Tag: agriculture

A Micasense RedEdge-M multispectral camera was mounted to a DJI Matrice 100 drone to collect field imagery. Flights occurred at solar noon the day prior to destructive harvests, and images were captured automatically using DJI Ground Station Pro. The drone was flown at an altitude of 15 m with 80 percent overlap between images. Raw images from each flight were built into field-scale orthomosaics and digital surface models using Pix4Dmapper version 4.3.31. The processed orthomosaics were loaded into QGIS version 3.22.1 for extraction of data at the plot level. First a grid layer was generated using the create grid function of QGIS overlaying each plot . Next a threshold layer was generated from the NDVI orthomosaic to differentiate between plants and soil. This threshold layer was then used to partition the DSM into two separate layers, a canopy DSM and a soil DSM that contained elevation information for each pixel. The zonal statistics plugin was then used to calculate the percent canopy area, mean canopy elevation, and mean soil elevation for each plot. From these statistics we calculated plant height by subtracting the average soil elevation from the average canopy elevation and a canopy volume index by multiplying the average plant height by the percent canopy area.All analyses were performed using R statistical software . A simple linear model was used to analyze alfalfa plant height from both ground measurements and remote sensing estimates in the 2018 dormancy trial. Least squares means for each entry in the trial were calculated using the emmeans package by Lenth . The Pearson correlation coefficient was then used to compare the two forms of data collection.

In the remaining trials, square plastic plant pots the Pearson correlation coefficient was again used to compare ground measured fresh weight with remote sensing estimates for biomass volume.We have flight data from a single harvest in the grass trial and alfalfa dormancy trial, from six harvests in the large sown alfalfa trial and from eleven harvests in the transplanted mini-sward alfalfa trial. The accuracy of drone-based remote sensing varied across the range of plot types and forage species, but overall, a high correlation between the two forms of data collection was observed . The Pearson correlation between the drone estimated volume index and ground measured plot fresh weight was greatest in the transplanted family rows used in the 2018 alfalfa fall dormancy trial and was the lowest in the transplanted mini-sward plots used in the 2020 genomic selection trial . In the large sown plots, there was a greater correlation in the grass trial than the alfalfa trial , although remote sensing was highly correlated with the ground measurements in both. The relationship between remote sensing and ground data appears to be linear in all instances except for the transplanted mini-sward plots. In this trial, differences in plots with high fresh weight were not as well identified by the drone as they were in plots with low fresh weight or in the other types of plots. In addition, there appears to be several instances of high biomass plots registering low volumes from the drone imagery, likely due to lodging.The process of phenotyping in plant breeding is expensive and laborious. In perennial forage breeding programs, which often have limited resources, evaluating large breeding populations is challenging due to repeated harvests across multiple years. Drone-based remote sensing offers a fast and effective method of assessing a large amount of material with little increase in labor. Incorporation of such technology in a breeding program enables breeders to increase selection intensity by increasing the scale of breeding trials and thus improving the rate of genetic gain or to replace manual measurements for laborious phenotyping tasks.

Drone estimated biomass volume serves as an effective proxy for biomass yield across a range of perennial forage breeding plot types as demonstrated in this study. Optimizing trials for the collection of remote sensing data and improving the high throughput phenotyping data analysis and curation pipelines could make the incorporation of this technology into breeding programs routine and could help to address the low rate of genetic gain observed in most perennial forage crops. Fall dormancy is a crucial trait in alfalfa that provides growers with information related to the potential of a cultivar to perform in specific environments. Alfalfa breeders must be aware of the degree of fall dormancy in their experimental populations to ensure their cultivars fit the target environments. The standard test for fall dormancy in alfalfa has a significant labor requirement and necessitates growing a dedicated trial that only provides dormancy data. Remote sensing estimates of plant height align very closely with ground-based plant height measurements for a single location and season and may be a more accurate way to assess plant height due to the large number of data points for each plant. When creating an orthomosaic from drone images, the digital surface model includes height information for each pixel; with the camera used in this trial and at a height of 15 m, each plant was represented by hundreds of data points per plant rather than a single measurement by hand of the tallest point of the plant. This will result in a more representative reading for the height of each plant and subsequently a better estimation of fall dormancy. We have previously shown that alfalfa fall dormancy characterization in sward plots is equivalent to the spaced plants used in the standard test .

Our results here suggest that modifying the fall dormancy standard test to enable remote-sensing height data collected as a matter of course in variety trials could be feasible, saving plant breeders time and money in evaluating dormancy response and providing growers with a more precise dormancy estimate. Although remote sensing has significant potential in forage breeding programs as they currently exist, a number of design and management modifications could be made to future breeding trials to maximize the quality of data being collected. First, anything that affects the soil level or plant height will be reflected in the data. Lodging was the most significant problem that we encountered over the course of this study and is a major issue concerning plant height or volume estimates from multi-spectral aerial imagery as it results in the underestimation of true biomass. Selecting a harvest interval that minimizes lodging is one method to mitigate this issue. Additionally, breeders can adjust flight schedules in anticipation of weather events that may result in lodging, such as high winds or heavy rain. As well as causing lodging, wind can influence the quality of aerial images. Plants that are moving in the wind may cause anomalies when stitching the raw images into an orthomosaic; therefore, weather monitoring is an important consideration in remote sensing as well as having a flexible flight schedule. Machinery traffic and mammalian pests are the main causeof soil level issues that we encountered. Wheel ruts from machinery may lead to overestimation of height and biomass, square pot plastic while mounding from mammalian pests such as gophers and ground squirrels will have the opposite effect. Thus, controlling mammalian pests and avoiding traffic on trials when the ground is soft will result in better remote sensing estimates. The method of data collection used in this study requires some reference to ground level within each plot and designing trials to enable clear ground level identification will improve the data collection process. Large gaps between plots will ensure that a ground reference can be found throughout the trial. A system that we recommend is that used in our grass trial, where alleys were mown between plots prior to flying. This has the advantage of maintaining the selection pressure from competition that plants would experience in a commercial field, while also providing a solid baseline for ground level. It also has the additional benefits of limiting the impact of traffic on the soil level and controlling weeds surrounding the plots. Finally, although not essential, ensuring trials are arrayed on a regular grid with straight lines and even plot sizes will streamline the data extraction process. Overlaying a grid in QGIS requires significant manual adjustment for trial layouts that are not uniform so additional care when planning and planting a trial, or ideally, using a GPS equipped planter will greatly simplify the pipeline. Though beyond the scope of this paper, a host of alternative applications for remote sensing data beyond plant height and volume estimation can be imagined. The non-destructive nature of remote sensing means that a breeder could measure the entire growth cycle of forage crops to make better informed selection decisions. There are wide range of vegetation indices other than NDVI to highlight various properties of vegetation that are not observable to the naked eye.

Forage quality is another key trait that breeders must evaluate for which different vegetation indices may prove useful. Remote sensing imagery also serves as a digital archive of trials that can be revisited to better understand trends in trial development over time.Despite the substantial range of applications for remote sensing data in perennial forage breeding, there are a number of limitations. High throughput data collection, storage, and processing require hardware and software investment and the knowledge to develop an analytical pipeline to extract actionable information. Also, weather plays a substantial role in data collection . Finally depending on the camera and drone, there may be a large initial investment to get set up. Remote sensing offers a promising method to reduce the costs of phenotyping in perennial forage breeding programs with the accurate estimation of important traits. The results from this study suggest that breeders could increase the size of breeding trials without a proportional increase in labor and consequently increase the rate of genetic gain for forage yield Breeders also have a new method of assessing fall dormancy in alfalfa that requires significantly less labor than traditional phenotyping. Ground-measurements supplemented with remote sensing data opens the door for smaller, resource limited breeding programs to adopt new methodology such as genomic selection to help bridge the gap in genetic gain between perennial forages and alternative crops thus ensuring continued inclusion in crop rotations.By value, tomato is the fourth most important commercial crop globally . Tomato is a rich source of minerals, vitamins, and phytochemicals. Post harvest deterioration is among the major challenges for fruit industry, accounting for up to 50% of harvested losses . The primary cause of post harvest deterioration is fruit softening, which decreases fruit shelf-life and increases susceptibility to pathogens . Theoretically, regulating the rate of softening would extend shelf-life and increase pathogen resistance and be an effective strategy to reduce post harvest losses . Fruit softening is result of destruction of the fruits wall’s structural polysaccharides and reduction in intercellular cell wall adhesion . The main components of the cell wall include cellulose, hemicellulose, pectin, and a small amount of protein . Due to the complex composition and structure, many enzymes have been reported to catalyse the fruit softening. The role of polygalacturonase , pectin methyl esterase , bgalactanase, expansin, and pectate lyases regulating fruit texture has been well investigated. Downregulation of the PG and PME genes does not affect tomato fruit softening . Silencing the PL gene in tomato delayed fruits softening and reduced susceptibility to grey mould, implying prolonging fruit shelf-life by genetic modification of cell wall-modifying enzymes is a potential approach . Ascorbic acid , vitamin C, a crucial compound is present in most living organisms . In higher plants, AsA functions as an antioxidant and enzymatic cofactor, playing a crucial role in multiple physiological processes including photoprotection, cell expansion and division, ethylene biosynthesis and abiotic stress responses . As a result of these critical functions in plants and its benefits to human health, AsA biosynthesis, recycling, and accumulation in plants have been extensively investigated. The current consensus is that the L-galactose pathway is the primary pathway for AsA accumulation in higher plants. The structural genes have been identified. L-galactose 1-phosphate phosphatase catalyses the conversion of L-galactose 1-phosphate to Lgalactose in AsA synthesis . It has been reported that the expression patterns of GPP are associated with AsA content in apple and tomato plants under abiotic stress . In Arabidopsis, the VTC4 gene encodes an enzyme catalysing the similarreaction with the GPP enzyme in AsA biosynthesis .

