不定芽与花苞中自然形成的营养芽发生部位一致。不定芽通过壮苗、生根培养可正常生根形成植株,如果继代培养周期超过21 d,鳞茎形成率可达90.56%。     

Regeneration of plants from Antirrhinum majus L. callus

Somaclone production in Antirrhinum majus plants by regeneration of plants from callus cultures has been achieved using three types of explant tissue. Regeneration from mature stem internode-derived callus was extremely poor. Callus derived from seedling shoot tips could be induced to form new shoots in six of seven cultivars tested. Regeneration was achieved in all seven cultivars when callus was produced from segments of hypocotyl and was most effective using agar-solidified medium containing 0.25 mgl^-1 naphthoxyacetic acid + 10% coconut milk. In this case, five of the cultivars produced shoots directly, one produced leaves from the petioles of which new shoots emerged, and one regenerated plants chiefly through the production of embryoids.     

Agrobacterium tumefaciens-mediated transformation of leek (Allium porrum) and garlic (Allium sativum)

Transgenic leek (Allium porrum) and garlic (Allium sativum) plants have been recovered by the selective culturing of immature leek and garlic embryos via Agrobacterium-mediated transformation using a method similar to that described by Eady et al. (Plant Cell Rep 19:376–381, 2000) for onion transformation. This method involved the use of a binary vector containing the m-gfp-ER reporter gene and nptII selectable marker, and followed the protocol developed previously for the transformation of onions with only minor modifications pertaining to the post-transformation selection procedure which was simplified to have just a single selection regime. Transgenic cultures were selected for their ability to express the m-gfp-ER reporter gene and grown in the presence of geneticin (20 mg/l). The presence of transgenes in the genome of the plants was confirmed using TAIL-PCR and Southern analysis. This is the first report of leek and true seed garlic transformation. It now makes possible the integration of useful agronomic and quality traits into these crops.     

Plant Regeneration from Mesophyll Protoplasts of Echinacea Purpurea

An efficient plant regeneration system was developed from isolated protoplasts of Echinacea purpurea L. using an alginate block/liquid culture system. Viable protoplasts could be routinely isolated from young leaves of Echinacea seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase and 0.3 mol l^–1 mannitol. Purified protoplasts were embedded in 0.6% Na-alginate block at a density of 1 × 10⁵/ml and cultured in a modified MS medium containing 0.3 mol l^–1 sucrose, 2.5 µmol l^–1 BA and 5.0 µmol l^–1 2,4-D. Cell colonies were observed after 4 weeks of culture, and the protoplast-derived colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS medium supplemented with 1.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Shoot organogenesis from protoplast-derived callus was induced on MS medium supplemented with 5.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Complete plantlets were obtained from the regenerated shoots on MS basal medium. The protoplast to plant regeneration protocol developed in this study provides the prerequisite for creating novel genotypes of this valuable medicinal species through genetic manipulation.