Abstract
Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange.
Highlights
Gene editing in sweet orange are usually faced with difficulties including low editing efficiency and low root formation rate, which severely limits the advancement of gene function studies and application in molecular breeding in sweet orange
The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90% (Fig. 1l)
We evaluated the editing efficiency induced by the three polycistronic tRNA-sgRNA (PTG)/CRISPRassociated protein (Cas)[9] constructs using Agrobacterium-mediated genetic transformation of sweet orange epicotyls
Summary
Gene editing in sweet orange are usually faced with difficulties including low editing efficiency and low root formation rate, which severely limits the advancement of gene function studies and application in molecular breeding in sweet orange. The application of Arabidopsis YAO promoter driven CRISPR/Cas[9] system in citrus rootstock cultivar Carrizo Citrange significantly improved gene editing efficiency than the 35S promoter driven CRISPR/Cas[9] system (Jia and Wang 2014; Zhang et al 2017; Alvarez et al 2021). Studies have shown that polycistronic tRNA-sgRNA (PTG) /Cas[9] is more efficient for multiplex gene editing than traditional CRISPR/Cas[9] system (Xie et al 2015; Wang et al 2018), and for Carrizo Citrange (Huang et al 2020).
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