Horticulture

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 .

Using the number of cities in which a tephritid species has been detected as a proxy for area infested, figure 2a shows that in 1960 there were only two California cities in which tephritids had been detected . However, by 1970 the number of cities with a tephritid detection had increased to 13, by 1990 to a remarkable 200 cities, and by 2010 to more than 300 cities. Although 10 different species contributed to these totals, A. ludens, C. capitata and B. dorsalis contributed the most, appearing in 77, 168 and 245 new cities, respectively, by 2012 . Reintroductions versus established populations A long-standing explanation for recurring fruit fly detections is that flies are continually being reintroduced, either in cargo shipments or by people carrying infested fruit from fruit-fly infested regions of the world. We test this hypothesis and an alternative one to account for the recurring detections; both hypotheses were originally framed by Carey for the medfly as reintroduction hypothesis—recurring tephritid detections are due to repeated introductions—and established population hypotheses—recurring detections are due to resident fly populations.

We assess the strength of these two hypotheses by comparing and identifying inconsistencies in relative numbers, square pots for plants diversity and frequency of detections in California and in other fruit-fly friendly regions.Tephritids are intercepted at all airports across the USA including all airports located in the southern states considered at risk for tephritid introductions. California ports of entry accounted for less than 20% of all insects intercepted in at-risk states . Assuming that insect interceptions can serve as proxies for the relative propagule pressure, if reintroductions were the primary source of detections, then the number of fruit fly detections in fruit-fly-friendly regions of the USA outside of California, compared with detections in California, should be roughly five to one, because California contributes about 20% of detections. Yet no tephritids were detected in the majority of states that are deemed at risk for fruit fly introduction and maintain robust monitoring programmes .Although the medfly and the olive fly are the only two tropical tephritid species that are long-term residents of fruitfly-friendly regions in the European Union , interception rates of other tephritid species at ports of entry throughout the EU are quite high. One source of evidence for this is EUROPHYT, the European notification system for plant health interceptions. This system’s database revealed that, of the total number of interceptions of harmful organisms in plants and plant products imported into the EU in 2011 , fully one-third were tephritids , and showed that, from 2007 to 2009, more than 700 individual tephritids in three genera and nine species not established in Europe were intercepted at Paris’s International Airport .

If the diversity and number of tephritid interceptions at the scores of international airports located in fruit-fly-friendly southern Europe, northern Africa and the Middle East are similar to those at the Paris airport, then the tephritid propagule pressure throughout this world region is far greater than in California. Yet, despite this pressure, with the exception of the peach fruit fly , discovered in 1998 in Egypt, no other tropical tephritids have been detected throughout the Mediterranean Basin for a century.Several lines of evidence support the hypothesis that from five to nine tephritid species have become self-sustaining populations in the state : their abrupt first appearance in the mid-1950s followed by high incidence of repeat detections, their marked seasonality and northward spread , the lack of new detections and/or introductions of new species in most other at-risk regions in the USA and Mediterranean Basin, and the high probabilities of repeatedly detecting many of the tephritid species in California while at the same time not detecting them in other at-risk areas. These findings do not rule out the possibility of multiple introductions into the state for tephritids such as the medfly. However, the multiple detections of several species in nearly the same location anywhere from 10 to 30 years after they were first detected, without any captures during interim years, suggests that, as for many other invasive species , tephritids can be present in low numbers for decades. Indeed, one of the important features of lags in invasion biology that probably also applies to the tephritid invasion of California is that invasions are often not recognized until they are over.

Our findings that multiple species of tropical tephritids have self-sustaining and thus established populations in California have profound economic implications. For example, a 1995 study estimated that medfly establishment alone would result in $493 million to $875 million in annual direct costs, and the imposition of an embargo would cause an additional loss of $564 million. The state economy could lose $1.2 billion in gross revenue and more than 14 000 jobs. However, two aspects of the invasions are advantageous for planners, programme directors and policy makers. The first is that local population sizes for all species are extremely small, and therefore likely to continue to be subdetectable. Therefore, based on phytosanitary standards of the International Plant Protection Convention, most regions of the state should continue to be classified as risk-free by trading partners. The second aspect of the invasions that can be exploited for the longer term involves the invasion lags, which imply that there can be relatively long windows of opportunity for developing new protocols and programmes. Commodity certification protocols can be developed for the creation of fly-free and low-prevalence zones, as can long-term research programmes on tephritid biology and management.Because the likelihood of slowing the spread of or eradicating an alien pest depends heavily upon its residency time, a basic invasion biology canon is that early detection is critical for rapid response . Our results reveal that, because the sources of repeat detections are captures from established populations rather from reintroduced ones, in most cases ‘early detection’ is a misnomer when applied to tephritid detections at all scales. Because this expression is often inaccurate, it is also misleading inasmuch as it implies that a policy primarily directed at preventing new introductions will solve the problem of recurrent detections or infestations.As is true for many alien insect populations, the majority of tephritid population growth and spread in the state is subdetectable because of the small size and cryptic habits of all life stages, the slow pace of naturalization processes, and suppression of populations by intervention programmes. In cancer diagnostics, this is referred to as the ‘rare-event detection problem’ ; in the context of fruit fly detections, the parallel concept is the difficulty in discovering exceedingly rare, scattered, ultra-small populations of tephritids that are mostly in pre-adult stages, hidden among millions of properties and tens of millions of micro-niches. The scores of examples of repeat tephritid finds within a small region of California, separated by decades, large square planting pots suggest that the efficiency of detecting small populations of fruit flies is grossly overestimated, and that the actual chances of discovering populations that are so tiny and scattered is vanishingly small. Our findings are consistent with two interrelated invasion biology principles that underlie the ability of tephritid populations to establish and maintain residency at ultra-low, cryptic and insidious population levels. The first involves what Simberloff refers to as the ‘mysterious lag phase’ in which new populations experience delayed growth. It is unlikely that the magnitude of the lag period in tephritids would be similar to the 150þ years reported for some introduced plant species. However, it is likely that tropical species of tephritids that are introduced to different climatic regions experience major population lags much like the multidecade lags observed in the melon fly in Africa and the cherry fruit fly in Europe. Recent studies suggest that many species’ invasion success may depend more heavily on their ability to respond to natural selection than on broad physiological tolerance or plasticity, and could also result from the need for multiple invasions to facilitate a sufficient evolutionary response.Although it is widely believed that human movement of infested plant material plays a major role in spreading introduced pests, capture patterns for the Mexican fruit fly suggest that this is not the case for this species and, by extension, may not be the case for many of the other invasive tephritids. For example, in 2011, 43 million vehicles and 17 million pedestrians crossed the six ports of entry from Mexico to California.

