Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation

Abstract

Background: Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible. Results: In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right-and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato. Conclusions: Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

Salinity Stress in Potato: Understanding Physiological, Biochemical and Molecular Responses

“…For instance, knockout of miRNA genes in rice [182] showed improved salt tolerance and targeting susceptibility (S) genes for pathogen re-sistance in Solanaceae crop tomato [183]. CRISPR/Cas system also successfully used to target potato genes, such as StIAA2 [184], granule bound starch synthase gene (GBSS) [185], coilin [186], acetolactate synthase genes (StALS1, StALS2) [187], StGBSSI, StDMR6-1 [188], and phytoene desaturase (PDS) [189]. Recently, non-browning potatoes were developed by knocking-out the polyphenol oxidases gene (StPPO2) [190].…”

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

“…Targeting the GUX1 gene with a single gRNA produced single nucleotide INDELS. We did not see large deletions using a single gRNA as are frequently seen in other plant species [58][59][60]. In single gRNA edited plantlets all the insertions were either a cytosine or an adenine.…”

Gene editing in tree and clonal crops: progress and challenges

“…These include the difficulties of obtaining nonchimeric plants without strong selection using antibiotic or herbicide resistance genes, and overall low rates of editing in regenerated plants (Chen et al 2018). The latter is a serious obstacle for species with low transformation rates, as normally tens to many hundreds of treated plants must be screened to find a small number of homozygous knockout edited plants (Bánfalvi et al 2020). However, because the gene-edited plants generated using these approaches will be exempt in many countries (discussed below), there has been a flurry of interest in their development.…”

“…Another option for improvement of transient editing is to have a short period of antibiotic selection when transient expression is highest. One study in potato saw no significant increase in edited, transgene-free plant recovery using this approach and was unable to recover non-chimeric edited shoots (Bánfalvi et al 2020). Another study in apple succeeded in retaining transgene-free edited shoots using a visual editing marker, but at a 0.26% rate compared with all regenerated shoots (Charrier et al 2019).…”

“…These studies were in potato, grape, rubber tree, apple, and banana (Malnoy et al 2016;Malnoy et al 2016;Andersson et al 2018;Fan et al 2020;González et al 2020;Wu et al 2020). Two studies employed transient delivery of editing reagents by Agrobacterium without integration and only one succeeded to obtain edited, transgene-free, nonchimeric regenerants (Charrier et al 2019;Bánfalvi et al 2020). These studies were in potato and apple.…”