The reasons for this are manifold and interconnected; however, most stem from two primary issues. First, many horticultural crops have been traditionally understudied because their lifecycle, genomic structure, or inability to regenerate via tissue culture, are not amenable to methods used in functional genomics. This leads directly to the second reason: many of the genes that contribute to the problems of PLW have not yet been identified. These two identified stumbling blocks will require new and significant financial investments to minimize their effect and accelerate the use of gene editing for reducing PLW .Although transgenic approaches for modifying plants have been advanced for three decades, transformation and regeneration are bottlenecks in using gene-editing tools to address crop improvements. The efficiency of Agrobacterium-mediated approach varies by Agrobacterium strain, and the plant species and tissue to be transformed. Almost 95% of woody fruit and nut crops are still recalcitrant to transgenic approaches because of poor transformation efficiencies using Agrobacterium. Transformation may be achieved using biolistic and electroporation approaches, which are non-tissue specific but lead to multiple insertions which can create additional non-intended genetic changes. Regeneration through tissue culture is even more challenging than the integration of foreign genes,blackberries in containers and the process is time-consuming . Regeneration takes 6–9 months for papaya, 5–8 months for kiwifruit, and 4–5 months for potato. Novel delivery approaches to bypass labor-intensive plant regeneration procedures are being developed. “Spray-on” gene editing involves coating nanosized carbon dots with plasmids containing gRNA and Cas9 cDNA, which are delivered directly to the cell.

Inducing meristem formation in tissues transformed with the CRISPR gene construct would produce edited plantlets without a callus explant step, thus saving time.Recent innovations promise to open up the number of crops that can be efficiently modified by gene editing. Specifically, transforming calli with a GRF4-GIF growth factor chimera has been used to accelerate regeneration efficiency more than 5-fold among some of the most recalcitrant species, and may be a major advance in crop improvement.Many post harvest traits are composite, with phenotypes that are the result of multiple environmental factors as well as genetics . As mentioned, common disorders such as PCI, blossom end rot, and superficial scald, are challenging to study at the molecular level. These traits are likely to be multigenic and multiallelic, with gene expression controlled in networks involving epistatic interactions and epigenetic mechanisms, that are influenced by environmental factors. For many years, environmental control, i.e., refrigeration and modified and controlled atmospheres, was the primary way of maintaining shelf-life and quality. Forward genetic approaches such as QTL and Association mapping, mutagenesis, and gene expression analysis, have been used to identify candidate genes that exert a significant degree of control over complex agricultural traits. However, introgressing favorable genes into a new cultivar may not always lead to the strong expression of a trait, because of environmental effects.There is much uncertainty about the right to market edited germplasm. In the United States, the Broad Institute holds the patent for CRISPR editing of eukaryotic cells. However, the University of California continues to challenge the 2018 ruling on multiple grounds. In most of Europe and the Pacific , the University of California holds the rights to CRISPR. As shown in Fig. 1, the perishable fruit, vegetable, and ornamental market is global. Those interested in growing “CRISPR’ed” produce in, e.g., California, and selling it in the US, Canada, China, and Australia, might need to invest millions to acquire permissions from both Broad Institute and the University of California.

This may increase the entry cost to commercialize post harvest gene-edited products, and limit the traits targeted to those with the highest profit-margin and simultaneously, push out smaller, “boutique” biotechnology firms. The public sector has not hesitated to use CRISPR. The Broad Institute allows unrestricted use of the Intellectual Property covered in its CRISPR patents for nonprofit and academic research uses, but commercial planting of the fruits of that research is open to legal challenge. Currently, in the US, commercial growers frequently invest in public breeding programs by providing land for field trials to state-level institutions. Material support from for-profit entities may become legally tenuous if a laboratory or facility is using CRISPR or any other patented technology. As CRISPR techniques become more refined and additional patents are submitted, these legal and financial contingencies may become more labyrinthine. However, the explosion in CRISPR research in plants, shows that the agricultural and horticultural world is eagerly embracing gene editing. The promised profit improvements currently outweigh the potential legal ramifications of patent infringements.Another roadblock to the commercialization of gene edited horticultural crops is their differing classification across the globe. The United States and China, which produce and consume a majority of the world’s fruit and vegetables , have readily embraced gene editing, and regulation in India is based largely on precedent. Modifications produced by gene editing vs. traditional breeding can be functionally identical, and distinguishing said modifications is near impossible. As a result, in June 2020 the United States announced the SECURE rule, stating that from April 2021, novel crops with DNA changes that could be introduced by traditional breeding can be fast-tracked for marketing. The European Union, however, has ruled that gene-edited crops are in the same classification as “traditional” GMOs. This places additional burdens on companies wishing to market edited produce in the EU and UK. European produce markets have high quality standards for flavor and texture, and new gene-edited crops could conceivably meet these criteria. The benefits of PLW reductions from gene editing may not be realized as quickly as in Europe.