Assuming that the direction of movement for roughly half of these vehicles and people was from Mexico to California, and if humans entering and dispersing around the state were responsible for the Mexico-to-California as well as the within-state movement of the Mexican fruit fly, then this species should have been detected more or less randomly throughout the state. But the vast majority of all A. ludens detections for nearly 60 years have been in the same areas in which this species continually reoccurs. At the same time, there have been virtually no discoveries of A. ludens in the regions of the state with extraordinarily high movement of Latino populations , such as the main agricultural areas in the Central, Salinas and Imperial Valleys .We know of no historical precedent in the invasion biology literature similar to the tephritid situation in California, where not only are several insect species within a single family invading a region at the same time, but the group also contains species within multiple genera. The California tephritid invasion thus provides unique opportunities to compare the invasive properties of species across different genera with similar life histories, to explore reasons why 17 tephritid species have been detected in California but few to none in many other fruit-fly-friendly regions of the USA and the world, and to develop new population theory for ultra-low, cryptic populations.CDFA and USDA declared 100% success for each of the several hundred eradication programmes that were launched against fruit flies in California . These declarations were accurate according to legal criteria specified by the USDA and the International Phytosanitary Commission; that is, a region is declared fruit-fly-free when no flies have been detected for a time period corresponding to three generations. Although these legal criteria are required for regulatory compliance to enable growers to ship their produce, our results reveal that the more stringent ecological requirements for eradication declaration were not met in the majority of cases. This underscores the continuing problem in the insect eradication literature of loosely and inaccurately applying a term that has a clear definition. Those interested in insect eradication can learn much from the epidemiological literature on eradication programmes regarding frameworks for evaluating systematically the potential for eradication, clear definitions of concepts and terms, and perspectives on the preconditions, difficulties and challenges of successfully eradicating insects.Although some authors have characterized population establishment as self-sustaining populations , none has attempted to specify criteria. The likely reason is that, because of the uncertainty resulting from a combination of demographic stochasticity and detection constraints, it is virtually impossible to define a precise point at which a small population becomes self-sustaining. In the light of this problem, we propose that early-stage invasions can be categorized using methods similar to those we used for Californian tephritids . The establishment category for each species is necessarily subjective and can be based on a combination of detection metrics, including capture span , total number of years captured, inter-year frequency of detections , total numbers of individuals detected, within-state distribution and spatial patterns of apparent spread.The organoleptic properties of tomato fruit are defined by a set of sensory attributes, such as flavor, fruit appearance and texture. Flavor is defined as the combination of taste and odor. Intense taste is the result of an increase in gluconeogenesis, hydrolysis of polysaccharides, a decrease in acidity and accumulation of sugars and organic acids, while aroma is produced by a complex mixture of volatile compounds and degradation of bitter principles, flavonoids, tannins and related compounds . Fruit color is mainly determined by carotenoids and flavonoids, while textural characteristics are primary controlled by the cell wall structure in addition to cuticle properties, cellular turgor and fruit morphology . In last years, tomato fruit organoleptic quality has been investigated both at the genetic and biochemical levels in order to obtain new varieties with improved taste. Recently, the genomes of traditional tomato cultivars such as San Marzano and Vesuviano , considered important models for fruit quality parameters, have been sequenced. SM, originating from the Agro Sarnese-Nocerino area in southern Italy, produces elongated fruits with a peculiar bittersweet flavor.

Taking dried peaches made without sulfur as an example, Salmonella survived the entire 180 days of storage with the final level of 4.38 ± 0.08 log CFU/g when the storage temperature was 5 °C. When the storage temperature was 20 °C, Salmonella fell below the limit of detection after 90 days of storage although they could still be recovered by enrichment from two of six samples that were tested. The presence of sand together with the presence of sulfur speeded up the die-off of Salmonella. The impact of dry inoculation might be due the low initial inoculation level or the limit nutrient available when Salmonella cells were dried on the surface of the sand. As shown in Figure 2.1D and Table 2.3, Salmonella fell below the limit of detection after 30 days of storage at 5 °C and could no longer be detected even by enrichment. When the storage temperature was at 20 °C, Salmonella fell below the limit of detection and could not be detected by enrichment after 15 days of storage. Injured cells were observed starting from Day 0. The difference between the counts obtained from TSAR and XLT-4R indicated the formation of injured cells. These differences were as big as 3.68 log CFU/g .Survival of E. coli O157:H7 on dried peaches. Figure 2.2 and Table 2.4 show the behavior of E. coli O157:H7 survival on the dried peaches. The E. coli O157:H7 population on the wet-inoculated peaches and peaches with sulfur immediately after inoculation were 9.40 ± 0.32 and 9.43 ± 0.45 log CFU/g respectively .

The initial E. coli population on the wet-inoculated dried peaches was 8.70 ± 0.26 log CFU/g . After 5 days of storage, blueberry packaging approximately 1 log reduction was observed from the inoculated dried peaches made without sulfur and stored at 5 and 20 °C. From Day 5 to Day 15, greater reduction was observed from peaches made without sulfur that were stored at 20 °C. A 3.53 log reduction was observed from 20 °C, while 0.97 log reduction was observed from samples stored at 5 °C . From Day 15 to 60, while E. coli O157:H7 further declined to 3.77 ± 0.40 log CFU/g on inoculated-dried peaches without sulfur stored at ambient temperature and maintained at similar levels from Day 15 to Day 60. The surviving E. coli O157:H7 on wet inoculated dried peaches made without sulfur fell below the limit of detection after 90 days of storage at both refrigerated and ambient temperatures and could no longer be detected by enrichment after 150 days of storage. The addition of sulfur speeded up the die-off of E. coli O157:H7. When the storage temperature was at 5 °C, E. coli O157:H7 on dried peaches made with sulfur decreased to 5.74 ± 0.12 log CFU/g after 15 days of storage. After additional 15 days , E. coli O157:H7 could not be detected by neither directly plating nor enrichment. When being stored at ambient temperature, E. coli O157:H7 fell below the limit of detection after 15 days of storage and could not be detected via enrichment after 30 days of storage. Similar to what was observed from Salmonella, lower initial inoculation levels were seen from sand-inoculated samples.

When the storage temperature was 5 °C, E. coli O157:H7presence on dry-inoculated peaches made without sulfur gradually decreased from 6.52 ± 0.45 log CFU/g to 3.27 ± 0.06 log CFU/g on Day 120. No E. coli O157:H7 could be detected via plating or enrichment on Day 180. When the storage temperature was 20 °C, E. coli O157:H7 only decreased by approximately 1.3 log by Day 60. After Day 60, a sharp decrease was seen on Day 90 as no E. coli O157:H7 can be detected by plating. The pathogen can be detected by enrichment until Day 150. No pathogen can be detected on Day 180. When looking at the dry-inoculated peach made with sulfur treatment, a 2.15 log reduction was observed in the first 5 days during the storage at 5 °C . While E. coli O157:H7 could still be detected by enrichment on Day 15, it could not be detected after Day 30. Storing at ambient temperature increased the reduction see on Day 5. Greater than 4.25 log reduction was observed in the first 5 days. E. coli O157:H7 could not be detected after 15 days of storage. The differences between counts obtained from TSAR and MACR were also observed due to the formation of injured cells during inoculation and storage. Survival of L. monocytogenes on dried peaches. Figure 2.3 and Table 2.5 show the behavior of L. monocytogenes survival on the dried peaches. The L. monocytogenes population on the wet-inoculated peaches and peaches with sulfur immediately after inoculation were 9.52 ± 0.70 and 9.54 ± 0.34 log CFU/g respectively . The initial L. monocytogenes population on the wet-inoculated dried peaches was 8.57 ± 0.04 and 8.20 ± 0.23 log CFU/g as determined on TSAR and MOXR respectively. When the storage temperature was 5 °C, L. monocytogenes decreased to 7.92 ± 0.06 and maintained at similar levels for the between Day 5 to Day 90. When the storage temperature was 20 °C, a 2-log reduction was observed during the first 5-day of storage. Another sharp decreasing of survival L.monocytogenes numbers was observed between Day 30 and Day 60.

