Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives

BACKGROUND: In order to produce an effective callus in Echinacea purpurea L.; determination of the explant type and growth regulators that best respond to callus induction and the optimization of the culture conditions to increase the amount of caffeic acid derivatives (CADs) in the obtained callus. CADs contents of callus cultures of E. purpurea were evaluated by establishing an effective callus induction system in vitro. RESULTS: Various medium containing different growth regulators were tested using leaf, petiole, cotyledon and root as the explants. The best callus development was achieved in MS medium with 1.0 mg l 1 2,4- D + 2.0 mg l 1 BAP in leaf, 1.0 mg l 1 NAA + 0.5 mg l 1 TDZ in petiole, 2.0 mg l 1 NAA + 1.0 mg l 1 TDZ in cotyledon and 0.5 mg l 1 NAA + 0.5 mg l 1 BAP in roots. Upon optimisation of callus growth, each type of explant was cultured for 4, 6, 8 and 10 weeks in medium for the analyses of caftaric acid, chlorogenic acid, caffeic acid and chicoric acid contents. The highest amounts of caftaric acid (4.11 mg/g) and chicoric acid (57.89 mg/g) were found from petiole explants and chlorogenic acid (8.83 mg/g) from root explants at the end of the 10-week culture time. CONCLUSIONS: As a result of the present study, the production of caffeic acid derivatives was performed by providing the optimization of E. purpurea L. callus cultures. Effective and repeatable protocols established in this study may offer help for further studies investigating the production of caffeic acid derivatives in vitro.

Effects of sampling time on polysaccharides of Dendrobium catenatum under temperature-controlled condition / 中国中药杂志

In order to reveal the accumulation trend of polysaccharides in Dendrobium catenatum and determine the effect of sampling time on polysaccharides, D. Catenatum D21 clone was harvested from January to December after culturing for 2 to 5 months in the growth chamber with constant temperature. Polysaccharides were determined by phenol-sulfuric acid method and the monosaccharide compositions were analyzed by pre-column derivative-UPLC. The results showed that the content of polysaccharide and its key component mannose was positively correlated with the culture time, but the contents of polysaccharides in all kinds of culture peaked from 5 to 6 months, which were consistent with the trend of field planting. The results suggested that the trend of polysaccharide accumulation in the plant could be related to the life rhythm of the sensory seasons of D. catenatum, which was significantly affected by the harvesting season, even under the constant condition of the culture chamber.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-gamma in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

Multiplicación en sistema de inmersión temporal del clon de Malanga Viequera -Xanthosoma spp / Multiplication in temporary immersion system cocoyam clone Viequera - Xanthosoma spp

El Sistema de inmersión temporal (SIT) constituye una alternativa en la micropropagación de plantas. El presente trabajo se realizó con el objetivo de establecer un protocolo para la multiplicación en SIT del clon de malanga “Viequera”. Se evaluó el efecto de tres tiempos de inmersión (7, 14 y 21 minutos), tres frecuencias de inmersión (2, 4 y 6 horas por día), cuatro volúmenes de medio de cultivo (5, 10, 15 y 20 ml por brote inicial) y cuatro tiempos de cultivo (15, 18, 21 y 25 días) en la multiplicación de los brotes de yemas axilares. Con tiempo de inmersión de 14 minutos cada 4 horas, un volumen de 15 ml de medio de cultivo por brote inicial y 18 días de cultivo, se logró el mejor comportamiento en la multiplicación de los brotes de yemas axilares, con un coeficiente de multiplicación de 10,50. El protocolo propuesto aumenta la productividad del material propagado en comparación con los desarrollados en medios de cultivo semisólidos, lo que representa una reducción en los costos de producción al introducir la multiplicación del cultivo en laboratorios comerciales de propagación.

Temporary Immersion System (TIS) is a alternative in the micropropagation of plants. This work was carried out to establish a protocol for the multiplication of clone TIS cocoyam “Viequera”. The effect of three immersion times (7, 14 and 21 minutes), three immersion frequencies (2, 4 and 6 hours per day), four volumes of culture medium (5, 10, 15 and 20 mL per shoot initial) and four times of cultivation (15, 18, 21 and 25 days) in the multiplication of shoots from axillary buds. With immersion time of 14 minutes every 4 hours, a volume of 15 ml of culture medium for initial outbreak and 18 days of culture, achieved the best performance in the multiplication of shoots from axillary buds, with a coefficient of multiplication 10.50. The proposed protocol increases the productivity of propagated material compared to those developed in semisolid culture media, representing a reduction in production costs by introducing the increase in cultivation in commercial laboratories.

Effect of two temperatures on in vitro nuclear maturation of bitch oocytes: relation to time culture intervals

In the present study, two commonly incubation temperatures used on in vitro oocyte maturation in canine species (Canisfamiliaris) were analysed with the aim to verify the rate of nuclear maturation of bitch oocytes after three culture intervals (48, 72and 96 h). The culture medium was TCM 199, supplemented with 25mM Hepes/l (v/v), with 10% heat inactivated estrous cowserum (ECS), 50mg/mL gentamicin, 2.2mg/mL sodium bicarbonate and 22mg/mL pyruvic acid, 1.0mg/mL oestradiol (E 8875Sigma), 0.5mg/mL FSH (Folltropin-V, Vetrepharm Inc. Ont, Canada) and 0.03IU/mL hCG (Profasi HP, Serono, Aubonne,Switzerland). Oocytes were recovered from ovaries of bitches at random estrous cycle stages by routine ovariohysterectomy(n=14) or by therapeutical ovariohysterectomy (n=8) at the veterinary hospital of the Universidade Federal do Rio Grande do Sul(UFRGS), Brazil. There was no statistical difference in the rate of meiosis of oocytes matured at 37oC or at 39oC at any time point.However, the proportion of oocytes that reached the metaphase II (MII) stage at 37o C after 72 h of in vitro culture (16/123; 13%),tended to be higher (P=0.064), when compared to that matured at the 39oC (6/148; 4,1%). The results demonstrate thattemperatures of 37oC and 39oC are equally effective for the in vitro culture of bitch oocytes.