Molecular, biochemical, and proteomic analyses of transplastomic tobacco plants expressing an endoglucanase support chloroplast-based molecular farming for industrial scale production of enzymes.

The successful production of recombinant enzymes by tobacco transplastomic plants must maintain compatibility of the heterologous enzyme with chloroplast metabolism and its long-time enzyme stability. Based on previous reports, it has been taken for granted that following biolistic-transformation, homoplasticity could be obtained from the initially heteroplastic state following successive rounds of selection in the presence of the selection agent. However, several studies indicated that this procedure does not always ensure the complete elimination of unmodified wild-type plastomes. The present study demonstrates that CelK1 transplastomic plants, which were photosyntetically as active as untransformed ones, remain heteroplastomic even after repeated selection steps and that this state does not impair the relatively high-level production of the recombinant enzyme. In fact, even in the heteroplastomic state, the recombinant protein represented about 6% of the total soluble proteins (TSP). Moreover, our data also show that, while the recombinant endoglucanase undergoes phosphorylation, this post-translation modification does not have any significant impact on the enzymatic activity. Biomass storage might be required whenever the enzyme extraction process could not be performed immediately following the harvest of tobacco mature plants. In this respect, we have observed that enzyme activity in the detached leaves stored at 4 °C is maintained up to 20 weeks without significant loss of activity. These findings may have major implications in the future of chloroplast genetic engineering-based molecular farming to produce industrial enzymes in transplastomic plants.

Toxicity effects of profenofos on embryonic and larval development of Zebrafish (Danio rerio).

The aim of the present study was to evaluate the developmental toxicity of profenofos to early developing Zebrafish (Danio rerio) embryos (4h post fertilization) in a static system at 1.0 to 2.25mg/L. Median lethal concentrations (LC50) of profenofos at 24-h, 48-h, 72-h and 96-h were determined as 2.04, 1.58, 1.57 and 1.56 mg/L, respectively. The hatching of embryos were recorded at every 12h interval and the median hatching time (HT50) was also calculated for each concentration. In a separate set of experiments, 96-h LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations were used to assess the developmental toxicity in relation to behavior, morphology, and interactions with the targeted enzyme acetylcholinesterase. Live video-microscopy revealed that the profenofos exposed embryos exhibited an abnormal development, skeletal defects and altered heart morphology in a concentration-dependent manner, which leads to alterations in the swimming behavior of hatchlings at 144-h, which indicate that developing zebrafish are sensitive to profenofos.

Developmental toxic effects of monocrotophos, an organophosphorous pesticide, on zebrafish (Danio rerio) embryos.

The present study examined the response of zebrafish embryos exposed to different concentrations (10, 20, 30, 40, 50, and 60 mg/L) of monocrotophos under static conditions for 96 h. We found that mortality had occurred within 48 h at all test concentrations, later insignificant mortality was observed. Monocrotophos (MCP) can be rated as moderately toxic to the Zebrafish embryos with a 96-h median lethal concentration (LC50) of 37.44 ± 3.32 mg/L. In contrast, it greatly affected the development of zebrafish embryos by inducing several developmental abnormalities like pericardial edema, altered heart development, spinal and vertebral anomalies in a concentration-dependent manner. A significant percent reduction in length by 9-48% and heart beats by 18-51% was observed in hatchlings exposed to LC10 and LC50 concentrations at 96 h when compared to controls. The process of looping formation of heart at embryonic stage was greatly affected by the LC50 concentration of MCP. The neurotoxic potentiality of MCP was assessed by using a marker enzyme, acetylcholinesterase in both in vitro and in vivo experiments. MCP was found to be the most potent inhibitor of AChE in vitro with an IC50 value of 4.3 × 10(-4) M. The whole-body AChE enzyme activity in vivo was significantly inhibited during the exposure tenure with the maximum inhibition of 62% at 24 h.

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin.

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants.

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress.

A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton ( Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20-24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.

In vitro segregation of marker genes in anther cultures of rice.

Segregation of genes controlling expression of anthocyanin pigmentation in rice (Oryza sativa L. subsp. indica) leaf blade and leaf sheath was examined in the microspore-derived plants. The segregation pattern of marker genes was found to fit closely the expected gametic segregation ratios among microspore-derived green as well as albino plants. Microspore-derived in vitro regenerated plants expressed genetic traits similar to seedlings. The results indicate that the germ cell culture technique can be of significance while monitoring gene action, i.e. anthocyanin synthesis at monoploid phase of plant development.

Somatic cell genetic studies inBrassica species : I. High frequency production of haploid plants inBrassica alba (L.) H. f. & T.

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.