The development of a long-term callus storage method for the commercially valuable flower bulb Lilium longiflorum is important for molecular breeding. A regeneration protocol was developed for undifferentiated cell callus culture lines of 'Osnat' (five transgenic and two non-transgenic lines), established in 2003 from bulb-scale segments. The transgenic plants were designed to produce pollen-less flowers and therefore carried the rolB gene under the control of the lat52 promoter, the nptII selection gene and the GUS reporter gene uidA, the last two genes driven by the CaMV35S promoter. After ten years of sub-culture, the calli retained kanamycin resistance but could not regenerate. GUS specific reaction and the presence of all three transgenes in the cultures were confirmed by PCR and RT-PCR. A protocol was developed to restore plantlet regeneration from the callus culture lines in five stages. Initially, 96 plantlets of ca. 10 mm tall with a 2-3 mm bulblet were isolated after regenerant development and transferred to the plantlet growth stage. At the end of the plantlet growth stage, 48% of the plantlets developed normally (5-8 mm bulblet), and were successfully acclimatized in the greenhouse. During the following season, 20-75% of plants flowered for each line. Another 214 plantlets from the same seven lines were studied after regenerant development and again displayed normal phenotypes after the plantlet growth stage (with variation between lines of 28-77%). The presence and activity of the transgenes in the phenotypically normal plantlets was confirmed by PCR and RT-PCR. Phenotypic characterization of the transgenic and control plants required more than one flowering season. These results suggest that this approach may be used as a long-term callus storage method.

Benzothiadiazole (BTH) is a structural analogue of salicylic acid (SA) which is widely recognized for its role in elicitation of systemic acquired resistance in a broad range of plant species. Here, BTH was applied to cell cultures of the bulbous ornamental plants Ornithogalum dubium and O. thyrsoides, showing a strong effect on rates of differentiation and morphogenesis. Morphogenic cell clusters in liquid Murashige and Skoog (MS) medium containing 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) were used for all treatments. The calluses were washed thoroughly and activated with increasing concentrations of BTH. Following the induction, calli were grown on a solid MS medium without growth regulators (MS) or on a comparable media with NAA and BAP (M-206). The calli treated with BTH displayed a dose dependent increase in formation of meristematic centres followed by enhanced shoot formation compared to controls. Microscopic analyses revealed increased differentiation to cell organelles and a strengthening of the cell wall. A stronger response to BTH was observed in MS than in M-206 medium. A similar effect on calli differentiation was obtained by three weeks darkness followed by light exposure. The dark/light positive effect on differentiation was further augmented by BTH in a synergistic fashion. It is suggested that BTH enhances the rates of morphogenesis in Ornithogalum cultures by triggering a plant regulator-like activity.

Bacterial soft rot is one of the most destructive diseases affecting Ornithogalum, and affects many other bulbous crops, ornamental plants and vegetable crops, as well. This disease is caused by Perctobacterium carotovorum (Erwinia carotovora), which penetrates through wounds or stomatal openings, spreads through the apoplast secreting cell-wall degrading enzymes and, eventually, macerates the entire plant and spreads to neighboring plants. There is no known resistance or effective control measure for use against this disease, making utilization of transformation technologies the favored choice for the introduction of disease resistance into this crop. Tachyplesin, a small antimicrobial peptide isolated from hemocytes of the Japanese horseshoe crab, has been reported to inhibit the growth of both Gram-negative and P P Gram-positive bacteria. Liquid-grown cell clusters of O. dubium were bombarded with gold particles coated with a plasmid carrying the nptII gene, conferring kanamycin resistance, a GUS reporter gene, and the Tachyplesin 1 (TPN1) target gene under the control of either the polyubiquitin (UBQ3) or strawberry vein-banding virus Delta SVB) promoter. Following prolonged selection in a liquid medium supplemented with 80 mg/L kanamycin in darkness, the cultures were transferred to regeneration medium in the light, where hundreds of transgenic plantlets developed. The presence of the target gene was confirmed in all of the transgenic plants tested. Upon infection with a highly virulent bacterial isolate from calla lily (Zantedeschia aethiopica), the control plantlets were completely macerated within a week; whereas the transgenic plants showed varying levels of resistance. Half of the transgenic plantlets remained vital and flourished for months following the Erwinia challenge, despite the continued presence of the plant pathogen on and around the plants.

Non-specific plant defense activators and multiple pathways for their transduction may induce resistance in plants against unrelated pathogens. This approach may reduce dependence on chemical pesticides and enhance utilization of relatively susceptible crops that maintain high marketing value. Zantedeschia is a growing crop in the ornamental bulb industry worldwide. The biggest problem in its development and production is the bacterial pathogen Pectobacterium carotovorum, the causal agent of soft-rot disease. Two plant defense activators, Bion, acting through the salicylic acid pathway, and methyl jasmonate, involving the jasmonate-dependent signaling pathway, differed in both their capacity to induce accumulation of polyphenols, and resistance against the pathogen. Methyl jasmonate elicitation brought about higher accumulation of free phenolics relative to Bion, only following challenge with P. carotovorum, suggesting that priming of bioactive polyphenols plays a role in calla lily defense against this pathogen. Results demonstrate enhanced biological activity against the necrotrophic P. carotovorum only in the methyl jasmonate induced plants. To further characterize the nature of induced compounds, two major compounds were collected and identified as c-glycosyl-flavonoids, swertisin and isovitexin by mass and NMR spectroscopies.

