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Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop’s genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.

Interest in the development of organically grown vegetable crops has risen over the past decades due to consumer preferences. However, most crops that have desirable consumer traits have been bred in conventional growing conditions, and their transfer to an organic setting is challenging. Here, the organically grown Hawaiian pumpkin (Cucurbita moschata) accession ‘Shima’ was crossed with the conventionally grown Puerto Rican variety ‘Taina Dorada’ to develop a backcross (BC1) population, where ‘Shima’ was the recurrent parent. A total of 202 BC1 (‘Shima’ X F1) progenies were planted in a certified organic field, and twelve traits were evaluated. We used genotype-by-sequencing (GBS) to identify the Quantitative Trait Loci (QTL) associated with insect tolerance along with commercially desirable traits. A total of 1582 single nucleotide polymorphisms (SNPs) were identified, from which 711 SNPs were used to develop a genetic map and perform QTL mapping. Reads associated with significant QTLs were aligned to the publicly available Cucurbita moschata genome and identified several markers linked to genes that have been previously reported to be associated with that trait in other crop systems, such as melon (Cucumis melo L.). This research provides a resource for marker-assisted selection (MAS) efforts in Cucurbita moschata, as well as serving as a model study to improve cultivars that are transitioning from a conventional to an organic setting.

Plant organ growth results from the combined activity of cell division and cell expansion. The co-ordination of these two processes depends on the interplay between multiple hormones that determine the final organ size. Using the semidominant Hairy Sheath Frayed1 (Hsf1) maize mutant that hypersignals the perception of cytokinin (CK), we show that CK can reduce leaf size and growth rate by decreasing cell division. Linked to CK hypersignaling, the Hsf1 mutant has an increased jasmonic acid (JA) content, a hormone that can inhibit cell division. The treatment of wild-type seedlings with exogenous JA reduces maize leaf size and growth rate, while JA-deficient maize mutants have increased leaf size and growth rate. Expression analysis revealed the increased transcript accumulation of several JA pathway genes in the Hsf1 leaf growth zone. A transient treatment of growing wild-type maize shoots with exogenous CK also induced the expression of JA biosynthetic genes, although this effect was blocked by the co-treatment with cycloheximide. Together, our results suggest that CK can promote JA accumulation, possibly through the increased expression of specific JA pathway genes.

The study of nitrogen fixation in sugarcane has a long history that has demonstrated high potential but with substantial variation in results. This 32-month study sought to assess the response of nitrogen fixation associated with sugarcane (Saccharum officinarum L. cvs. ‘Akoki, Honua‘ula, and ‘Ula) to available soil nitrogen. Plants were grown in large pots of perlite along with a fixing and a non-fixing plant control and administered liquid fertigation with varying amounts of isotopically enriched nitrogen. Assessment of nitrogen fixation utilized nitrogen isotope tracing and acetylene reduction assay in the target and control plants. Isotope enrichment and acetylene reduction assay both indicated that nitrogen fixation peaked under low nitrogen application, and declined with higher application rates, with agreement between the two methods. These results suggest that sugarcane engages in a downregulation of nitrogen fixation under high nitrogen availability, potentially explaining the high variation in published experimental results. This suggests that nitrogen management and fertilization strategy can impact the atmospheric inputs of nitrogen in sugarcane cultivation, and the potential to improve nitrogen application efficiency in cropping systems utilizing sugarcane.

Aeroponic cloning is a great strategy to maintain desired genotypes by generating a whole new plant from cuttings. While this propagation technique has been demonstrated for tomatoes (Solanum lycopersicum) and potatoes (Solanum tuberosum), no protocol has been developed for peppers (Capsicum spp.). The ability to clonally propagate different Capsicum holds promise for domestic and industrial growing operations since elite cultivars with desirable traits (e.g., high capsaicin levels, nutrient content, and striped fruit) can be perpetuated without the need of planning a nursery. We tested six Capsicum species for their feasibility of aeroponic cloning by stem cuttings. All domestic species were successfully regenerated under aeroponic conditions but not for Capsicum eximium, a wild species. Of the species analyzed, Capsicum annuum peppers had the fastest node formation (11.6 +/− 0.89 days, P ≤ 0.01) and obtained a larger volume of roots (P ≤ 0.01) after node formation as compared to C. baccatum, C. frutescens, and C. pubescens. This study presents a cost-effective strategy to clonally propagate peppers for personal, industrial, and conservation purposes.