Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal -endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal -endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The -endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutella xylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.     

Induction of somatic embryos of celery by control of gaseous compositions and other physical conditions

The celery (Tall Utah, Apium graveolens var. dulce) was adopted as a model system to investigate effective methods for enhancing embryogenesis. The focus was placed on attaining the optimum gaseous composition and the mixing intensity and viscosity of culture systems. The mixing intensity was investigated by using creased flasks and evaluated by shear force index (SFI). By using different closures with different ventilation coefficients, K w, to adjust the gaseous composition in the flasks, it was found that embryo frequency (EF) increased with high or low ethylene concentration in the headspace of the culture flasks. Cultivating the embryogenic cells under controlled ethylene concentrations depressed EF at ethylene concentrations higher than 0.08 ppm. Moreover, 0.1 μM of CoCl₂ remarkably repressed the ethylene formation in the culture medium and enhanced EF. and around 3% and 30%, respectively, favored EF. For non-creased flasks, addition of CMC (carboxymethyl cellulose) decreased the EF and DO level of the medium, due to lowered mass transfer rate attributed to high viscosity. For creased flasks, supplementation of 0.4 g/ 100 ml CMC reduced the ill-effects of high shear stress and maximized EF (7.22%). Our results are potentially applicable to other species and large-scale embryo production systems.