AIMS:Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta.METHODS AND RESULTS:Several calibration steps were required to distinctly gate the GFP-labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads.CONCLUSIONS:The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta.SIGNIFICANCE AND IMPACT OF THE STUDY:The combination of time- and cost-saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild‐type Pectobacterium carotovorum was examined in white‐flowered calla lily (Zantedeschia aethiopica). Following a 24‐h induction period, plants were challenge‐inoculated with P. carotovorum, originally isolated from calla lily or potato plants, previously transformed using a gfp broad‐host‐range promoter‐probe vector. After another 24 h, Bion treatment (10 µg mL−1, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10 µg mL−1, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 mm, applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long‐lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA‐signalling pathway in the short term, but the jasmonate/ethylene‐signalling pathway is required for durable protection.

Ornithogalum mosaic virus (OrMV) causes flower deformation and deterioration of planting stocks in species and hybrids of Ornithogalum and Lachenalia. No resistance to viral infection by OrMV is known, making utilization of transformation technologies the natural choice for the introduction of virus resistance. Transformation with viral coat protein (CP) and replicase genes has been shown to confer resistance to viral infection in many plant species. Liquid-grown cell clusters of O. dubium were bombarded with gold particles coated with a plasmid carrying nptII gene, conferring kanamycin resistance, GUS reporter gene, and either CP gene or the viral replicase (N1b) target genes under the control of either polyubiquitin (UBQ3) or the strawberry vein-banding virus deleted (Delta SVB) promoters. Following prolonged selection in a liquid medium supplemented with 80 mg/1 kanamycin in darkness, the cultures were transferred to regeneration medium in the light, where hundreds of putative transgenic plantlets developed. Most of the regenerated plants were GUS-positive. PCR analysis indicated the presence of GUS reporter gene and nptII selectable gene, and either the CP or replicase transgenes. Transgenic plants are being propagated vegetatively before being challenged with virus infection to confirm their state of resistance.

Genetic transformation mediated by bombardment with microscopic metal particles carrying target genes is the preferred method for the introduction of foreign genes into monocotyledonous plants. The fact that most flower bulbs are monocotyle-donous and that almost all commercial cultivars are propagated vegetatively makes them good candidates for molecular breeding through microprojectile bombard-ment. We report here on a development of a reliable method for an efficient genetic transformation of both Lilium longiflorum and Ornithogalum dubium using a particle inflow gun to deliver gene constructs into the target plant tissue, followed by a prolonged selection in the dark in liquid medium supplemented with kanamycin. The system was first optimized for Lilium longiflorum 'Snow Queen'. Based on the level of transient GUS expression, liquid-grown cell clumps are more competent than leaves. Large cell clusters (2-10 mm) maintain their organogenic potential while smaller clusters (<2 mm) cease to grow and die. The liquid-grown tissue cultures have a level of competence for transformation about 50-70 times greater than that of solid-grown callus cultures, and compact cell clusters are more competent than loose clusters. The cells were bombarded with a pCAMBIA2301 vector, carrying nptII gene conferring kanamycin resistance and GUS reporter gene. Following selection for 4-6 months in a liquid medium supplemented with 80 mg l(-1) kanamycin in the dark, the cell clusters were transferred to a regeneration medium in the light where hundreds of transgenic plantlets developed. The plants retained their stable transgenic state when grown in the greenhouse for two seasons. The transformation of O. dubium was similar in principle to that of L. longiflorum with three major differences: lily liquid-grown cultures grew more rapidly and had a higher potential for somatic embryo development. Ornithogalum cultures under selection took longer to develop into semi-organized cell clumps of sufficient size to allow continued shoot regeneration, were mostly organogenic, and the regenerated plantlets had higher rate of vitrification.

Lilium species (Liliaceae) are a significant floriculture commodity and one of the three major bulb crops in the commercial market. Lilies are monocotyledonous plants that have been generally recalcitrant to molecular genetic manipulation because of limitations that restrict utilization of transformation technologies that are routinely applied to dicotyledonous plants. Cucumber mosaic virus (CMV) causes a serious disease of lily. It is known that transgenic plants bearing a disabled CMV replicase gene can be resistant to that virus. About 5000 pieces of morphogenic calli, 3-4 mm in diameter, derived from segments of sterile bulblet scales of L. longiflorum Thunb. cv. 'Snow Queen' were microprojectile bombarded, using a Finer-type of bombardment apparatus, followed by bialaphos selection. The plasmid p35SAc containing PAT selectable marker gene encoding phosphinothricin-N-acetyltransferase (that detoxifies the herbicide "Basta") under the control of 35S promoter and the nopaline synthase (NOS) terminator; and the plasmid pSAN101 containing the CMV defective replicase gene under the control of Act promoter and NOS terminator, were used. After several rounds of callus selection, plants were regenerated in vitro. PCR analysis indicated that these plants contain both the PAT and the disabled CMV replicase transgenes.