Recent Progress in Amaryllidaceae Biotechnology.

Plants belonging to the monocotyledonous Amaryllidaceae family include about 1100 species divided among 75 genera. They are well known as medicinal and ornamental plants, producing pharmaceutically important alkaloids, the most intensively investigated of which are galanthamine and lycorine. Amaryllidaceae alkaloids possess various biological activities, the most important one being their anti-acetylcholinesterase activity, used for the treatment of Alzheimer's disease. Due to increased demand for Amaryllidaceae alkaloids (mainly galanthamine) and the limited availability of plant sources, in vitro culture technology has attracted the attention of researchers as a prospective alternative for their sustainable production. Plant in vitro systems have been extensively used for continuous, sustainable, and economically viable production of bioactive plant secondary metabolites. Over the past two decades, a significant success has been demonstrated in the development of in vitro systems synthesizing Amaryllidaceae alkaloids. The present review discusses the state of the art of in vitro Amaryllidaceae alkaloids production, summarizing recently documented plant in vitro systems producing them, as well as the authors' point of view on the development of biotechnological production processes with a focus on the future prospects of in vitro culture technology for the commercial production of these valuable alkaloids.

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes.

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.

Phytochemical and biological study of callus cultures of Tulbaghia violacea Harv. Cultivated in Egypt.

As in vitro plant cultures are used extensively to produce bioactive metabolites, our goal was to establish calli from Tulbaghia violacea Harv. flowers and assess the tissue phytochemically and biologically. Murashige & Skoog medium(MS) + 22.6 µM 2,4-dichlorophenoxyacetic acid +2.2 µM benzylaminopurine induced callus from flowers. Gas chromatography/mass spectrometry(GC/MS) analyses of n-hexane extracts of calli(HC) and flowers(HF) revealed 33 and 32 components(92.6 and 98.5%, respectively). Hydrocarbons were predominant in HC (55.0%), whereas a higher percentage of oxygenated compounds was found in HF(74.6%). Trans(E)-anethole(39.1%) and 16-hentriacontanone (30.3%) dominated in HF and HC, respectively. However, sulphur compounds were only detected in HF. Quantitative estimation of thiosulphinates, phenolics, flavonoids and saponins in ethanolic extracts of calli(EC) and flowers(EF) showed much higher contents in EF. Antioxidant, antimicrobial and cytotoxic screening of extracts demonstrated that EF was the most potent, followed by HF and EC; conversely, HC was inactive. Although HC and EC were less biologically active, these calli could be an alternative source of bioactive metabolites.

Ultrastructural aspects of pollen ontogeny in an endangered plant species, Pancratium maritimum L. (Amaryllidaceae).

Pollen ontogeny in Pancratium maritimum L. was studied from the sporogenous cell to mature pollen grain stages using transmission electron, scanning electron, and light microscopy to determine whether the pollen development in P. maritimum follows the basic scheme in angiosperms or not. In the course of microsporogenesis and microgametogenesis, special attention was given to the considerable ultrastructural changes that are observed in the cytoplasm of microsporocytes, microspores, and mature pollen grains throughout the successive stages of pollen development. Microsporocyte differentiation concerning number and ultrastructure of organelles facilitates the transition of microsporocytes from the sporophytic phase to the gametophytic phase. However, cytoplasmic differentiation of generative and vegetative cells supports their functional distinctness and pollen maturation. Although microsporogenesis and microgametogenesis in P. maritimum generally follow the usual angiosperm pattern, abnormalities such as formation of unreduced gametes were observed. During normal microsporogenesis, meiocytes undergo meiosis and successive cytokinesis, resulting in the formation of isobilateral, decussate, and linear tetrads. Subsequent to the development of free and vacuolated microspores, the first mitotic division occurs and bicellular monosulcate pollen grains are produced. Pollen grains are shed from the anther at binucleate stage. During pollen ontogeny, three periods of vacuolization were observed: in meiocytes, in mononucleate free microspores, and in the generative cell.

Leaf patterning of Clivia miniata var. variegata is associated with differential DNA methylation.

KEY MESSAGE: Leaf patterns (yellow, green and striped) of Clivia miniata var. variegata might be caused by differential DNA methylation in CCGG sites in response to heterogeneous environmental pressure. Clivia miniata is an important ornamental plant.Clivia miniata var. variegata (Cmvv) is a variegated leaf mutant of C. miniata. Typical Cmvv has attractive green and yellow-stripped leaves. The study has revealed that an explant of Cmvv, even a full-green explant, could regenerate plants of three different types: yellow, green, ands triped; normal-appearing chloroplasts were found in guard cells but not in mesophyll cells of all the three types of Cmvv using confocal laser scanning microscopy (CLSM).Thus, we speculated that cells of the three types of Cmvv had an identical mutation and the mutation might disturb mesophyll cell chloroplast biogenesis after symplastic isolation of guard cells. Using CLSM and methylation sensitive amplification polymorphism (MSAP), we found that (a) striped leaves of Cmvv are due to sectorial decreases in chlorophyll levels and the decreases are associated with CG hypermethylation; (b) extent of epigenetic divergence among the three types of Cmvv leaves is positively correlated with intensity of leaf-color difference; and (c) green stripes of two plants are clustered in one group based on the MSAP profiles, but green and yellow stripes of a plant are not. Sequencing analysis indicated that CG hypermethylation in gene bodies of CPSAR1 and ycf2 might lead to gene silencing and yellow leaves/stripes of Cmvv. All together, it is possible that cytosine methylation involved regulating leaf color of Cmvv, also striped pattern of Cmvv might be caused by differential DNA methylation in response to heterogeneous environmental pressure. Furthermore, a novel leaf-color epigenetic hypothesis was proposed in this article.