All systems with size-controlling rootstocks were irrigated and fertigated using sub-surface drip to maintain a soil moisture between -20 and -60 cbar throughout the growing season. Microsprinklers were used for irrigation and fertigation in the Nema Quad system. The soil at the site is a well-drained Hanford, fine sandy loam. Weeds were controlled by mowing the row middles and applying herbicides to maintain a 1.5m wide weed-free strip down the tree rows. All systems received a light summer pruning and heavy dormant pruning to establish desired structure and improve light interception. Approximately a week before harvest, total canopy light interception using a ACCUPAR LP- 80 meter was measured in each plot. Harvest occurred on two or three separate days, depending on rootstock and cultivar, during the growing season due to variance in fruit maturity, as is common in stone fruit production. Each data tree was harvested individually, total quantity of fruit produced, and total fruit weight data were recorded which enabled calculation of mean fruit weight per tree. Harvest data were collected for growing seasons 2017-2019. A linear model was created in R markdown for each season and cultivar’s harvest. With each linear model, an ANOVA test was conducted using a 95% confidence level and Dunnett’s method adjustments to identify significant differences among the four orchard systems for both scion cultivars. A true significant difference was concluded if the comparison between two systems had a p-value less than 0.05, a t-ratio greater than 1.68 , black plant pots plastic and a confidence level range that did not include 0.During the 2017 harvest season for the June Flame cultivar, trees in all systems produced commercially acceptable mean fruit size, >200g per individual fruit .

The C-6 Quad system produced significantly larger fruit when compared to the Nema Quad system . In the June Flame 2018 harvest, mean individual fruit weights in all systems were again > 200g, . Although the C-9 Quad system produced large enough fruit for fresh market sale, the mean individual fruit weight was significantly less compared to fruit in the Nema Quad system . The harvest season of 2019 for June Flame had individual fruit weight above 200g in all systems . However, it should be noted that the C-6 V and C-9 Quad systems produced significantly smaller fruit compared to the Nema Quad system . In all three seasons for the June Flame cultivar, the C-6 Quad system produced fruit of equal or larger size than the Nema Quad system. In the 2017 harvest season for the August Flame cultivars, all systems with size-controlling rootstocks produced significantly larger fruit than the Nema Quad system . In a few cases, fruit size exceeded 300g per fruit in systems with size controlling rootstocks, >50% larger than the minimum requirement for large sizing in the fresh market . The harvest season of 2018 for August Flame may have been the most productive of all years for both cultivars, all systems exceeded 250g in mean individual fruit weight . The C-6 Quad system had significantly larger fruit than the Nema Quad system while C-9 Quad had significantly smaller fruit . In the 2019 harvest season all systems produced fruit sizes above 200g but were smaller than fruit from previous seasons . Although the C-6 V and C-9 Quad systemsdid not differ significantly from the Nema Quad system, the C-6 Quad system produced significantly smaller fruit compared to the Nema Quad system .During the 2017 harvest season of the June Flame cultivar, the C-6 V and C-9 Quad systems produced significantly fewer fruit per hectare compared to the Nema Quad system.

There was no significant difference between the C-6 Quad and Nema Quad systems . For June Flame in 2018 there were no significant differences in yield per hectare among the C-6 V, C-6 Quad and Nema Quad systems. The C-9 Quad system produced significantly fewer fruit per hectare compared to the Nema Quad system . The 2019 June Flame harvest had close to identical fruit count per hectare between the C-6 Quad and Nema Quad systems .Once again, the C-9 Quad was the only system that produced significantly fewer fruit per hectare compared to the Nema Quad system . In August Flame’s harvest of 2017 there were no significant differences among systems using size-controlling rootstocks and the Nema Quad system for fruit produced per hectare . In the 2018 harvest season for August Flame, the C-9 Quad system produced significantly fewer fruit per hectare compared to the Nema Quad system . Meanwhile the C-6 V and C-6 Quad systems maintained similar fruit counts per hectare as the Nema Quad system. In the 2019 harvest for August Flame the C-6 V system produced significantly fewer fruit per hectare than the Nema Quad system. The C-6 Quad and C-9 Quad systems did not differ significantly for fruit count per hectare compared to the Nema Quad system . It should be noted that during the 2019 harvest season some trees displayed signs of water stress in the field which may have hindered production and skewed results for that season.During the 2017 harvest of the June flame cultivar, there was a significant difference in the slope of the relationship between fruit size and fruit per hectare among the C-6 V and the Nema Quad systems . Data from all systems fit a linear model that had a negative correlation between fruit size and fruit per hectare. Although a negative correlation was visible between fruit size and fruit per hectare in the C-6 V system, its magnitude was not as steep as with other systems in the same season . The following season, 2018, for June Flame there were significant differences in the fruit size vs. fruit per hectare relationship among systems. . Even though no significant differences were detected with the ANOVA analysis, linear models were weak at representing the relationship between fruit size and fruit per hectare and all systems using size-controlling rootstocks had an R-squared value <0.15 .

Continuing the trend from the previous season, in 2019 for June Flame, there were no significant differences in the slope of fruit size vs fruit per hectare relationship for any of the systems . The contrast between the C-6 Quad system and Nema Quad system did have a t.ratio with a greater absolute value than 1.68, however the P.value for the same comparison was still greater than the designated alpha, > 0.05. In this same season the C-6 Quad system had the best fit for the linear model showing a negative correlation between fruit size and fruit per hectare. All other systems fit the model poorly and also did not indicate a clear negative correlation between fruit size and fruit count per hectare . For the August Flame harvest of 2017, data from all systems fit linear models that showed a negative correlation between fruit size and fruit per hectare . Values for the t. ratio between the C-9 Quad and Nema Quad systems were beyond the absolute limit but had a P. value greater than the declared alpha, thus no significant differences were confirmed .For the 2018 harvest of August Flame there were no significant differences in the fruit size vs. fruit per hectare relationships detected among systems . Linear models fit 2018 August Flame data better than other years and showed a clear negative correlation between fruit size and fruit per hectare . In 2019 there was a wide spread of mean fruit sizes per tree in the August Flame data and no significant differences occurred among systems for the relationship between fruit size and fruit per hectare . Although the ANOVA analysis did not indicate differences among systems, linear models indicated a weak negative correlation between fruit size and fruit per hectare with all systems having near horizontal models accompanied by Rsquared values <0.1 .Although R-squared values for the linear models representing the relationship between fruit size and fruit per hectare were identical to those for fruit size and fruit per tree , large plastic pots for plants there were differences detected in the contrast analysis for slopes . Data for the June Flame 2017 and 2018 harvest seasons indicated no significant differences in the relationship for fruit size vs fruit count per tree among any of the systems . In 2019 there was a significant difference in the data for the June Flame cultivar between the C-6 Quad system and the Nema Quad system . In the 2017 harvest data of August Flame there was a significant difference in the fruit size vs. fruit per tree relationship among C-6 V and Nema Quad systems . The difference in 2017 data was visually apparent in the steeper slope indicated in the C-6 V system but that might be a result of the narrow range of fruit loads per tree in that system . No significant differences in the fruit size vs. crop load per tree relationship were detected in the harvest season of 2018, however both, C-6 Quad and C-6 V systems, had t. ratios indicating one may exist, but p-values remained above alpha, therefore a difference was not conclusive . Data for the 2019 harvest of August Flame indicated no significant differences in this relationship between systems, and in fact, the fruit size vs. crop load per tree relationship were most similar among systems in this year compared to other years .

