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Updated: Jan 29, 2026

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses
Published on: January 25, 2018
Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts
D Rangasamy1, C Ratledge1, C J Woolston1
1Department of Applied Biology, University of Hull, Hull, HU6 7RX, UK Fax no.: +44-1482-465458 E-mail: c.ratledge@biosci.hull.ac.uk, , , , , , GB.
Abstract:
Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide.
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