An approximately 2.4 log reduction was seen from TSAR. Starting from Day 90, L. monocytogenes could not be recovered by either direct plating nor enrichment on peaches made without sulfur. The pre-drying sulfur treatment sped up the die-off of L. monocytogenes. Starting from Day 15, L. monocytogenes could not be recovered from any dried peaches made with sulfur treatment stored at both temperatures. Dry inoculation yielded lower initial inoculation levels compared with wet inoculation. There were 6.56 ± 0.09 log CFU/g of L. monocytogenes on peaches made without sulfur treatment and 5.81 ± 0.03 log CFU/g of L. monocytogenes on peaches made with sulfur treatment. This lower inoculation level together with the presence of sulfur led to a significant reduction of L. monocytogenes during the first 5 days of storage regardless of the storage temperatures. Starting from Day 5, L. monocytogenes could only be detected from dry-inoculated dried peaches made with sulfur treatment by enrichment. With dried peaches made without sulfur treatment, bluleberry packaging box the surviving L. monocytogenes gradually decreased from Day 0 to Day 120 when the storage temperature was at 5 °C. Starting from Day 150, L. monocytogenes could no longer be recovered from dry-inoculated dried peaches without sulfur treatment. When the storage temperature was at 20 °C, L. monocytogenes could not be detected by either plating nor enrichment starting from Day 120. Sulfur measurement. The amount of total sulfur dioxide and free SO2 was measured during the first 30 days of ambient storage . The measurement was suspended after Day 30 due to the “shelter-in-place” order placed in March 2020. As shown in Table 2.6, the initial level of free SO2 and total SO2 in the dried peaches made with sulfur treatment were 830± 32 mg/Kg and 2,108 ± 32 mg/Kg respectively. The wet inoculation led to a loss of approximately 122 mg/kg of free SO2 and approximately 73 mg/kg of total SO2. The 2-day drying after inoculation only impacted the total SO2 level and didn’t impact the free SO2 level . No significant change was observed in free SO2 from Day 0 to Day 5. A significant loss of free SO2 was seen from Day 5 to Day 15 . On Day 30, there were 393 ± 46 mg/kg of free SO2 and 1,544 ± 12 mg/kg of total SO2 present in these dried peaches.The survival of common bacterial pathogens, Salmonella, E. coli O157:H7, and L. monocytogenes, was monitored on dried peaches made without and with sulfur pre-drying treatment. Two inoculation carriers were applied, and the two storage temperatures were tested. This impact is applicable to all pathogens tested. For example, Salmonella could not be recovered by enrichment from wet-inoculated dried peaches made with sulfur treatment after 60 days of storage at 5 °C and 15 days of storage at 20 °C. When they were inoculated onto dried peaches made without sulfur treatment, there were 4.95 ± 0.07 log CFU/g of Salmonella survived on these samples by the end of 180 days of storage at 5 °C. Even when the storage temperature was at 20 °C, Salmonella was still detected via enrichment from dried peaches made without sulfur.

The impact of sulfur treatment on the survival of pathogens was also reported by Liu et al. . Similarly, no Salmonella cell was recovered from sulfurtreated apricots via enrichment after 90 days of storageat 22 °C, while ~2.5 log CFU/g of Salmonella was recovered from dried apricots made without sulfur dioxide . Although sulfur dioxide treatment facilitates bacterial die-off, it has the potential to induce asthmatic reactions in some people . The free SO2 levels detected in dried peaches made with sulfur used in this study were higher than previously reported numbers. Although the processors did label the packages with “made with sulfur treatment”, additional studies may be needed to gain insight into sulfur levels present in samples sold at the farmers markets and the changes of free and total SO2 during storage. Storage temperature is another factor that generates a significant impact on pathogen survival. In general, pathogens survived at a higher level for longer period of time at low temperatures than ambient temperature. For example, Salmonella survived on dried peaches made without sulfur at 5 °C for up to 180 days with a final level of 4.59 log CFU/g on wet-inoculated ones and 4.38 ± 0.08 log CFU/g on dry-inoculated ones. On the same samples stored at ambient temperature, Salmonella could only be detected via enrichment after 90 days of storage, indicating the surviving level was below 1.9 Log CFU/g. Cuzzi, et al. found similar results. In their study, L. monocytogenes was inoculated onto dried applies, strawberries and raisins with sand and stored at 4 °C and 23 °C . Since L. monocytogenes could not be recovered from inoculated dried apples at Day 0 , only the survival in dried strawberries and raisins were monitored in this study . When the storage temperature was at 23 °C, L. monocytogenes decreased rapidly by greater than 4 and 3.6 log CFU/g after 14 and 7 days of storage on raisins and dried strawberries. However, when the storage temperature was at 4 °C, L. monocytogenes only decreased by approximately 0.1 and 0.2 log CFU/g/month. After 336 days of storage, L.monocytogenes only decreased by 1.4 and 3.1 log CFU/g on raisins and strawberries, respectively. The impact of inoculation carriers on the survival of pathogen was completed by the fact that different carriers led to, sometimes, different initial inoculation levels before storage. For example, the wet inoculation brought 9.45 ± 0.06 log CFU/g of Salmonella on dried peaches made without sulfur on Day 0, while the dry inoculation had an initial inoculation level of 7.26 ± 0.14 Log CFU/g to dried peaches. Lower initial inoculation levels from sand inoculated samples were also seen for E. coli O157:H7. In this case, the impact of carriers can not be fully studied. In the study conducted by L. R. Beuchat and Mann , two inoculation methods were used to inoculate the dried fruits. One was misting dried fruits with an aqueous suspension of a 5-serotype cocktail of Salmonella, and the other was mixing the dried fruits with sand on which a 5-serotype cocktail had been dried. Authors found that the survival of Salmonella on dried cranberries, raisins, strawberries, and date paste inoculated using the dry carrier and wet carrier followed similar trends. In the study conducted by Feng et al. , plate-grown E. coli O157:H7 were inoculated onto in-shell hazelnuts via wet or dry carriers . After that, samples were stored at 24 ± 1 °C for 12 months. Their results showed that E. coli O157:H7 reduced rapidly on sand-inoculated hazelnut than wet-inoculated ones, although the initial inoculation levels before storage were similar . In the study conducted by Liu et al. , Salmonella was inoculated onto dried apricots made with or without sulfur. The liquid inoculum was diluted for wet inoculation so that the same initial inoculation levels were achieved for both wet- and dry-inoculated dried apricots. Based on their results, Salmonella survived at higher levels for longer period of timeon sand-inoculated dried apricots.

The ABA-sprayed plants had an average fruit phloem sap uptake of 0.778ml fruit–1 d–1 and an average phloem sap solute concentration of 301mg ml–1 . Therefore, the solute accumulation per day was 1.04×144.3=150.07mg fruit–1 d–1 in non-ABA-sprayed plants and 0.778×301=234.18mg fruit–1 d–1 in ABA-sprayed plants. Therefore, the results also showed that ABA-sprayed plants had higher phloem solute accumulation per fruit than non-ABA-sprayed plants from 15 to 30 DAP. According to the present data and other studies, ABA could also be acting at the whole-plant level as a signal triggering carbohydrate accumulation and osmotic adjustment in sink organs . In addition, spraying peach fruit with ABA has been shown to increase the activity of sorbitol oxidase, a predominant enzyme in the metabolism of the translocated sugar alcohol sorbitol, which was followed by an enhanced sugar accumulation in the fruit . The higher phloem sap solute concentration in ABA sprayed plants can decrease fruit apoplastic solute potential, which is then equilibrated by a parallel decline in fruit total water potential . Under these conditions, plastic growing bag higher fruit solute accumulation can increase the water potential gradient between the fruit and stem, favouring fruit xylem sap uptake .

Accordingly, the present results show that whole-plant ABA spray treatment not only increased phloem solute accumulation per fruit, but also decreased leaf transpiration, maintaining a higher stem water potential and higher total fruit water uptake, compared with non-ABA sprayed plants.Following irrigation in the morning, tomato plants treated with ABA had a smaller increase in xylem sap flow rate intothe leaves compared with non-treated plants, presumably due to suppression of stomatal opening . In all treatments, the increase in xylem sap flow after the time of irrigation in the morning probably reflected the combined effects of leaf rehydration as well as increasing light intensity stimulating stomatal opening, and increasing VPD from increased air temperatures and decreased relative humidity in the greenhouse environment. The reduction in leaf xylem sap flow after 15:30h to 16:30h was presumably a result of the reverse changes in the environmental conditions observe dearly in the day after irrigation. These results and other studies also show a direct relationship between high leaf transpiration and higher leaf Ca2+ uptake , suggesting that leaf Ca2+ accumulation is dependent only on leaf xylem sap uptake triggered by leaf transpiration rates. In addition, the data also show that Ca2+ concentration in the leaf xylem sap extracted by pressurizing the leaves or by inducing leaf guttation were similar, suggesting that there is no significant Ca2+ contamination of leaf xylem sap when the leaves are cut and pressurized in the pressure chamber for xylem sap extraction.