A relationship between light interception and yield was most apparent in the June Flame cultivar with the C-6 Quad and C-9 Quad systems which produced data that fit linear models with the highest R-squared values. The linear model for the Nema Quad system had the steepest slope but not a very strong R-squared value. Data from the C-6 V system had a poor fit with a linear model. Interestingly the systems with data that had a poor fit to the model also had the highest % light interception, often >50% . August Flame cultivars showed a similar pattern for the relationship between amount of light intercepted and yield. Data from the Nema Quad and C-6 V systems had poor fits to the linear models but also had the highest light interception. Data from the C-9 Quad system had a moderate correlation between PAR and yield, fit the model best. The C-6 Quad system is an apparent outlier, having a value of almost 5 Kg/m2 yield with only about 40% light interception, and a very slight negative correlation between the two parameters . Both of the C-6 V systems with the June and August flame cultivars had trends as shown in previous research, higher density systems were able to intercept a higher proportion of light during earlier years because the trees fill their allotted space more quickly, .The mean fruit size for the June Flame cultivar in 2017 was similar among all systems, most likely a result of consistent thinning resulting in the desired crop loads per tree. In 2018 the mean fruit size for June Flame systems was exceptionally large, especially for an early bearing cultivar. Considering that the C-6 Quad and C-6 V systems had some of the largest fruit sizes provides strong evidence that size-controlling rootstocks are not always associated with reductions in fruit size. The C-9 system had poor performance in the trial but, with its success in previous studies and how well systems with the more size controlling rootstocks performed in this trial, it is likely not due to the reduced hydraulic conductance associated with size controlling rootstocks . June Flame systems in 2019 closely mirrored fruit sizes from the previous season, providing more confidence that any reduction in fruit size compared to the Nema Quad system is unlikely a result of size controlling rootstocks. The results from the June Flame cultivar are most promising because there were concerns that the size-controlling rootstocks may have the potential to have negative effects on fruit size in early maturing cultivars. With how quickly early bearing cultivars must set and mature fruit during the spring flush growth, there was concern that reduced hydraulic conductance associated with undeveloped xylem would influence fruit size . However, this trial did not provide evidence that early maturing cultivars on the size-controlling rootstocks produce smaller sized fruit compared to those on more vigorous rootstocks. With the August Flame cultivar, systems using size-controlling rootstocks also were not found to diminish fruit size in this later maturing cultivar. In 2017 all dwarfing systems performed beyond expectations.

Furthermore, Burkholderia seminalis strain ASB21 was found to be able to produce the plant hormone auxin, promote rice seedling growth, and reduce aluminum toxicity symptoms in host plants. Similarly, a Burkholderia seminalis strain isolated from Bangalore, India can produce indole acetic acid and enhance tomato seedling growth. Although it is known that Burkholderia seminalis belongs to the plant-growth-promoting rhizobacteria , only limited strains and their promoting abilities are well characterized. In this study, we examined the amounts of IAA produced by B. seminalis strain 869T2 in various growth conditions, detected the strain’s siderophore synthesis and phosphate solubilization abilities, and demonstrated its growth-promoting abilities in several leafy vegetables, including pak choi, lettuce, and amaranth.Various growth parameters of different plant species were measured at selected days, ranging from 14 to 80 days, after inoculation with strain 869T2. The fresh weight, dry weight, and length of leaves and roots as well as the width, number, and surface area of leaves were measured in harvested pak choi, lettuce, and Chinese amaranth as described previously. The fresh weight, length, number, and color of fruits of hot pepper and okra were recorded following previously described methods . Chlorophyll was extracted from the leaves with N, N-Dimethylformamide for 1 hour in the dark, square plant pot and chlorophyll a and b concentrations were calculated from the absorbance of the crude extract at 647 and 664 nm. Anthocyanin concentrations were determined using a published acidified methanol method. Hot pepper fruits were first ground with liquid nitrogen.

Acidified methanol was then mixed with the ground materials for 10 min in darkness with shaking. These crude extracts were subsequently mixed with an extraction solvent containing 1:1 chloroform:water to isolate anthocyanins. After centrifugation, the absorbance of the supernatant was read at 530 and 657 nm by the spectrophotometer, and anthocyanin contents were calculated from these values.The effects of pH were also examined by culturing strain 869T2 in LB media at 30 ◦C over a pH range of 4 to 9. Strain 869T2 was able to grow over this entire pH range . The results shown in Figure 1D demonstrate that IAA production was at a similar level when bacteria were grown at pH 6 to 9, whereas the IAA amount decreased 44.0% when bacteria were grown at pH 4. Additionally, three different sugars, glucose, fructose, and sucrose, were used in the minimal medium to examine the effects of different carbon sources on IAA production. Strain 869T2 grew similarly in the M9 salt media with different kinds of sugars . The results shown in Figure 1F indicate that when strain 869T2 was grown in the media with two kinds of monosaccharide, glucose and fructose, the IAA amounts were higher than for the bacteria grown in the media with sucrose. We further investigated whether strain 869T2 had other plant-growth-promoting traits, including siderophore production and phosphate solubilization abilities, with agar plate assays. Supplementary Materials Figure S1A shows that the strain 869T2 colonies exposed to CAS agarose turned yellow, indicating the siderophore production ability of strain 869T2. Furthermore, Figure S1B reveals that the formation of halos around the strain 869T2 colonies grown in Pikovskaya’s agar medium with 0.5% tricalcium phosphate suggests that strain 869T2 may have the ability to solubilize phosphate.Seedlings of ching chiang pak choi and pak choi from the Brassica genus were also inoculated with strain 869T2 to examine its effects on plant growth.

At 27, 33, and 40 days after inoculation with strain 869T2, the average fresh weight and dry weight of above ground leaves of ching chiang pak choi were higher than those of the control plants . Furthermore, the average leaf length and width, petiole length and width, number of leaves per plant, total leaf area per plant, and leaf area per leaf were greater in the 869T2-inoculated ching chiang pak choi compared to the control plants . The results shown in Figure 3J,K demonstratethat the average plant height and width of the 869T2-inoculated ching chiang pak choi were also greater compared to the control plants. Similarly, after the ching chiang pak choi was inoculated with strain 869T2, the average values of root fresh weight, dry weight, and length were higher in comparison to control plants . Figure 3O–Q indicate that both the aerial and below ground parts of ching chiang pak choi were larger after inoculation with strain 869T2. Figure 3R also shows that the ching chiang pak choi inoculated with strain 869T2 grew faster and flowered earlier than control plants 53 days after inoculation. Similarly, after inoculation with strain 869T2, the pak choi grew larger, including larger and more numerous leaves, larger aerial parts overall, and longer and heavier roots . These data indicate that inoculation of strain 869T2 in two vegetables from the Brassicaceae family significantly improved their growth.Because B. seminalis strain 869T2 successfully colonized Arabidopsis and two types of plants from the Brassicaceae family and promoted their growth, we further examined whether strain 869T2 could promote the growth of plants from the Asteraceae and Amaranthaceae families. At 35, 43, 50, and 56 days after inoculation with strain 869T2, the fresh weight of the aerial parts of inoculated loose-leaf lettuce plants increased 12.7- to 46.6-fold compared to the 0-day post-inoculation plants . By comparison, in the mock-inoculated control plants, the fresh weight increased 8.0- to 36.0-fold over the same period .

