Establishment Of Methods For Viscoelastic Measurements For Arabidopsis Thaliana Cells

At present, the study of animal cell mechanics has entered the mainstream of bioscience. However, the study and detection methods of plant cell mechanics are lagged far behind. Cell viscoelasticity is an important biophysical parameter related to the cellular structures and functions. In this work, we optimized the conditions of plant regeneration and suspension cell culture of Arabidopsis thaliana. Quartz crystal microbalance (QCM) technique was used for the study of dynamic viscoelastic properties of protoplasts, calluses and suspension cells of Arabidopsis thaliana for the first time.5 MHz SiO2-deposited quartz crystals were modified with the positively charged poly-L-lysine(PLL), low melting agarose/PLL. The adhesion processes of Arabidopsis mesophyll protoplasts, calluses and suspension cells to the modified QCM crystals were monitored in real time and the effects of osmotic pressure and adhesive substrate to the viscoelastic responses were studied. The main results are as follows:1. The optimized media for inducing callus tissues were experimentally determined from different Arabidopsis thaliana sources and are summarized below:B5+2 mg/L 2,4-D +1 mg/L 6-BA+2 g/L phytagel (pH 5.6) for Arabidopsis thaliana seeds; B5+0.5 mg/L 2, 4-D+0.05 mg/L 6-BA+2 g/L phytagel (pH 5.6) for Arabidopsis thaliana roots and leaves. The optimized medium for inducing buds with Arabidopsis calluses is MS solid medium+5 mg/L 6-BA+0.5 mg/L NAA (pH 5.8); and the optimized medium for inducing roots with Arabidopsis calluses is MS solid medium+0.25 mg/L NAA (pH 5.8).2. The dynamic viscoelastic properties of Arabidopsis protoplasts and their changes under different osmotic stresses were studied for the first time using the QCM technique. The results indicated that the viscoelasticity of Arabidopsis protoplasts is determined by the cytoskeleton and membrane. Under hypertonic conditions, the protoplasts became stiffer as the increase of osmotic pressure when QCM crystals of 5 MHz or lower overtones were used for the measurement, which coincides with what was found in animal cells; whereas the results obtained with higher overtones showed that the protoplasts became softer. Under hypotonic conditions, we discovered the unique viscoelastic behavior of protoplasts compared to animal cells, the protoplasts could become softer or stiffer when 5 MHz crystal was used, the protoplasts became stiffer as the decrease of the osmotic pressure when overtones of 5 MHz crystal were used. It is therefore concluded that the membrane of a protoplast can still maintain its functions of contraction and softening under hypertonic conditions, and expansion and stiffening under hypotonic conditions even it is free of its cell wall.3. The dynamic differentiations of Arabidopsis calluses were monitored by the QCM technique and the preliminary results showed that viscoelastic changes and stiffening were accompanied with the differentiations of both roots and buds, the callus differentiated into roots became stiffer than the callus differentiated into the buds.4. The growth of Arabidopsis cells was dynamically monitored with QCM and the preliminary results indicated increase in stiffness during the growth of the cells, which coincides with what was found for cells during exponential growth in the literature.