In that case, ABA treatment may have also reduced the hydraulic resistance within the fruit, favouring xylemic water movement in the fruit towards the blossom-end tissue, provided a hydrostatic gradient responsible for xylem sap flow was present in the fruit . Since Ca2+ is believed to be mobile in the plant exclusively through the xylem vessels , the observed increase in xylem sap flow towards the fruit in the pedicle, and a reduced hydraulic resistance within the fruit, may explain the observed higher fruit Ca2+ accumulation in ABA-treated plants. Neither effect was observed in ABA-dipped fruit, suggesting that changes in Ca2+ partitioning in the plant are responsive only to whole plant ABA treatment. The pattern of fruit xylem sap uptake followed an increase after irrigation in all treatments until 15:30h to 16:30h, decreasing thereafter at both 15 and 30 DAP. Similar to the leaves, this pattern could be explained by the combined effects of an increase in plant water content right after irrigation and an increase in VPD early during the day that increased the evaporative demand due to increasing air temperatures and decreasing relative humidity durig the day time. Late in the day, decreasing SWP due to plant water loss and decrease in the VPD and consequently the evaporative demand due to decreasing air temperatures and increasing relative humidity could limit xylem sap flow into the fruit, resulting in the observed decrease in fruit xylem sap uptake. At night, the VPD was low but not zero, and continued plant water loss under these conditions may have been associated with the observed reverse flow of fruit xylem sap at 15 DAP for non-ABA-treated plants. Although a reverse xylem sap flow was observed later in the irrigation cycle, the fruit growth rate was always positive, indicating that phloem sap uptake maintained the positive growth rates even under reverse xylem sap flow conditions. The reverse flow of fruit xylem sap was not observed at 30 DAP, possibly because of higher fruit solute content compared with 15 DAP. The higher solute content decreased fruit water potential , which possibly increased the strength of the fruit as a sink for xylemic sap uptake under limited xylem conductivity conditions. The present results showed that fruit xylem sap uptake decreased from 15 to 30 DAP in tomato.

Previous studies have shown that phloem may represent 76–83% and xylem may represent 17–24% of fruit peduncle water uptake at early stages of growth and development . Consistent with the data presented, other studies have also shown that at later stages of growth and development, the xylem contribution to fruit water uptake decreases due to loss of xylem functionality and/or reduction in the hydrostatic gradient responsible for xylemic sap uptake and movement in the fruit . However, other studies have shown that xylem transport into trusses of tomato fruit cultivar Gourmet remained functional throughout the first 8 weeks of growth. In addition, these studies showed that ~75% of water net influx into the fruit occurred through the external xylem and ~25% via the perimedullary region, which contains both phloem and xylem . Differences in the phloem/xylem ratio of fruit sap uptake presented in the literature could be attributed to different genotypes and/or the growing conditions of each study. In future studies, direct measurements of phloem sap uptake into the fruit using nuclear magnetic resonance should be carried out for the same tomato cultivar and growing conditions as used in the present study to compare precisely the methods and the results obtained .Although no statistically significant changes in Ca2+ concentrations in stem xylem sap were observed among the treatments, spraying plants with ABA increased the Ca2+ concentration in the xylem sap moving into the fruit. The movement of Ca2+ in the xylem vessels depends on adsorption and desorption of Ca2+ from active exchange sites within the cell walls . In that case, fruit of ABA-sprayed plants possibly had exchange sites within the xylem cell walls that were more saturated with Ca2+, wholesale grow bags maintaining higher levels of soluble Ca2+ in the xylem sap stream. In addition, evidence suggests that special nutrient transport systems exist at the interface between living cells and xylem vessels . The higher Ca2+ concentration observed in the xylem sap of the peduncle of fruit from ABA-sprayed plants could be the result of the higher flow rate of xylem sap into the fruit leading to a higher saturation of Ca2+ binding sites in the xylem vessels and cell uptake requirements that reduced Ca2+ binding to active exchange sites in the cell walls as well as the Ca2+ uptake into living cells at the interface with the xylem vessels.Spraying tomato plants with ABA increased the Ca2+ concentration and Ca2+ accumulation in the pericarp tissue at the fruit peduncle end by increasing fruit xylem sap uptake, decreasing fruit phloem sap uptake, increasing Ca2+ concentration in the xylem sap moving into the fruit, and possibly by increasing phloem Ca2+ transport into the fruit. The results show that ABA spray treatment increased fruit xylem sap uptake4.72-fold, fruit xylem sap Ca2+ concentration 1.28-fold, and fruit growth 1.41-fold, compared with water spray treatment, respectively. These results suggest that the increase in fruit xylem sap uptake was the most important effect of ABA spray treatment leading to the observed higher fruit Ca2+ accumulation from 15 to 30 DAP. The Ca2+ accumulation in fruit tissue estimated by multiplying the xylem sap Ca2+ concentration in the fruit peduncle by its respective flow rate into the fruit from 15 to 30 DAP was ~84% of the Ca2+ accumulation quantified by the difference in total fruit Ca2+ content observed at 30 DAP minus the total fruit Ca2+ content observed at 15 DAP.

Considering that fruit water uptake is via the xylem and phloem, the results suggest that the phloem may have also contributed to fruit Ca2+ uptake under the experimental conditions described in this study. The results also show a greater difference between the quantified and estimated Ca2+ accumulation in the fruit of ABA-sprayed plants than in the fruit of other treatments , suggesting that spraying plants with ABA also enhanced fruit phloem Ca2+ uptake. Considering that spraying tomato plants with ABA decreased fruit phloem sap uptake, it is possible that this treatment increased Ca2+ concentration in the phloem sap to increase fruit Ca2+ uptake to compensate for the reduction of phloem sap uptake. These results agree with previous studies suggesting that phloem can also have an important contribution to fruit Ca2+ uptake depending on the phloem sap Ca2+ concentration and phloem sap flow rate into the fruit . In the present study, it was assumed that fruit transpiration rates were similar among all treatments. Future studies related to the effect of ABA on xylem and phloem fruit water uptake should include direct measurements of fruit transpiration rates. In the xylem vessels, after reaching the peduncle end of the fruit, Ca2+ can be taken up by the cells, bind to active exchange sites within the cell walls, or remain soluble in the xylem vessels to be translocated towards the blossom-end tissues of the fruit . Accordingly, the present results show that higher xylem sap and tissue Ca2+ content at the fruit peduncle end resulted in higher fruit Ca2+ translocation to and Ca2+ accumulation in the blossom-end tissues in response to whole-plant ABA treatment. Dipping the fruit in ABA did not affect xylem sap or tissue Ca2+ content at the fruit peduncle end, but resulted in higher Ca2+ accumulation and higher Ca2+ in the apoplast in the blossom-end tissue at 15 DAP, suggesting that ABA also triggered a fruit-specific mechanism that favoured Ca2+ translocation from the peduncle end towards the blossom-end region of the fruit. This latter effect was not observed at 30 DAP. According to the present data, spraying the whole plant with ABA or dipping the fruit in ABA maintained a higher number of functional xylem vessels that reduced the resistance to xylemic water and Ca2+ movement into the blossom-end tissue, which could help to explain the observed higher Ca2+ content in the distal end of the fruit. In ABA-dipped fruit, the increase in Ca2+ concentration in the blossom-end tissue was only observed at 15 DAP, possibly due to the reduction in any ABA effect on maintaining a higher number of functional xylem vessels at late stages of fruit growth and development. It is possible that ABA could also increase the number of functional xylem vessels connecting the fruit to the plant, which should be determined in future studies. In addition, higher cuticular wax content in epidermal cells at 30 DAP compared with fruit at 15 DAP could limit fruit ABA uptake during the later dip treatments.At the whole-plant level, ABA treatment triggered stomatal closure, decreasing xylemic water and Ca2+ flow to the leaves, which maintained higher stem water potential. Under such conditions, whole-plant ABA treatment favoured xylemic water and Ca2+ movement into the rapidly expanding fruit, resulting in higher Ca2+ content reaching the fruit peduncle end. However, the data suggest that xylem sap uptake could not fully explain fruit Ca2+ accumulation due to the difference between the observed total fruit Ca2+ accumulation and the estimated fruit Ca2+ accumulation based on the Ca2+ concentration in the xylem sap and xylem sap flow rate into the fruit. These results suggest that phloem could have acted as a source of Ca2+ to the fruit under the experimental conditions described in this study. More detailed studies should include direct measurements of fruit transpiration rates to better characterize the role of phloem in fruit Ca2+ uptake. In addition, a better understanding of phloem contributions to fruit Ca2+ uptake can be accomplished by developing efficient methods to extract and quantify Ca2+ in the phloem sap moving into the fruit.

