Artificial symbiotic association and growth induction of embryogenic calli (Elaeis guineensis Jacq.) inoculated with Herbaspirillum seropedicae (Z78)

Aims: The purpose of this experiment was to determine the artificial symbiosis interaction of Herbaspirillum seropedicae (Z78) on oil palm embryogenic calli. Methodology and results: For this purpose, symbiotic associations were established between Z78 and embryogenic calli of oil palm tissue cultured. A total of five treatments involved, in particular: i) + 3.0 mg/L 2,4-D + 100% N MS medium (control), ii) + Z78 pellet cells (1 mL) + 25% N MS medium, iii) + Z78 supernatant (1 mL) + 25% N MS medium, iv) + Z78 broth culture (1 mL) + 25% N MS medium, and v) + Z78 sonicated cells (1 mL) + 25% N MS medium. All treatments were supplied with minimal N sources (25% N), ammonium nitrate and potassium nitrate, while the control was treated with 100% N sources. Treated samples were harvested on D80 and observed for biomass and diameter increment (%), formation of embryoids, and Z78 colonization. The results showed embryogenic calli in the inoculated treatments that contained depleted N produced similar result to the control treatment which contained 100% N nutrients. Positive interactions occurred between the diazotroph and host plant tissues as viewed under FESEM and EFTEM. Among the treatments, Z78 sonicated cell showed better growth of embryogenic calli compared to others. Conclusion, significance and impact study: The in vitro nitrogen-depleted artificial symbiosis environment allowed the diazotroph (Z78) to be expressed and provide the nitrogen sources and indole-3-acetic acid for cell growth. This study represents beneficial co-culture interaction effects of different inocula of diazotrophic bacterial cells with in vitro embryogenic calli of oil palm.

Growth curve, biochemical profile and phytochemical analyses in calli obtained from the procambium segments of Bacupari

Garcinia brasiliensis, popularly known as Bacupari, is native to the Amazon and commonly used in folk medicine for its therapeutic properties. This plant is rich in bioactive compounds like benzophenones. However, there are no works about the in vitro establishment and achievement of secondary metabolites in this plant. Thus, the aim of this work was to determine the growth curve and to perform the biochemical and phytochemical analyses in calli obtained from the procambium segments of Bacupari. The growth curve of calli followed a sigmoidal pattern, with four distinct phases (lag, exponential, linear, deceleration). Total soluble sugars were higher on the inoculation day and the reducing sugars on the 20 th day. Amino acids increased from the 60 th day up to the stabilization on the 120 th day. The protein content varied, but it seemed to be related to the amino acids metabolism. The phytochemical screening showed the presence of phenolic and flavonoid compounds in the calli and the HPLC analysis allowed the identification of Fukugetin, Guttiferone A and 7-epiclusianone.

Study of Genetic Fidelity of Somatic Embryos - Derived Plants of Gentiana kurroo Royle from Western Himalayas - Plant of Great Medicinal Value.

Aim: To study the effect of plant growth regulators on direct and indirect somatic embryogenesis on Gentiana kurroo Royle – an endangered medicinal plant. Place and Duration of Study: Department of Biotechnology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan-173230, Himachal Pradesh, India, 2010-2013. Methodology: For induction of direct and indirect somatic embryogenesis petiole, leaf and petiole derived callus (after incubation in activated charcoal) were cultured on MS medium supplemented with a range of combinations of auxins (2,4-D, picloram, dicamba) and cytokinins (zeatin, kinetin). Results: Somatic embryos were observed from both direct and indirect method. The best performance was observed on MS basal medium supplemented 1.0mg/l dicamba. Development of somatic embryos was observed on MS basal+0.50mg/l GA3+1.0 mg/lKn and the maximum plantlets formation was achieved when somatic embryos (directly and indirectly induced) were shifted to half strength MS basal medium. Conclusion: In direct pathway somatic embryos were in contact with maternal tissue in a suspensor like structure. In indirect pathway, the explants first proliferated to give rise to callus before embryoids were induced. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.

