Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3–60% per transformation and from 0–29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87–100%. This demonstration of CRISPR/Cas in potato extends the range of plant species modified using CRISPR/Cas and provides a framework for future studies.

Potato is the world's most important vegetable crop, with nearly 400 million tons produced worldwide every year, lending to stability in food supply and socioeconomic impact. In general, potato is an intensively managed crop, requiring irrigation, fertilization, and frequent pesticide applications in order to obtain the highest yields possible. Important traits are easy to find in wild relatives of potato, but their introduction using traditional breeding can take 15-20 years. This is due to sexual incompatibility between some wild and cultivated species, a desire to remove undesirable wild species traits from adapted germplasm, and difficulty in identifying broadly applicable molecular markers. Fortunately, potato is amenable to propagation via tissue culture and it is relatively easy to introduce new traits using currently available biotech transformation techniques. For these reasons, potato is arguably the crop that can benefit most by modern biotechnology. The benefits of biotech potato, such as limited gene flow to conventionally grown crops and weedy relatives, the opportunity for significant productivity and nutritional quality gains, and reductions in production cost and environmental impact, have the potential to influence the marketability of newly developed varieties. In this review we will discuss current and past efforts to develop biotech potato varieties, traits that could be impacted, and the potential effects that biotech potato could have on the industry.Resumen La papa es el cultivo hortícola más importante en el mundo, con cerca de 400 millones de toneladas producidas a nivel mundial anualmente, acreditando la estabilidad en el suministro de alimentos e impacto socioeconómico. En general, la papa es un cultivo manejado intensivamente, que requiere riego, fertilización y aplicaciones frecuentes de plaguicidas para obtener los más altos rendimientos posibles. Los caracteres importantes son fáciles de encontrar en parientes silvestres de la papa, pero su introducción usando el mejoramiento tradicional puede llevar de 15 a 20 años. Esto es debido a la incompatibilidad sexual entre algunas especies silvestres y cultivadas, el deseo para eliminar características indeseables de las especies silvestres del germoplasma adaptado, y la dificultad en la identificación de marcadores moleculares aplicables ampliamente. Afortunadamente, la papa es receptiva a la propagación por cultivo de tejidos y es relativamente fácil la introducción de nuevos caracteres usando técnicas biotecnológicas de transformación actualmente disponibles. Por estas razones, la papa es probablemente el cultivo que se puede beneficiar mayormente por la biotecnología moderna. Los beneficios de la papa biotecnológica, como el flujo genético limitado a cultivos que se siembran convencionalmente y a los parientes como malezas, la oportunidad para productividad significativa y logros en calidad nutricional, y las reducciones en los costos de producción e impacto ambiental, tienen el potencial para influenciar la comercialización...

HighlightPTB proteins of potato bind to the mobile RNA, StBEL5, and enhance stability and trafficking of the RNA to select organs. This protein–RNA interaction leads to enhanced tuber production.

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.