Cnr also displayed similar functional enrichments to WT among their respective ripening-related DEGs, including photosynthesis-related pathways, carbohydrate, and amino acid metabolism, and plant hormone signal transduction . Compared to Cnr, rin shared a smaller number of ripening-related DEGs and functional enrichments with WT fruit . The number of ripening-related DEGs shared between nor and WT fruit was negligent, and no functional enrichments were detected in this set of DEGs. Similar to our previous analysis, we mined the ripening-related DEGs to determine the patterns of expression of key genes involved in fruit quality traits . We observed that Cnr and WT showed similar gene expression of SlPSY1, SlLCY1, POLYGALACTURONASE 2A , pectate lyase , PECTIN METHYLESTERASE 1 , and ACTINATE HYDRATASE . Fruit from nor and rin did not have similar ripening expression patterns to WT fruit for those genes, except for the SlPG2A and SlPME1 in rin. Altogether, these data indicate that Cnr fruit undergo the most similar ripening progression to WT fruit, plastic nursery plant pot while nor and rin fruit have moderate to minimal changes between the MG and RR stages.

The changes in gene expression of CNR, NOR, and RIN in the ripening mutants indicate that the genes are interconnected during fruit development. In addition, Cnr consistently showed earlier defects in fruit traits, gene expression, and hormone pathways. To characterize the combined genetic effects of the mutations on tomato fruit, we generated homozygous double mutants through reciprocal crosses of the single mutants. We then phenotyped the double mutants for fruit traits and ethylene production . Because the reciprocal crosses produced fruit indistinguishable from each other, we reportthem as only one double mutant . Fruit of nor/rin double mutants were almost indistinguishable from both nor and rin fruit in appearance and external color. Fruit resulting from any cross with Cnr as a parent presented similar visual characteristics . We also performed a PCA of the color measurements to compare the double mutants to their parental lines at the RR stage and confirmed this observation . Based on these observations and our earlier phenotypic and transcriptional data, we confirmed that the Cnr mutation affects early fruit development. In contrast, the nor and rin mutations act during fruit ripening. If defects in Cnr occur earlier in fruit development than those caused by nor or rin, we expected the Cnr/rin and Cnr/nor double mutants to behave similarly to Cnr anddisplay similar phenotypes . Cnr/rin fruit were significantly less firm than either parent at the MG stage but performed most similarly to Cnr at the RR stage. Cnr/nor fruit was not distinguishable from either parent in firmness at MG but was firmer than Cnr RR fruit. Interestingly, Cnr/nor fruit exhibited high ethylene production at the MG stage like the Cnr fruit. At the RR stage, Cnr/nor showed a less pronounced decrease in ethylene production, resulting in higher hormone levels than either parent. Although some phenotypic differences were detected, we verified that Cnr/rin and Cnr/nor resembled the Cnr parent for most of the fruit traits measured.

If nor and rin act synergistically during ripening, the rin/nor double mutants would have a more extreme phenotype than either on their own. At the MG stage, rin/nor fruit firmness was statistically similar to rin but became an intermediate phenotype at the RR stage. For ethylene, rin/nor fruit produced less than either parent at both stages, although not significant, suggesting a combined effect of both mutations.The spontaneous ripening mutants, Cnr, nor, and rin, are essential genetic tools to untangle the complexity of climacteric fruit ripening and to breed for extended shelf-life or field harvest traits in tomato . However, thorough phenotyping of the fruit traits affected by these mutants using plants grown under field conditions has been neglected. Here, we produced an extensive quantitative study of fruit quality in the tomato ripening mutants and corroborated it across multiple field seasons. We were able to carefully describe physiological and molecular differences between the mutants by sampling large numbers of fruit and surveying distinct stages through ripening in ways not feasible with greenhouse experiments.We determined that some ripening events in the mutants nor and rin were not completely blocked but severely delayed. By examining the OR stage, we found that the mutation in nor may strongly affect firmness and taste while pigment accumulation was only delayed and slightly perturbed . These phenotypes were supported by higher expression of carotenoid biosynthesis genes in nor RR than WT and an increase in SlPSY1 between the MG and RR stages . The accumulation of pigments in nor fruit, particularly at late stages in development, has gone unnoticed in previous studies, but it partially resembles the CRISPR-NOR mutants . In contrast, rin fruit showed strong inhibition of pigment accumulation but less dramatic alterations to fruit taste-related traits, only delaying the accumulation of sugars and decrease in acidity . The lack of upregulation of SlPSY1 in rin appears to contribute to the color defects, consistent with evidence that RIN directly regulates this gene . Both nor and rin exhibited severe delays or inhibition of ripening-related gene expression changes. While highly similar to WT at the MG stage, nor and rin fruit showed large deviations from WT at the RR stage . In fact, the gene expression profiles of nor and rin RR fruit remained similar to those from WT MG fruit. The physiological data generated in this study show nor and rin mutations have different impacts on fruit quality traits. Soluble solids and acid accumulation are negatively impacted in both mutants, but more dramatically in nor fruit. In addition, previous reports have demonstrated a similar pattern among volatile profiles of the mutants at the red ripe stage, with rin again showing more similarity to WT in flavor related traits . This suggests rin fruit are less likely to hinder flavor profiles than nor fruit when breeding for fresh-market hybrid varieties with extended shelf-life. Although nor showed lower quality flavor attributes, its coloration at overripe stages was most similar to WT compared to rin; and thus, it can be useful in breeding hybrid varieties when coloration is a critical fruit trait, such as in the case of processing tomato varieties. Overall, this knowledge will provide valuable information on these tradeoffs of using either loci for breeding programs. Because the Cnr, nor, and rin mutants never acquire equivalent colorations to WT, their ripening stages have been determined based on the fruit’s age expressed as days after anthesis or days after the breaker stage. Sometimes described as BR + 7 days, seedling starter pot the RR stage has been the primary developmental time employed for studying the ripening mutants. As we showed here, the OR stage could provide better comparisons against WT RR fruit for mutants with delayed ripening phenotypes. We demonstrated that in the nor fruit, the RIN and CNR genes only begin to increase in expression in a way comparable to WT at the OR stage . This observation corresponds to over a 10-day delay for some of the ripening processes to begin. The delayed ripening events observed in the OR fruit have not been described before in the spontaneous normutant.Although the Cnr mutant has been assumed to have normal fruit development before ripening , there have been indications that the Cnr mutant displays defects that are not ripening-specific, such as earlier chlorophyll degradation and altered expression of CWDE . We showed that the Cnr mutation causes substantial defects in fruit prior to ripening as seen through statistically significant deviations in fruit size, color, firmness, and TA, ethylene production, and gene expression at the MG stage .

