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Three new homoisoflavonoids, (3R)-3,5-dihydroxy-7-methoxy-3-(4’-hydroxybenzyl)-4-chromanone, 4, and its 5-O-β-D-glucopyranoside and 5-O-β-D-gentiobiose derivatives, 5 and 6, along with the known homoisoflavonoids, (3S)-5-hydroxy-7-methoxy-3-(4-methoxybenzyl)-4-chromanone, 1, (3R)-5-hydroxy-7-methoxy-3-(4-methoxybenzyl)-4-chromanone (2) and (3S)-5-hydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone, 3, were isolated from the bulbs of Ornithogalum dubium Houtt.. In addition, three known cardenolides, 3β-(O-α-L-rhamnoside)-5β,14β-dihydroxy-19-oxocardenolide 7, 3β-(O-α-L-rhamnoside)-5β,11α,14β-trihydroxy-19-oxocardenolide, 8 and 3β-(O-α-L-rhamnoside)-5β,11α,14β,19-tetrahydroxycard-20(22)-enolide, 9, were isolated from Ornithogalum ponticum ‘Sochi’. Yields of homoisoflavonoids were low (0.0002 % - 0.0003 %) so stereospecific synthetic methodology needs further refinement to produce antiangiogenic (3R)-3-benzylchromanones and (3S)-3-hydroxyhomoisoflavonoid analogues for further evaluation. This work confirms that, apart from producing cardenolides, the absolute configuration at C-3 of homoisoflavonoids produced by Ornithogalum is 3R (H-3β) for the 3-benzylchromanones and 3S (3-OH β) for the 3-hydroxy benzylchromanones, in agreement with our earlier reports.

The phytochemical study of Ornithogalum dubium Houtt. (Asparagaceae) led to the isolation of five undescribed steroidal glycosides together with two known ones. Their structures were established by using NMR analysis and mass spectrometry as (25R)-3β-hydroxyspirost-5-en-1β-yl O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside, (25S)-3β-hydroxyspirost-5-en-1β-yl O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside, (22S)-16β-[(α-L-rhamnopyranosyl)oxy]-22-hydroxycholest-5-en-3β-yl O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside, (22S,23S)-1β,3β,11α,16β,23-pentahydroxy-5α-cholest-24-en-22β-yl β-D-glucopyranoside, (22S,23S)-3β-[(β-D-glucopyranosyl)oxy]-22,23-dihydroxy-5α-cholest-24-en-16β-yl O-α-L-rhamnopyranosyl)-(1 → 4)-β-D-glucopyranoside. Their cytotoxic activities against two human cells, a lung carcinoma A-549 and a promyelocytic leukemia HL-60 cell lines, were evaluated by using the XTT method. The results showed no significant cytotoxicity on the tested cells. The influence of the potentiation of cisplatin cytotoxicity in A-549 cells was also investigated and a slight effect was observed only for the (25R) spirostane-type derivative.

Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible to tissue culture, and often complex media and hormone requirements are needed to achieve successful plant propagations. The availability of new hormones or chemicals acting as hormones are critical to the expansion of tissue culture potentials. Phloroglucinol has been shown to have certain hormone-like properties in a variety of studies. Ornithogalum dubium, an important geophyte species, was used to characterise the potential of phloroglucinol as the sole plant-like hormone in a tissue culture experiment. Tissue culture, plant regeneration, total phenolic and genetic variability were established by applying a variety of methods throughout long-term experiments. Phloroglucinol did induce callus formation and plant regeneration when used as the sole supplement in the media at a rate of 37%, thus demonstrating auxin/cytokines-like properties. Callus formation was of 3 types, friable and cellular, hard and compact, and a mixture of the two. The important finding was that direct somatogenesis did occur albeit more frequently on younger tissue, whereby rates of induction were up to 52%. It is concluded that phloroglucinol acts as a &ldquo, hormone-like&rdquo, molecule and can trigger direct embryogenesis without callus formation.

Ornithogalum dubium is a popular ornamental monocot native to South Africa with flower colors ranging from pure white to deep orange. Gene editing based on the CRISPR/Cas9 system has recently been shown to hold potential for color improvement in ornamental flower crops. To apply this approach to Ornithogalum color manipulation, genomic or transcriptomic data must first be collected. Here, cDNA libraries of O. dubium leaves and flowers were constructed and sequenced using the Illumina HiSeq 2500. Over 155 million 100-bp paired-end reads were assembled into a transcriptome database of 360,689 contigs, of which 18,660 contigs were differentially expressed between leaves and flowers. Carotenoids are the main pigment imparting spectrum of orange hues to O. dubium flowers. By querying our database, we identified a total of 16 unique transcripts (unigenes) predicted to be involved in the carotenoid biosynthesis pathway of Ornithogalum. Combining carotenoid profiles, we further inferred several key unigenes responsible for floral coloration and accumulation in O. dubium, of which the gene LCYB/comp146645_c0 was found as a suitable target to generate potentially red flower varieties of O. dubium. Our research thus provides a framework for the application of CRISPR/Cas9 technology to improve this ornamental crop.

