DNA-free transcriptional activation of cabbage (Brassica oleracea L.) using CRISPR/DCAS9 ribonucleoproteins to enhance heat stress tolerance based on morphophysiological plant traits

Red cabbage (B. oleracea) is one of the most distinct species among the numerous species of Brassica genus due to having high level of anthocyanins. Brassica species are widely consumed vegetable crops with great health benefits. However, they are highly vulnerable to high temperature and their production are limited to highland areas in Malaysia. Understanding how plants adjust their developmental programs in response to temperature variations is central to sustain crop productivity in the modern agriculture facing global climate change. Global climate change has generated significant fluctuations of ambient growth temperature, which can profoundly influence diverse developmental, physiological, and morphological responses, including modulations in plant growth and yield. In recent years, many crop genomes have been sequenced and innovative biotechnological approaches allowed to take a step forward towards the development of new improved cultivars harboring precise genome modifications. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, represents the main methods available for plant genome engineering through targeted modifications. Such technology, however, requires efficient transformation protocols and extensive genomic resources and accurate knowledge before they can be efficiently exploited in practical breeding programs. This study investigated on heat-tolerant/sensitive cultivars based on morphophysiological indicators and the action and interaction of different genes in the molecular network to serve as critical tools for genetic improvement in cabbage. The feasibility of DNA-free transcriptional activation method through delivery of CRISPR/dCas9-based transcriptional activation domains (TADs) ribonucleoproteins (RNPs) into the red cabbage protoplasts was also evaluated. To screen the morphophysiological indicators, the morphological and physiological performance of two different varieties of white and re