An In Vitro Cucumber Chloroplast Transformation Method

Abstract: Chloroplasts are semi-autonomous organelles in which photosynthesis takes place and chloroplast transformation is an important technique for investigating regulation mechanism of gene expression in the chloroplast. Transformation of chloroplasts by cells or tissues normally requires a long experimental period for achieving homoplasmic lines. In this paper， a chloroplast transformation method is reported， by which chloroplasts are isolated by differential centrifugation from green cotyledons of cucumber cultivated under sterile conditions，washed and suspended in 0.33 mol/L sorbitol， and then transformed by electroporation at 13 kV/cm voltage. The presence of exogenous gene and its expression in chloroplasts after transformation were confirmed by PCR， RTPCR and observation of GFP fluorescence by fluorescence microscopy. It is hopeful that this method can provide a quick way for identification of efficient chloroplast expression vectors and other basic research.

CSTR: 32200.14.cjcb.2016.11.0008