Similarly, the dry weight of the inoculated loose-leaf lettuce increased more than that of the control plants at 35, 43, 50, and 56 days after inoculation . These data indicate that inoculation of the loose-leaf lettuce with strain 869T2 significantly enhanced plant growth. The weight increases of the inoculated loose-leaf lettuce plants were due to increases in average leaf width and length , the number of leaves per plant , total leaf area per plant and per leaf , and plant height and width . Furthermore, the root fresh weight of the inoculated loose-leaf lettuce plants increased 4.5- to 12.4-fold at 35, 43, 50, and 56 days after inoculation compared with the 0-day post-inoculation plants ; in contrast, that of the mock-inoculated control only increased 2.5- to 8.5-fold compared with the 0-day post-inoculation plants . Additionally, the root dry weight and length increased more in the inoculated loose-leaf lettuce plants than in the control plants . As seen in Figure 4M–O, overall plant size and leaf size increased after inoculation with strain 869T2, suggesting that strain 869T2 improves loose-leaf lettuce growth.We also inoculated strain 869T2 into romaine lettuce and red leaf lettuce. The results shown in Figures S4 and S5 demonstrate that both kinds of lettuce grew taller and wider, had more and larger leaves, and had heavier aerial and below ground tissues after inoculation with strain 869T2 compared with the control plants. The chlorophyll contents of red leaf lettuce leaves were also higher in the 869T2-inoculated plants than the control plants . These data collectively indicate that the three evaluated kinds of lettuce can grow significantly better after inoculation with strain 869T2. We also selected Chinese amaranth of the Amaranthaceae family to test the effect of strain 869T2 on its growth. At 36, 43, and 50 days after inoculation, the fresh weight of the 869T2-inoculated Chinese amaranth exhibited a 20.0- to 56.6-fold increase when compared to the 0-day post-inoculation plants, square pot whereas the control plants only showed an 8.3- to 33.5-fold increase when compared to the 0-day post-inoculation plants . Other plant growth parameters of the 869T2-inoculated and control plants were also examined 36, 43, and 50 days after inoculation . Figure 5 illustrates that the 869T2-inoculated Chinese amaranth individuals had more and larger leaves, were taller and wider, and had heavier and longer roots than the control plants. These data show that inoculating strain 869T2 into Chinese amaranth promoted its growth.Because B. seminalis strain 869T2 promoted the growth of several leafy vegetables, we next tested the effects of the strain 869T2 on the flowering and fruit production of hot pepper and okra . Hot pepper plants, from the Solanaceae family, were inoculated with strain 869T2 but we did not observe significant growth promotion effects on the aerial and root parts of the plants. However, we did observe that the 869T2-inoculated hot pepper plants flowered 20 days after inoculation; the number of flowers continually increased and had more than a 7-fold increase at 37 days after inoculation . In the mock-inoculated control plants, we observed flowering 21 days after inoculation, and the number of flowers had only increased 5-fold at 37 days after inoculation . The average number of fruits on the 869T2-inoculated plants was higher than that on the control plants at 30, 37, 44, and 51 days after inoculation . The average numbers of flower buds, flowers, and fruits per plant were higher in the 869T2-inoculated plants than in the control plants beginning 21 days post-inoculation . Furthermore, the percentages of hot pepper fruits with red and green/yellow coloring were higher in the 869T2-inoculated plants than in the control plants 59, 66, 73, and 80 days after inoculation . Similarly, the average anthocyanin contents of the 869T2-inoculated plants were significantly higher than those of the control plants at 66, 73, and 80 days after inoculation . However, the average length, width, and fresh weight of the fruits were not significantly different between the inoculated and control plants .

Collectively, these data suggest that the inoculation of hot pepper with strain 869T2 could increase flowering and fruiting in hot pepper plants and accelerate fruit maturation.We subsequently examined the effects of strain 869T2 on okra, which belongs to the Malvaceae family. The overall plant size and weight were not significantly different between the 869T2-inoculated and control okra plants. We observed, however, that the number of nodes of the first flower was smaller in the 869T2-inoculated okra than in the control plants, suggesting that the 869T2-inoculated okra plants flowered earlier than the control plants . In addition, the average fresh weight and diameter of the fruits from the 869T2-inoculated plants were greater than those of the control plants , although the average fruit lengths were similar. These data demonstrate that the okra fruits became heavier and wider after inoculation with strain 869T2. In summary, inoculation of strain 869T2 into hot pepper and okra plants could cause plants to flower at earlier growth stages.The members of the genus Burkholderia belong to the class β-proteobacteria and have a broad distribution, residing universally in soil, water, and in association with plants, fungi, animals, and humans. Some Burkholderia species are plant pathogens in many vegetables and fruits, while others have been reported as opportunistic pathogens of humans and other animals. However, many other Burkholderia species are beneficial to plants, suppressing plant diseases and promoting plant growth by various processes, including the production of antibiotics, secretion of allelochemicals, induction of pathogen resistance in plants, nitrogen fixation, or enhancing nutrient uptake by host plants. These beneficial Burkholderia species are free-living or endophytic and form mutualistic associations with their host plants. Burkholderia species’ high versatility and adaptability to different ecological niches rely on the high genomic plasticity of their large multichromosome genomes and the production of various bacteria secondary metabolites. In this study, we characterized the endophytic bacterium Burkholderia seminalis strain 869T2 isolated from vetiver grass, which was recently described and included in the Burkholderia cepacia complex . We have documented the IAA production, siderophore synthesis, and phosphate solubilization abilities of B. seminalis strain 869T2. Inoculations of strain 869T2 into tested plants demonstrated the plant growth promotion ability of this bacterium in several plant species from the Brassicaceae, Asteraceae, and Amaranthaceae families. Plant endophytic bacteria can increase the nutrient uptake and biomass accumulation of host plants through the production or regulation of various plant hormones, such as auxin, cytokinin, gibberellins, and ethylene. Indole acetic acid is a naturally occurring auxin produced by several endophytic bacterial species through the L-tryptophan metabolism pathway. Tryptophan can exist in the exudates of plants and is utilized by the bacteria to synthesize auxin, which enhances the growth of host plants. Auxin is the major plant hormone that regulates various aspects of plant growth and development, such as root initiation and development, leaf formation, fruit development, floral initiation and patterning, phototropism, and embryogenesis. Several plant-growth promoting bacteria can synthesize IAA, including Bacillus, Burkholderia, and Pseudomonas species. In this study, Burkholderia seminalis strain 869T2 was able to synthesize approximately 2.0 to 2.2 µg mL−1 IAA in the presence of tryptophan and increased both the above ground and below ground biomass of tested plant tissues.

The extracted phytochemicals created inhibition zones against Bacillus subtilis, Staphylococcus aureus, E. coli, and Salmonella spp. of 13, 15, 9, and 11 mm respectively . These results suggest that the phytochemicals found in dried fruits could play a role in the survival of pathogens. Dry fruit related outbreaks. While not many, there have been several foodborne outbreaks associated with low moisture foods . In 2020, an outbreak of the hepatitis A virus associated with dates occurred in the United Kingdom . Twenty eight people were infected and the dates, which were imported from Jordan, were subsequently voluntarily recalled. Another outbreak of hepatitis A occurred in England from semi-dried tomatoes, which infected two people . An outbreak of salmonellosis in Norway was associated with consumption of a Salmonella Agbenicontaminated dried fruit and nut mix . In this outbreak, 39 people were infected. Another outbreak was linked to Salmonella Phage type 13a in a dried vegetable spice mix, in which 108 people were infected in Sweden . Objectives. As discussed above, dried fruits are of great economic importance to California and different processors follow distinct protocols to prepare their dried fruits. In addition to the various drying methods used, pre- and post-drying treatments can also be applied. Unfortunately, there has not been a systematic evaluation of the antimicrobial efficacy of different drying methods or pre- and post-drying treatments or a combination of them.Pathogen contamination of the final products can happen at any processing point. Once happened, it is critical to better understand the behavior of these pathogens in dried fruits. The goal of this study is to fill in the current knowledge gaps associated with microbial safety risks of dried fruit by conducting a challenge study. The three pathogens selected include Salmonella, E. coli O157:H7, and L. monocytogenes. Dried fruits, including peaches, pluots, tomatoes, blueberry grow bag and dates were purchased from local farmers markets for this study.When conducting challenge studies and preparing artificially contaminated food items, there are different carriers that can be used.

The currently available inoculation methods can be grouped into three major categories: a liquid-carrier method, a dry-carrier method, and a no-carrier method. Examples of liquid carriers include peptone water, saline buffers, and ultrapure water. Cocoa butter oil has also been used to carry out inoculation . Dry carriers that have been tested include sand, chalk, and talc . The no-carrier method utilizes freeze- or vacuum-dried cultures or cell pellets and directly applies them to products . Choosing the correct inoculation carrier is critical. Although liquid carriers, such as buffered peptone water or ultrapure water, have been more widely used for delivering pathogens onto product surfaces, the addition of liquid or the introduction of additional moisture into the products changes the moisture content and aw of a dry substrate and may requires additional or extended drying steps . As indicated by Palipane and Driscoll , moisture adsorption/desorption isotherms are inherently non-equivalent, the aw of the product after an additional drying step may not be the same as the original food. Beuchat and Mann used two different methods for inoculating dried cranberries, raisins, and strawberries and date paste. No difference in Salmonella behavior was observed between two inoculation methods . Similar observation was made by Blessington et al. , in which no difference in Salmonella decline was observed between dry-inoculated and wet-inoculated nut kernels. Both studies indicated that when choosing the proper carriers fordried products, the key features or changes that need to be monitored are the physical or chemical properties. In addition, inoculation methods should try to mimic real life contamination scenarios to give the most accurate representation of survival after a contamination event. It is a water-based salt solution containing sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate. It helps maintain the osmolarity balance of bacteria when being used as the carrier for inoculation. However, since another key function if this buffer is to help maintain a constant pH, there is a concern over the use of PBS for inoculation and sample homogenization when measuring the pH of inoculated dried fruits. In this case, before the long-term survival study, the impact of PBS on the pH measurement needs to be studied. An efficient recovery method that can release and recover target bacteria from food surfaces, is the foundation for accurate detection and enumeration.