The olive tree, olive fruit and olive oil have been at the core of Mediterranean agriculture and trade since early cultivation times, providing sustenance to various cultures and civilizations of the Mediterranean Basin. Over the last few decades, olive cultivation has undergone important technological changes, which have involved a reduction in the number of olive oil varieties used, and an increase in the density of new plantations that is linked to improvements in harvesting machinery and irrigation systems. In the early 1990s, a new design and management strategy for olive orchards, the super-high-density hedgerow system, appeared in Catalonia . Later it was introduced into other Spanish regions and other countries. Clonal selections of local varieties were planted in new olive orchards with tree densities ranging from 600 to 1,000 trees per acre to test the suitability of the plants to mechanization and the production of high-quality, extra-virgin olive oil. Traditional olive orchards have 80 to 200 trees per acre . Just a few olive varieties have been compared for their adaptability to high-density plantings and continuous mechanical harvest. Our program at the Institut de Recerca i Tecnologia Agroalimentaria screened three old Mediterranean olive varieties — ‘Arbequina’ and ‘Arbosana’ from Catalonia and ‘Koroneiki’ from Crete — to identify those with outstanding characteristics such as compact growth habits, low-medium vigor, early maturity and excellent oil quality . Agronomical evaluations in Spain and other countries have shown that these IRTA clones are precocious , achieve higher yields earlier after planting and produce extra-virgin oil of excellent quality. Olives were introduced in California by the Catalan Franciscan fathers, who planted olive trees in gardens adjacent to their missions. Their olives and oil were appreciated not only as food but also as an element in liturgical celebrations. Today,blackberry container the predominant table olive industry in California is supported by classic cultivars introduced for their suitability to traditional and intensive table olive orchard systems . In California, the industry generally plantsfive cultivars to produce black-ripe olives . ‘Mission’ trees were likely introduced to California during Franciscan times, via Mexico in 1769 . Olive planting for oil production, by contrast, has grown from negligible acreage in 1996 to approximately 16,000 acres by 2008. Most of this acreage, 12,000 acres , is planted in super-high-density orchards with 560 to 870 trees per acre . Most high-density California olive plantings are three releases of IRTA’s clonal plant material from the Mas de Bover research station in Catalonia, Spain, the initial selections from their olive improvement program started in the mid-1980s. The IRTA clonal varieties currently available are ‘Arbequina i-18’, ‘Arbosana i-43’ and ‘Koroneiki i-38’, propagated in California by a few authorized nurseries. The success of these early clonal selections and the super high-density system is also evidenced by their early adoption in traditional olive oil–producing countries, such as Spain, Portugal, Tunisia and Morocco, as well as diverse, nontraditional olive growing regions that are beginning to produce extra-virgin olive oils of remarkable quality such as California, Chile and Australia. We describe the performance and limitations of these olive oil clones in comparative fi eld trials performed in an irrigated, super-high-density system in Catalonia, which supports their adoption in modern orchards. We also contribute additional information to help define the suitable orchard design and management of super-high-density plantings in California.Differences between varietals in cumulative fruit yield became evident during early years of the trial .

