With global warming, environmental stress has become increasingly serious. Gene editing technology based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has gained widespread use in plants. However, commonly used gene editing methods carry the risk of integrating exogenous genes into the genome of the recipient plant, thereby limiting their application in crops. To address this limitation, researchers merged the Cas9 protein and gRNA utilized in CRISPR/Cas9 technology into an Ribonucleoprotein (RNP) complex in vitro, enabling direct introduction into recipient cells for gene editing. Protoplast, devoid of cell wall, is more likely to absorb foreign genetic material, making it an ideal receptor for the RNP complex. Nevertheless, the technology of protoplast regeneration in plants is still immature, and the mechanism of protoplast division and dedifferentiation is still unclear.
Previous research demonstrated that protoplast regeneration is controlled by many factors, such as light, temperature, and phytohormones. Nicotiana tabacum, as a model plant of the Solanaceae family, is frequently employed in genome editing and molecular mechanism studies. Presently, we are employing transcriptome analysis of N. tabacum protoplasts subjected to phytohormone treatments at distinct time points throughout the isolation and regeneration period, to gain deeper insights into the mechanisms governing protoplast regeneration. If successful, it will facilitate the subsequent regeneration of the N. tabacum protoplast gene edited by the RNP complex to obtain tobacco plants that can tolerate environmental stress.