Stomaching, shaking, rubbing, sonication, pummeling, pulsing, as well as blending have been tested and used for releasing and recovering bacteria from food or environmental samples . Based on the various physical and chemical properties of the samples, the efficacy of these cell recovery method change. As shown by Kim et al. , bacterial populations recovered from pummeled and pulsed iceberg lettuce, perilla leaves, cucumber, and green pepper samples were higher than those recovered from sonicated and hand-shaken samples. However, this trend was not observed on cherry tomatoes. Thus, which bacteria recovery method is more appropriate for sand-inoculated dried fruit needs to be determined.Dried fruits. Dried fruits used for this survival study were purchased from local farmers markets. Fruits used included sundried tomatoes, peaches, peaches processed with sulfur, pluots processed with sulfur, low-moisture Medjool dates, and high-moisture Medjool dates . High-moisture dates are harvested directly from the tree; low-moisture dates are dates allowed to continue drying in nets after falling off the tree and have a harder texture compared to the high-moisture dates. Tomatoes and the sulfured peaches and pluots were sun dried. The peaches that were not processed with sulfur were dried using a dehydrator. Once purchased, the dried fruits were stored at room temperature for up to 1 week prior to use in experiments. Inoculation with wet and dry carriers. Dried fruits were combined with either water or sand by the following methods. Briefly, ultrapure water was added to every 100 g of each of dried fruits and massaged by hand for 1 min. The dried fruits were then transferred to plastic containers with drying racks lined with filter paper . The lids of the containers were taped down slightly ajar with a piece of mesh to cover the opening . The containers of fruit were set out to dry at room temperature for 48 h.

For sand inoculation, 20 g of sand was added to 100 g of each dried fruit and massaged and shaken for 1 min. The dried fruits were then transferred to gallon storage zipper bags and stored at room temperature. For the water-inoculated samples, the pH and aw was measured before and right after the inoculation. After 48 h, the pH and aw of both the wet and dry inoculated dried fruits were both measured. A pH meter and water activity meter were used to take the measurements. Effect of phosphate buffered saline on pH measurement of dried fruits. Phosphate buffered saline was chosen as the wet carrier with which to inoculate the dried fruits. Since PBS is a buffer solution, the impact of PBS on the pH measurement of dried fruits was tested. To do so, dried fruits were combined with either ultrapure water or PBS and massaged and shaken by hand for 1 min. The pH of the sample was measured before the addition of the liquid, immediately after massaging, and after 48 h of drying. To measure the pH, blueberry grow bag size each fruit sample was combined with water or PBS equal to 40% of the sample mass and then stomached for 1 min at the fast setting to homogenize. The pH meter was used to take the measurements. Bacterial cultures and inoculum preparation. The strains of bacteria used for this study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains of rifampicin-resistant Salmonella were used. The strains are summarized in Table 1.1.Individual frozen stock cultures were streaked onto tryptic soy agar , Sparks, MD, supplemented with 50 µg/mL of rifampicin , and incubated at 37 °C overnight. Each isolated colony was transferred into 10 mL of tryptic soy agar supplemented with rifampin at 50 µg/mL , and then incubated at 37 °C overnight. One 10-µL loopful of the overnight culture was transferred to 10 ml of fresh TSBR and incubated at 37 °C for another 24 h. The newly inoculated broth was spread onto TSAR plates, 250 µL per plate, one plate per strain, and incubated for 24 h at 37 °C. To recover bacterial lawns from plates, 1 mL of phosphate-buffered saline was pipetted onto each plate, and an L-shaped plastic cell spreader was used to loosen and scrape the lawn. The re-suspended cells were then pipetted into a 15- mL Falcon™ tube . The addition of PBS and lawn scraping was repeated two more times for each plate, for a total of 3 mL of PBS used per plate. Approximately 2.5 mL of culture was recovered from each plate. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail of Salmonella. The cocktail was diluted and plated onto TSAR for calculating the inoculum level. Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. Salmonella-inoculated sand was used to test the recovery method used for dried fruit . Twenty grams of sand was inoculated with 1 mL of the 5- strain Salmonella cocktail and they were mixed together by hand for 1 min. Samples of the inoculated sand were sampled immediately after mixing and after 48 h of drying. The drying process was done at 40 °C for 48 h in a gravity oven . At each sampling point, three 10-g sub-samples were taken for the analysis of Salmonellacounts.

Each 10-g inoculated sand sample was divided into two portions . These two portions were added to two 24-oz filter bags together with 95 mL of PBS in each bag. One bag was stomached for 1 min using a smasher , while the other bag was shaken by hand for 1 min. The contents were then serially diluted appropriately with PBS and two 100 µL suspensions from each dilution were spirally plated onto TSA with rifampicin and Xylose Lysine Tergitol 4 agar with 50 µg/mL rifampicin . After 24 h of incubation at 37 °C, colonies were counted and the populations determined. Statistical Analysis. One trial was conducted for every test performed in this section. At each sampling point, three samples were analyzed . Means comparison were performed using Excel to determine whether carrier type had a significant impact on pH and water activity of dried fruits as well as if homogenization methods for inoculated sand had a significant impact on recovery of pathogenic cells. Differences between mean values were considered significant at P < 0.05.Inoculation with a wet or dry carrier. Table 1.2 shows the pH and water activity of dried fruits before and after the addition of either water as a wet-carrier or sand as a dry carrier. In low-moisture dates, the initial pH was 5.83 ± 0.06. With both wet and dry-inoculation, the change in pH was statically significant, dropping to 5.24 ± 0.05 and 5.59 ± 0.04 respectively. The initial aw of the low-moisture dates was 0.62 ± 0.03 and showed no significant change after either wet or dry-inoculation, with their values being 0.64 ± 0.00 and 0.61 ± 0.00, respectively. In high-moisture dates the initial pH was 5.59 ± 0.04. The pH did not change significantly afterwet-inoculation. The pH dropped after the dry-inoculation to 5.39 ± 0.04. The initial aw of the high-moisture dates was 0.55 ± 0.02. No significant change was observed after either wet or dry inoculation. The initial pH of the dried peaches was 3.94 ± 0.07, and neither the wet nor the dry inoculation generated significant change on the pH. Additional loss of moisture might have occurred during the 48-h of drying after wet inoculation, which might be the reason why there was differences between the aw before and after wet-inoculation. A similar observation was made in dried peaches made with sulfur treatment, as the aw decreased after wet-inoculation. Both the wet- and dry-inoculation slightly reduced the pH value of the products, changing from 3.59 to 3.51 and 3.48 respectively. In dried pluots, although neither wet- nor dry-inoculation generated any impact on aw, dry-inoculation reduced the pH of the products . For sundried tomatoes, inoculation had no impact on pH but the dry inoculation significantly reduced the aw of the products .

In addition to ABA, other hormone biosynthesis genes exhibited a similar expression such as auxin and cytokinin, both of which have been implicated in non-climacteric fruit maturation and ripening . Thus, pistachio should be considered a non-climacteric fruit.While ethylene was not important for ripening in pistachio, ethylene may be more critical prior to kernel development at the end of Stage II where a rise in biosynthesis occurs . The exact function of the hormone at this stage of development is unknown but has previously been suggested to be involved in bud abscission in alternate bearing years and needs further investigation . JA-related genes were also elevated during Stage II and were among the highest expressed hormone-related genes. JA is best known as a stress hormone involved in many responses to abiotic and biotic stress but can also function in fruit development . JA biosynthesis genes were previously shown to be differentially expressed in pistachio vegetative tissues that underwent a salinity treatment, compared to the control . However, there were no known stresses occurring during our samplings to explain elevated expression. This suggests JA is a critical hormone in pistachio fruit Stage II development prior to kernel initiation, or other events occurring concurrently. As pistachio is known to be tolerant to environmental stress, JA and ethylene levels may play a role evolutionarily to adapt to these extreme environments.The hull functions as a protective tissue encapsulating the shell and kernel. The breakdown of the hull caused by senescence can lead to a lower quality commodity; for example, plastic grow bag the shell becomes more vulnerable to staining from the hull and the kernel becomes more accessible to pests. The hull is rich in volatile compounds mainly composed of terpenes . We examined the dynamic changes of volatile compounds in the hull during Stage III and IV to gain insight into the events leading to hull ripening and senescence.

