Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2019, Vol. 40 ›› Issue (8): 1537-1545.DOI: 10.3969/j.issn.1000-2561.2019.08.012

• Biotechnology and Tissue Culture • Previous Articles     Next Articles

Optimization of Extraction Conditions and Methods for Sugarcane Protoplast RNA

LI Suli,LIU Fangjun,LI Zhigang,LAI Peiheng,LONG Ansi   

  1. College of Agricultural, Guangxi University, Nanning, Guangxi 530005, China
  • Received:2019-01-18 Revised:2019-04-13 Online:2019-08-25 Published:2019-08-20


In order to obtain high-yield and high-purity sugarcane protoplast RNA, this study investigated the osmotic pressure and protoplast viability on sugarcane protoplasts using the sugarcane protoplasts of ROC22 and GT28, and compared the Trizol method, improved Trizol method, improved CTAB method and kit method. The protoplast osmotic pressure was positively correlated with RNase activity, and the protoplast activity was significantly negatively correlated with RNase activity. Higher RNase activity resulted in a worse RNA extraction effect. Using 0.5 mol/L mannitol as an osmotic pressure regulator, the most active sugarcane protoplasts could be obtained. When the protoplast vigor was 70% and 90%, the RNA integrity was good and the purity was high, which met the requirements of subsequent molecular experiments. The RNA yield was the highest when the protoplast activity was 90%. Therefore, when extracting sugarcane protoplast RNA, the activity of protoplast should be at least above 70%. Using the improved Trizol method, a large number of high-quality RNA could be prepared for subsequent molecular experiments. Using this condition and method, it was ensured that a large amount of high quality sugarcane protoplast RNA was obtained. The results of the study provided methods and technical support for the molecular biology research in the somatic cell fusion breeding of sugarcane.

Key words: Saccharum officinarum, protoplast, RNA, viability, RNase

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