Efeito de diferentes meios nutritivos e fitorreguladores visando à otimização da calogênese de Annona mucosa (Jacq. ) / Effect of different culture media and plant growth regulators for the optimization of callus induction of Annona mucosa (Jacq. )

Annona mucosa é uma árvore frutífera da família Annonaceae, produtora de importantes metabólitos secundários de interesse medicinal, como lignanas, acetogeninas e alcaloides. A cultura in vitro de calos representa um importante recurso para a produção contínua de metabólitos, viabilizando a conservação da biodiversidade química e a obtenção controlada de material para estudos biológicos e fitoquímicos. O objetivo deste trabalho foi otimizar a produção de calos friáveis de A. mucosa, avaliando o efeito de diferentes meios nutritivos e fitorreguladores. Segmentos de folha e de hipocótilo de plântulas germinadas in vivo foram utilizados como explantes e inoculados nos meios de cultura MS, WPM e B5 suplementados com picloram (2 - 20µM) isolado ou combinado com as citocininas BAP, KIN ou TDZ (0,2 - 1µM). As culturas foram mantidas a 26±2ºC, no escuro, com subcultivos mensais. A produção de calos foi avaliada por aferição do peso dos calos, após 90 dias. Em todos os tratamentos na presença da auxina picloram, o cultivo de hipocótilos resultou em maior porcentagem de formação de calos, sobretudo no meio de cultura WPM. A associação com TDZ produziu massa calogênica friável altamente proliferativa e ausente de oxidação, alcançando valores superiores àqueles obtidos em trabalhos prévios com a espécie. Os resultados viabilizam o uso do material em suspensões celulares e posterior caracterização fitoquímica para a exploração da produção in vitro de metabólitos da espécie.

The Annona mucosa is a fruit tree of the Annonaceae family that produces a range of secondary metabolites of medicinal interest, such as lignans, acetogenins and alkaloids. The callus culture represents a renewable source of valuable medicinal compounds and controlled supply of material for biological and phytochemical studies. Therefore, this study was carried out to investigate the effects of three nutrient media, different concentrations of picloram and cytokinin types, in order to optimize the biomass yield and friability of calluses of A. mucosa. Leaf and hypocotyl segments from seedlings produced from in vivo seed germination were used as explants, which were inoculated in MS, WPM and B5 culture media supplemented with picloram (2-20µM) only or in addition to the cytokinins BAP, KIN or TDZ (0,2 - 1µM ). Cultures were maintained at 26±2ºC in the dark, with monthly subcultures. After 90 days, biomass production was evaluated. In all treatments, hypocotyl explants provided the highest percentage of callus formation, particularly in WPM. The association with TDZ produced highly proliferative friable callus, with no oxidation, reaching higher values than the previous works with this species. The results enable the use of the calluses produced in cell suspensions and the subsequent phytochemical characterization, in order to explore the in vitro production of metabolites of the species.

Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae) / Establecimiento de suspensiones celulares de dos especies Jatropha (Euphorbiaceae) de Costa Rica

J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.

J. curcas es un importante recurso alternativo de biocombustible. Por otro lado, propiedades de interés agronómico, farmacológico e industrial han sido reportadas para esta especie. El objetivo de este estudio fue el establecimiento y mantenimiento a largo plazo de callos y cultivos celulares en suspensión de J. curcas y J. gossypifolia, con el objetivo de permitir futuros estudios para nuevos compuestos con aplicaciones farmaceúticas e industriales. Los callos friables fueron exitosamente inducidos a partir de segmentos de hipocótilos J. curcas and J. gossypifolia cultivados en medio MS semisólido suplementado con 1.5mg/L y 0.5mg/L of 2,4-D, respectivamente. Los cultivos celulares en suspensión de J. curcas fueron establecidos utilizando 1g de callos de 35 y 60 días de edad en 50mL de medio MS líquido adicionado con 1.5mg/L de 2,4-D. Después de 35 días, los cultivos en suspensión celular iniciados con callos de 35 días, mostraron mayor crecimiento celular con una biomasa máxima de 194.9g/L de peso fresco y 6.59g/L de peso seco y 17.3% de volumen empacado. La fase exponencial finalizó al día 35 en los cultivos iniciados con callos de 35 días, y al día 21 en los cultivos iniciados con callos de 60 días. Dos fuentes de carbono fueron evaluadas: sacarosa y maltosa. La producción de mayor biomasa fue obtenida con sacarosa. Los cultivos celulares se establecieron con callos de 35 días cultivados en medio MS con la misma concentración de 2,4-D utilizada para la inducción de callos y 30g/L de sacarosa. Este medio fue considerado el óptimo para el mantenimiento y crecimiento de suspensiones celulares en ambas especies con subcultivos cada 20 días. El potencial biotecnológico para la producción de compuestos bioactivos en estas especies, para aplicaciones farmacológicas, agrícolas e industriales está siendo evaluado.