We saw a rise of volatile compounds at the onset of ripening that may offer a signal of developmental changes occurring We observed limonene and alpha-terpinolene to be the highest produced monoterpenes consistently across years. Alpha-terpinolene had also been found in high proportions of Tunisian pistachio variety but did not have as high of concentrations of limonene, which could be due to varietal differences . Limonene has been shown to accumulatein orange peel with ripening in order to attract insects and pathogens . This relationship between limonene and other organisms was proposed to have evolved to facilitate seed dispersal, opening up the fruit to expose the seeds. This could also be the case for pistachio, in which the volatile production at the start of ripening signals that the kernel is mature and attracts seed dispersing organisms. Volatile signals further define the events leading to ripening in pistachio and have additional implications for management practices to time treatments against insects.Like canonical fleshy fruit, pistachio fruit quality is determined during fruit ripening. Ripening changes in the hull coincide with important quality traits and can be used to anticipate the best harvest time. Therefore, understanding the timing and relationship between the hull and kernel during Stage IV allows for increased quality. Our study integrates multiple approaches, including physiology, biochemistry, and genomics, to provide the most thorough understanding of pistachio fruit development to date. Fruit ripening in pistachio to our knowledge has not been previously explored. The hull undergoes changes in composition preceding harvest consistent with fruit ripening including, softening and color change which we define as an additional stage, Stage IV . These changes are important attributes that help determine harvest time and maximize fruit quality. For example, hull softening allows fruits to be detached from the tree, however, if overripe the hull senesces and the degradation can cause shell stain and make kernels vulnerable to pests and disease decreasing its nutritional and market value.

We integrated gene expression data with the observed physical changes to inform the events occurring leading to fruit ripening. Color change is a characteristic of ripening and provides a visual indication of when fruit are ready for harvest. Previous studies have identified anthocyanin, carotenoids, and chlorophyll compounds in pistachio hulls . However, the proportion of these compounds present depended on the stage sampled and variety, with measure-ments limited to pistachio green hulls prior to ripening. Thus, it is unclear which specific compounds lead to the pink colorations in the hull during ripening. Our gene expression analysis indicated that the flavonoid pathways were active in the hull, however there was not a strong expression of anthocyanin reductase genes, the critical final step for anthocyanin production, giving the compounds their pigmentation. It is clear that the fruit produce some anthocyanins because they have been identified in the purple colored seed coat surrounding the kernel . We found stronger gene expression of carotenoid biosynthesis. Among the pigments identified in Grace et al., lutelin was the highest measured in pistachio hulls . We observed high expression of ctrZ which is annotated to act in the step leading to lutein biosynthesis. The change in green coloration to yellow at the end of Stage III also indicates chlorophyll degradation may be occurring. We identified several chlorophyll degradation genes expressed in modules with ripening patterns, such as STAY GREEN , a chlorophyllide reductase that regulates chlorophyll protein degradation . Thus, from our analysis we hypothesize that hull color becomes yellow from chlorophyll degradation and shades of pink from carotenoids. It is well known that fruit softening is mediated by cell wall degrading enzymes in both climacteric and non-climacteric fruits, such as tomato and strawberry . Cell wall degrading enzymes acting on the backbone of pectin molecules, such as polygalacturonase and pectin lyase, are highly expressed in other fruit and exhibit a ripening-specific gene expression pattern . Further, α-LAFase is highly expressed and acts as a catalyst with other coexpressed cell wall degrading enzymes . Thus, we were interested if these enzymes were expressed in a ripening-specific pattern. Pectate lyase was among the highest expressed CWDEs annotated from the CAZy database in pistachio and began to rise in expression at Stage IV .

Consistent with this, pectins have been measured in pistachio hulls and were proposed as a potential source of commercial pectins . The presence of a large proportion of pectins in green hulls and the high expression of pectin-degrading enzymes suggest these enzymes promote pectin degradation and softening of the tissue. The mechanisms involved in this still need to be explored. Overall, knowing how and when hull softening occurs during the growing season can help advise the optimal time for harvest.Kernel growth during Stage III leads to the maturity of the seed and ripening of the hull. Understanding when the kernel is most desirable for consumption and when the fruit is ready for harvest can improve management practices and fruit quality. Maturity can be observed when kernels reach their maximum size and fat content at the start of Stage IV, as ripening progresses . From our gene expression and metabolite data we see that fatty acid biosynthesis occurs early on in kernel development and is primarily composed of unsaturated fatty acids, with much of the production reaching its maximum during ripening . Further, pe grow bag the kernel shows hormonal indications of seed maturity with an increase of GA at the start of Stage IV and ABA increasing throughout Stage IV. Pistachio kernels are consumed for their unique flavor and nutritional benefits. Kernels are made up of primarily unsaturated fats, including both poly- and mono- unsaturated fatty acids . Although PUFAs provide essential fruitrients to the human diet, they make kernels more vulnerable to rancidity, reducing their shelf life . Thus, the ratios of PUFA and MUFA are important for considering nutritional benefits and shelf life. We identified important enzymes in our gene expression data explaining the accumulation of specific unsaturated fats . Interestingly, our study showed fluctuations in the composition of unsaturated fatty acids through kernel development similar to a previous study . The mono-unsaturated fatty acid oleic acid increases through time while the poly-unstaturated linoleic acid decreases. These fluctuations were not completely explainable with our expression data, but are likely caused by other fluxes in the fatty acid metabolism downstream of these compounds.Calcium is an essential plant nutrient required for proper plasma membrane function, in storage organelles to counterbalance anionic charges, in the cytosol for cellular signalling responses, and in the apoplast for cell wall structure . Ca2+ deficiency disorders in fruit have been attributed to lower total tissue Ca2+ content, as well as abnormal regulation of cellular Ca2+ partitioning and distribution . The symptoms of Ca2+ deficiency disorders in fruit start with cell plasmolysis and the water-soaked appearance of blossom-end tissues that eventually becomesdark brown as cells die . Although Ca2+ is believed to move in the plant exclusively through the xylem vascular tissue , the mechanisms regulating Ca2+ partitioning and allocation in tomato plants and fruit remain poorly understood. Consistent with xylem sap flow, the direction and rate of xylemic Ca2+ flow in the plant should be determined by water potential gradients in response to different tissue transpiration and growth rates . In that case, higher transpiration and growth rates can reduce water potential and increase tissue strength as sinks for xylemic Ca2+. Therefore, the partitioning of Ca2+ flowing from the roots toward leaves and fruit will depend on the xylem sap Ca2+ concentration, as well as leaf and fruit transpiration and growth rates. Accordingly, leaves have much higher transpiration rates than fruit, which results in much higher Ca2+ content in the leaves than in the fruit .

Previous studies have shown that specifically reducing leaf transpiration by decreasing atmospheric vapour pressure deficit or treating tomato plants with abscisic acid can potentially decrease xylemic Ca2+ movement into the leaves, and increase its movement into the fruit . However, direct measurements of xylemic Ca2+ concentration and xylem sap flow rates into leaves and fruit in response to reduced leaf transpiration rates have not been reported. Spraying whole plants with ABA increases fruit total tissue and apoplastic Ca2+ concentrations, and reduced fruit cell membrane leakage and the incidence of blossom-end rot . These studies suggest that ABA may affect not only total fruit tissue Ca2+ concentration but also the regulation of cellular Ca2+ distribution, which could affect fruit susceptibility to Ca2+ deficiency disorders such as BER . Since these studies were based on whole-plant ABA sprays, the results cannot be specifically attributed to whole plant or fruit responses to ABA . Fruit-specific ABA studies are still needed to understand if the prevention of BER development is a whole-plant, a fruit specific, or a combination response to ABA. The objectives of this study were to determine Ca2+ partitioning and allocation in tomato plants and fruit in response to whole-plant and fruit-specific ABA treatments, as well as to analyse the effect of changes in Ca2+ partitioning and allocation on fruit susceptibility to BER under water stress conditions. The HRM was developed to measure low net sap flow rates that can take place in either direction in the vascular tissue , but for the current study only the xylem sap flow rate was determined by heat girdling the middle leaf pedicel or fruit peduncle. The heat girdling was accomplished by passing an electrical signal for 20 s across a constant an wire with 0.8mm diameter looped twice around the pedicel or peduncle 1 cm upstream of each heat sensor before starting the sap flow measurements . Heat girdling destroys the phloem cells, obstructing phloem sap movement, while the xylem sap flow remains intact and functional due to its non-living cells. This technique has been used to isolate and quantify phloem and xylem sap flow rates . After heat girdling, sap flow measurements were made over a 24h period. After xylem sap flow measurements, zero sap flow readings were determined by cutting the middle leaf pedicel or fruit peduncle 1 cm downstream of each sensor. The zero xylem sap flow readings were used to determine the baseline accurately for each sap flow sensor after sap flow measurements. After determining the zero sap flow rate, the middle leaf pedicel or fruit peduncle was cut at the heat sensor region to measure the diameter of the xylem vascular tissue, which was used to calculate the volume of xylem sap moving into the leaf and fruit over time. One fully expanded top leaf and one tagged fruit on each plant replication were used for the sap flow analysis.

