Alternatively, protoplasts can be transformed with ribonucleoprotein complexes, consisting of Cas9 associated with the gRNA ( Svitashev et al., 2016). However, CRISPR/Cas9 can also be expressed through transformation with mRNA encoding the Cas9 enzyme along with the desired guide RNA (gRNA) ( Zhang et al., 2016). Although relatively infrequent, the use of DNA (often in the form of plasmids) does not fully preclude the random integration of transgenes ( Lin et al., 2018). The enzymatic removal of the plant cell wall allows for the introduction of foreign DNA, RNA, or protein into protoplasts through either polyethylene glycol (PEG) treatment or electroporation. Advantages of Using Protoplasts in NPBTĪs stated above, the use of transient transformation of protoplasts can circumvent transgenesis (the integration of genetic material from one organism into the genome of another organism). Aside from the potential to speed up the application of NPBT, the use of protoplasts may have numerous other advantages. In contrast to conventional breeding or transgenic CRISPR/Cas9 approaches, gene editing through transient transformation and regeneration of protoplasts can achieve the desired genetic outcome within a single clonal generation by avoiding the integration of foreign DNA into the host genome ( Figure 1C). This is especially true for highly heterozygous and/or outcrossing crops. However, the current most commonly used NPBT method of inserting a transgenic CRISPR/Cas9 construct into the host genome and then crossing it out again to obtain transgene-free progeny still requires multiple rounds of selection ( Figure 1B). The ability to efficiently modify crop genes can save several years over conventional breeding approaches and phenotypic recurrent selection ( Bull et al., 2017). In contrast to transgenic approaches, NPBT can avoid costly and time-consuming regulatory hurdles and accelerate the introduction of new crop lines to the ag market ( Lassoued et al., 2021).īreeding for the introgression of new traits from a wild relative or mutagenized population into an elite crop cultivar is a lengthy procedure, requiring numerous rounds of selection to regain the characteristics of the parental strain ( Figure 1A). These new plant breeding technologies (NPBT) have opened avenues of fundamental and translational research that were previously inaccessible. Since the advent of CRISPR/Cas9 and related gene-editing technology, direct modification of crop genomes has become the way of the future for advanced breeding techniques in agriculture ( Zhang et al., 2019). Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. School of Plant and Environmental Sciences, College of Agriculture and Life Sciences, Virginia Tech, Blacksburg, VA, United States.
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