Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

ABSTRACT The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

BACKGROUND: Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, ß-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, ß-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. RESULTS: Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 µg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, ß and γ tocopherols (145.61, 25.52 and 18.56 µg/100 g) were detected in the cell cultures collected at day 6. CONCLUSIONS: As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.

Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures

BACKGROUND: The aim of the present work was to examine the role of UV-C irradiation on the production of secondary metabolites (total phenolic, total flavanols, total flavonols, catechin, ferulic acid and trans-resveratrol in phenolic compounds and α-, ß-, γ- δ-tocopherols) in callus cultures. Studies on the effects of UV-C treatment on callus culture are seldom and generally focused on UV-B. However UV-C radiation play an important role in accumule secondary metabolites. RESULTS: In this study, callus cultures from Öküzgözü grape cultivar were initiated from leaf petiole explants. Calli formed after 6 weeks on the medium supplemented with 0.5 mg L-1 benzylaminopurine (BA), 0.5 mg L-1 indole acetic acid (IAA) on B5 media. Callus tissues were exposed to UV-C irradiation at 10, 20 and 30 cm distances from the UV source for 5 and 10 minutes and samples were collected at hours 0, 24 and 48. CONCLUSIONS: The greatest total phenolic content (155.14 mg 100 g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. 24 h and 48 h incubation times, 30 cm and 5 min were the most appropriate combination of UV-C application in total flavanol content. Maximum total flavonol content (7.12 mg 100 g-1) was obtained on 0 h, 5 min and 20 cm combination. The highest (+)- catechin accumulation (8.89 mg g-1) was found in calli with 10 min UV-C application from 30 cm distance and sampled after 48 h. Ferulic acid content increased 6 fold in Öküzgözü callus cultures (31.37 µg g-1) compared to the control group. The greatest trans-resveratrol content (8.43 µg g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. The highest α-tocopherol concentration was found in calli exposed to UV-C for 10 min from 30 cm distance and sampled after 24 h. As a conclusion, it was showed that UV-C radiation had remarkable promoting effects on the accumulation of secondary metabolites in the calli of Öküzgözü grape cultivar.

Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars.

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.

Regeneración vía embriogénesis somática de una variedad colombiana élite de Theobroma cacao L / Regeneration through somatic embryogenesis of an elite colombian Theobroma cacao L variety

El objetivo de esta investigación fue evaluar dos protocolos de propagación vía embriogénesis somática a partir de explantes florales en dos clones élite BIOB e ICS95 de Theobroma cacao L. Se obtuvo un 50 y 32% de callo embriogénico en ICS95 y BIOB respectivamente con el protocolo de Fontanel et al. (2002), modificado después de un periodo de cultivo de tres meses. Los embriones pasaron por fases que se correspondieron con medios de cultivo diferenciales: Inducción, Formación, Maduración y Mantenimiento. Para la embriogénesis somática secundaria se obtuvo un 23% de embriones a partir de embriones somáticos primarios en un medio, conteniendo 1mg/L de 2,4,5 T (2,4,5 Triclorofenoxiacético). Se logró, además, desarrollar enraizamiento adventicio aplicando pulsos de IBA (Ácido Indol Butírico) a 0.5mg/L y 0.5g/L durante un minuto. Las plantas enraizadas se llevaron a una mezcla de tierra: arena (1:1) para su adaptación ex vitro, obteniéndose un 66% de plantas aclimatadas. Los estudios histológicos mostraron diferentes características típicas del desarrollo embriogénico. Este es el primer reporte en el que se logra de manera exitosa la conversión hasta plántula (68%) y la adaptación ex vitro de una variedad colombiana de cacao vía embriogénesis somática primaria y secundaria.

In this research we evaluate two protocols of propagation via somatic embryogenesis from floral explants using two elite clones BIOB and ICS95 of Theobroma cacao L. We obtained 50 and 32% of embryogenic callus on ICS95 and BIOB respectively with Fontanel et al., (2002) protocol modified after three months of culture. The embryos went through four phases; Induction, Formation, Maduration and Mantenimiento which corresponded each one with different media culture. For secondary somatic embryogenesis we obtained 23% of embryos from primary somatic embryos in a medium with 1mg/L of 2,4,5 T (2,4,5 Triclorofenoxiacetic). Also we obtained plants that developed new roots applying pulses with IBA (Indol Butiric Acid) 0.5mg/L and 0.5g/L for a minute. The developed plants were moved to a mix of potting soil and sand (1:1) for their ex vitro adaptation, getting 66% of acclimatized plants. The histological analysis showed the typical characteristics of the embryogenic development. This is the first report where it is achieved the successful conversion to plantlets (68%) and ex vitro adaptation of a colombian cocoa variety via primary and secondary embryogenesis.