Fruit are rich in essential nutrients, yet most people do not consume the recommended amount of fresh produce to sustain healthy diets and reduce disease risks ERS — Food Availability and Consumption. To promote consumption, expand access, and reduce waste, fruit quality and shelf life need to be in- creased through breeding and biotechnology, alongside adequate harvest practices, transportation logistics, and post harvest treatments. Commercial fruit crops are primarily bred for high yield and extended shelf life to meet the expectations for mass production and global markets, however, recently, there has been a shift of focus toward developing new crop varieties that meet consumer demands for better flavor and nutrition. Balancing shelf life with consumer-based quality traits is perhaps the biggest challenge breeders and researchers face in the quest for better quality fruit, mainly because these attributes appear to have negative genetic correlations in many crops . Quality peaks when fruit reach their optimum ripeness. Many studies on fruit ripening of various plant species have emerged in the past decade, helping to identify genetic pathways and molecular regulators that can be manipulated for crop improvement . Moreover, biotechnology advances have provided access to high- quality genomic resources and tools, supporting breeding strategies, genetic modification, and gene editing in traditional and nontraditional fruit crops. Here, we review current knowledge of the genetics of fruit traits and argue that manipulating transcription factors is a promising approach to enhance fruit quality. We discuss how pleiotropic effects could potentially be avoided by targeting TFs that exclusively regulate specific pathways instead of master regulators. However, ripening master regulators may remain useful if their effects on gene expression can be fine-tuned . Similarly, plastic square flower bucket the timing and coordination of regulators need to be considered to achieve desired effects on fruit traits .

Finally, we consider the current climate surrounding consumer acceptance of genetically modified and gene-edited fruit.Fruit are used as food source for macronutrients and micronutrients . Macronutrients are essential to provide energy and maintain the body’s structure and functions. Vitamins are required for various bodily functions and can only be found in food sources such as fruit. Antioxidants inhibit cell damage caused by oxidative agents. Carotenoids , and phenolics , reviewed above, are all antioxidants, as well as vitamin C . Vitamin-D deficiency is a global health problem due to few dietary sources of this vitamin. Bio-fortification of vitamin D in tomatoes has recently become possible by engineering its biosynthesis from a pre-existing pathway . Owing to partial duplication of the pathway, a single enzyme could be knocked out with CRISPR–Cas9 to convert the precursor into vitamin D without an expense to other metabolites. This discovery has further implications for other Solanaceae plants. Ascorbic acid, an important antioxidant and nutrient for immune health and wound healing, has proven to be less easily bio-fortified into fruit because increasing biosynthesis also leads to activation of catabolic and recycling pathways . Post transcriptional regulation of an ascorbic acid biosynthesis enzyme from kiwifruit has been demonstrated using tobacco leaves . Removing the upstream open reading frame that repressed translation increased ascorbic acid concentration in the leaves. Function-specific TFs can increase ascorbic acid in fruit without negative impact on quality as demonstrated in tomato. SlHZ24, a bHLH TF, regulates ascorbic acid biosynthesis and catabolism genes, and its transient over expression has been reported to increase the accumulation of this vitamin . Other TFs, SlNL33 and SlNFYA10, have been found to regulate the pathway negatively, and silencing them also increased ascorbic acid level . Understanding regulatory pathways governing nutrients can also facilitate traditional breeding programs. For example, a genome-wide association study led to the discovery and validation that alleles in SlbHLH59 determine ascorbic acid content in tomato cultivars . A less specific effect on nutrient accumulation can be achieved with hormone applications. For example, ethylene application in kiwifruit, a climacteric fruit, increased ascorbic acid and other antioxidants during ripening . Ascorbic acid also increased after nitric oxide application in sweet pepper, a non-climacteric fruit .Fruit flavor is a critical quality trait for consumer acceptance. Flavor includes all sensations experienced when eating, consisting of taste, aroma, and texture . In fruit, taste is mainly defined by a balance between sweetness and acidity but can include bitterness and umami.

Fruit aroma comes from specific classes and combinations of volatile organic compounds . For example, the unique kiwifruit flavor is associated with esters, mainly ethyl butanoate and methyl butanoate . Modulation of function-specific transcriptional and post-transcriptional regulators offers an effective solution for flavor improvement. A bHLH TF in banana activates 11 starch-degrading genes expressed during fruit ripening and is a likely candidate for increasing sugar and sweetness . In strawberry, editing the uORF of a bZIP TF that controls sucrose biosynthesis led to its translational activation and higher sugar content in fruit . Combining datasets generated through genomic, transcriptomic, metabolomic, and consumer panel studies has proven to be an effective strategy for identifying flavor-related genes and TFs. In tomato, metabolite data, associated loci, and consumer panels were analyzed across hundreds of varieties to determine key genes contributing to flavor . Coupling metabolites relevant to flavor with transcriptomic analyses can produce gene networks and identify regulatory TFs involved in flavor pathways. This approach was used in kiwifruit, where the AcNAC4 TF regulating a key gene in ester biosynthesis was validated . Homologs of these NAC TFs have been implicated in ester formation in peach and apple , suggesting that their functions are conserved across diverse families of climacteric fruit . Flavor is impacted by post harvest handling. Chilling during transportation and storage can alter fruit flavor. Epigenetic factors have been shown to regulate the suppression of VOC biosynthesis in chilled tomatoes . Most climacteric fruit are picked unripe and later treated with ethylene to induce ripening. However, this practice has been associated with poor flavor development. A study in off-vine ripened tomatoes confirmed that the fruit presented reduced VOC emission and a low sugar/acid ratio, due to alterations in gene expression and decreased methylation of their promoters . Genomic resources can help anticipate consumer preference and assist in breeding to select fruit with enhanced flavor. A population genomic study revealed that distinct consumer preferences between eastern and western countries drove selection for peach cultivars with different acidity . Fruit VOC profile data can help predict consumer liking before performing sensory panels, allowing for a more efficient selection of high-flavor fruit, as seen in tomato and blueberry . Moreover, the tomato pan-genome helped identify a rare favorable allele selected against during domestication that could be incorporated back into new cultivars to improve fruit flavor .Fruit texture is associated with freshness, flavor, and shelf-life potential. Texture involves many attributes, such as firmness, juiciness, crispiness, and meltiness . Fruit softening is mainly attributed to the remodeling and degradation of the polysaccharides in the primary cell walls .