Therefore we propose Cnr may be more accurately described as a developmental mutant and not exclusively a ripening mutant. Further complementing these results, the Cnr fruit displayed large transcriptional deviations from WT that can be traced back as far as 7 dpa . These early development defects are likely a result of reduced CNR expression in the mutant, which is typically expressed in locular tissue before fruit maturity . Our analysis of ripening-related gene expression in Cnr showed striking similarities to WT in the number and functions of genes changing between stages. Moreover, 69.5% of ripening-related DEGs in Cnr were shared with WT . These results further support the hypothesis that Cnr is not exclusively a ripening mutant. Instead, Cnr fruit undergoes gene expression changes consistent with WT “ripening.” However, the ripening related changes in gene expression that occur in Cnr are not enough to compensate for the large defects accumulated in the fruit during growth and maturation. In a recent report, a knockout mutation to the gene body of CNR yielded little visible effects on fruit development and ripening , which suggests that the Cnr mutant phenotype may result from more than just a reduced expression of the CNR gene as previously reported . It has also been demonstrated that Cnr fruit have genome-wide methylation changes that inhibit ripening-related gene expression . The developmental defects observed in Cnr are likely caused by these methylation changes, directly or indirectly caused by the Cnr mutation . Thus, to better understand the Cnr mutation, more physiological data at earlier stages of development needs to be analyzed and complemented with more in-depth functional analysis of gene expression alterations at the corresponding stages. In addition, further molecular and genetic studies need to be performed and compared against complete CNR knockout mutants.Previous reports have shown ethylene levels to be very low or even undetectable in the ripening mutants . Our data support that the mutants never produce a burst in ethylene production, even at the OR stage where more ripening phenotypes are observed . The orange-red pigmentation in nor OR fruit and the similarities of OR fruit in texture and taste-related attributes to WT RR fruit occur independently of an ethylene burst. These observations evidence that other regulatory mechanisms exist to initiate ripening events outside of ethylene . Unlike previous reports, our data consistently showed that Cnr presented increased ethylene levels at the MG stage compared to WT . Interestingly, Cnr fruit produced more of the ethylene precursor ACC than WT at the RR stage. Also, rin made equivalent levels to WT fruit. Ethylene biosynthesis is divided into two programs: System 1 produces basal levels of the hormone during development, and System 2 generates the climacteric rise in ethylene during ripening . Each of these systems is catalyzed by a different set of ethylene bio-synthetic enzymes . It is clear that all mutants show defects to System 2 of ethylene biosynthesis, but they also appear to have alterations specific to System 1. For example, we observed that SlACO3, a System 1-specific ACC oxidase, was higher expressed in Cnr fruit than WT .The role of ABA in climacteric ripening is not as well explored but has been reported to be complementary to ethylene . Previous reports in WT fruit have shown that ABA increases until the breaker stage, just before the ethylene burst . ABA has also been shown to induce ethylene production and linked to the NOR transcription factor . We found that nor and rin fruit did not show decreases in ABA concentration during ripening like WT did . For nor, the constant levels of ABA between MG and RR stages are another example of how fruit ripening events are delayed or inhibited. RIN and ABA have been demonstrated to have an inverse relationship where RIN expression is repressed with the induction of ABA . The significant increase of ABA accumulation in rin during ripening suggests that ABA biosynthesis and metabolism are misregulated in this mutant. rin fruit appear to present a delayed peak in ABA levels compared to WT fruit. More developmental stages, genetic manipulations, and exogenous hormone treatments are needed to investigate further the trends of ABA accumulation seen in the ripening mutants.The interactions between the CNR, NOR, and RIN in ripening have been debated in the literature . The TF RIN directly interacts with NOR and CNR, binding to their respective promoters, and therefore has been proposed to be the most upstream TF among the three regulators . Here we provided evidence that the three TFs display at least indirect effects on each other. We have argued that the Cnr mutant shows a wide breadth of defects across fruit development before ripening begins, and thus, we propose the Cnr mutation is acting before NOR or RIN. This further supports the hypothesis made in Wang et al. that Cnr acts epistatically to nor and rin .

Consequently, recombination between the causal and marker loci will occur during the breeding process and as allele frequencies change with each selection cycle, LD shifts impacting the accuracy of predictive models over time. GS models therefore requires regular updating and as such, model training becomes an important component of a modern breeding system. With the cost of genotyping rapidly decreasing and the recent release of multiple chromosome-scale, haplotype-phased genome assemblies for alfalfa , genomics is becoming a viable option for many smaller breeding programs. Recently the use of GS has been investigated by alfalfa breeders for biomass yield , forage quality and salinity tolerance . However, these studies were based on phenotypic and/or genotypic data at the individual plant level. Although useful, alfalfa is often evaluated at the family level using half- or fullsib families and then marketed as a synthetic population. Andrade et al. proposed GS may be better incorporated into an alfalfa breeding program by genotyping pooled families to obtain allele frequency marker data rather than individual genotyping calls. One major takeaway from much of the work in GS is the size of the training population plays a key role in the predictive ability of the final model . However, bucket flower for lesser funded breeding programs, genotyping and phenotyping can quickly become prohibitively expensive with the inclusion of more material.

Another is incorporating remote sensing and high throughput phenotyping to reduce the expensive labour component of phenotyping the training population.Plant phenotyping is a core foundation of plant breeding and has evolved through the years. Accurate and rapid measurement of phenotypic data is essential to understanding the genetic basis for plant traits and for the subsequent generation of improved germplasm. For biomass yield in alfalfa this traditionally required the destructive sampling, drying and weighing of hundreds to thousands of experimental units multiple times over the 2-4 year lifespan of alfalfa breeding trials.This process is labor-intensive, time-consuming, and costly. Recently, improvements in camera technology, aerial photography, and data processing have resulted in the broad adoption of remote sensing and high throughput phenotyping in agriculture which can significantly reduce the high labor cost of phenotyping. Remote sensing allows for the accurate, efficient, and non-destructive estimate of biomass and has been shown to be useful for high throughput phenotyping in breeding applications , including the prediction of biomass yield in large alfalfa breeding plots . What is not yet clear however, is whether the same predictive ability transfers to the variety of other plot types used in alfalfa breeding; family rows and minisward plots.

This is of particular interest for training a genomic selection model where upwards of 1000 families need to be evaluated for optimal predictive ability . The benefits of successfully incorporating HTP into an alfalfa breeding program will be twofold. Firstly, it will enable trial sizes to increase, benefitting not only training populations for genomic selection, allowing greater prediction accuracies, but will be useful to upscale traditional evaluation trials. Secondly, non-destructive biomass measurement will allow the tracking of growth rates throughout the season and other temporal traits, something that is not currently feasible with traditional destructive harvest methods.Alfalfa is one of the most widely grown perennial forage legumes in temperate and Mediterranean-climate regions worldwide , owing to its exceptional yield, high nutrition, broad adaptability, nitrogen fixation, and host of beneficial ecosystem services . Alfalfa hay grown in California predominantly supports the largest dairy sector in the USA, but also provides forage for sheep, beef, and horse production as well as a growing export market . Alfalfa is an allogamous autotetraploid and is characterized by severe inbreeding depression . Alfalfa is highly heterozygous, and cultivars are synthetic populations that exhibit high variability . Most breeding programs currently utilize recurrent phenotypic selection, where the best genotypes are recombined following evaluation trials that typically last 2-4 years . Despite the numerous benefits of alfalfa, the economic viability of alfalfa is under threat from an increasing yield gap relative to major cereal crops and other potential substitutes in the dairy ration . This yield gap has developed due to low rates of genetic gain for forage yield in alfalfa, particularly over the last 30 years where progress has stalled completely . This lack of yield improvement can be ascribed to a range of factors common in outcrossing perennial forages, namely long selection cycles, multiple harvests per year,small breeding investment, the inability to develop hybrids, the harvesting of all above ground biomass , the need to maintain forage nutritive value, and significant genotype by environment interaction . However, yield improvement has occurred in other perennial forages such as perennial ryegrass and white clover ; therefore, progress should be possible in alfalfa. Lamb et al. suggested that the lack of yield improvement in alfalfa is because less breeding focus has been placed on yield, instead there has been a focus on improving tolerance to biotic and abiotic stresses.

