Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

Plants often encounter challenging environmental factors, including intense sunlight, drought, extreme heat, cold temperatures, salinity, excessive metals, and nutrient shortages, which can heavily affect their growth and survival. In this regard, L-ascorbic acid (AsA) is not only an essential nutrient for human health but also plays a significant role in plant responses to environmental stresses, regulating various functions during growth and development, redox signaling, and phytohormone biosynthesis. The growing need to cope with climate change, together with the advancement of CRISPR/Cas9-editing technologies, stimulated new opportunities to enhance AsA biosynthesis to improve crop stress tolerance. In this review, we discuss the biosynthesis and regulation of AsA in abiotic stress response mechanisms. We also explore the latest advancements of CRISPR/Cas9 technologies, their applications, and their challenges as tools for modifying genes associated with AsA metabolism, aiming to develop crops more tolerant and resilient to environmental changes.

Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.

The worldwide agricultural system confronts a significant challenge represented by the increasing demand for food in the face of a growing global population. This challenge is exacerbated by a reduction in cultivable land and the adverse effects of climate change on crop yield quantity and quality. Breeders actively embrace cutting-edge omics technologies to pursue resilient genotypes in response to these pressing issues. In this global context, new breeding techniques (NBTs) are emerging as the future of agriculture, offering a solution to introduce resilient crops that can ensure food security, particularly against challenging climate events. Indeed, the search for domestication genes as well as the genetic modification of these loci in wild species using genome editing tools are crucial steps in carrying out de novo domestication of wild plants without compromising their genetic background. Current knowledge allows us to take different paths from those taken by early Neolithic farmers, where crop domestication has opposed natural selection. In this process traits and alleles negatively correlated with high resource environment performance are probably eradicated through artificial selection, while others may have been lost randomly due to domestication and genetic bottlenecks. Thus, domestication led to highly productive plants with little genetic diversity, owing to the loss of valuable alleles that had evolved to tolerate biotic and abiotic stresses. Recent technological advances have increased the feasibility of de novo domestication of wild plants as a promising approach for crafting optimal crops while ensuring food security and using a more sustainable, low-input agriculture. Here, we explore what crucial domestication genes are, coupled with the advancement of technologies enabling the precise manipulation of target sequences, pointing out de novo domestication as a promising application for future crop development.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are revolutionizing precision genome editing and gene expression control in crop plants. While effective CRISPR-Cas applications traditionally rely on labor-intensive stable genetic transformation to deliver Cas nucleases and guide RNAs into plant cells, plant viruses have emerged as a faster and efficient alternative, a strategy known as virus-induced gene editing (VIGE). Cas12a, Class 2 Type V CRISPR nucleases, are an alternative to broadly used Cas9 for plant genome engineering. Both kind of nucleases offer precise editing, but some Cas12a unique features make them particularly well suited for VIGE. In this study, we first used a tobacco rattle virus vector to compare editing efficiency of various target sequences and CRISPR RNA (crRNA) architectures in Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a)-expressing Nicotiana benthamiana plants, evaluating results in infected tissues and seeds. Next, we developed a tobacco etch virus (genus Potyvirus)-derived vector efficiently delivering crRNAs throughout the plant. This approach enabled generation of plants with all four edited alleles in the allotetraploid N. benthamiana through in vitro regeneration from infected leaves, and to produce edited non-infected progeny, although at a very low frequency. We then demonstrated the successful application of the potyviral vector for VIGE in agronomically important crops, such as tomato or cultivated tobacco. Finally, we replicated this design using two other potyviral vectors, turnip mosaic virus, and lettuce mosaic virus. Given the conserved biological properties among potyviruses, we believe these findings are broadly applicable to the largest genus of plant RNA viruses, significantly expanding the host range of the VIGE technology.