Bulbs, corms and other subterranean storage organs are commonly used as explant source material for the establishment of geophytes in vitro. The inflorescence stalk was found to be a good alternative source of explants to overcome explant contamination originating from underground storage organs. Inflorescence explants of Allium, Dichelostemma, Eucrosia, Gladiolus, Haemanthus, Hyacinthus, Narcissus, Nerine and Ornithogalum were used to establish cultures in vitro. The regeneration potential of the inflorescence was compared with regeneration from bulb twin scales or from apical buds isolated from corms. Gladiolus (Iridaceae) explants isolated from the floral stem just below the expanding florets, still enclosed in the bracts, were highly regenerative in the presence of naphthalene acetic acid (NAA) and kinetin. In the presence of 2,4-dichlorophenoxyacetic acid and benzyl aminopurine (BA) in the medium, explants isolated from the tissue at the junction between the peduncle and the pedicels of a young Nerine (Amaryllidaceae) inflorescence regenerated several buds. The scapes of young unemerged inflorescences taken from sprouting bulbs of Narcissus (Amaryllidaceae), following a 15 °C storage treatment, regenerated buds in the presence of NAA, BA, elevated phosphate and adenine sulfate in the medium. The number of buds regenerated depended on the location on the scape from which the explant was isolated, and on the duration of the 15°C treatment. In Allium (Alliaceae), capitulum tissue between the flower pedicels regenerated buds. Explants excised from the peduncle, as well as the pedicel-peduncle junction of Dichelostemma (Alliaceae), Ornithogalum, Hyacinthus (Hyacinthaceae) and Eucrosia (Amaryllidaceae) regenerated several buds in each type of explant. In the case of Haemanthus (Amaryllidaceae), pedicel-peduncle junction explants regenerated buds only when excised from inner whorl florets. Propagation protocols and the potential use of expediently isolated inflorescence explants for efficient micropropagation of geophytes are discussed.

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 caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most devastating diseases of Ornithogalum species. No effective control measures are currently available to use against this pathogen; thus, introduction of resistant genes via genetic transformation into this crop is a promising approach. Tachyplesin I, an antimicrobial peptide, has been shown to effectively control numerous pathogenic bacteria, including Pcc. In this study, liquid-grown cell clusters of Ornithogalum dubium and Ornithogalum thyrsoides were bombarded with a pCAMBIA2301 vector containing a cel I leader sequence fused to a gene encoding tachyplesin I, a neomycin phosphotransferase ( npt II) gene that served as a selectable marker and a β-glucuronidase (GUS) gene that served as a reporter. Selection was carried out in the dark in liquid medium containing 80 mg/L kanamycin. Regeneration was executed in the light after 6–14 months depending on the cultivar. Hundreds of transgenic plantlets were produced and their identity was confirmed through GUS activity assays. PCR and RT-PCR were used to confirm the presence of the target, reporter and selection genes in the divergent lines of plantlets. The resistance of the O. dubium plants to Pcc was evaluated in vitro, following infection with a highly virulent isolate from calla lily. Although control plantlets were completely macerated within a week, 87 putative transgenic subclones displayed varying levels of disease resistance. During three growing seasons in the greenhouse, the transgenic O. dubium lines grew poorly, whereas the transgenic O. thyrsoides plants grew similarly to non-transgenic plants.

The ovule of Ornithogalum dubium is anatropic, bitegmic and crassinucellate. The integuments are biseriate. Starch grains occur in the outer integument, the funicule and in the intercellular space surrounding the embryo-sac. The embryo-sac development is of the Polygonum type. The synergids are conspicuous and contain a large filiform apparatus. Fertilization is porogamic and occurs 24 h after pollination. The pollen tube enters the egg apparatus in the region of an intercellular space between the micropylar ends of the synergids. Sperm cells in the pollen tube appear to be released through small protuberances of the pollen tube. Migration of the sperm cell to the egg cell is possibly through a gap in the wall between the egg cell and the receptive synergid. Changes in the synergids occur only after a pollen tube has entered the receptive synergid. Changes occurring in the persistent synergid suggest that this cell may be involved in the nutrition of the pro-embryo up to the eight-cell pro-embryo stage. Syngamy is of the post-mitotic type. The first division of the zygote is transverse. The basal cell gives rise to the multicellular suspensor, and the terminal cell to the embryo. The endosperm is of the Helobial type.