ABSTRACT
Abstract The influence of silver nitrate (AgNO3), benzyladenine (BAP), and indole-3-acetic acid (IAA) on low frequency somatic embryogenesis (LFSE) induction in Caturra and Catuaí arabica coffee was evaluated. For the Caturra cultivar, the production of somatic embryos was significantly increased by adding AgNO3 to the semisolid culture medium. The highest average number of somatic embryos for this cultivar was obtained using 6.6 μM BAP, 2.85 μM IAA, and 40 μM AgNO3. In contrast, for the Catuaí cultivar, the highest average number of somatic embryos was obtained using semisolid medium supplemented with 8.8 μM BAP, and 2.85 μM IAA. Using these protocols, somatic embryos were directly induced using leaf sections of in vitro plants of both coffee cultivars within 8 weeks. The somatic embryos developed into rooted plants with a 100% survival rate upon transfer to the greenhouse.
Subject(s)
Plant Growth Regulators , Seeds/chemistry , Silver Nitrate/administration & dosage , Coffea , Tissue Culture TechniquesABSTRACT
A protocol for Coffea arabica L. cvs. Caturra and Catuaí plant regeneration via indirect somatic embryogenesis (ISE) was established. Furthermore, a biolistic mediated genetic transformation protocol was optimized for Catual callus aggregates. Maximum callus induction was obtained when Caturra (87 percent) and Catuaí (67 percent) leaves were cultured on Murashige and Skoog medium with 18.56 micronM kinetin and 4.52 micronM2,4-dichlorophenoxyacetic acid (2,4-D). Catuaí suspension cultures were established from embryogenic callus using liquid proliferation CP and Sli media and diffused light and darkness. The higher suspension cultures fresh weight was obtained using Erlenmeyer (1425.4 +/- 354.9 mg) than Recipient for Automated Temporary Immersion System (RITA®) (518.6 +/- 55.1 mg), whereas the dry weight of suspension cultures was not significantly affected by the culture system used. Higher number of embryos per vessel (307.6 +/- 49.0) and their fresh weight (9.6 +/- 1.5 mg) were obtained with semisolid R medium than S3 medium. The highest somatic embryo development (25.0 +/- 2.7) and fresh weight (780.0 +/- 85.4 mg) were obtained with 1 min of immersion every 8 hrs. Higher fresh weight of regenerated plantlets was obtained with liquid Yasuda medium in RITA® (124.6 +/- 16.3 mg) than semisolid media (36.3 +/- 11.3 mg). For genetic transformation, the effect of helium pressure (900 and 1550 psi), and target distance (9 and 12 cm) and plasmid (pCAMBIA 1301, pCAMBIA 1305.2 and pCAMBIA 1301-BAR) on transient uidA expression Catuaí suspension cultures were evaluated. The highest number of blue spots was obtained using 900 psi and 9 cm (125.8 +/- 17.3). Stable uidA expression was observed on Catual callus aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l-1 of kanamycin.
Subject(s)
Coffea Cruda , Embryonic Development , Transformation, Genetic , Culture Media , SuspensionsABSTRACT
The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.
Subject(s)
Animals , Rabbits , Gene Expression , Oryza/virology , Viral Nonstructural Proteins/genetics , Tenuivirus/chemistry , Plant Viruses/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Microscopy, Immunoelectron , Viral Nonstructural Proteins/metabolism , Plant Viruses/metabolism , Blotting, WesternABSTRACT
Genetic engineering and the food derived from genetically modified crops (GMCs) have been the center of debate worldwide, as has occurred historically with the advent of new technologies. Questions are derived from the potential impact of GMCs to the environment and the safety of the products to the consumers. In relation to the first inquiry, practice has been oriented to a case-by-case-study, according to the own characteristics of the GMC, in order to minimize its impact in the environment. Scientific studies in diverse latitudes of the world have demonstrated that GMCs in the market showed no adverse effects related to this issue. In relation to food derived from the GMCs, rigorous evaluation protocols have been developed and approved by FAO and WHO to guarantee the innocuousness of these products. Up to the moment, no contraindications for human health have been pointed out for the products that are available today in the market. In the particular case of Costa Rica, the country has established since the 90s a regulatory biosafety framework for the management of the GMCs, safeguarding the biodiversity of the country and the health of consumers. At the same time the country has made significant public and private investments in the field that allowed the country to obtain a leading position in biosafety in the region and genetic engineering research at national research centers. Any attempt to restrict or prohibit these activities in the country, will put in risk the previously described investment, will affect the generation of new knowledge for decision making and the leadership in the field, preventing the benefits derived from this promising technology.