Effect of integrated bombardment and Agrobacterium transformation system on transient GUS expression in hypocotyls of rapeseed (Brassica napus L. cv. PF704) microspore-derived embryos.

A new method for transformation of rapeseed microspore-derived embryos (MDEs), based on microwounding of MDEs by particle bombardment prior to inoculation with an Agrobacterium suspension was reported. In this study, effects of two transformation systems (integrated bombardment and Agrobacterium transformation system and the singular bombardment) on transient GUS expression in hypocotyls of rapeseed (Brassica napus L. cv. PF704) MDEs were studied. Bombardment parameters were: helium pressure, 1350 psi; distance between stopping screen and target tissue, 9 cm, gold particles size of 1.0 microm and chamber vacuum pressure, 24 in Hg. In integrated system A. tumefaciens strain AGL1 carrying the binary vector pCAMBIA3301 was used. Bombarded hypocotyls of MDEs were inoculated with Agrobacteria at OD600 = 1.0 for 10 min or OD600 = 0.25 for 24 h. Integrated transformation system increased the mean number of blue spots about 2-2.5 fold compared to singular bombardment and inoculated hypocotyls with OD600 = 1.0 for 10 min produced the highest number of blue spots per bombardment (743 +/- 23.67).

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

Cold-induced accumulation of protein in the leaves of spring and winter barley cultivars.

Electrophoretic pattern and quantitative changes in soluble proteins were determined in the leaves of spring and winter cultivars of barley (Hordeum vulgare L., cv. Makouei and cv. Reyhan, respectively) exposed to 4 degrees C for 14 d. Seedlings were grown in a controlled growth chamber for 2 weeks at a constant air temperature of 20 degrees C and then transferred to constant 4 degrees C for 14 d followed by returning to 20 degrees C (cold treatment), or they were maintained throughout at 20 degrees C during the experimental period of 40 d (control treatment). Plants were sampled every 48 h for leaf fresh weight measurements. Total leaf soluble proteins were extracted and their concentration was either determined by a colorimetric method, or size-fractionated on SDS-PAGE. Low temperature-induced increases in protein amount occurred over the second week of exposure to cold treatment irrespective of cultivar: the winter cultivar was 2 d prior in this response. The protein patterns and their density showed differences between-cultivars and between-temperature treatments. A new cold-induced polypeptide was recognized in the leaves of winter barley cultivar on day 22 (8 d at 4 degrees C) compared to the control. This polypeptide was produced earlier over the first 48 h of low temperature in the winter cultivar compared with the spring one, recognizing in the leaves of cold-treated seedling until day 26. This more rapid response to a low temperature by the winter barley cultivar indicates a more sensitive response compared with the spring barley, probably cold-shock protein is a component of this cold-induced response.