In Tarragona, the highest cumulative yields were measured for ‘Arbequina i-18’ followed by ‘Arbosana i-43’ and ‘Koroneiki i-38’ . In a similar trial in Cordoba , ‘Arbequina i-18’, ‘Arbosana i-43’ and ‘Koroneiki i-38’ showed higher mean harvest yields than other varieties tested ; in this southern location ‘Koroneiki i-38’ was the most precocious . During the first years of both trials, the influence of environment on precocity and average crops achieved was larger in Cordoba due to the higher vegetative tree growth in this province. The mean harvest of super-highdensity cultivars in Tarragona was 4,397 pounds per acre , 4,205 pounds per acre , 10,344 pounds per acre and 7,291 pounds per acre , all similar to harvests obtained in other high-density orchards in Spain. The high yields observed in early years of the Spanish trials and commercial orchards are not sustainable. Under the favorable growing conditions that foster vigorous tree growth, a reduction in potential production occurs in the 6th to 8th years, with averages of 7,138 to 8,030 pounds per acre, usually due to shade and limited ventilation in the tree canopies . The yields of 7- to 10-year-old orchards are more variable and depend on management of the canopy volume, which should not exceed 143,000 to 171,500 cubic feet per acre to facilitate movement of the over-the-row harvesters.We observed the lowest tree vigor in ‘Arbosana i-43’ and ‘Arbequina i-18’ . ‘Koroneiki i-38’ is notorious for being more vigorous and producing more suckers than the other cultivars. The yield efficiency of each varietal clone was measured to determine the balance between productive and vegetative activity during the early bearing phase. The highest index scores were observed in ‘Arbequina i-18’ and ‘Arbosana i-43’ , followed by ‘Koroneiki i-38’ . ‘Koroneiki i-38’ showed a higher tendency to vegetative growth, and the crop was irregular among trees during the first years of the trial.Several intrinsic varietal characteristics, such as growth habit and canopy width, influence the efficiency of fruit removal during mechanical harvest. Our selections display two growth habit categories: semi-erect and open canopy . Straddle machines or grape harvesters perform better than trunk shakers for these cultivars. More than 90% of the fruit was removed in all cultivars, independent of their size, position in the canopy and maturation index. By contrast, the efficiency of trunk-shaking harvesters is clearly influenced by growth habit , and yield is improved with an erect or semierect tree shape, large fruits and low fruit removal force.Gradual fruit ripening and maturation is commonly observed in the three cultivars, although this parameter is highly influenced by tree fruit load and seasonal conditions as well as geographical location. Optimal harvest time is different for each of the cultivars: ‘Arbequina i-18’ is optimal in Catalonia from mid-November to mid December, ‘Koroneiki i-38’ matures in late December and ‘Arbosana i-43’ in mid-January.‘Arbequina i-18’ produced larger fruits than the other two cultivars . The pulp/stone ratio was higher for ‘Arbosana i-43’ and ‘Arbequina i-18’, followed by ‘Koroneiki i-38’. Fruit water content ranged between 56.0% in ‘Koroneiki i-38’ and 61.1% in ‘Arbosana i-43’. Oil content expressed as percentage of dry weight was higher in ‘Arbequina i-18’ , followed by ‘Koroneiki i-38’ and ‘Arbosana i-43’ . The three clonal selections produced extra-virgin olive oil of excellent quality. The fatty acid composition of ‘Arbequina i-18’ and ‘Arbosana i-43’ oils was similar . ‘Koroneiki i-38’ oil is characterized by a higher content of oleic acid at the expense of palmitic and linoleic acid, which contribute to longer shelf life. ‘Arbosana i-43’ and ‘Koroneiki i-38’ oils were consistently richer in polyphenols than ‘Arbequina i-18’. When compared at the organoleptic sensory level , ‘Arbequina i-18’ oil was the most balanced of the three, with a medium fruity intensity, balanced in the palate and an outstanding sweetness. ‘Koroneiki i-38’ produced the most fruity, green, bitter and pungent oil of the three, and ‘Arbosana i-43’ oils had an intermediate palate profile. Oil composition and flavor change as the olive fruit develops. The distinctive and contrasting sensory attributes of the extravirgin oils from each varietal allows for unique blends with a wide range of interesting sensory characteristics.