Protoplast Regeneration and Its Use in New Plant Breeding Technologies

Abstract

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. 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. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. 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. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. 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.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

“…Temperature is another crucial factor for maintaining the viability of the isolated protoplasts. Most plant protoplasts are stable at room temperature (23 °C–28 °C) [ 37 ]. In contrast, however, our experiments showed that all the peanut protoplasts died at 23 °C after 48 h. Therefore, we tested the viability of protoplasts at 4 °C and 13 °C.…”

“…Our tests indicated that 13 °C was the ideal temperature for the PEG-mediated transformation. The optimum concentration of PEG and the duration of the PEG incubation time are other criteria that need to be considered for increasing transformation efficiency in protoplasts; it varies from plant to plant [ 27 , 37 ]. Our data showed that 50% of PEG and 5 min incubation time gave the best results for peanut protoplast transformation.…”

Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants

“…For example, Agrobacterium- and virus-mediated transformation methods are ineffective in many crop varieties lacking the necessary host susceptibility factors ( Hwang et al, 2017 ; McLeish et al, 2019 ). Biolistic bombardment and regeneration from protoplasts are also challenging in many elite crop cultivars ( Kelliher et al, 2019 ; Ren et al, 2020 ; Reed and Bargmann, 2021 ). Since these recalcitrant elite cultivars cannot be directly transformed with the relevant constructs, one proposed method calls upon crossing elite cultivars with other varieties that are more amenable to transformation with the constructs needed for CRISPR/Cas9-mediated genome editing, in sum using one plant as a delivery vehicle.…”

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

“…For leguminous crops such as chickpea and soybean, fully expanded leaves are the best choice for protoplast isolation [ 158 , 159 ]. Temperature is another crucial factor for maintaining the viability of the isolated protoplasts: most plant protoplasts are stable at room temperature (23 °C–28 °C) [ 160 ]; however, cold storage is sometimes necessary. The optimum concentration of PEG and the duration of the PEG incubation time are other criteria that need to be considered for increasing transformation efficiency in protoplasts [ 160 , 161 ].…”