Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

The relationship between morphology changes and antioxidant enzymes activity during somatic embryogenesis of long-term-maintained callus of Zoysia matrella [L.] Merr.

A callus line of manila grass (Zoysia matrella [L.] Merr.) has been maintained for 8 years in our lab. The present study investigated changes in ultrastructure and antioxidant enzyme activity during regeneration of the callus and examined the correlation between these changes and regeneration ability. The changes in fresh weight and diameter of the callus over time could be described by a sigmoidal growth curve with different stages. Electron microscopy revealed small embryonic callus cells, isodiametric in shape, with large, obvious nuclei and dense cytoplasm. The cellular structures and morphology changed dramatically as regeneration proceeded. Of particular note was the formation of folded scutellum-like embryos at 14 d, which might be the turning point for morphological differentiation. Catalase (CAT) and peroxidase (POD) activities were the lowest at 14 d, the same time when superoxide dismutase (SOD) activity was the highest. Thus, we speculate that the formation of the scutellum-like structures is associated with higher activity of SOD and lower activities of CAT and POD.

The Impact of Different Cultivation Systems on the Content of Selected Secondary Metabolites and Antioxidant Activity of Carlina acaulis Plant Material

Roots and leaves of Carlina acaulis L. are still used in ethnomedicine in many European countries; however, the limited occurrence of the plants and protection of this species necessitate a search for alternative ways for obtaining this plant material. In this study, in vitro cultures, hydroponic cultures, and field cultivation were applied to obtain the C. acaulis plant material. Its quality was evaluated using antioxidant activity tests and high performance liquid chromatography analysis. Our study showed that the antioxidant activity and the content of chlorogenic and 3,5-di-caffeoylquinic acid in roots of plants cultivated in hydroponics and field conditions were comparable. However, the amount of carlina oxide was significantly higher in plants from the field. The flavonoid content in leaves obtained from both cultivation systems was at the same level; however, the antioxidant activity and the content of the investigated metabolites were higher in the soil cultivation system. The callus line exhibited high differentiation in phytochemical compositions depending on the treatments and medium compositions.

Ginsenoside and phenolic compounds in hydromethanolic extracts of American ginseng cell cultures and their antioxidant properties

The present study was performed to investigate the antioxidant properties of callus and suspension culture extracts of <em>Panax quinquefolium</em> (American ginseng). The ginsenoside content and the total phenolic content (TPC) in these cultures were also examined. The total amount of the nine studied saponins was found to be 2.08, 1.69, and 0.202 mg g⁻¹ dry weight in red callus line (RCL), green callus line (GCL), and suspension cultures, respectively, by HPLC analysis. The TPC was estimated using the Folin–Ciocalteu method. The TPC of the suspension culture extracts was approximately 36.7% and 17.6% higher than that of the RCL and GCL, respectively. The antioxidant activity of the extracts was evaluated using the in vitro ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and FRAP (ferric reducing antioxidant power) assays; the methanolic <em>P. quinquefolium</em> suspension culture extracts demonstrated stronger antioxidant activity than that of the callus cultures.

Variability of rice haploids obtained in anther culture in vitro

Morphological variability of haploid plants and doubled haploids of rice obtained on one callus line in anther culture in vitro was studied. The work was carried out on rice plants Oryza sativa L. subspecies japonica Kato varietу Cascade. Regenerant plants of one callus line obtained from one rice anther (four in total) were divided into two or three groups of 20-30 plants, depending on the sample size in order of their differentiation on callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) formed half of the haploids, half of the doubled haploids, and two other callus lines (5.1 and 7.2) numerous haploids. On callus lines with numerous haploids (5.1 and 7.2), as the regenant number increases, the size of plants decreases (plant height, number of flowers on the main panicle, number of panicles). On the lines 15.1 and 18.1 between groups of haploids and between groups of doubled haploids statistically significant differences absent. In breeding purposes for the induced doubling of the number of chromosomes in haploid regenerants with antitubulin substances such as colchicine, it is advisable to use plants that form on callus among the first. Between haploids of four callus lines and doubled haploids of two callus lines, statistically significant differences (at p=0.001) were revealed using the Hotelling's T2-criterion, calculated for the whole complex of biometric features. Haploids of different lines differed in three or four of them, doubled haploids on three of the five signs (length of panicle, productive bushiness and plant height). Varieties of interest to breeders may be improved by anther culture in vitro.

In Vitro Organogenesis of a Slipper Orchid, Paphiopedilum ‘Alma Gavaert’

The aim of the present study was to improve the regeneration efficiency of callus lines in a slipper orchid, Paphiopedilum ‘Alma Gavaert’. Three kinds of vegetative tissues, root, stem and leaf segments, were used as explants to induce callogenesis; out of these, only root explants formed callus and was subcultured in the presence of 5 mg/L dicamba and 5 mg/L 2,4-D combined with 1 or 2 mg/L TDZ. The resulting four callus lines, assigned as 5Di1T, 5Di2T, 5D1T and 5D2T, respectively, were used to test the effect of NAA to BA ratios on re-differentiation, wherein the highest number of shoots (approximately 2 shoots/0.1 g callus clump) were obtained in callus line 5D2T at ratios of 0.001 and 0.002. A largely improvement of shoot regeneration efficiency was obtained by continuous selection of callus lines which derived from different explant positions. Eventually, six callus lines, including 5D2T-T6-G5 to 5D2T-T6-G10, were able to produce approximately 10 times of shoots per callus clump when compared with the parental callus line 5D2T.

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone ‘Mediterraneo’. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the ‘Mediterraneo’ embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen (“two-step freezing”).

Cellular Calcium Distribution Modulates the Growth of Callus and Protoplasts of Halophyte Mangrove Plant, Avicennia Alba - an X-ray Microanalysis

Two cultured cell lines were developed from cotyledons of a halophyte mangrove, Avicennia alba.In the high-Ca callus line, which was sub-cultured in amodified amino acid medium containing 3 mM CaCl2, growth of calluses and their protoplasts were both inhibited by low concentrations of CaCl2 in the culture medium. Removal of Ca2+ from the culture medium stimulated callus growth and the calluses could be sub-cultured without CaCl2 (low-Ca callus line). The intra- (cytoplasmic matrix and vacuole) and extra- (cell wall) cellular concentrations of elements, i.e., [Ca], [K], [Cl], [Na], [Mg], [P] and [S] were investigated using quantitative X-ray microanalysis of cryosections of calluses from bothcell lines. [Ca] was high in the cytoplasmic matrix and cell wall of the high-Ca line. [Ca] was lowered in the low-Calineinall cell compartments, though still detected. Ca-containing electron-dense precipitates were accumulated in the middle lamella of cell walls in resin-embedded sections of the high-Ca line. CaCl2 in the medium stimulated protoplast growth only in the low-Caline. These results suggested that a low cellular [Ca] is needed for protoplasts growth of A. alba. The importance of cellular [Ca] for the growth of halophilic mangrove plant cells was discussed.

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex.

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex.