Horticulture

Alternate bearing is also perhaps one of the oldest known plant behaviors, as written descriptions of it can be found going back over several millennia. Most often these references are simple observations of trees that flower in one year but not the next , or as one early account of cider production in England elegantly described it, the trees would “. . . provide a full and complete blessing every second year” . Other written accounts offer advice on how to get sterile trees back into production, though it is not always clear if these early authors were aware of the full two-year cycle. Nevertheless, the prescribed regimes of girdling, branch twisting, and pruning are well-matched to its existence nonetheless: by removing a lot of branches or inflicting specific types of damage, this advice would stimulate the trees to produce a dense crop of new branches, which go on to bear fruit in the following year. Other accounts instead try to explain the biennial pattern by speculating that the trees require a period of “rest” before or after a large crop. Alternatively, vertical tower for strawberries the cycle has been suggested to be the result of rainfall, variation in cross-pollination efficiency, and/or total the flower numbers.

Not everyone was inclined to such speculation though, as Japanese farmers reportedly accepted the cycle as nature’s way, without trying to intervene at all.Despite the long history of this tree behavior, there have been relatively few attempts to learn how or why the plants produced the cycle. Even when descriptions of such experiments have survived, they tend to be hearsay accounts published by 3rd parties, making it difficult to determine what exactly transpired. For example, one horticultural manual from the 1800’s recognized that an unequal growth trade-off between the fruits and branches was a significant symptom of alternate bearing trees, and recommended that farmers artificially restore that balance by cutting off excess vegetative growth. In another example twenty five years later, a reporter for a popular horticultural magazine visited a nearby orchard and held a walking interview with the owner. In the process, we learn that the proprietor had performed an experiment some years earlier, in which he had removed all the flowers from a single tree one season, and found an abundance of fruit in the next. In contrast, academic interest in alternate bearing was essentially non-existent until the late 1800’s, when the subject apparently received a major boost from the establishment of state sponsored agricultural experiment stations. This was apparently a global trend, as England adopted a private research station that began as early as 1843, Japan obtained one by official decree in 1871, and United States began building its research stations following the Hatch Act in 1887.

By the turn of the century, these and other experiment stations had begun to produce a large number of agricultural publications, quickly establishing the foundation of crop science as we know it today. Research in the alternate bearing field lagged behind somewhat, perhaps by the need to establish experimental orchards, but began to produce a burst of new research starting around 1900. Thus began a roughly 30 year period of clever experimental designs and careful scholarship, ultimately producing the tools and basic concepts of alternate bearing research. Beginning with the development of chemical sprays in the 1940’s, the focus of this research began to shift away from basic inquiries and instead frequently focused on practical farming issues. Over the next few decades, this work made slow but steady progress towards understanding the physiological mechanism that propagates the cycle, while also identifying a broad array of species with similar physiological symptoms. The past few decades were also an extremely productive time for alternate bearing research, as this student estimates that the field has accumulated more than 1200 papers that mention the cycle by name, as well as another 3- 4000 papers in closely related subjects, with less obvious key words. In contrast, work to incorporate modern genetic tools is a much more recent phenomenon, beginning with just a handful of papers in the early 2000’s , then escalating rapidly by the end of the decade.

Generally such genetic work has attempted to observe when and where key developmental genes respond to the cycle, because it is not yet possible to make an entirely predictive model of alternate bearing, based on known developmental pathways from other species. Such knowledge is however, is expected to help farmers precisely control the behavior in popular tree crops, either through selective breeding , transgenic modification, or predictive mathematical models. A genetic understanding may even provide a way to clarify current ideas about the ecology and evolution of the cycle, while also identifying key developmental differences that separate annual and perennial species. In many cases alternate bearing is described largely in terms of fruit production, but close observations have shown that the actual phenotype is much more complicated. Similar two-year cycles have also been found in leaf area, flower numbers, branch lengths, and trunk diameter, just to name a few . Several physiological parameters have also been found to change in phase with the cycle, including mineral nutrients and carbohydrates, which tend to accumulate in the fastest growing organ, and are depleted in slower growing organs. Plant hormones have also been correlated with specific parts of the cycle, and are important regulators of floral induction, dormancy, fruit ripening, and abscission. Collectively, these findings outline an inverse relationship between fruit and vegetative growth that is broadly applicable to all species: When fruits are abundant, the vegetative growth is reduced. Conversely, when fruit are rare, the vegetative growth is abundant. This pattern is commonly known as a growth trade-off. Such trade-offs have been known to plant specialists for almost two centuries, and there are numerous documented examples where the fruits are negatively correlated with growth elsewhere in the plant, container vertical farming and occasionally with tissue death, especially of the meristem region. The existence of a growth trade-off however, does not immediately explain how this contributes to a biennial cycle. Most temperate trees shed their fruits in the fall, and there is usually nothing left to affect growth at the start of the second season. With the exception of more tropical trees like Avocados, this suggests the plants actually retain a “memory” of the fruit load from the previous season, a hypothesis that immediately raises questions about how that information is stored and transmitted over time. While the idea that plants may retain that memory in terms of their gene expression patterns has never been tested, the available evidence instead suggests that this information is recorded in the plant’s physical anatomy. For example, most trees produce flowers from their axillary buds a few months to a year after the buds are first produced. The number of such buds is proportional to the vegetative growth in the previous season, and since vegetative growth is often coeval with immature fruit, it is easy to see how a growth trade off might be recorded directly by the number of axillary buds. In addition, immature flowers and inflorescences typically begin growing while still inside the buds many months before they burst open, so the growth trade-off has the potential to affect the size of the inflorescence at a very early stage in their development.

Any initial differences in size incurred in this way are then likely magnified in the second season, when both inflorescence and vegetative growth resumes at an accelerated rate. Together the combination of inflorescence size and number can substantially change the number of flowers in subsequent season, and thus have an indirect control over number of subsequent fruits. In fact, when branch lengths and flower numbers are mapped to a generic branch structure, it is possible to derive a reasonably descriptive model of alternate bearing anatomy, summarized in Figure 1.0. In the “ON” year, the presence of a large number of fruit is thought to cause the reduction in vegetative branch length and leaf numbers, providing fewer axillary buds for the return bloom in the next season. In the following “OFF” year, the axillary buds produce fewer flowers and fruit, allowing the vegetative longer branches to grow longer and bear more leaves. The cycle then repeats itself when the axillary buds produce a large number of flowers in the 3rd season, creating yet another “ON” year. Once started, this mechanism is thought to be sufficient to propagate the biennial cycle indefinitely, barring intervention by the environment or anthropological factors. The variation in branch length is also permanently recorded in the plant anatomy, and with a few caveats, several years worth of cycles can be seen simply by noting the distance between successive sets of bud scars, or the remnants of old cones or inflorescences. The simplicity of this model is perhaps its greatest selling point, as it can readily incorporate information from several other areas of research. Following the discovery of plant hormones for example, it was soon found that gibberellic acid was an important regulator of flower numbers in many species, either as an activator or a repressor, and has its strongest effect during the period of floral induction. The response to auxin apparently changes over the course of the season, as mid-season auxin treatments can increase fruit abscission rates, while applications to immature inflorescences and mature fruit tend to prevent abscission. Ethylene seems to have a dual function, enhancing the number of flowers in some cases, while stimulating abscission of young fruits in others. The role of abscisic acid is not quite as clear, but the concentration of this hormone is known to fluctuate with the cycle in citrus trees, yet it was not correlated with abscission of immature pistachios nuts. In addition to the hormones, the distribution of starches, soluble carbohydrates and even inorganic mineral content can be mapped to anatomically different phases of the cycle. The result is a comprehensive physiological model of alternate bearing, which can be used to make reliably accurate predictions for both practical and theoretical applications. The basic physiological model is also surprisingly compatible with more recent concepts of plant development. For example, most vegetative branches do not grow continuously, but are instead produce in regular bursts commonly known as a “flush”. Also variously known as a “growth unit” “iterative growth” or even polycyclic growth, a flush typically occurs once per year in temperate climates, but tropical trees can produce as many as 3-4 flushes per year. Each flush is composed of at least two discrete phases of growth, known as the juvenile and adult phases. Confusingly, these two terms are also used to describe growth patterns related to the age of the plant in years, so for the sake of clarity it is necessary to borrow a slightly different terminology and refer to the variation along a single branch as “seasonal heterophylly”. The first leaves of the flush are distinguished from the later leaves by subtle-to-significant differences in trichome density, color, size, and leaf morphology, and typically, the first leaves are also abscised while still immature. Typically a flush is thought to begin and end with SAM dormancy, and as a result, alternate bearing anatomy can easily be outlined as the sum of two consecutive flushes, attached end to end.The flowers in contrast, are born on a completely different type of flush, usually called an inflorescence. These are typically produced from dormant axillary buds, though some plants also use the SAM as well. All such buds have the option of producing vegetative or reproductive structures, and the deciding factor is determined by a combination of information derived from the external and internal environments. Once the correct combination for each species is recognized though, the buds are said to be “induced”, irrevocably committing them to a reproductive fate. The resulting Inflorescence Meristem then produces the branches, bracts and flowers of the mature inflorescence. While the flowers are an obvious indication of reproductive growth, the inflorescence branches may also be distinguished from vegetative branches by their unique patterns in color, diameter, and trichomes. In addition, the inflorescence is also a determinate structure that senesces at maturity. The resulting abscission zone or boundary with dead tissue then provides a clear indication of which tissues were reproductive and which were vegetative .

Pruning mature open center fruit trees involves keeping the center free of vigorous upright shoots, reducing tree height, and thinning out branches to reduce crowding. For peaches and nectarines, select 1-year-old lateral fruiting branches that originate close to main branches. Thin these fruiting branches and head them by one-third if they are longer than about 1 to 2 feet . Remove or cut back 2-year-old fruiting shoots or cut them back to 1- year-old shoots. On other species, old fruiting spurs should be renewed periodically. Table 1 shows the location of fruiting buds, the longevity of spurs, and the desired severity of pruning of selected species. When dormant pruning, become familiar with the plump fruiting buds and prune accordingly to ensure adequate flowering the following spring. Pruning of mature almonds involves few or no heading cuts; simply thin out branches—often fairly large branches—to prevent crowding.Central leader training is often used for apples and sometimes for pears, Asian pears, pecans, and quince. These trees tend to have dominant central leaders, a characteristic that lends itself to the central leader training method. However, because many apple and pear varieties are susceptible to fire blight, plastic pots 30 liters open center or other multiple-leader methods are often preferred so that if a major limb is lost the tree can be more easily redeveloped.

Central leader training involves keeping trees shaped somewhat like Christmas trees, with lateral branches arranged in separate layers, or “tiers,” separated by open areas of canopy, and branches in lower tiers wider than those in upper ones. Instead of sunlight reaching lower fruiting branches through the center, as with the open center method, it reaches them from the sides and between branches. For young central leader trees, the goal is to create three or four tiers of lateral branches, with about four branches per tier. This is done preferably during spring and summer by heading back or bending down any vigorous shoots that grow upright and compete with the central leader. Create the first tier of four lateral branches by tying or staking branches outward at an angle just above horizontal after they have grown 2 to 3 feet long. When the central leader has reached about 21⁄2 to 3 feet past the first tier, usually in the first dormant season after planting, head it just below this point and train a second tier of four branches outward from the cut leader. These branches should be offset vertically from those of the first tier. Then create the third tier in a similar manner.

Avoid bending one branch directly over another; also, maintain the tree’s pyramidal shape by keeping lower branches longer than upper branches. The entire process will take 3 to 4 years, depending on the tree’s vigor. Some side branching of these main lateral branches should be encouraged. Vigorous upright shoots should be removed or headed back during the growing season to only three to six buds, although some shoots should be left longer if fruit are exposed to hot afternoon sun. Heading these shoots a couple of times during the growing season creates fruit-bearing spurs if the tree is not too vigorous. Heading may also encourage the growth of another set of vigorous shoots; simply remove these shoots or prune them back to create more spurs.With walnuts, the first main branch originates higher than on most trees, about 5 to 8 feet , and the spacing between branches is about 3 to 5 feet . Pruning of mature walnut trees is not essential, but thinning of branches will keep trees healthy and productive. Persimmons bear on current-season shoots that originate from buds produced near the ends of 1-year-old lateral branches. For this reason, once the tree is developed, avoid heading these shoots and be sure that sunlight reaches lower shoots, or they will become less productive or die. Also, each limb must be kept strong from the start by ensuring adequate sunlight and shortening branches if necessary by cutting back to upward- and outward-growing lateral branches.

Avoid heading cuts except to stimulate branching, such as on young trees; such heading cuts are best made in late spring and summer to reduce strong upright growth of “whip” branches. Persimmons often grow taller than is desired for picking fruit. Trees that are allowed to grow unchecked can make large, beautiful trees; however, branches often break with the weight of fruit. If they are headed at a given height each year, the subsequent vigorous shoots will shade lower shoots by midsummer, so these shoots must be thinned by summer pruning. Where possible, cut to lateral branches rather than making heading cuts.“Fruit bushes” are standard trees or, preferably, trees on dwarfing rootstock that are kept small by periodic summer pruning. This method can work for nearly all fruit species. The beauty of this system is its simplicity and ease of management. Pruning begins in about late April or early May of the first growing season, when new growth is about 2 feet long. At this time, cut the new growth in half, aiming for a uniform, bushy appearance. Hedge-trimming shears may be useful in this. In about late June, cut the subsequent new growth in half. If new growth is vigorous, it may need to be cut once more during the season. These heading cuts promote an excess of branches, so thin them by removing some shoots to allow sunlight to reach the lower branches. If needed, thin out additional crowding branches in the dormant season when they are more visible. In the second year, continue cutting new growth in this manner until the trees reach 5 to 7 feet tall, a height at which you can easily prune the top. Pruning in subsequent years involves cutting off any shoots above the tree’s permanenth eight two to three times per year. Also, thin crowding branches, especially at the top of the tree, and remove unproductive fruiting wood in early spring when branches without flowers are visible.No matter which training method you choose, do some pruning in the spring and summer to train young trees and shorten the time to full fruit production. When necessary, bend and stake shoots of young trees during the spring and summer so they will grow in the desired direction. Bending branches in this manner develops the scaffold structure faster than heading them and waiting for new lateral branches to form. On mature trees, summer pruning mainly involves removing vigorous upright shoots that are not needed as permanent branches and heading or thinning shoots to control tree height and develop branches. If trees receive appropriate summer training and pruning, far less dormant pruning is necessary. However, the absence of leaves in winter provides a clear view of the framework of the tree. At that time, thin or head any branches that were not adequately pruned during the growing season. One notable feature about apricots is that they are susceptible to infection by the branch-killing disease eutypa dieback, round plastic pots which is a particularly serious problem in Northern California around the San Francisco Bay Area. Infection occurs on wounds made in fall or early winter, causing severe gumming at pruning wounds and branch dieback.

Therefore, it is best to prune apricots either in late summer, so that at least 6 weeks of rain-free weather follow the pruning, or late in the dormant season , by which time far fewer viable spores are present. If pruned in summer, be sure to prevent sunburn by leaving enough foliage to cover limbs or by painting exposed limbs white with a 50-50 mixture of interior white latex paint and water.Refrigeration is indisputably the most effective strategy to prolong shelf-life, preserve quality and delay the deterioration of many fruits and vegetables . However, in cold sensitive commodities such as tomato , storage at temperatures between 0-12°C induces the onset of molecular, biochemical, and physiological alterations known as post harvest chilling injury , which are manifested when fruit are rewarmed to room temperature . PCI is a complex and multilayered phenomenon. Its early stages are temperature-dependent and are mediated by physical changes in cellular membranes . Loss of membrane stability triggers the activation of a signal transduction cascade that transmits the cold stimulus downstream through a series of molecular players, e.g., second messengers, eliciting symptoms characteristic of this disorder . These symptoms include modifications in respiration and ethylene production, disruption in the synthesis of aroma volatiles, accumulation of reactive oxygen species , lipid peroxidation, and DNA and protein damage . These molecular and cellular processes ultimately lead to failures in fruit ripening, the development of surface pitting, seed browning and higher susceptibility to post harvest decay . Most of what is known about the cold signal transduction pathway in plants, comes from studies of Arabidopsis thaliana, which is able to cold-acclimate and endure freezing temperatures . The C-Binding Factor gene family of transcription factors are positive regulators of the cold response that interact with the cis-elements of downstream cold-responsive genes . These target genes, also known as the CBFregulon, encode protective proteins and enzymes, and are involved in the synthesis of metabolites that enhance the plant’s fitness during cold stress . In tomato fruit, SlCBF1-3 genes are also induced by cold , but the size and types of genes comprising the CBF regulon are not the same as in Arabidopsis . This might partly explain tomato’s inability to cold acclimate . Different members of the Arabidopsis CBF gene family have been ectopically expressed in tomato plants under the control of the constitutive CaMV35S promoter, resulting in increased stress tolerance, but with concomitant growth reduction and flowering delay . This, due to CBF’s involvement in gibberellin repression, and DELLA protein accumulation . A transgenic phenotype over expressing AtCBF1 in tomato fruit was characterized, and revealed this gene influenced ripening as well as fruit’s response to post harvest cold stress . In this work, we hypothesized that CBF1 over expression in tomato cv. Micro-Tom fruit during post harvest chilling would enhance fruit tolerance to cold stress and reduce the incidence of PCI. We cloned this gene from two sources: cultivated tomato and the wild tomato relative Solanum habrochaites . S. habrochaites has been extensively studied due to its tolerance to cold stress . ShCBF1 has been cloned and expressed into Arabidopsis plants conferring tolerance to freezing and salinity but displaying phenotypic abnormalities . In this study, both ShCBF1 and SlCBF1 genes were driven by the stress-inducible promoter RD29A . Our goal was to specifically induce CBF1 expression in harvested fruit stored in the cold, as well as minimize pleiotropic effects caused by constitutive over expression. To test our hypothesis, transgenic fruit were cold-stored, which elicited ectopic CBF expression, and their post harvest performance was examined and compared to wild-type fruit under the same conditions. To broaden the scope of traditional studies of chilling injury beyond fruit post harvest, we also tested if the photosynthetic responses of CBF1-overexpressing seedlings would be affected by cold stress. The goal was to understand the physiological effects of additional CBF1 transcripts at different phases of the plant life cycle. Finally, we evaluated the influence of post harvest fruit chilling on seeds and seedling traits that were measured under control or cold conditions, to explore the concept of transgenerational adaptive mechanisms transmitted from fruit to progeny.Fruit were cold-stored and transferred to 20°C for three additional days to induce PCI. The presence of surface lesions or pitting and deterioration in the form of decay, were recorded. Wild-type fruit consistently had the lowest CII scores among all genotypes, maintaining levels below 30% after 2 or 3 weeks of cold storage, and rewarming . In contrast, transgenic fruit developed pits after just one week at 2.5°C, especially in Sh-36 and Sl-2. There was a slight decrease in CII between weeks 2 and 3, which was linked to the pitted lesions becoming ‘swollen’ in appearance . The highest incidence of decay was observed in Sh-36, Sl-2, and Sl-12 . A myriad of symptoms was recorded, i.e., severe discoloration, wrinkles around the stem scar, and surface ‘translucency’ , but they were absent or minimized in the WT. Degradation of RNA samples obtained from the transgenic fruit was observed and verified during agarose gel electrophoresis and may be connected to the phenotypic deterioration induced by CBF1 over expression. Based on their contrasting PCI phenotype, further analyses were conducted on Sl-2 and Sh-13 fruit in addition to the WT.

Daily cycles of light and darkness also promoted maintenance of glucosinolate content during post harvest storage of cabbage . Total glucosinolate content in the cabbage leaf disks stored under light/dark cycles remained stable with no significant fluctuation in levels over the 21 days of analysis . In comparison to total glucosinolate levels in light/dark-stored cabbage, the glucosinolate levels were significantly lower by 7 days when cabbage leaf disks were stored under constant darkness and by 21 days when stored under constant light . Total glucosinolate levels declined by 70% and 88%, respectively, in cabbage disks stored at 22°C under constant light or constant darkness. Remarkably, glucosinolate levels of the cabbage leaf disks stored at 4°C also showed a significant decrease by 21 days, with a loss of 50% of the initial glucosinolate levels , indicating that storage under cycle of light/darkness led to enhanced retention of this valuable phytochemical even relative to refrigeration.In this work, hydroponic bucket we examined whether kale, cabbage, lettuce and spinach leaf tissue maintain the ability to respond to light/dark cycles during post harvest storage and whether under these conditions that better mimic the natural light cycles of the environment tissue deterioration would be reduced.

Our goal was to expose plant tissues to diurnal conditions known to maintain the functioning of the circadian clock and thereby capitalize on physiological enhancements conferred by robust circadian rhythms. Plants grown under light/dark cycles that match the endogenous cycling of their internal circadian clock have a growth and reproductive advantage over plants exposed to light/dark cycles that do not match their internal oscillator . Furthermore, phasing of circadian rhythms so as to be synchronized with the external environment promotes biotic stress resistance. We found that storing green leafy vegetables in cycles of 12 hours of light followed by 12 hours of darkness improved several post harvest performance markers compared to post harvest storage of the leaf tissues under constant light or constant darkness. Similarly, a modest reduction in senescence was noted for post-harvest broccoli stored under natural light/dark cycles. Perhaps surprisingly, we found that storage in light/dark cycles resulted in several aspects of post harvest performance being comparable to storage under refrigeration, a commonly practiced method of post harvest storage thought to slow down cellular breakdown. The longevity of kale and lettuce leaf color, chlorophyll levels, and tissue integrity, which are important contributors to the appeal of green leafy vegetables to consumers, were largely indistinguishable whether the kale and lettuce leaf samples were stored at 22°C under light/dark cycles or were stored under refrigeration in constant darkness .

Spinach leaf samples also maintained green coloration and chlorophyll levels under light/dark cycles at 22°C as well as when refrigerated, but refrigeration was more successful at preventing spinach leaf tissue breakdown. Significant improvement of green coloration and chlorophyll content was seen when cabbage leaves were stored under light/dark cycles at 22°C compared to refrigeration, demonstrating that light may not only be important for clock entrainment but also can provide the additional benefit of promoting continued photosynthesis during post harvest storage. Promotion of photosynthesis and/or chlorophyll levels was previously observed in post-harvest crops stored under light. However, constant light during post harvest storage can also cause detrimental physiological activity, such as respiration leading to browning and transpiration contributing to weight loss. Therefore, cycling of light treatment with darkness periods may not only maintain clock function but may also avoid physiological damage that may occur in plant tissues under too much light. In addition to improvement of green leafy vegetable appearance by post harvest storage under light/dark cycles, we found that this post harvest storage treatment of plant crops may improve human health benefits through maintenance of phytochemical content .

Chlorophyll, responsible for the visual appeal of green leafy vegetables, also has beneficial impact upon human health upon ingestion. Chlorophyll can limit efficacy of carcinogens, such aflatoxin B1 and can activate Phase II detoxifying enzymes. Glucosinolates, sulfurcontaining compounds that play a major role in Brassicaceae plant herbivore defense, also underlie the human health benefits attributed to Brassicaceae vegetable consumption. For example, the glucosinolate glucoraphanin has potent anticancer activity. Previous studies have shown that glucosinolate levels can be maintained by refrigeration or exposure to radiation; here we find that post-harvest storage under light/dark cycles can also lead to sustained glucosinolate levels .Fruits are a good source of compounds with phenolic functionality including phenols, lignins, lignans, coumarins, tannins, phenolic acids, and Žavonoids which are important in the human diet. ese phenolic secondary metabolites comprise of an aromatic benzene ring with one or more hydroxyl groups that can exist as a simple monomeric phenolics or a complex polymerized polyphenolic molecules. ese molecules generally play a critical role in plant defense mechanisms including pathogen or insect attack, ultraviolet light, and mechanical damage in plants. ey also play an important role in human health, protecting against damage induced by reactive oxygen or nitrogen species. Phenolics are classified based upon their carbon skeleton and in accordance with the number of phenol units present. Phenolics arise from either phenylalanine or tyrosine amino acids that are deaminated into cinnamic acids and are primarily derived from the phenylpropanoids. Simple phenolic acids are divided into two groups: hydroxybenzoic acids and hydroxycinnamic acids. ese molecules play a vital function to protect the organism against to biotic or abiotic stress factors. Most phenolic compounds are highly reactive, are therefore not stabile, and can degrade into a wide variety of products. In the last decade, many epidemiological studies indicate a direct relationship between consumption of fruits and the prevention of diseases such as cancer and those related to inŽammation and oxidative stress. Monomeric phenolic compounds can polymerize to form more complex phenolic compounds. ese include Žavonoids, tannins, and lignin. Complex phenolics have a range of biological activities and functional characteristics and are of great interest to the food industries, cosmetic industry, and allied health fields.Flavonoids are complex phenolics that can be classified into six subclasses that include flavonols , flavanones , flavones , isoflavones , anthocyanins , and flavanols and are present principally as glycosylated, esterified, and polymerized derivative forms in fruits. Flavonols play an important role as antioxidants; for example, they protect ascorbic acid from autoxidation in juices and which can lead to juice discoloration. Although flavonoids are abundant in fruit, and fruits or beverages can be a significant source of dietary flavonoids, levels will vary depending on the varieties, environmental conditions, soil, and climatic factors. Berries are a good source of quercetin and its derivatives , stackable planters whereas the most abundant dietary flavanone glycoside is hesperetin-7-O-rutinoside present in citrus fruits. Peterson et al. reported that the most prevalent dietary flavanone aglycones are naringenin, hesperetin, isosakuranetin, and eriodictyol. ,e same authors demonstrate that a citrus fruit is also a primary source of narirutin, eriocitrin, didymin, neohesperidin, naringin, hesperidin, neoeriocitrin, and poncirin. ,e ratio of these compounds to each other can vary. For example, narirutin and naringin were detected in grapefruit in high ratios, while the levels of hesperidin and narirutin in oranges and eriocitrin in lemons were even higher. In addition, some flavanone glycosides such as 7-rutinoside are tasteless, in contrast to neohesperidin , naringin, and hesperetin which have an intense bitter taste isolated from bitter oranges and grapefruit. Apigenin is another key flavone found in fruits, vegetables, spices, and herbs and is abundant in grapefruit, beverages, some vegetables, and herbal plants such as chamomile.

Isoflavones are present in plants in the glycosylated forms but are converted to aglycone forms through the action of intestinal microflora. Isoflavones are detected commonly in legumes such as green beans, fava, and soybeans, and among them, genistein -4H-1-benzopyran-4- one and daidzein -4H-1- benzopyran-4-one are the two major forms of dietary isoflavones and are consumed in soy products. Due to the structural similarities to human hormone estrogen, isoflavones have potent estrogenic properties. Anthocyanins are another important class of flanovids that are colorful water-soluble glycosides and acylglycosides of anthocyanidins. 3-O-glycosides or 3,5-di-O-glycosides of malvidin, delphinidin, pelargonidin, cyanidin, petunidin, and peonidin are known as the most common natural anthocyanins and are classified based on the number and position of hydroxyl and methoxy groups. Anthocyanins are responsible for the brilliant colors of various plant parts including flowers and leaves and especially fruits having red, blue, purple colors, particularly strawberries, blueberries, black currants, cherries, raspberries, and red and purple grapes. Anthocyanidins are also responsible for the color of red wines. ,eir color based upon the degree of methylation and with pH is discrete from other phenolics by the range of colors each forms . Color differences of anthocyanins depend on the substitutions of the B ring, the pattern of glycosylation, and the degree and nature of esterification of the sugars with aliphatic or aromatic acids, and also on the pH, temperature, type of solvent, and the presence of copigments. Berries are a good source of anthocyanins, and 100 g of berries can provide up to 500 mg of anthocyanins. Flavan-3-ols are the core structure of condensed tannins and are the most complex subclass of flavonoids. Flavan-3-ols have been previously reported as an antioxidant, chemopreventive, and immunoregulation agents. Procyanidins exist in a wide range of foods and often exist in foods in a range of galloylated forms. Most widely used techniques for phenolics are HPLC , LC/MS, GC , GC/MS, UV-Vis spectrophotometry, mass spectroscopy, electrochemical, and fluorometric methods. Liquid chromatography mass spectrometry is used to determine phenolics in both APCI and ESI techniques, ABTS+ and DPPH.Sample preparation and extraction methods varied widely based on the nature of the sample matrix of the fruit or vegetable and based on the chemical structures of the phenolic compounds being extracted. As most samples contain a mixture of simple and complex polyphenolic compounds, such as phenolic acids, flavonoids, anthocyanins, and proanthocyanins, it is critical to choose a suitable method for sample preparation and extraction. Proteins, carbohydrates, lipids, or other elements may play negative effect to extraction of phenolics. In addition, it is not always possible to extract fresh samples, and special preparation techniques such as lyophilization, nitrogen pulverization, or drying may be needed. Particle size of extracted material and solvent-to-solute ratios need to be considered. As seen in Figure 1, there are many reliable qualitative and quantitative methods available for the measurement and characterization of the phenolic content in different natural products. Moreover, the success of these techniques will depend on the most effective sample preparation and extraction methods. Extraction efficiency is greatly influenced by solvent choice and composition and plays a critical role in the extraction yield of phenolics from fruits and vegetables. Generally, for the extraction of phenolics, water, acetone, ethyl acetate, alcohols , and their various percentages of mixtures are used. In addition to the solvent type extraction conditions, parameters such as temperature and duration also influence the yield of phenolics.Khoddami et al. previously reported that recovery of phenolics varied from one sample to another sample. It is also reported that acid- or base-catalyzed hydrolysis is also an important consideration for the stability of the phenolics in extracts. Davidov-Pardo and Marn-Arroyo reported that the extraction pH plays an important role in the extraction efficiency of phenolic compounds, and the same authors implied that catechins and their isomers are detected more efficiently in alkaline conditions as compared with acidic ones. Extraction of phenolic compounds are commonly done using either liquid-liquid or solid-liquid extraction technique. However, liquid-liquid extraction has some disadvantages because of using costly and potentially toxic solvents. For this reason, improved extraction methods such as solid-phase microextraction and solid-phase extraction techniques are used to extract phenolics from liquid samples. In general, inexpensive and simple methods such as soxhlet, reflux, and maceration processes are the more conventional procedures used to recover phenolics from solid samples. In addition, ultrasound-assisted extraction , microwave-assisted extraction , ultrasound microwave-assisted extraction , supercritical fluid extraction , subcritical water extraction , and high hydrostatic pressure processing are the methods that help us to shorten extraction times and decrease the release of toxic pollutants through reducing organic solvent consumption and are relatively simple to perform.

However, the production of H2O2 and phenolic compounds, and higher PPO activity in the translucent tissue were detected specifically in the VB tissues, not in other living non-VB cells after chilling . These data indicated that IB did not randomly occur in any living parenchyma cell but is specific to the phloem. Therefore, if the phloem is the site of IB initiation in PCI of pineapple fruit, then future studies should focus on how different traits ofthephloem, e.g.,membrane properties, antioxidant system, etc., vary among cultivars differing in IB response.In all IB-occurring cultivars studied, the VBs found at the F/C boundary contained fewer sclerenchyma fiber cells than did the VBs found at the C region, except in the PTV cultivar . The sclerenchyma fibers are composed of lignified cells, which offer protection to the VBs . When these anatomical features of the pineapple are considered, the link between VB number and the appearance of IB at the F/C is clear. This occurrence may be explained by the morphological structure of the pineapple fruit. The fruit is a composite of multiple individual fruitlets, fused to each other and to the C.

At maturity, hydroponic nft system the fruitlets become the ediblefleshly part ofthe fruit composed predominantly of parenchyma cells and some VBs containing lignified cell walls . The C of the fruit consists mainly of VBs with connections to the stem. The C region constitutes a stele of VB, one connected to the other vertically. From the C region,theVBs are distributed to the F/C of the fruit . The VBs in the F/C region form the denser network that grows both vertically and horizontally toward the F regions . These data suggested that the higher number of VBs at the F/C regions might explain why IB symptoms are easily detectable to the naked eye. However, as mentioned previously, the properties of phloem, e.g., their membrane properties and antioxidant system, at the F/C region might play a role in IB development.Consistent with published data,the IB-resistant MD2 showed no IB and TS after being stored at 10 ◦C for three weeks . It is possible to hypothesize that differences invascular structure may play a role in the observed lack of IB in resistant cultivars. Among all four cultivars studied here,the number and size of the xylem elements and phloem per VB did not correlate with varietal IB resistance . It is noteworthy,however,thatthe IB-resistantMD2hadfewerVBs but more sclerenchyma fiber cells surrounding the phloemand xylem, resulting in a larger size of VBs compared with other cultivars. In addition, the number and size of VBs and the number of sclerenchyma cells in all regions of the MD2 fruit were similar . In MD2, there were the multiple layers of the sclerenchyma cells on the lateral sides of VB. In contrast, in PTV and SV, there were no more than two layers of the sclerenchyma cells on the lateral sides ofVB, and only a single one in TST.

These special anatomical features may serve a protective purpose, preventing separation of the VBs themselves and limiting further damage to the tissue from chilling injury . Interestingly, MD2 stored at 10 ◦C for six weeks exhibited chilling injury at the exocarp . These data suggested that there are potentially different physiological and biochemical processes occurring in the fruit of the IB-resistant MD2 including: different physiological and biochemical properties of phloem cell ,fewer metabolically active phloem cells due to the lower number of VBs compared to the susceptible lines , and very low levels of PPO activity or phenolics so that any browning compounds produced were not visibly detectable .A plant-based diet is defined as the consumption of plants such as fruits, vegetables, nuts, seeds, oils, whole grains, legumes, and beans. It may or may not include small amounts of animal products such as meat, fish, sea-food, eggs, and dairy, but these foods tend to be avoided. Plant foods are rich in phytochemicals, antioxidants, vitamins, and minerals which prove beneficial to health on a micronutrition level, especially when plants are consumed in their whole, unprocessed form. When brought to the forefront of a patient’s dietary pattern, these naturally nutritious foods may translate into clinically significant improvements to patients’ quality of life and disease prognosis, and mitigate disease complications.

Historically, these foods have been avoided in patients on dialysis due to concerns of hyperkalemia and protein deficiency. However, the recent recognition of their potential benefits and improved understanding of potential pitfalls have favored the reintroduction of these foods into the diet of patients on dialysis.Traditional dietary interventions in patients with kidney disease encourage the restriction of phosphorus, potassium, sodium, and fluid as well as tailoring protein intake to the patient’s CKD stage and whether they require kidney replacement therapy. These guidelines tend to result in a diet devoid of fruits and vegetables, lacking in variety and resulting in limited satisfaction. Broadening the diet of patients with kidney disease to include these otherwise healthy foods may not only improve satisfaction and increase variety but may also carry additional benefits. A recent multinational cohort study found that higher consumption of fruits and vegetables in patients on dialysis was associated with lower all-cause and non-cardiovascular death. In this study, patients receiving long-term hemodialysis completed a food frequency questionnaire for a median follow-up of 2.7 years . Only 4% of the patients consumed at least 4 servings of fruit and vegetables per day as recommended in the general population. Plant-based diets, a fiber- and polyphenol-rich intervention, may attenuate cardiovascular disease risk markers in patients on hemodialysis. Polyphenols are a diverse group of micronutrients only consumed through plants. A systematic review and meta-analysis of 12 studies found that polyphenol-rich interventions in patients on hemodialysis improved diastolic blood pressure , triglycerides , and myeloperoxidase. Fiber supplementation was found to significantly lower total cholesterol, LDL cholesterol, and inflammatory markers – all of which are known cardiovascular risk factors. Plant fats, which are rich in unsaturated fatty acids, have shown to be beneficial in the diets of patients on hemodialysis by improving inflammatory markers. Vegetable oils are rich in polyunsaturated fatty acids, such as linoleic acid, nft channel which are known to have cardioprotective and anti-inflammatory properties. In one study, linoleic acid levels, as a proportion of plasma fatty acids, were shown to be inversely associated with IL-6 and all-cause mortality in a cross-sectional study of hemodialysis patients.Dietary fiber is a nondigestible, nonabsorbable carbohydrate polymer that may be used to treat chronic kidney disease by exploiting its ability to reduce uremic toxin production and, perhaps, postpone dialysis. Since dietary fiber is exclusively found in plants, there may be a unique advantage associated with the use of plant-based diets . Fermentable fibers are metabolized into short-chain fatty acids, especially acetate, propionate, and butyrate. Short-chain fatty acids may play a role in kidney health by modulating systemic inflammation and maintaining the integrity of the gut mucosal barrier. Today, plant-derived complex carbohydrates are recognized as prebiotics for their role in promoting the growth and metabolic activity of beneficial saccharolytic organisms like Bifidobacteria and Lactobacillus.

The protective effects of gut microbiota have been widely studied in chronic kidney disease, but there is a paucity in data regarding the bidirectional gut-kidney axis in hemodialysis patients. Compared to healthy individuals, a cross-sectional study found end-stage kidney disease patients on hemodialysis to have an increased relative abundance of Proteobacteria , Actinobacteria, and Firmicutes by phylogenic microarrays. Of note, these taxa are not usually associated with uremic toxin production. Indoxyl sulfate and p-cresol are uremic toxins generated by colonic bacteria in response to food that is not absorbed in the small bowel. Uremic toxins accumulatein patients with chronic kidney disease and may contribute to the progressive loss of glomerular filtration rate by increasing the expression of transforming growth factor beta which increases fibrogenesis. A cross-sectional study of patients undergoing hemodiafiltration found that vegetarian patients had 47% lower levels of indoxyl sulfate and 67% lower levels of p-cresyl sulfate than the nonvegetarian patients. In another study of patients on hemodialysis, an increase in fiber intake was shown to significantly reduce plasma levels of indoxyl sulfate by 17% and non-significantly reduce plasma levels of p-cresol by 8% in a randomized control trial . Another cross-sectional study found that increased levels of p-cresol were associated with an increased risk of death in patients on maintenance hemodialysis. Outside of decreasing uremic toxins, fiber supplementation in patients on hemodialysis also improved lipid profiles and oxidative status and decreased systemic inflammation when compared to a placebo in a 6-week randomized control trial. The results of the trial revealed a significant decrease in total cholesterol level, high-density lipoprotein level, and TC:LCL ratio; a significant decrease in total antioxidant capacity and malondialdehyde; and a significant decrease in tumor necrosis factor-α, interleukin-6, interleukin-8, and C-reactive protein in the 10 and 20 g per day fiber supplement groups. A long term prospective cohort study of patients on peritoneal dialysis showed an independent association between fiber intake and all-cause mortality wherein each gram per day increase in fiber intake correlated with a 13% reduction in all-cause mortality. Contrarily, low dietary fiber intake has been shown to negatively impact patients on hemodialysis on multiple occasions. In a prospective cohort study of patients on dialysis , lower fiber intake was associated with an increased risk for major adverse cardiovascular events when compared to patients with higher fiber intake. Another study on maintenance hemodialysis patients found that dietary fiber level was independently correlated with advanced glycation end products and C-reactive protein levels.In hemodialysis patients, the incidence of constipation varied according to the definition of constipation, but it was noted to be 71.7% in a 2013 cross-sectional study using the Roma III criteria. As a chronic or frequently recurrent symptom, constipation impairs patients’ health-related quality of life and may result in a substantial emotional and social burden. The issue may be easily treated by emphasizing the consumption of high-fiber foods, like plants. It may be possible that the historically low consumption of fiber-rich foods of patients on hemodialysis may be to blame for the high rate of constipation in this population. In the general population, patients with constipation had 12% higher all-cause mortality, 11% higher incidence of coronary heart disease, and 19% higher incidence of ischemic stroke compared to patients without constipation. US Veterans using one or >2 types of laxatives experienced a similarly higher risk of all-cause mortality and cardiovascular and cerebrovascular burden when compared to US veterans not taking any laxatives. A recent multi-center study evaluated the prevalence and correlates of constipation in hemodialysis patients from 4 dialysis centers using a questionnaire based on the Roma IV criteria to assess constipation status and lifestyle habits along with a food frequency questionnaire focused on the main sources of dietary fiber and found that >30% of patients had constipation at a given point in time. In this study, the independent predictors of constipation symptoms included diabetes and lower frequency of fruit intake. Moreover, constipation can worsen hyperkalemia, while having soft bowel movement may be associated with a lower likelihood of hyperkalemia.Although phosphorus is an essential nutrient, it is difficult for patients to avoid states of phosphate excess in advanced CKD. Consequently, patients must be placed on phosphorus-restricted diets to reduce the risk of hyperphosphatemia, which has been associated with an increased risk of death and cardiovascular disease. Given the varying bio-availability of phosphorus from foods, dietary changes may be made to take advantage of low phosphate-containing foods. In the gut, 40–60% of phosphate in animal protein is absorbed by the intestinal tract compared to only 10–30% of phosphorus in plant protein. This is partly because the bulk of phosphorus in plants is bound to phytate, which is difficult for humans to digest due to lack of phytase to release the phosphorus. In a cross-over trial in patients with CKD, the effects of vegetarian and meat diets on phosphorus balance showed that 1 week of a vegetarian diet led to significantly lower serum phosphorus and significantly lower fibroblast growth factor-23 levels than being on a meat diet . This finding has also been seen in a small observational study by Wu et al. wherein serum phosphate levels were significantly lower in vegetarian patients than in non-vegetarian patients on hemodialysis.When diagnosed with end-stage kidney disease, traditional dietary interventions may place patients at risk for vitamin deficiencies.

There are striking examples in tomato and other plants where identified genes regulate the development of fruit shape. For example, the OVATE gene in tomato regulates the phenotypic transition from round to pear-shaped fruit . If large-effect mutations underlie differences in strawberry fruit shape, the ordinal classification system proposed here should enable the discovery of such effects. Furthermore, quantitative phenotypes were linked to genetic features that interact with large-effect genes, i.e., suppressors of OVATE , through bulk segregant analysis and quantitative trait locus mapping . In woodland strawberry , fruit size and shape are linked to the accumulation and complex interaction of auxin, gibberellic acid, and abscisic acid, mediated by the expression and activity of FveCYP707 and FveNCED, as well as other genes. Because of the high H2 estimates for several of the newly created phenotypic variables , we hypothesize that quantitative, nft hydroponic latent space phenotypes can yield a more comprehensive understanding of the underlying genetic mechanisms of fruit shape in garden strawberry through GWAS and other quantitative genetic analyses. We anticipate that the analyses in this study will enable us to discover and study the genetic determinants of fruit shape in strawberry and other specialty crops.

Over the past few decades, models of peach fruit growth and plant development have identified useful principles for assisting growers in making horticultural management decisions. For example, harvest-date prediction models are now available to aid in managing fruit crops . The unusually early harvest of California’s peach crop in 2004 — which had record high temperatures during bloom time — and attendant difficulties in attaining the fruit sizes desired by the market have increased interest in using physiological concepts to understand the effects of early-spring temperature on peach fruit growth and in anticipating fruit size at harvest. The dependence of peach fruit development on spring temperatures during the first 30 days after bloom has been established; there is a strong correlation between the sum of growing degree hours accumulated in the 30 days after bloom and the number of days between bloom and harvest for several stone-fruit cultivars . Traditionally, the California cling stone peach industry has used reference-date fruit size to predict what the fruit size potential will be for a given year, and then peach growers conduct fruit-thinning accordingly. Reference-date fruit sizes are known to vary from year to year, but the reasons for this variation were previously unclear.

Lopez and DeJong compared a 20-year cling stone-peach data set collected and archived by the California Canning Peach Association, which included full bloom date , reference date and fruit size at reference date from different locations in California. When the data — collected from orchards near the Central Valley cities of Kingsburg, Modesto and Yuba City — was correlated with seasonal weather data available through the California Irrigation Management Information System , a strong correlation was found between accumulated GDH30 and the number of days between full bloom date and reference date . The number of days between full bloom date and reference date decreased as accumulated GDH30 increased . There was also a strong correlation between the number of days from full bloom date to reference date, and fruit size at reference date. Fruit size at reference date increased with an increase in the number of days from full bloom date to reference date . This study indicated that peach trees apparently could not supply resources rapidly enough to support the potential maximum fruit growth rates when accumulated GDH30 was higher than a threshold value . Consequently, fruit size at reference date in years with very warm spring temperatures was less than in years when accumulated GDH30 was below that threshold value. Furthermore, previous research has documented that fruit growth potential unfulfilled in early spring cannot be compensated for later in the season .

Although the effects of early-spring temperature on fruit size have been quantified for cling stone peaches at reference date, and fruit size at reference date is thought to be a good indicator of fruit size at harvest, industry wide data on fruit size at harvest is not available from the canning cling stone peach industry. However, the industry wide data situation is the opposite for California fresh market peaches. This industry does not keep representative seasonal data on full bloom date or reference date, but general fruit-size data is available from industry records maintained by the California Tree Fruit Agreement . At the outset of this study, we anticipated that the environmental factors influencing fruit growth and development rates for canning cling stone peaches would be the same as for fresh-market freestone peaches. Thus we initiated a follow-up study using relationships we had established for cling stone peaches to analyze the seasonal environmental effects on industry wide data for the fruit size of freestone peach cultivars. When fresh-market peaches are packed for shipping, the fruit are separated into different size categories related to the number of fruit that will fit into a standard-size lug box. Fruit packed in a size 30 lug are larger than those in a size 40 lug, while the fruit in a size 40 lug are larger than those in a size 50 lug, and so on. The CTFA annually reports the percentage of the total number of lugs for specific fruit-size categories that are shipped for each major cultivar at harvest. Data from three different fresh market peach cultivars were used for this study and compared with full bloom and fruit reference-date data for cling stone canning peach cultivars . However, the average fruit size lug category of the fresh-market cultivars was not correlated with the number of days between full bloom date and reference date for the cling stone cultivars in a given year in the Kingsburg area . When we analyzed historical trends in fruit size, there was a clear trend toward lower average lug categories; over the 20 years of this study, the average size of the fruit packed for each cultivar increased significantly . Although improvements in cultural practices may account for some of the increases in packed fruit size, this long-term trend presumably can be attributed to marketing pressures. Consumer acceptance of California peaches has been related to soluble solid concentration, acidity or soluble solid concentration/acidity ratio, but the major quality factor is fruit appearance . Likewise, market pricing has consistently favored larger-sized fruit. The percentage distribution trends of average fruit-size categories over the 20 years were similar for the three cultivars, and there were no significant differences in the slope of the regressions between Elegant Lady and O’Henry, or between Elegant Lady and Flavorcrest. However, the slope of the response for Flavorcrest was steeper than for O’Henry .When the effect of the year on the percentages of four fruit-size lug categories at harvest was analyzed independently, different patterns were observed among the cultivars and categories . Although all the cultivars increased the percentages of larger fruit-size lug categories and decreased the percentages of the smaller fruit size lug categories , nft system the slopes of the 30s and 80s categories of Flavorcrest relationships were clearly different from those of Elegant Lady and O’Henry . The change in Flavorcrest peaches observed in figure 4 could be primarily explained by a drastic reduction in the percentage of fruit packed in the smallest fruit-size lug category . However, the change observed in Elegant Lady and O’Henry was mostly related to an increase in the percentage of fruit packed in the largest fruit-size lug category . Although industry wide data on fruit packed per acre is not available for these cultivars, one practical implication of these results is that the average yield of packed fruit has likely declined over the same 20-year period, since, on average, fruit size is generally correlated with crop load . For example, a California thinning study showed a substantial effect on yield for both O’Henry and Elegant Lady .

For O’Henry, the change in lug size from 1985 to 2004 required average fruit weights to increase from 0.48pound to 0.56 pound . To obtain these fruit weights, a typical tree would need to be thinned to 976 and 523 fruit in 1985 and 2004, respectively. Thus, yields would have dropped from 464 pounds per tree to 291 pounds per tree , a 37% decrease over the 20 years. The results for Elegant Lady were similar. However, the profitability of early cultivars such as Flavorcrest was probably affected more by market pressures than Elegant Lady and O’Henry, since decreases in the amount of fruit in small size categories were not offset as much by increases in fruit packed in the large-size categories . When the data in figure 4 was used to normalize the fruit-size lug data to account for the long-term general trend, the deviation of a given year’s average fruit-size lug category from the long term trend was clearly related to spring weather patterns. The average, long term, trend-adjusted fruit-size lug category for the fresh-market cultivars in a given year decreased, with an increase in the number of days of fruit growth between full bloom and reference date recorded in the same year for cling stone peaches . Although the variability in this relationship among the different cultivars could have been related to the inherent variability in sources of fruit that were packed, the similarity of the slopes of the relationships for the three cultivars indicates that the conditions driving the relationships were likely similar for all three cultivars .Previous research with cling stone peaches, combined with this analysis of fresh-market peach data, indicate that early fruit development rates are clearly related to heat accumulation, and that high early-spring temperatures tend to decrease the average size of fruit packed in a given year. This is apparently because in especially warm springs, the tree cannot supply resources rapidly enough to support the potential fruit growth rates associated with high rates of phenological development. The relationships between fruit developmental patterns, fruit growth potentials and spring temperatures are even more important in light of the clear long-term marketing trends toward packing larger-sized fruit. Grower success will depend upon the ability to anticipate yearly fruit-sizing potential for individual cultivars and make the appropriate, cost-effective adjustments in cultural practices. It is well documented that early and heavy fruit thinning can increase average fruit size, but may cost more and/or reduce overall yields . However, these practices may be particularly useful in difficult fruit sizing years in light of the increasing market pressure for large-size fruits.California’s coastal districts, where 86% of the nation’s strawberries are produced on 38,600 acres, are the most productive strawberry-growing areas in the United States . To achieve this level of productivity, California strawberry producers need effective soil disinfestation, productive varieties and cultural practices such as polyethylene mulch and drip irrigation . Strawberries are very sensitive to soil pathogens, and growers with these highly productive systems have become dependent on preplant fumigation. Traditionally, they used methyl bromide plus chloropicrin as the basis for soil pest control. Fumigation with these chemicals controls soilborne pathogens such as Verticillium dahliae, Phytophthora species, Pythium species, Rhizoctonia species, Fusarium oxysporum and Cylindrocarpon species, as well as nematodes, soilborne insects and weed seeds in the soil seedbank . In 1992 methyl bromide was classified as a Class I stratospheric ozone-depleting chemical. Since 2005, under the Montreal Protocol, the use of methyl bromide for fumigation in the United States has been permitted only through critical use exemption . The methyl bromide phase-out and other regulatory limitations make research on alternative pest control measures essential. Currently, some California strawberry fields can still be treated with methyl bromide under the critical use exemption, which is subject to annual review by the parties of the Montreal Protocol.However, methyl bromide costs have been increasing, and its use in strawberry production has been decreasing . In traditional fumigation of California strawberry fields, beginning in the 1960s and continuing until recently, growers applied methyl bromide plus chloropicrin to the total field area. This process was called flat fumigation; the entire field was covered with polyethylene film to hold the fumigant at the concentration needed to kill soil pests . In the last decade, a sizable portion of the strawberry acreage has been treated with fumigants applied to the strawberry bed by drip fumigation .

New LED exit signs are inexpensive, with prices typically starting at around $20. The U.S. EPA’s ENERGY STAR program website provides a list of suppliers of LED exit signs. Tritium exit signs are an alternative to LED exit signs. The advertised lifetime of these signs is around 10 years and prices typically start at around $150 per sign. Electronic ballasts. A ballast regulates the amount of electricity required to start a lighting fixture and maintain a steady output of light. Electronic ballasts can require 12% to 30% less power than their magnetic predecessors . New electronic ballasts have smooth and silent dimming capabilities, in addition to longer lives , faster run-up times, and cooler operation than magnetic ballasts . New electronic ballasts also have automatic switch-off capabilities for faulty or end-of-life lamps. Replacement of T-12 tubes with T-8 tubes. In many industrial facilities, it is common to find T-12 lighting tubes in use. T-12 lighting tubes are 12/8 inches in diameter . T-12 tubes consume significant amounts of electricity, hydroponic channel and also have extremely poor efficacy, lamp life, lumen depreciation, and color rendering index.

Because of this, the maintenance and energy costs of T-12 tubes are high. T-8 lighting tubes have around twice the efficacy of T-12 tubes, and can last up to 60% longer, which leads to savings in maintenance costs. Typical energy savings from the replacement of a T-12 lamp by a T-8 lamp are around 30% . Replacement of mercury lights. Where color rendition is critical, metal halide lamps can replace mercury or fluorescent lamps with energy savings of up to 50%. At a Basic American Foods facility in Shelley, Idaho, the production area lighting system was upgraded using metal halide lamps. According to the company, the improved color rendition and increased light levels offered by the metal halide lamps helped production workers better detect deffects in the plant’s potato products. Plant sanitation was also improved, because staff could better see debris on the equipment and floors . Where color rendition is not critical, high-pressure sodium lamps offer energy savings of 50% to 60% compared to mercury lamps . High-intensity discharge voltage reduction. Reducing lighting system voltage can also save energy. A Toyota production facility installed reduced-voltage HID lights and realized a 30% reduction in lighting energy consumption .

Commercial products are available that attach to a central panel switch and constrict the flow of electricity to lighting fixtures, thereby reducing voltage and saving energy, with an imperceptible loss of light. Voltage controllers work with both HID and fluorescent lighting systems and are available from multiple vendors. High-intensity fluorescent lights. Traditional HID lighting can be replaced with high intensity fluorescent lighting systems, which incorporate high-efficiency fluorescent lamps, electronic ballasts, and high-efficacy fixtures that maximize output to work areas. These systems have lower energy consumption, lower lumen depreciation over the lifetime of the lamp, better dimming options, faster startup and re-strike capabilities, better color rendition, higher pupil lumens ratings, and less glare than traditional HID systems . Daylighting. Daylighting involves the efficient use of natural light in order to minimize the need for artificial lighting in buildings. Increasing levels of daylight within rooms can reduce electrical lighting loads by up to 70% . Unlike conventional skylights, an efficient daylighting system may provide evenly dispersed light without creating heat gains, which can reduce the need for cooling compared to skylights.

Daylighting differs from other energy efficiency measures because its features are integral to the architecture of a building; therefore, it is applied primarily to new buildings and incorporated at the design stage. However, existing buildings can sometimes be cost effectively refitted with daylighting systems. Daylighting can be combined with lighting controls to maximize its benefits. Because of its variability, daylighting is almost always combined with artificial lighting to provide the necessary illumination on cloudy days or after dark . Daylighting technologies include properly placed and shaded windows, atria, clerestories, light shelves,and light ducts. Clerestories, light shelves, and light ducts can accommodate various angles of the sun and redirect daylight using walls or reflectors. More information on daylighting can be found at the website of the Daylighting Collaborative led by the Energy Center of Wisconsin .The use of on-site electricity generation appears to be quite limited in the U.S. fruit and vegetable processing industry. In 2002, only 5% of the industry’s electricity was generated at individual facilities . The use of on-site generation was confined almost exclusively to the fruit and vegetable canning sub-sector, where the extensive use of steam in blanching, evaporating, pasteurizing, and sterilizing applications makes combined heat and power systems particularly attractive. Self generation can be an attractive option for many facilities for reducing the energy intensity of utilities services. This chapter provides a brief overview of several self-generation measures applicable to the U.S. fruit and vegetable processing industry. Combined heat and power . For industries like fruit and vegetable processing that have simultaneous requirements for process heat, steam, and electricity, the use of CHP systems may be able to save energy and reduce pollution. Combined heat and power plants are significantly more efficient than standard power plants because they take advantage of waste heat. In addition, electricity transmission losses are minimized when CHP systems are located at or near the facility. Often, utility companies will work with individual companies to develop CHP systems for their facilities. In many cases, the utility company will own and operate the facility’s CHP system, allowing fruit and vegetable processors to avoid the capital expenditures associated with CHP projects while reaping the benefits of a more energy-efficient source of heat and electricity. In addition to energy savings, CHP systems also have comparable or better availability of service than utility generation. In the automobile industry, for example, typical CHP units are reported to function successfully for 95% to 98% of planned operating hours . Many large-scale CHP systems use steam turbines. Switching to natural gas-based systems is likely to improve the power output and efficiency of the CHP system, due to increased power production capability. Although the overall system efficiency of a steam turbine-based CHP system is higher than that of a gas turbine-based CHP system , the electrical efficiency of a gas turbine-based CHP system is superior . Furthermore, modern gas-based CHP systems have low maintenance costs and will reduce emissions of NOx, SO2, CO2, and particulate matter from power generation considerably, especially when replacing a coal-fired boiler . In general, the energy savings of replacing a traditional system with a standard gas turbine-based CHP unit is estimated at 20%-30% . However, hydroponic dutch buckets savings may be greater when replacing older or less maintained boilers.Additional advantages are that the amounts of power and thermal energy produced by the turbine can be adjusted to meet current power and thermal energy loads. If steam loads are reduced, the steam can then be used for power generation, increasing output and efficiency . Drawbacks include the additional complexity of the turbine’s design. The economics of a CHP system depend strongly on the local situation, including power demand, heat demand, power purchasing and selling prices, natural gas prices, as well as interconnection standards and charges, and utility charges for backup power. In some states, programs may offer support for installation of CHP systems .

Tri-generation. Many new CHP systems offer the option of tri-generation, which provides cooling in addition to electricity and heat. Cooling can be provided using either absorption or adsorption technologies, which both operate using recovered heat from the co-generation process. Absorption cooling systems take advantage of the fact that ammonia is extremely soluble in cold water and much less so in hot water. Thus, if a water-ammonia solution is heated, it expels its ammonia. In the first stage of the absorption process, a water-ammonia solution is exposed to waste heat from the co-generation process, whereby ammonia gas is expelled. After dissipating the heat, the ammonia gas—still under high pressure—liquefies. The liquid ammonia flows into a section of the absorption unit where it comes into contact with hydrogen gas. The hydrogen gas absorbs the ammonia gas with a cooling effect. The hydrogen-ammonia mixture then meets a surface of cold water, which absorbs the ammonia again, closing the cycle. One food company that has successfully implemented absorption technology is the Ghirardelli Chocolate Company, a California based manufacturer of chocolate products. Ghirardelli’s manufacturing facility in San Leandro, California, uses an on-site electricity generating system, which is powered by four 350 kW natural gas-fired reciprocating engines. In 2003, the company installed a single-stage 145 ton absorption chiller that runs entirely on heat from the engines’ exhaust and jacket water. According to the company, the combined area of the buildings being cooled by the absorption chiller is approximately 35,000 square feet . In contrast to absorption cooling, adsorption cooling utilizes the capacity of certain substances to adsorb water on their surface, from where it can be separated again with the application of heat. Adsorption units use hot water from the co-generation unit. These systems do not use ammonia or corrosive salts, but use silica gel . Adsorption units were originally developed in Japan and are now also marketed in the United States. The thermal performance of absorption and adsorption systems is similar, with a coefficient of performance between 0.68 and 0.75. The capital costs of both systems are also comparable. However, the reliability of an adsorption unit is expected to be superior and its maintenance costs are expected to be lower . Back pressure turbines. At many facilities, steam is produced at a higher pressure than is demanded by process requirements. Often, steam pressure is reduced for process use by passing steam through pressure reducing valves, essentially wasting thermal energy. A back pressure steam turbine can perform the needed pressure reduction while converting this otherwise wasted thermal energy to electricity for use throughout the facility. According to the U.S. DOE, back pressure turbines can be considered wherever a pressure reducing valve has constant steam flow of at least 3,000 pounds per hour and when the steam pressure drop is at least 100 psi . Morning Star Packing Company, a manufacturer of tomato paste and other canned tomato products located in Williams, California, uses back pressure turbines to generate 100% of facility electricity needs . In the mid- to late- 1990s, the company installed three 1 MW back pressure turbines at a cost of around $847,000, including capital costs and installation expenses. Reported electricity cost savings have totaled nearly $500,000 per year. The company projected that over the 20-year lifetime of the back pressure turbines, they expect to save almost $9 million in total energy bills and realize a compound annual rate of return of more than 60% .Photovoltaic panels. Photovoltaic panels convert sunlight directly into electricity and can provide a reliable and renewable source of electricity to facilities with ample sunlight. Photovoltaic panels, which are typically mounted on the roof of a facility, convert electricity to DC current, which is subsequently sent through an inverter and transformer and converted into AC power. The AC power can be fed directly into a facility’s power supply. While the capital and installation costs of photovoltaic systems are currently somewhat high , manufacturers can often receive substantial rebates and tax credits from state and federal agencies that can help make photovoltaic investments more economically attractive. Inverters typically last 10 to 20 years, while photovoltaic panels can typically generate power for 25 to 40 years . Kettle Foods, a producer of all natural snacks based in Salem, Oregon, installed a 114 kW photovoltaic power system on the roof of its processing plant and headquarters in 2003. Reportedly, the system saves the company $8,400 in energy costs each year, while also avoiding around 2,500 tons of CO2 emissions. The initial capital and installation costs totaled $675,000, but the company received over $400,000 in clean energy incentives, Oregon energy tax credits, and U.S. federal energy tax credits, which helped to make the project more economically viable . Over the 40-year life of the system, the company estimated a 7% average rate of return and a net present value of $55,000. However, the project has also helped reinforce Kettle Foods’ image as an environmental steward and has reportedly led to good corporate publicity.

In the absence of a steam trap maintenance program, it is common to find up to 15% to 20% of steam traps malfunctioning in a steam distribution system . Energy savings for a regular system of steam trap checks and follow-up maintenance is conservatively estimated at 10% . One industrial case study indicates a payback period of less than four months . Although this measure offers a quick payback period, it is often not implemented because maintenance and energy costs are generally separately budgeted. In addition to energy and cost savings, proper functioning of steam traps will reduce the risk of corrosion in the steam distribution system. At a Land O’Lakes dairy facility in Tulare, California, a U.S. DOE sponsored energy assessment estimated that implementing a steam trap maintenance program would save nearly 20,000 MBtu of natural gas per year and lead to annual energy savings of around $278,000 .Steam trap monitoring. Attaching automated monitors to steam traps in conjunction with a maintenance program can save even more energy without significant added cost.

This measure is an improvement over steam trap maintenance alone, large round plant pots because it gives quicker notice of steam trap failure and can detect when a steam trap is not performing at peak efficiency. Employing steam trap monitoring has been estimated to provide an additional 5% in energy savings compared to steam trap maintenance alone, at a payback period of around one year . Systems that are able to implement steam trap maintenance are also likely to be able to implement automatic monitoring. Leak repair. As with steam traps, steam distribution piping networks often have leaks that can go undetected without a program of regular inspection and maintenance. The U.S. DOE estimates that repairing leaks in an industrial steam distribution system will lead to energy savings of around 5% to 10% . At a Land O’Lakes dairy facility in Tulare, California, the U.S. DOE estimated that natural gas savings of $18,000 per year could be realized by implementing a steam leak maintenance program . Additionally, regular inspection and leak repair can reduce the likelihood of major system leaks, which can be very costly to repair. Flash steam recovery. When a steam trap purges condensate from a pressurized steam distribution system to ambient pressure, flash steam is produced.

As with flash steam produced by boiler blow down, steam trap flash steam can be recovered and used for low grade facility applications, such as space heating or feed water preheating . The potential for this measure is site dependent, as its cost effectiveness depends on whether or not areas where low-grade heat is useful are located close to steam traps. Where feasible, this measure can be easy to implement and can save considerable energy. For example, an analysis of a U.S. based food processing facility predicted that the installation of a flash steam recovery system used for feed water preheating would save the plant around $29,000 in fuel costs annually at a payback period of less than 1.8 years . Based on the reduction in boiler fuel use, it was further estimated that the plant’s carbon emissions would be reduced by 173 tons per year.Process integration. Process integration refers to the exploitation of potential synergies that might exist in systems that consist of multiple components working simultaneously. In facilities that have multiple heating and cooling demands, like those in the fruit and vegetable processing industry, the use of process integration techniques may significantly improve facility energy efficiency by linking hot and cold process streams in a thermodynamically optimal manner. For example, the heat rejected in a facility’s cooling process can be recovered and used in process heating applications . Developed in the early1970s, process integration is now an established methodology for improving the energy efficiency of continuous industrial processes . At Elite Salads and Snacks, a Dutch producer of pre-cooked foods for the catering industry, continuous demand for both heating and cooling provided an attractive opportunity to integrate both functions into one common system.

The company used rejected heat from its cooling system in combination with recovered heat from its flue gas condenser to pre-heat process water. The rejected heat from the cooling system was also raised to a higher temperature via the addition of a heat pump. The process integration initiative led to natural gas savings of approximately 120,000 cubic meters per year with a payback period of around 2.5 years . McCain Foods, a major producer of frozen French fried potatoes, installed an integrated heat recovery system in its Scarborough, England, facility in 1995. Heat was recovered from fatladen fryer exhaust gases via a vapor condenser and from boiler flue gases via economizers. The recovered heat was used to pre-heat air for potato chip dryers, to provide hot water for potato blanching, and to provide hot water for miscellaneous processes around the facility. The project led to annual energy savings of £176,000 and a simple payback period of 3.6 years . Pinch analysis. Pinch analysis takes a systematic approach to identifying and correcting the performance limiting constraint in any manufacturing process system. It was developed originally in response to the “energy crisis” and the need to reduce steam and fuel consumption in oil refineries and chemical plants by optimizing the design of heat exchanger networks. Since then, the pinch analysis approach has been extended to resource conservation in general, whether the resource is capital, time, labor, electrical power, water, or a specific chemical species such as hydrogen. The critical innovation in applying pinch analysis was the development of “composite curves” for heating and cooling, which represent the overall thermal energy demand and availability profiles for the process as a whole. When these two curves are drawn on a temperature-enthalpy graph, they reveal the location of the process pinch , and the minimum thermodynamic heating and cooling requirements. These are called the energy targets. The pinch analysis methodology involves first identifying the targets and then following a systematic procedure for designing heat exchanger networks to achieve these targets. The optimum approach temperature at the pinch is determined by balancing capital and energy tradeoffs to achieve the desired payback. The procedure applies equally well to new designs and retrofits of existing plants. Energy savings potential using pinch analysis far exceeds that from well-known conventional techniques such as heat recovery from boiler flue gas, insulation, and steam trap management. At the Nestle Svenska food processing facility in Bjuv, Sweden, a pinch analysis study was performed in 1993 to optimize facility-level energy consumption. The pinch analysis identified improvements to the facility’s steam system—specifically, plant pots round heat recovery opportunities in the facilities soup, baby foods, and vegetable departments—that would reduce the facility’s annual energy consumption by 10% with an expected payback period of around three years . The expected annual savings in energy costs were estimated at around 300,000 Swedish Kronor .Motors are used throughout a typical fruit and vegetable processing facility to drive process equipment , conveyors, ventilation fans, compressors, and pumps. According to the U.S. DOE, the typical industrial plant in the United States can reduce its electricity use by around 5% to 15% by improving the efficiency of its motor-driven systems . Pumps are particularly important pieces of motor-driven equipment in many fruit and vegetable processing plants. Pumps are used extensively to pressurize and transport water in cleaning, water fluming, and wastewater handling operations, for transporting liquid food streams between processes, and for circulating liquid foods streams within the processes themselves . Studies have shown that as much as 20% of the energy consumed by pumping systems could be saved through changes to pumping equipment and/or pump control systems . This chapter presents some of the most significant energy efficiency measures available for motors and pumps in industrial applications.When considering energy efficiency improvements to a facility’s motor systems, it is important to take a “systems approach.” A systems approach strives to optimize the energy efficiency of entire motor systems , not just the energy efficiency of motors as individual components.

A systems approach analyzes both the energy supply and energy demand sides of motor systems as well as how these sides interact to optimize total system performance, which includes not only energy use but also system uptime and productivity. A systems approach typically involves the following steps. First, all applications of motors in a facility should be located and identified. Second, the conditions and specifications of each motor should be documented to provide a current systems inventory. Third, the needs and the actual use of the motor systems should be assessed to determine whether or not motors are properly sized and also how well each motor meets the needs of its driven equipment. Fourth, information on potential repairs and upgrades to the motor systems should be collected, including the economic costs and benefits of implementing repairs and upgrades to enable the energy efficiency improvement decision-making process. Finally, if upgrades are pursued, the performance of the upgraded motor systems should be monitored to determine the actual costs savings .Strategic motor selection. Several factors are important when selecting a motor, including motor speed, horsepower, enclosure type, temperature rating, efficiency level, and quality of power supply. When selecting and purchasing a motor, it is also critical to consider the life cycle costs of that motor rather than just its initial purchase and installation costs. Up to 95% of a motor’s costs can be attributed to the energy it consumes over its lifetime, while only around 5% of a motor’s costs are typically attributed to its purchase, installation, and maintenance . Life cycle costing is an accounting framework that allows one to calculate the total costs of ownership for different investment options, which leads to a more sound evaluation of competing options in motor purchasing and repair or replacement decisions. A specific LCC guide has been developed for pump systems , which also provides an introduction to LCC for motor systems. The selection of energy-efficient motors can be an important strategy for reducing motor system life-cycle costs. Energy-efficient motors reduce energy losses through improved design, better materials, tighter tolerances, and improved manufacturing techniques. With proper installation, energy-efficient motors can also run cooler and have higher service factors, longer bearing life, longer insulation life, and less vibration.The choice of installing a premium efficiency motor strongly depends on motor operating conditions and the life cycle costs associated with the investment. In general, premium efficiency motors are most economically attractive when replacing motors with annual operation exceeding 2,000 hours/year. However, software tools such as MotorMaster+ can help identify attractive applications of premium efficiency motors based on the specific conditions at a given plant. Sometimes, even replacing an operating motor with a premium efficiency model may have a low payback period. According to data from the Copper Development Association, the upgrade to high-efficiency motors, as compared to motors that achieve the minimum efficiency as specified by EPACT, can have paybacks of less than 15 months for 50 hp motors . Payback times will vary based on size, load factor, running time, local energy costs, and available rebates and/or incentives . Given the quick payback time, it usually makes sense to by the most efficient motor available .NEMA and other organizations have created the Motor Decisions MatterSM campaign to help industrial and commercial customers evaluate their motor repair and replacement options, promote cost-effective applications of NEMA Premium® motors and “best practice” repair, and support the development of motor management plans before motors fail. At the Odwalla Juice Company’s facility in Dinuva, California, an IAC energy assessment found that the installation of more energy efficient motors would lead to $6,300 in annual cost savings with a simple payback period of only eight months . Similarly, in energy audits of seven fresh fruit and vegetable processing facilities in California, the installation of premium efficiency motors as motors wear out was expected to yield simple payback periods ranging from 0.7 to 1.6 years . Stahlbush Island Farms, a grower, canner, and freezer of fruits and vegetables in Corvalis, Oregon, also replaced targeted motors with higher efficiency models as motors wore out. The expected average payback period was estimated at 2.7 years . When all targeted motors are replaced over a 12-year period, the company expects to save 50,000 kWh of electricity per year and to cut their electricity bill by around $2,300 per year. In some cases, it may be cost-effective to rewind an existing energy efficient motor, instead of purchasing a new motor.

Decide if you need to apply by noting the amount of insect and disease damage during the previous growing season. Treat at the onset of dormancy in late November until delayed dormancy, just before buds begin to open in February or early March. The exact timing during the dormant period can vary depending on the disease being controlled. Once flower buds begin opening, you risk damaging the fruit and may kill pollinating bees with certain insecticidal sprays. Spraying after pruning allows maximum coverage as there are no leaves to block the spray. Pump sprayers or compressed air sprayers can be used. Avoid making applications on water-stressed trees to avoid injury. A good time to spray is right after a period of rain or foggy weather. Do not spray during fog, rain, or during or prior to freezing weather . To avoid damage to deciduous trees, oils should not be applied within 30 days before or after applications of sulfur or certain other fungicides. Oils are generally not recommended for use during the dormant season on walnut trees. Always read the label carefully for restrictions and follow product directions.

Fixed copper fungicides contain some form of elemental copper, such as tribasic copper sulfate, large round plant pots copper oxychloride sulfate, or cupric hydroxide. For some diseases it may be necessary to make several applications to protect newly emerging shoots and flowers, especially during rainy weather. Do not apply copper compounds after bloom because it will cause russetting of the fruit. Lime sulfur is less widely recommended as a dormant spray for general use; however, it is a useful tool for apple or pear scab problems when applied just as buds swell. Lime sulfur and other sulfur-containing compounds should not be applied within 3 weeks of an oil application or tree damage may result. Avoid using lime sulfur on apricot trees, since they are particularly sensitive to sulfur. Bordeaux is a mixture of copper sulfate, hydrated lime, and water. Bordeaux has been an outstanding fungicide and bactericide that has been used for decades mainly because it is very persistent on the trees and able to withstand winter rains. However, it requires careful preparation, takes longer to mix, stains surfaces blue, and is incompatible with other pesticides. Currently there are no premixed Bordeaux products registered in California. Additionally, no lime products are registered for mixing with copper to make a Bordeaux mixture. Dormant treatments may not always be required. For some insect pests and diseases, one dormant application may be adequate with good spray coverage.

For other problems, and depending on pest pressure, up to three applications may be necessary for good control. Decide if, and how many applications, you need to apply by noting the amount of insect and disease pressure during the previous growing season.As U.S. manufacturers face an increasingly competitive environment, they seek out opportunities to reduce production costs without negatively affecting the yield or the quality of their finished products. The volatility of energy prices in today’s marketplace can also negatively affect predictable earnings. The challenge of maintaining high product quality while simultaneously reducing production costs can often be met through investments in energy efficiency, which can include the purchase of energy-efficient technologies and the implementation of plant-wide energy efficiency practices. Energy-efficient technologies can often offer additional benefits, such as quality improvement, increased production, and increased process efficiency, all of which can lead to productivity gains. Energy efficiency is also an important component of a company’s overall environmental strategy, because energy efficiency improvements can often lead to reductions in emissions of both greenhouse gases and other important air pollutants. Investments in energy efficiency are therefore a sound business strategy in today’s manufacturing environment. ENERGY STAR® is a voluntary program operated by the U.S. Environmental Protection Agency . The primary purpose of the ENERGY STAR program is to help U.S. industry improve its competitiveness through increased energy efficiency and reduced environmental impact.

Through ENERGY STAR, the U.S. EPA stresses the need for strong and strategic corporate energy management programs and provides a host of energy management tools and strategies to help companies implement such programs. This Energy Guide reports on research conducted to support the U.S. EPA’s ENERGY STAR Fruit and Vegetable Processing Focus, which works with U.S. fruit and vegetable processors to develop resources and reduce information barriers for energy efficiency improvement. This Energy Guide provides a detailed overview of available measures for energy efficiency in the U.S. fruit and vegetable processing industry. Given the importance and rising costs of water as a resource in fruit and vegetable processing, this Energy Guide also provides information on proven measures for improving plant-level water efficiency. Moreover, water efficiency improvement can also reduce energy use for water heating, treatment, and pumping. The fruit and vegetable processing industry in the United States—defined in this Energy Guide as facilities engaged in the canning, freezing, and drying or dehydrating of fruit and vegetable products—is an important industry from both an economic and energy use perspective. In 2004, the industry generated nearly $38 billion in product shipments and employed nearly 112,000 people directly in over 1,300 different facilities . Although fruit and vegetable processing facilities can be found throughout the United States, the states of California, Oregon, Washington, and Wisconsin account for roughly one half of total industry employment. The industry spent nearly $810 million on energy costs in 2002: $370 million for purchased electricity and $440 million for purchased fuels, which consisted primarily of natural gas . Because the costs of electricity and natural gas are rising rapidly in the United States, energy efficiency improvements are becoming an increasingly important focus area in the U.S. fruit and vegetable processing industry for managing costs and maintaining competitiveness. This Energy Guide begins with an overview of the trends, structure, and production characteristics of the U.S. fruit and vegetable processing industry in Chapter 2. A description of the main production processes employed in fruit and vegetable processing is provided in Chapter 3. In Chapter 4, the use of energy in the fruit and vegetable processing industry is discussed along with an overview of the main end uses of energy in typical canning, freezing, and drying or dehydrating facilities. Chapters 5 through 13 describe a wide range of available measures for improving energy efficiency in U.S. fruit and vegetable processing facilities, with a focus on energy-efficient technologies and practices that have been successfully demonstrated in facilities in the United States and abroad. Although new energy-efficient technologies are developed continuously , this Energy Guide focuses primarily on those technologies and practices that were both proven and currently commercially available at the time of this writing. However, plant pots round because emerging technologies can often play an important role in reducing industrial energy use, Chapter 14 offers a brief overview of selected promising emerging energy-efficient technologies of relevance to fruit and vegetable processing. Given that the U.S. fruit and vegetable processing industry manufactures a wide variety of products and employs a diversity of production methods, it is impossible to address all end uses of energy within the industry. This Energy Guide therefore focuses on only the most important end uses of energy in typical canning, freezing, and drying or dehydrating facilities. Lastly, recognizing the importance of water as a resource in fruit and vegetable processing as well as its rising costs, this Energy Guide concludes with information on basic, proven measures for improving plant-level water efficiency in Chapter 15. Many of the water efficiency strategies discussed in Chapter 15 can lead to energy savings as well. Table 1.1 provides a summary of key economic and energy use data presented in this Energy Guide for the U.S. fruit and vegetable processing industry.This Energy Guide defines the U.S. fruit and vegetable processing industry as facilities engaged in the canning, freezing, and drying or dehydrating of fruits and vegetables, which constitute the three major methods of fruit and vegetable preservation employed by the U.S. food industry today.

More specifically, this Energy Guide considers the four U.S. food industry sub-sectors defined by the North American Industry Classification System codes listed in Table 2.1. Also summarized in Table 2.1 are the key products manufactured by each sub-sector. It can be seen in Table 2.1 that the U.S. fruit and vegetable processing industry manufactures a wide variety of products, many of which are staples in the typical American home. Such staples include frozen concentrated orange juice, canned tomato sauces, ketchup, frozen French fried potatoes, canned soups and stews, frozen fruits and vegetables, dehydrated potatoes, and fruit jams and jellies.The primary purpose of fruit and vegetable processing is to preserve fruits and vegetables in a stable form that enables extended storage and shipment to distant markets, which allows consumers to purchase a wide variety of fruit and vegetable products at all times of year. Fruit and vegetable processing can also be used to provide consumers with food products that are more convenient to prepare and consume. Of all the fruits and vegetables consumed in the United States each year, roughly one half are processed into canned, frozen, or dehydrated consumer products. In 2003, around 370 pounds of fruits and vegetables per capita were processed for consumption in the United States . Americans purchased nearly $21 billion worth of processed fruit and vegetable products directly in 1999, or nearly 10% of their total grocery budget .Fruit and vegetable canning is the largest sub-sector of the U.S. fruit and vegetable processing industry in terms of both economic output and employment. In 2004, U.S. fruit and vegetable canneries generated over $18 billion in product shipments, roughly one half of the of the industry’s total economic output . Fruit and vegetable canneries in the United States employed nearly 48,000 people directly in 2004 at 764 different facilities .In the canning process, fruits and vegetables are sterilized and preserved in hermetically sealed containers that prevent microbial spoilage. Common container materials include enamel-coated steel, tin-coated steel, aluminum, plastic, and glass . Fruit and vegetable canneries in the United States manufacture a wide variety of products, including canned tomato sauces, ketchup, fruit and vegetable juices, canned vegetables and fruits, fresh fruit juices, pickles, and fruit jellies and jams. However, canned tomatoes and tomato-based products represent the most important products from this sub-sector from an economic perspective, accounting for over $5.5 billion in product shipments in 2002 . Other major sub-sector outputs from an economic perspective are canned orange juices, pickles and pickled products, canned jellies, jams, and preserves, fresh orange juices, canned corn, and canned beans. A summary of key products manufactured by U.S. fruit and vegetable canneries is provided in Appendix A. Although fruit and vegetable canneries are located across the United States, the greatest number of canneries is found in California due to the state’s large agricultural industry. According to the California League of Food Processors, California canneries produce 33% of the world’s processed tomatoes , 100% of the U.S. supply of canned peaches and fruit cocktail, and 100% of the U.S. supply of black ripe olives . In 2002, nearly 16,000 people were directly employed in 145 fruit and vegetable canneries in California . After California, the states with the highest employment in fruit and vegetable canneries are Wisconsin , Florida , and New York . Major U.S. based companies in this sub-sector include H.J. Heinz , Del Monte Foods , J.M. Smucker , ConAgra Foods , Ocean Spray Cranberries , and Seneca Foods . Frozen fruit, juice, and vegetable manufacturing is the next largest subsector of the U.S. fruit and vegetable processing industry after canning. This sub-sector generated $8.7 billion in product shipments in 2004, or roughly one quarter of the industry’s total economic output . Frozen fruit, juice, and vegetable manufacturers in the United States employed over 35,000 people directly in 2004 at 247 different facilities . Freezing preserves fruits and vegetables by lowering their temperature to a point at which the growth of micro-organisms is severely limited . Key products manufactured by the frozen fruit, juice, and vegetable manufacturing sub-sector include frozen French fried potatoes, frozen concentrated orange juices, frozen potato patties and puffs, frozen sweet yellow corn, frozen onions , and frozen strawberries. From an economic perspective, frozen French fried potatoes represent the most significant product manufactured by the frozen fruit, juice, and vegetable manufacturing sub-sector.

The SlIMP3 has potential as an important candidate gene for molecular breeding with the goal of improving shelf-life of tomato fruit. Interestingly, treating tomato fruits with myoinositol also improved cell wall bio-genesis, delayed softening, and extended shelf-life in fruits. Litchi fruit lost less water after myoinositol treatment . Collectively, all these results demonstrate that myoinositol has strong potential for improving tomato post harvest life. Does the increased AsA biosynthesis contribute to the cell wall bio-genesis and delayed fruit softening? The SlDHAR gene, that is responsible for recycling of AsA, was overexpressed in AC tomato. The SlDHAR-overexpressed plants exhibited higher AsA contents compared with the WT plants. However, cell wall thickness, fruit firmness, wall loss, and softening time in the SlDHAR-overexpressed plants were similar to those in WT plants . The data presented here corroborates that the increased AsA content does not delay softening and or prolonged shelf-life in tomato fruit.B. cinerea is a necrotrophic fungal pathogen leading to grey mould rot. It is among the most destructive postharvest pathogens of fruit .

Causing huge economic losses, plant plastic pots genetic modification has been attempted to control this postharvest pathogen. The cell wall is an important barrier to pathogen infections . Simultaneous downregulation of PG and Exp1 genes in tomato fruit reduced cell wall breakdown and susceptibility to B. cinerea . Suppression of SlPL in tomato resulted in increased fruit firmness and reduced susceptibility to B. cinerea . Overexpression of the carbohydratebinding module of expansin 1 in tomato increased fruit firmness and decreased susceptibility to B. cinerea . In this study, overexpression of SlIMP3 increased cell wall thickness and improved fruit resistance to B. cinerea . The increased cell wall thickness serves to retain inhibition of B. cinerea infection. Our data corroborated that the cell wall modification is an effective strategy for improving fruit tolerance of postharvest pathogens. In addition, the increased cell wall thickness was not only found in SlIMP3-overexpressed fruit, but also in SlIMP3-overexpressed leaf and stem . Therefore, we speculate that SlIMP3 may confer resistance to broad-spectrum pathogens in tomato. In conclusion, SlIMP3 is a bifunctional enzyme with the ability to regulate AsA and myoinositol biosynthesis. Overexpressing SlIMP3 in tomato not only increased AsA accumulation, but also delayed the fruit softening and enhanced tolerance to B. cinerea, suggesting the potential value of SlIMP3 in plant improvement programmes with the goal of improving postharvest fruit life.

Citrus is one of the most important and widely grown commodity fruit crops . Citrus has a non-climacteric fruit maturation behaviour and a unique anatomical fruit structure . The fruit contains two peel tissues, flavedo and albedo. The flavedo accumulates pigments and compounds which contribute to the fruit aroma, while the albedo comprises spongy cells rich in pectin. During the early stages of fruit development the albedo occupies most of the fruit volume and it becomes gradually thinner during fruit development as the juice cells in the pulp grow . Growth and development of the citrus fruit can be divided into three major stages . Stage I starts immediately after fruit set and is characterized by extensive cell division. During the transition to stage II, cell division ceases in all fruit tissues except the outermost flavedo layers and the tips of the juice sacs. During this stage, citrus fruit grows through cell expansion. Juice sac cell enlargement is mostly driven by the expansion of the vacuole, which occupies most of the cell volume. Stage III is the fruit maturation and ripening stage when fruit growth slows down and the pulp reaches its final size. Citrus fruit development is characterized by changes in primary and secondary metabolite content, with sugars and citric acid being the major components of the juice sac cells. Sucrose is translocated to the fruits from the leaves throughout fruit development, and constitutes about 50% of the total soluble sugars.

The anatomy of the citrus fruit, where the juice sacs are disconnected from the vascular bundles present in the albedo, suggest apoplastic sucrose downloading . Sucrose can then be hydrolysed by cytosolic invertases or stored inCitrus is one of the most important and widely grown commodity fruit crops . Citrus has a non-climacteric fruit maturation behaviour and a unique anatomical fruit structure . The fruit contains two peel tissues, flavedo and albedo. The flavedo accumulates pigments and compounds which contribute to the fruit aroma, while the albedo comprises spongy cells rich in pectin. During the early stages of fruit development the albedo occupies most of the fruit volume and it becomes gradually thinner during fruit development as the juice cells in the pulp grow . Growth and development of the citrus fruit can be divided into three major stages . Stage I starts immediately after fruit set and is characterized by extensive cell division. During the transition to stage II, cell division ceases in all fruit tissues except the outermost flavedo layers and the tips of the juice sacs. During this stage, citrus fruit grows through cell expansion. Juice sac cell enlargement is mostly driven by the expansion of the vacuole, which occupies most of the cell volume. Stage III is the fruit maturation and ripening stage when fruit growth slows down and the pulp reaches its final size. Citrus fruit development is characterized by changes in primary and secondary metabolite content, with sugars and citric acid being the major components of the juice sac cells. Sucrose is translocated to the fruits from the leaves throughout fruit development, and constitutes about 50% of the total soluble sugars. The anatomy of the citrus fruit, where the juice sacs are disconnected from the vascular bundles present in the albedo, suggest apoplastic sucrose downloading . Accumulation of citric acid in the vacuole of the juice sac cells is correlated with vacuole acidification mediated by the proton pumping activity of the tonoplastic H+ -ATPase. Citrate begins to accumulate during the second phase of fruit development. The accumulation continues for a few weeks, reaching a peak when the fruit volume is about 50% of its final value and then acid declines gradually as the fruit matures . Citrate decline during the second half of fruit development is associated with the activity of CsCit1, a H+ /citrate symporter . It has been suggested that some of the citrate is targeted for amino acid biosynthesis generally induced during the second half of fruit development . Indeed, black plastic pots there is an increase in some amino acid metabolizing genes, including those of the GABA shunt, and their corresponding enzymes during the citrate decline stage . In the last few years, studies using transcriptome analysis and metabolite profiling demonstrated a tight regulation of fruit metabolism during fruit maturation . However, comparison of mRNA expression levels, proteins amounts, and enzymatic activities have revealed low correlations between metabolome and transcriptome, indicating that transcriptome analysis was not sufficient to understand protein dynamics or biochemical regulation . A more direct correlation is expected for proteins and metabolites and, therefore, quantitative mass spectrometric proteomics and metabolomics are becoming attractive approaches. Quantitative proteomics has been used for the quantification of complex biological samples . Previously, LC-MS/MS was used to identify the proteome of various cellular fractions of the juice sac cell . More recently, a label-free differential quantitative mass spectrometry method was developed to follow protein changes in citrus juice sac cells. Two alternative methods, differential mass-spectrometry and spectral counting were used to analyse the protein changes occurring during the earlier and late stages of fruit development . Along with the generation of a novel bio-informatics tool, iCitrus, the above method enabled the identification of approximately 1500 citrus proteins expressed in fruit juice sac cells and the quantification of changes in their expression during fruit development. In this study, label-free LC-MS/MS-based shot-gun proteomic and metabolomic approaches were utilized to investigate citrus fruit development.

These tools were used to identify and evaluate changes occurring in the metabolic pathways of juice sac cells which affect citrus fruit development and quality. Integration of proteomic and metabolomic analyses created a more comprehensive overview of changes in protein expression and metabolite composition of primary metabolism during citrus fruit development and maturation.An extensive comparative proteomics study was conducted in order to identify protein changes occurring during citrus fruit growth and development. Samples were collected from three developmental stages; early stage II, stage II, and stage III . For proteomics analysis, two biological repetitions from two consecutive years were collected from at least 20 pooled fruits for each stage . For gene expression, enzyme activities, and metabolome analysis, three biological repetitions of three consecutive years were analysed. For better identification of differentially expressed proteins during fruit development and to decrease sample complexity, the juice sac cells were fractionated into soluble and membrane-bound proteins . Changes in protein expression were revealed by comparisons between spectra originated from fruit juice sac cells at different stages: stage II versus early stage II and stage III versus stage II. The complete data of the differential proteins detected can be found in Supplementary Tables S1 and S2 at JXB online. The analysis revealed a significant metabolic change occurring during the transition from early stage II to stage II and from stage II to stage III . Although these changes involved a wide range of processes, this study focuses on protein changes related to primary metabolism. Processes involving sugar metabolism, the TCA cycle, amino acid metabolism, energy production, and cell wall related metabolism changed significantly in citrus juice sac cells during fruit development. Citrus fruit accumulate large amounts of sugars, mainly sucrose, glucose, and fructose. Enzymes participating in sucrose metabolism were highly represented in the proteome analysis of citrus fruit juice sac cells. Most of the enzymes involved in sucrose degradation and glycolytic pathways were up-regulated during the transition from early stage II to stage II and were upregulated toward maturation, emphasizing the regulatory role of glycolysis in sugar utilization to drive fruit growth during citrus fruit development . Hexokinase, fructokinase, glucose-6-phosphate isomerase, fructosebisphosphate aldolase, ATP-dependent 6-phosphofructose- 1-kinase, triosephosphate isomerase, and enolase protein expression did not change significantly during the early stages and were up-regulated during the transition from stage II to stage III. UDP-glucose pyrophosphorylase, phosphoglucomutase, glyceraldehyde-3-phosphate dehydrogenase, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerate kinase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase were up-regulated throughout fruit development. Two pyruvate kinases were identified: iCitrus ID 52671 that did not change during the transition from early stage II to stage II and iCitrus ID 28935 that was up-regulated at stage II compared witho early stage II. Both proteins were upregulated during the transition from stage II to stage III. Sucrose synthase was found to be an interesting exception, since it was down-regulated during the transition from early stage II to stage II, and was up-regulated nearer to maturation . Four citrus sucrose synthase isoforms derived from four different unigenes were identified and clustered into three groups according to their sequence homology. Group 1 consisted of isoforms with homology to unigenes related to CitSUSA , group 2 consisted of proteins derived from unigenes related to CitSUS1 , and group 3 comprised CitSUS4, shown in this study to be expressed in the fruit. The expression patterns of CitSUS1 and CitSUSA were in agreement with their corresponding transcripts and with previously characterized enzymatic activities . The CitSUS1 gene was shown to be expressed in the early stages of fruit development and its expression decreased towards maturation, while the CitSUSA gene was up-regulated towards maturation.In this study, it is shown that the amounts of both CitSUS1 isoforms decreased in the transition from early stage II to stage II while that of iCitrus ID 33038 increased during the transition from stage II to stage III similar to CitSUSA which was up-regulated towards maturation, in agreement with the gene expression profiles and enzyme activity . In addition, CitSUS4 was found to be significantly down-regulated between early stage II and stage II and was not detected in the later stage of fruit development , thus indicating that its amounts remained constant. This reaction catalyses the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate. While most glycolytic enzymes are highly conserved between organisms, two types of phosphofructokinase isoforms exist in plants . In addition to the ATP-dependent phosphofructokinase , a pyrophosphate-fructose-6-phosphate-phosphotransferase uses pyrophosphate as the phosphoryl donor.

The Cnr mutation is also thought to be a gain of function mutation, although the mechanism has yet to be understood . The Cnr mutation results from hypermethylation upstream of the gene near the promoter and has been shown to inhibit the genome-wide demethylation cascade associated with normal tomato ripening . Previously, these TFs were regarded as master regulators of ripening; however, given the new information about the nature of the mutations in Cnr, nor, and rin, it is less clear the precise roles the TFs are playing in ripening . The nor and rin mutants have been utilized in breeding for developing tomato hybrids with extended shelf life or extended field harvest depending on their purpose for the fresh market and processing tomato industries . Hybrids between elite varieties and the ripening mutants have a delayed ripening progression, but with the tradeoff of decreased fruit quality attributes, such as color, taste, and aroma . Although there are some publications dedicated to evaluating the physiological characteristics of mutant or hybrid fruit , up to this point, large plastic growing pots much of what we know about the ripening mutations is based on controlled greenhouse experiments with limited fruit and few ripening stages examined.

A complete dataset of phenotypic data produced from large-scale field trials evaluating fruit ripening and senescence is lacking to provide information relevant to breeding, particularly in the new context of the molecular mechanisms behind the nor and rin mutations. The Cnr mutant provides a unique opportunity to study the role of epigenetics in fruit ripening but is not used in breeding because the mutant phenotype is dominant. Cnr has been regarded as a ripening mutant due to its unique colorless phenotype and additional ripening defects . It has been suggested that Cnr fruit undergo normal growth and development ; however, fruit appear different from wild type even before ripening, with a smaller size, alterations in cell wall enzyme expression, and earlier chlorophyll degradation . To better utilize Cnr as a tool for studying fruit development and ripening, a broader understanding of the physiological and transcriptomic alterations in this mutant is necessary. These spontaneous single mutants need to be reevaluated as tools to understand the wide-ranging biological processes regulated by each TF. Previous literature has generally assumed that the mutations block ripening, resulting in similar processes affected . This study demonstrates that each mutant has a unique ripening phenotype, resulting from a combination of inhibited and delayed developmental processes.

We integrated phenotypic data with gene expression data and hormone measurements in the Cnr, nor, and rin mutants across ripening and senescence to characterize the extent and timing of the ripening defects. Tomatoes grown under field conditions were assessed for fruit traits over multiple seasons. We then performed a transcriptomic analysis to gain more definition of the timing in which mutant fruit deviated from WT in their development and to determine specific molecular functions altered in each mutant. Due to their pivotal role in regulating ripening, we focused on defects in hormone networks, including biosynthesis and accumulation. We analyzed the influence of each mutation on the expression of the other TF throughout ripening and senescence. Finally, to better understand the combined genetic effects of the mutants on fruit ripening, we generated homozygous double mutants of Cnr, nor, and rin and used phenotyping and transcriptional data to evaluate the relationships between the mutants.Tomato plants of c.v. ‘Alisa Craig’ and the isogenic ripening mutants Cnr, nor, and rin were grown in randomized plots under standard field conditions in Davis, CA, United States, during the 2016, 2017, 2018, and 2020 seasons. Fruit tagged at 10 days post-anthesis , which corresponds to 7 mm in fruit diameter, were harvested at stages equivalent to the WT fruit.

Fruit were sampled at the mature green , turning, red ripe , and overripe stages, corresponding to 37, 45, 50, and 57 dpa, respectively. The term “RR” is used throughout the manuscript to refer to the 50 dpa stage of all genotypes, even when the mutant fruit do not turn red. Fruit stages for each of the mutants were further validated by external color analysis . Double mutant fruit were generated through reciprocal crosses: Cnr × nor, nor × Cnr, Cnr × rin, rin × Cnr, nor × rin, and rin × nor. Fruit were selfed after the initial cross to generate an F2 segregating generation. The double mutants were initially selected in the F2 generation through genotyping and phenotyping. At least two additional generations after F2 were obtained through selfing to ensure the stability of the double mutations and to perform the experiments in this study. Three seasons of data were collected for the Cnr/nor fruit while only one season of data was collected for the rin/nor and Cnr/rin crosses.The mutant lines were genotyped for their respective mutations. For nor, the Phire Plant Direct PCR Kit was used to extract DNA and amplify the region of the gene containing the 2 bp mutation using the primers listed in Supplementary Table 1. The PCRs were run on a SimpliAmp Thermal Cycler with the following conditions denaturation: 99◦C for 5 min; 35 cycles of 98◦C for 5 s, 56◦C for 25 s, and 72◦C for 25 s; with a final extension of 72◦C for 1 min. The PCR products were purified using Wizard SV Gel and PCR Clean-Up System and then sequenced with Sanger technology to confirm the absence of the two nucleotides. For rin, the Phire Plant Direct PCR Kit was used to extract DNA and perform end-point PCRs using primers specific for the mutant and WT alleles . The following PCR conditions were used for the WT allele primers: denaturation 99◦C for 5 min; 35 cycles of 98◦C for 5 s, 55◦C for 25 s, and 72◦C for 25 s; with a final extension of 72◦C for 1 min. The PCR conditions for the mutant allele primers were: denaturation 98◦C for 5 min; 40 cycles of 98◦C for 5 s, 58◦C for 25 s, and 72◦C for 25 s; with a final extension of 72◦C for 1 min. The PCR products were visualized as bands using a 1% agarose gel. The Cnr epimutation was genotyped by bisulfite sequencing. Extracted DNA was treated with the Zymo Gold bisulfite kit . Bisulfite treated-DNA was PCR amplified for the CNR promoter region containing the methylation changes using the primers listed in Supplementary Table 1. The following PCR conditions were used: 94◦C for 2 min; 40 cycles of 94◦C for 30 s, 54◦C for 30 s, and 60◦C for 45 s, and a final extension of 60◦C for 10 min. The PCR products were then Sanger sequenced and compared to the same region amplified in untreated controls with primers . The following conditions were used to amplify the untreated DNA: 95◦C for 2 min; 35 cycles of 95◦C for 30 s, 56◦C for 30 s and 72◦C for 1 min, and a final extension of 72◦C for 10 min. To ensure mutants were homozygous for the locus, we confirmed the double mutants by allowing the plants to self for at least two additional generations and checking that the progeny were not segregating for any fruit phenotypes.Fruit trait data were collected across four field seasons . The genotypes, large plastic pots developmental stages, number of biological replicates, and number of field seasons used for fruit trait phenotyping can be found in Supplementary Table 2. One season of phenotyping was performed for Cnr/rin and rin/nor double mutant fruits for color, firmness, and ethylene. Three seasons of data were collected for the Cnr/nor double mutant fruit for ethylene and two seasons of data for color and firmness. Fruit were collected from multiple plots or harvests to capture environmental variability.

Fruit trait measurements were taken on the same day of harvest for all samples unless noted. Intact and halved fruit were imaged using the VideometerLab 3 facilitated by Aginnovation LLC1 . External color measurements were obtained from individual fruit with the CR-410 Chroma Meter and recorded in the L∗ a ∗b ∗ color space, where L∗ quantifies lightness, a∗ quantifies green/red color, and b∗ quantifies blue/yellow color. Principal component analysis of the color parameters was performed with the FactoMineR package and graphed with the FactoExtra package in R . Non-destructive firmness measurements were taken on the TA.XT2i Texture Analyzer using a TA-11 acrylic compression probe, a trigger force of 0.035 kg, and a test speed of 2.00 mm/sec with Exponent software . Firmness values are reported as kilograms force. The size was measured by taking the largest diameter of the fruit with a handheld caliper. Tomato juice was produced by pressing the fruit tissues with a juicer and filtering with cheesecloth to measure total soluble solids and titratable acidity . At least five biological replications of tomato juice were obtained from independent pools of 10–12 fruit from distinct plots in the field or at different harvest dates within the field season. TSS were measured as percent Brix with a Reichert AR6 Series automatic bench refractometer from the prepared juice with three technical replicates. Four grams of juice were diluted with water in 20 mL of deionized water to measure TA based on citric acid equivalents. Significant differences in fruit traits across genotypes and ripening stages were determined in R using Type I analysis of variance tests, followed by a post hoc test using the R package agricolae .On the day of harvest, the fruit pericarp tissues were dissected and flash-frozen in liquid nitrogen. Frozen tissues were then ground to a fine powder with the Retsch Mixer Mill MM 400. One gram of ground tissue was used for RNA extractions as described in Blanco-Ulate et al. . RNA concentrations were quantified with Nanodrop One Spectrophotometer and Qubit 3 . RNA integrity was then assessed on an agarose gel. Six biological replicates composed of 8–10 independent fruit were extracted per genotype and ripening stage from the 2016 and 2018 seasons.Four biological replicates each of Cnr/nor MG and RR fruit RNA were used to prepare cDNA libraries. cDNA libraries were prepared with Illumina TruSeq RNA Sample Preparation Kit v.2 from the extracted RNA. The quality of the barcoded cDNA libraries was assessed with the High Sensitivity DNA Analysis Kit in the Agilent 2100 Bioanalyzer and then sequenced on the Illumina HiSeq 4000 platform by the DNA Technologies Core at UC Davis Genome Center. Raw RNAseq data from WT, Cnr, nor, and rin at MG and RR were obtained from a published dataset by our group , GEO accession GSE148217, while raw RNAseq data from the immature stages of the ripening mutants were extracted from Lü et al. . The RNAseq datasets for the Cnr/nor double mutant were generated in this study. The raw sequencing reads from the different datasets were analyzed de novo following the bio-informatics pipeline described below. Ethylene production measurements were taken from MG, RR, and OR fruit on the day of harvest. At least five biological replicates of 5–7 fruit were used for the measurements. The genotypes, developmental stages, and number of biological replicates used for ethylene analysis in each field season can be found in Supplementary Table 2. Fruit were weighed and placed in 1 L airtight glass jars. Headspace gas was extracted from the sealed containers after 60 min and was injected into a Shimadzu CG-8A gas chromatograph . Sample peaks were measured against an ethylene standard. The rate of ethylene production was calculated from the peak, fruit mass, and incubation time. Frozen ground tissue prepared from the tomato fruit pericarp was lyophilized, weighed, and extracted in isopropanol:H2O:HCL1MOL with 100 l of internal standard solution as described in Casteel et al. . Abscisic acid and 1-aminocyclopropane-1-carboxylate were measured using liquid chromatography coupled totandem mass spectrometry and internal standards as described in Casteel et al. . The hormone concentrations were expressed as ng/g of dry weight. Four to six biological replicates composed of 8–10 fruit were used for these measurements for the 2017 season. Significant differences in hormone accumulation across genotypes and ripening stages were determined using Type I ANOVA in R, followed by an HSD test using the R package agricolae . In some cases, pairwise comparisons in hormone accumulation were also conducted by Student’s t-test in R.Fruit from the Cnr, nor, and rin mutants fail to acquire most ripening-associated traits that make them appealing for consumption. Yet, each mutant can be distinguished by their unique phenotypes .