Desinfestação superficial, germinação e regeneração in vitro a partir de sementes de calêndula / Superficial disinfestation, germination and regeneration in vitro from marigold seeds

Os objetivos deste trabalho foram: desenvolver um protocolo de desinfestação superficial de sementes, selecionar uma metodologia de germinação in vitro, avaliar o potencial de regeneração no cultivo in vitro e a influência do tempo de cultivo no tipo de calo formado e na regeneração a partir de calos de calêndula. Para a desinfestação superficial, foram testados diferentes tempos de imersão em solução de hipoclorito de sódio a 2,5 por cento. Na germinação in vitro, foram testados: imersão em ácido sulfúrico absoluto; imersão em ácido clorídrico absoluto; retirada do tegumento e embebição das sementes; e embebição das sementes sem a retirada do tegumento. Para avaliar o potencial de regeneração in vitro, foram testadas diferentes concentrações de 6-benzilaminopurina (BAP) e ácido alfa-naftaleno acético (ANA) e tempos de cultivo. A imersão em solução de hipoclorito de sódio a 2,5 por cento por 30 minutos aliada à remoção do tegumento promove a germinação in vitro de sementes de calêndula, efetuando uma desinfestação superficial satisfatória. Para a regeneração de partes aéreas e raízes a partir de sementes de calêndula não é necessária a suplementação com fitorreguladores. Na presença de BAP, independentemente da presença ou não de ANA, calos primários induzem à formação de calos esponjosos e friáveis e de calos verdes e rígidos, estes em menor número; na ausência de BAP é induzida a formação de calos pequenos. Calos jovens são mais eficientes em regenerar partes aéreas em calêndula.

The aims of this paper were: to develop a protocol of superficial disinfestation of marigold seeds; to select a methodology of germination in vitro of marigold seeds; to evaluate the potential regeneration of in vitro culture of marigold; and the influence of in vitro culture time in the kind of callus formed. The superficial disinfestation was perfomed by different times of immersion in 2.5 percent sodium hypoclorite solution. The methodology was tested by immersion in sulfuric acid; immersion in chloridric acid; removal of tegument and soaking of seeds; and just seeds soaking. To evaluate the potential of in vitro regeneration were tested different concentrations of growth regulators and culture times. The immersion in 2.5 percent sodium hypochlorite solutions for 30min coupled with the removal of tegument promote the in vitro germination and superficial disinfestation of marigold seeds satisfactorily. Primary calli in BAP presence, with or without ANA, induce formation of spongy and friable calli and of green and hard calli, these are in least amount. Young calli are more efficient to regenerate aerial parts.

Characterization of The Na~+/H~+ Antiporter in Tonoplast Vesicles From Populus tremula. calli / 生物化学与生物物理进展

The Na+/H+ antiporter in vacuolar membranes transports Na+ from the cytoplasm to vacuoles using a pH gradient generated by proton pumps, which could reduce Na+ toxicity. It is uncertain that whether the woody plants have the same mechanism. Through differential centrifugation and sucrose density gradient centrifugation, tonoplast vesicles were isolated from Populus tremula calli broken by blender. After establishing pH gradient by V-ATPase, Na+ could dissipate the pH gradient, which indicates that there is Na+/H+ antiporter in the tonoplast vesicles from Populus tremula calli (Km=11.4 mmol/L). Amiloride could inhibit the Na+/H+ antiporter activity. The antiporter could transport Na+ and K+, the affinity for Na+ is higher. Salt stress decreased Km and Vmax.

Comparison of the relative content of trichosanthin between root and callus derived from Trichosanthes kirilowii / 中国药学杂志

OBJECTIVE：The relative content of trichosanthin (TCS) of the calli induced from the leaves of Trichosanthes kirilowii Maxim. was measured and a comparison between the calli and the root was made. METHODS：TCS was obtained by the fractional precipitate with acetone from the homogenate of the root or the calli. To examine and measure TCS, several methods, such as immuno-precipitation reaction, SDS-PAGE and electrophoregram scanning, were usde. RESULTS：The results of immuno-precipitation reaction and SDS-PAGE showde that TCS existed in the calli and in the root of T.kirilowii Maxim.. It was found that TCS was the richest component in the acetone precipitated crude extract of the calli with a relative content of 44.22% TCS in the extract, though the absolute content of TCS in the calli was less than that in the root. CONCLUSION:Extracting TCS from the calli derived from leaves has not been reported previously. The absolute content of TCS in the root is 2.66 times more than that in the calli.