Although this enables alfalfa to reach its yield potential, it is not increasing yield per se in populations under improvement. Furthermore, alfalfa yield is often selected indirectly based on evaluation of vigor on spaced plants or on family rows , which has been shown to be a poor proxy for forage yield in the dense swards used in commercial alfalfa production . Marker-assisted selection is a useful tool for plant breeding programs and may be one way to improve the rate of genetic gain. Early research enabled breeders to identify molecular markers strongly linked to quantitative trait loci for a variety of important traits in alfalfa . However, MAS is primarily effective for traits controlled by relatively few genes with large effects. Complex traits, including yield, are usually controlled by many loci with small effects . In this case, genomic selection offers a compelling alternative to MAS by using a model that includes the effect of all markers in computing a genomic estimated breeding value for each individual in the population. Genomic selection can address one of the largest impediments to faster genetic gain in alfalfa – the need for multi-year evaluations that extend the length of each selection cycle. Selection can be made on genotypic information alone without the need for phenotypic evaluation, reducing the cycle time length from3-5 years to less than 6 months. With the cost of high-throughput sequencing decreasing and the recent publication of multiple chromosome-scale, haplotype phased genome assemblies for tetraploid alfalfa , the prospect of a robust genomic selection program is now possible for many alfalfa breeding programs. Various studies have investigated the use of GS in alfalfa breeding for a range of traits including biomass yield, cut flower bucket forage quality and salinity tolerance. Moderate prediction accuracies were obtained for biomass yield, stem digestible neutral detergent fiber , and leaf protein content, ranging from 0.3-0.4 . The vigor of alfalfa under salt stress has also been assessed and a predictive model developed with a prediction accuracy of 0.793 . Although the results of these studies suggest GS could be used to increase the rate of genetic gain for a rage of traits in alfalfa, no empirical demonstration of GS has been published to date. We hypothesized that using genomic selection for high yield based on a model developed from the phenotypic evaluation of clonally replicated genotypes would result in higher yield than a population selected by genomic selection for low yield or than phenotypic selection. The objective of this study was to empirically test populations developed from a genomic selection model for forage dry matter yield in densely sown sward plots of alfalfa.The germplasm used for genomic selection derived from a population created by Dr. Don Viands at Cornell Univ. in the 1990s called NY0358. We previously described this population, the NE-1010 clonal selection population, in an experiment using SSR markers for association analysis . Briefly, NY0358 was formed by intercrossing three elite, semi-dormant cultivars and recombining the resulting population twice. The NY0358 population underwent two cycles of selection for biomass yield using clonal evaluations at multiple locations .

About 200 individual plants were included at the beginning of each cycle. These plants were clonally propagated using stem cuttings, and three replications were planted to the field at each location. In each replication, three clones were included in a plot; thus, each individual genotype was replicated nine times at each location in each cycle. Yield data were collected across multiple harvests and multiple years on individual plots, bulking the biomass of the three clones within the plot. In the first cycle, data were obtained from Ithaca, New York; Ste.-Foy, Québec; and Ames, Iowa. The top yielding 10% of genotypes selected based on an across location analysis of total annual yield were recombined to form NY0847. A second cycle of phenotypic selection was conducted using NY0847; genotypes were clonally propagated as for Cycle 1 and yield data collected from Ithaca, NY and Ste.-Foy, Québec. The best 10% of genotypes from NY0847 based on a statistical analysis of total annual biomass yield from NY only were intercrossed to form NY1221 .For genomic selection, we used a model developed from the initial clonal evaluation cycle total annual yield measured in NY only, because these data were more robust than those from the other locations . We based the model on total annual yield, which is a more important trait than yield of any individual harvest. We applied the model to seedlings from the population NY0847 and subsequently conducted a second cycle of genomic selection. We grew 19 or 20 individual seedlings from each of the 20 maternal families composited to create NY0847, for a total of 384 individual seedlings genotyped using GBS, as described previously , multiplexing 100 genotypes in a single lane of a HiSeq 2000 DNA sequencer. We aligned sequences with previously determined sequence tags to only analyze SNP that had been part of the model . Following SNP scoring and imputation, we computed GEBVs for each individual plant. Based on GEBVs, we selected the top 20 genotypes, restricting selections to no more than four individuals from any given maternal half-sib family to maintain variation in the population. These 20 individuals were intermated in the greenhouse by hand without emasculation to form the GSC1H population. An analogous population, GSC1L, based on the lowest 20 GEBVs was also formed. In addition, a random selection of 20 plants from the 400 plant population was intermated as a control population, GSC1R. For the second cycle of selection, seeds of each maternal half-sib family used to form GSC1H were germinated and DNA from 19 or 20 plants from each of the 20 families was isolated for a total of 384 plants analyzed with GBS markers. We again selected the top and bottom 20 individuals based on GEBVs as done for Cycle 1 and intercrossed them separately in the greenhouse to create GSC2H and GSC2L, respectively.Experiments were established in April 2017 at two locations in the United States each consisting of ten replications laid out in a randomized complete block design. The two locations were the Cornell University Research Farm in Ithaca, NY on a Niagara silt loam and 973 mm average annual rainfall; and Tulelake, CA , on a Tulebasin mucky silty clay loam . The sowing rate was 20 kg ha-1 with plots measuring approximately 1.5m × 5m. Each plot contained 8 rows spaced 17cm apart. An alfalfa border was sown around the entire experimental plot area. Soil tests were conducted at each location and fertilizer applied to maintain P and K at recommended levels for high yielding perennial forages . Trials were monitored for weeds, insects, and mammalian pests, with control measures conducted accordingly. Forage yields were estimated by mowing a swath through each plot leaving a residual of 7 cm. Prior to harvest, alleyways between plots were mown to remove edge effects and ensure plots were of uniform length. The target maturity for harvest was bud to early flowering stage. In Ithaca the harvest area was 1- by 4m. There was a total of nine harvests, three in each of 2018, 2019 and 2020, with no data collected in the establishment year. In Tulelake the harvest area was 0.9- by 4-m. There was a total of 12 harvests, three in 2017, four in 2018 and 2019, and a single harvest in 2020.

To avoid contamination when handling anthers and pollen, I cleansed our fingers and tweezers by splashing fresh alcohol before and after every use. When possible, each of the mix pollen crosses was replicated at least twice on each plant. Seed paternity – We collected at most five young leaves when available or any leaves from all father, mother, and offspring plants. The tissue collected in the field or at the greenhouse was immediately packed inside labeled clear plastic envelopes, placed inside a cooler with dry ice and promptly transferred to be stored in -80° C until initiating the DNA extractions. We determined paternity by genotyping microsatellite loci or short tandem repeats previously used for Brassicaceae species . Total genomic DNA was extracted from 300 mg of leaf tissue collected using the DNAeasy Plant Mini kit . We followed the kit’s instructions only modifying the elution step by reducing the amount of buffer to 50 µl to yield 100 µl of final product. Ten pairs of previously developed primers for Brassicaceae were initially tested and screened for amplification and detection of polymorphism among the five fathers. DNA concentration was quantified using a micro-volume UV-vis spectophotometer Nanodrop 2000 . Among those ten primers I chose the four most informative and polymorphic comparatively among the five fathers .Polymerase chain reactions amplifications of the four loci were performed in a 20 µL total volume with X 0.3 U of Taq polymerase , 2 µL of 10X buffer , 10 mM dNTP, 10 µM/L primers, 10 µM M13 dye and 1.2 µL of ~5-40 ng total DNA. For each locus, procona London container the forward primer had a M13 tail labeled with a fluorescent dye .

A pigtail sequence was attached to each reverse primer to avoid scoring problems due to genotyping errors as a result of adenosine addition artifacts . Amplification was performed as follows: 94o C for 5 min, 30 cycles of 94o C for 30s, 56o C for 45s, and 72o C for 45s followed by 8 additional M13 tail cycles of 94o C for 30s, 53o C for 45s and 72o C for 45s and a final extension of 72o C for 10 min. Analysis of microsatellite fragment size for all four loci were done in a Big Dye Terminator v3.1 sequencing chemistry .Within-fruit seed characteristics – Data were normalized when needed and feasible with log-normal or Box Cox transformations . Significant probability values were adjusted a posteriori with sequential Bonferroni tests to adjust for type I error . One-way analyses of variance were used to compare seed weight at the lineage and population level. Significant results were followed by TukeyHSD post hoc tests for multiple paired comparisons of means at the lineage and populations levels. To compare within-fruit seed characteristics among different seed positions I used a Kruskal-Wallis tests to compare: seed weight, within-fruit seed weight percentage, and the relative within-fruit seed fecundity. For the purpose of these tests, I discerned among the three types of crosses: either mix or single hand pollination or the control open pollinated plants. Multiple regressions followed by the associated ANOVA were performed to assess the effect of lineage, population, type of cross, maternity, paternity and seed weight.

