OBJECTIVETo develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions.

METHODSTo establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA.

RESULTSOur data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration.

CONCLUSIONWe have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

Gene Expression Regulation, Plant ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hydroponics ; methods ; Plants, Genetically Modified ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Tobacco ; genetics ; growth & development

Contracture and spasticity of ankle joints were major sources of disability in neurological impairment including stroke and cerebral palsy, etc. The manual stretching used in physical therapy might be laborious and time-consuming to the therapists and the outcome was dependent on the experience and the subjectiveend feelingof the therapists. A device was developed that could safely stretch the an-kle joint to its extreme positions with quantitative control of the resistance torque and stretching velocity. Furthermore, it could satisfy a strong need for quantitative and objective measures of the impairment and rehabilitation outcome. This was just the meaning intelligent stretching referred to. This article described the origin of the concept of intelligent stretching and its definition, operational principle, and su-periority and weakness, as well as its application in ankle joint spasticity and contracture in patients with stroke and cerebral palsy.