Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.
This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.
['Gao|Xiao-Yu|XY|', 'Liao|Xing-Cheng|XC|', 'Wu|Ruo-Lai|RL|', 'Liu|Ting|T|', 'Wang|Hai-Xing|HX|', 'Lu|Ling-Li|LL|']
D002104:Cadmium D002498:Centrifugation D002499:Centrifugation, Density Gradient D002681:China D018525:Germination D008293:Malates D008353:Mannitol D018515:Plant Leaves D018517:Plant Roots D011523:Protoplasts D031963:Sedum D013696:Temperature D014617:Vacuoles
Purified isolation Vacuole Sedum alfredii Protoplast Hyperaccumulator plant