Comparisons among cross types and paternal siring frequencies at different fruit positions were assessed with goodness of fitness chisquare tests, which were followed by Pearson’s chi-square with 10000 permutations the differences among row were lower than 5. All statistical tests were implemented using the R statistical program and extra statistical R packages were downloaded from the Comprehensive R Archive Network . Paternity – We scored genotypes of father, mother and offspring individuals by visualization of the results in GeneMapper Software 3.7 . A genotype with a single PCR fragment was considered a homozygote having two identical alleles. Visual inspection of allele assignments and manual corrections were systematically done. We employed the exclusion parentage analysis to determine from the pool of fathers used, which one sired a particular seed by comparing the genotype of the three or four father candidates and the known mother to the focal progeny. We determined multiple paternity by comparing the siring fathers at different seed positions within the same fruits. We also assessed whether the fathering occurred in a non-random manner by measuring the frequency at which the siring occurred. Finally, I compared the performance of the fathers by calculating at the offspring siring times, seed weight, within-fruit seed weight percentage and within-fruit relative fecundity and per fathers.Knowing that lineages and populations have a significant influence on weight, I moved on to compare, within lineages, if the type of crosses and within-fruit seed position influence seed weight, within-fruit seed weight percentage and within-fruit seed fecundity . We tested this by using Kruskall-Wallis tests independently for each seed position and seed position bin. The tests were done for each type of cross individually within each lineage. Figures 3.2, 3.3, and 3.4 graphically show the average values from our data set. Within-fruit seed positioning has no statistical significant influence on weight. A trend for heavier seeds at peduncular positions for the hybrid derived lineage CAwr in control fruits can be visualized in figure 3.2. The opposite trend seems to be true for the wild radish Rr. The cultivar Rs has a sinusoidal trend. In the case of the percentage of within-fruit seed weight, seed position per se : influences significantly control, single and mixed crosses fruits of CAwr, and influences significantly mixed Rr and slightly single Rr fruits. Seed position bins only moderately influence within-fruit seed weight percentage in control CAwr fruits. No effects of seed position were found on within-fruit seed fecundity. One-way analysis of variance followed by Tukey post-hoc tests were performed to test if seed position bins for each lineage and type of cross had an effect on seed weight .

The results suggest that only in the case of CAwr control plants is there a significant effect of the bins on seed weight. In this particular case, the Tukey post-hoc test reveals that it is the stylar end bin compared to the peduncular end were the difference lies with a significant negative effect on seed weight. Fecundity and relative fitness – A total of 540 crosses formed viable fruits out of the 595 crosses that I performed. Among the 949 seeds found viable after extraction and first visual inspection, 312 seeds were transplanted to the common gardens at AgOps and among those 247 survived to the end of the experiment. Within lineages, cut flower transport bucket multiplicative fitness functions for mixed and single crosses reveal that total relative fitness was not significantly different among seeds from either cross for both progenitors and marginally different for the hybrid-derived lineage . For Rs the difference was significant as a result of lower viable seed/pod. When the fitness functions for mix and single crosses were compared within each lineage differences were found in all three cases. Surprisingly, mix crosses had lower fitness than single crosses for Rr and CAwr, and the opposite is true for Rs. Differences in fecundity, number of viable pods per pollination and seed viability, while not always significant, affect the overall fitness. Paternity – We found evidence of within-fruit multiple paternity, i.e. seed sired by different fathers within the same fruit, for all three lineages: 11 out of 11 fruits for CAwr, 9 out of 11 for Rr and 1 out of 2 for Rs. Fruits from single crosses had all seed sired by the chosen father. As mentioned before, seed viability was an issue for our experiment. Very few fruits with all or most seeds survived to the end of the experiment, in particular for Rs . For this reason I cannot accurately assess if the multiple paternity is or is not random. Also, because I had so few plants from Rs that survived until the end of the experiment, I eliminated them from the rest of the analysis . The percentage of siring, seed weight average, within-fruit seed percentage average, and within-fruit seed fecundity average for CAwr and Rr for mixed and single crosses are represented in appendices I.1, I.2, J.1, and J2. The siring success of the five CAwr fathers is provided in table 3.7. Each column contains the results by individual father, and each row within a division is the value per seed position bin. All of the values in table 3.7 pertain only to mixed crosses with CAwr and Rr mothers because there was insufficient sample size to analyze the crosses with Rs mothers.

The number of seeds sired in each of the three seed position bins did not differ significantly by father. However, when the number of seeds sired by each father was expressed relative to the number of times a particular father was included in a mixed pollination, the percentages of siring were significantly different. Father 1 and father 2 had higher success siring than the other 3 fathers . Their total percentage of siring exceeds 100% because they sired seeds at least twice within same fruits. Father 2 was the most successful siring at both stylar and peduncular portions of the fruits. Seed weight average and within-fruit seed weight did not differ significantly among fathers. In contrast, the highest average within-fruit seed weight was found on seeds sired by father 2 at peduncular position. The within-fruit fecundity did differ significantly among fathers. Father 2 had higher fecundity relative to the other fathers, at all seed position bins . In mixed crosses, when I assessed each father at different seed position bins, I found that father 2 has higher and moderately to significantly different: siring percentage, average seed weight and percentage of seed weight at peduncular portions of the fruits However its within-fruit relative fecundity was the same in all seed position bins. Father 3 has a moderately higher average seed weight at stylar positions and significantly higher within-fruit relative fruit of seed at peduncular positions. In single crosses, the only significantly different performances across seed position bins happened for average seed weight of seeds sired by father 4 at middle positions and within-fruit relative fecundity of seeds sired by father 4 and father B at peduncular positions. Father performances vary when pollinating in single and mixed pollen crosses. When I assessed lineage-by-lineage, fathers and seed position bins, I found that CAwr offspring resulting from mixed crosses fruits, father 2 appears to have the highest siring percentage at seeds in peduncular ends, with highest average within-fruit seed weight and fecundity at all sections of the fruit. Father 3 has the highest seed weight at stylar and peduncular ends but high average fecundity only at stylar end seeds . These results are not replicated in the case of single pollen crosses. In the case of Rr offspring resulting from mixed crosses, father 2 also sired the most seeds but this time at the stylar end with highest average within-fruit seed fecundity. Here also the results were not replicated at the single pollen crosses. Allele frequencies in father, mother and offspring are compiled in appendix K. Maternal and paternal effects – Maternal effects are significant at phenological life cycle level with significant effect on days to germination, days to first true leaf emergence, and final plant weight. Paternal effects significantly influence reproductive output such as total fruit production and potential reproduction as well as offspring seed weight . Fathers also marginally influence cotyledon width and days to flower buds emergence. Lineage and population influence both morphological as well as fitness related characters including seed weight, which is consistent with our previous results. Seed weight is also influenced by the type of cross but not by seed position bins. Seed weight influences cotyledon width and days to first true leaf emergence.Previous studies have demonstrated non-radom multiple paternity for the hybrid derived CAwr fruits . Our results show that multiple paternity also occurs in both progenitor lineages. Because very few whole fruits were represented in the offspring that survived until the end of the experiment, I were unable to determine if the distribution of paternal DNA is non-random with respect to seed position within the pod. Across lineages, for mixed crosses only, the father that sired most seeds was the one from which offspring were the most fecund. Mixed and single hand pollinations gave different results for individual fathers at different sections of the fruits.