Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.
J Zhejiang Univ Sci B
; 18(1): 85-88, 2017.
Article
em En
| MEDLINE
| ID: mdl-28071001
ABSTRACT
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Protoplastos
/
Vacúolos
/
Folhas de Planta
/
Sedum
País/Região como assunto:
Asia
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article