The cuticle layer, deposited on the CWs of epidermal tissue, also contributes to fruit firmness by preventing water loss and maintaining cell turgor pressure . Traditional breeding has focused on creating firmer fruit that withstand transportation and have longer shelf life. This has been accomplished in tomato by developing hybrid lines between elite varieties and non-ripening mutants such as ripening-inhibitor and non-ripening . These mutants have defects in TFs considered master regulators of many ripening processes,plastic plant pot including the induction of cell wall-degrading enzymes and changes in cuticle composition . Other efforts to improve firmness and shelf life in climacteric fruit have taken advantage of mutations affecting ethylene biosynthesis and perception, as this hormone is also known to regulate genes encoding CWDEs, among others . However, as already discussed, modulating master regulators or hormone pathways has numerous drawbacks to other quality traits, such as color and flavor. Thus, downstream TFs controlling specific CW enzymes, such as LOB TF in tomato, may prove to be better targets . Another possibility is to leverage the availability of natural or induced allelic variants in ripening master regulators to produce a range of fruit phenotypes. For example, spontaneous or equivalent delayed fruit deterioration and CRISPR–Cas9-generated mutations in the ripening regulator NOR can extend shelf life with minimal impact on other fruit attributes , compared with the canonical mutant nor . RNAi and gene editing approaches have been used to target ripening-specific CWDEs that influence fruit firmness, such as polygalacturonase , pectate lyase , and pectin methyl esterase . The first GM fruit product, the FLAVR SAVR™ tomato, was engineered with antisense RNA against SlPG, however, it did not show a phenotype for fruit firmness . In contrast, the CRISPR–Cas9 SlPL knockout in tomato significantly improved fruit firmness and shelf life . Similar observations were previously reported in strawberry using RNAi knockdowns of FvPL and FvPME. In addition to firmer fruit, tomato and strawberry SlPL mutants have reduced fruit susceptibility to fungal disease . Breeding strategies and molecular studies have also focused on ameliorating textural defects such as fruit mealiness caused by cold storage. Mealiness, considered the opposite of juiciness, occurs when neighboring cells lose adhesion and detach while remaining intact . In peach, quantitative trait loci associated with cold-tolerant varieties have been identified to support breeding for less mealy fruit . Beyond QTLs, understanding the genetic mechanisms behind the trait provides avenues for targeting breeding and genetic modifications. For instance, peach mealiness appears to be associated with increased DNA methylation, leading to the downregulation and hypermethylation of mealy-associated genes such as PpCYP82A3 . Finding molecular approaches to avoid the deposition of methyl groups in the promoters of key ripening genes in response to cold storage can serve as a potential solution to mealiness.The FLAVR SAVR™ tomato hit the market in the early 90s promising a product with longer shelf life. However, this GM fruit had high production costs and was not well accepted by consumers, which led to its removal from the marketplace . Since then, other bioengineered crops with improved plant disease resistance or production-related traits have become available worldwide . These products have not sparked much enthusiasm mainly because they were not generated considering consumer-based traits or due to public fear of GM organisms. Recently, two fruits bio-engineered for quality attributes were approved by both US and Canadian regulatory entities and are available to consumers. These are the Pinkglow™ pineapple and the Arctic™ apple. The latter was bioengineering to reduce oxidative browning in the cut fruit . These fruit are considered novelty items because they are less available in the marketplace and significantly more expensive than traditional cultivars. Both fruits are primarily sold online and offered in limited supplies. The Pinkglow™ pineapple costs nearly ten times more than a common yellow pineapple. Despite their limitations, these fruits were developed with the consumer in mind, which may entice the public more than previous GM products.In 2022, the USDA deregulated the purple tomato developed with the expression of two snapdragon TFs. Novel fruit colors and potential higher nutrition may draw consumers to a new emerging category of bio-engineered produce. These fruit will also need to meet high consumer expectations of flavor, affordability, and food safety to ensure their success. Gene editing techniques such as CRISPR–Cas9 enable the fine-tuning of quality traits in a variety of fruit crops and may be more well received than previous GM products. For example, Japan started selling the first Cas9-edited fruit in the world in 2021, a health-promoting γ-aminobutyric acid -enriched tomato . Gene-edited fruit without foreign DNA have a more straightforward regulatory path in the United States than GM products, increasing the speed to market and reducing costs associated with authorization. However, this is not the case in other parts of the world, such as the European Union. In conclusion, researchers are armed with knowledge on fruit ripening and tools to improve fruit quality and generate greater access to fresh, flavorful, and nutritious food. Demands for better-tasting, more sustainable fruit, are in reach . We sit on the edge of an era where gene-edited and bio-engineered commodities can become a new category in the market, if the consumer will allow it.Fleshy fruit gain most of their quality traits, such as color, texture, flavor, and nutritional value, as a result of physiological and biochemical changes associated with ripening.

Firms that had made investments that led to higher worker productivity should have wanted to advertise that information in order to convince workers that they should accept a lower piece rate, while firms that had not should have wanted to hide the fact. Newman and Jarvis also found that piece rates were adjusted in many packing sheds if the quantity and/or quality of the grapes being processed changed. Although strikes were legally prohibited, female workers were able to “stop” production and successfully negotiate an increase in the piece rate, and the reverse was also true. Indeed, several male shed managers said that they found it easier to negotiate a “fair” piece rate with women than to negotiate pay with men. The evidence in Newman and Jarvis suggested that women workers were both aware of the importance of firm-influenced productivity differences and able to obtain information about what firms actually provided. Thus, there was evidence that female workers operated within and were part of a highly sophisticated labor market in which firms and workers obtained and used information regarding about their heterogeneity. Neither workers nor firms thought that firms or workers were homogeneous.Each worker was asked how many months he/she would have liked to work during 1991 and 1992. The mean response by females was 11.1 months and 9.7 months, respectively. The responses were surprisingly high, round flower buckets given that most women had worked much less than this in 1991. The question did not specify a wage at which they would be employed, but the responses suggested that most women wanted to work most of the year.

To further explore workers’ preferences for seasonal as opposed to permanent employment, including a specified wage, each worker was asked to indicate her/his preference for one of three employment options: 1) 3 months employment per year at100,000 pesos per month, 2) 6 months employment at 60,000 pesos per month, and 3) 12 months employment at 35,000 pesos per month. Option 2) required twice the time to obtain an additional 60,000 pesos of annual income, as did option 3). When the options were designed, it was believed that most women preferred to work only the summer months and thus would choose the first option. In fact, 50% of workers chose permanent employment as their preferred option and an additional 25% choose the 6-month option. Only 25% choose the 3-month high-salary option, even though its 300,000-peso salary turned out to significantly exceed the mean annual earnings of workers. What do these answers mean? We believe, based on respondents’ anecdotal comments, that most female temporary agricultural laborers want to work most of the year. First, most women wanted to earn more, even if the marginal earnings associated with longer employment were low. Second, some women expressed a desire to spread their income throughout the year and thought that a permanent salary would help achieve this. They commented on how hard it was for them to adequately carry funds from one month to the next given a lack of accessible savings institutions1 and constant pressure from friends and relatives for loans and assistance. Third, many women said that they enjoyed working, particularly as it allowed them to feel productive, benefit economically, and have greater social contact with others. When asked what they liked about their work, some women responded simply that they enjoyed it. However, most provided additional insight into what the work experience meant for them. Some responded that work allowed them to financially help their families, others that they enjoyed socializing with other women, having an opportunity to talk about their problems, possible solutions, and simply their concerns, and to form personal connections that they could not otherwise make.

Others were glad simply to feel useful. Having said that women wanted additional employment, they were not idle. Combining employment and family responsibilities placed a harsh demand on women’s time and energy. Their summer work schedule was especially grueling. They worked very long hours. Processing sheds began work at 2:00 p.m. and continued until all of the grapes picked had been packed. On average, cleaners worked 9.6 hours per day and packers 10.3 hours, not counting time waiting for the sheds to open, or for coffee breaks and dinner. Work commonly finished well after midnight and, during the seasonal peak, as late as 6:00 a.m. Many females liked beginning work in the early afternoon because it gave them time to take care of family responsibilities in the morning. Women also mentioned the burden that they faced in meeting these dual responsibilities. Some women indicated that their husbands did not want them to work and a few said they had obtained agreement to work only during peak season when earnings were high. However, more women indicated that their husbands appreciated their income, recognized that it improved family welfare, and supported their working. A few women said that their husbands had accepted some household tasks to help. All of the women interviewed worked at a task that was paid on a piece rate basis most of the time. Despite the increased pressure and expenditure of effort that piece rate work required, when asked their preference for summer work, 58% of workers expressed a preference for piece rate pay when working in a packing shed during the summer. The overwhelming reason for preferring piece rate pay was that it allowed the worker to earn more. Nonetheless, a significant proportion of these workers would have preferred a salaried job during the summer and most did not want piece rate employment on a year round basis. Indeed, 61% of workers preferred a fixed wage if they could get a year around job. Roughly half said they did not want to work at the intense pace required by piece rate work on a continuing basis. Others commented that they felt that they would earn more if working for a wage, including some who said that they would earn a higher rate for overtime, while others said their earnings would be more stable. The workers interviewed understood the advantages and disadvantages of their various employment options. Their decision to accept piece rate work depended partly on a lack of other remunerative employment during the rest of the year. Their relative poverty contributed to their desire to work hard when an employment opportunity presented itself. One might expect the supply of labor for temporary agricultural work on a piece rate basis will decrease if and when other employment opportunities materialize.Many women entered the labor force to supplement the income of other family members and/or to satisfy their own special needs. However, a majority of women in our data set were the major income earner in their household. Approximately 180 women lived in households that contained no male workers. Another 25% of women were the primary earner in their households even though their households included an adult male who worked at least part of the time. We lacked data to test whether women’s acceptance of temporary work in the fruit sector had improved household income. We only observed the households in which female workers resided, not the households they resided in prior to obtaining employment, plastic flower buckets wholesale and we had no information on rural families that did not have any members working in the fruit sector. It is nonetheless instructive to see how the income earned by women in our sample affected their current households.It is often tempting, after eating a particularly good orange or avocado, to plant the seed and grow your own tree full of these delicious fruit. Trees grown from these seed, however, may produce fruit that are not edible at all, or the trees may not bear fruit for many years.

The best way to produce good-quality fruit is to grow seedlings from them and then attach, by budding or grafting, material from trees that are known to be good producers. Budding and grafting can also be used to change or add varieties to mature citrus or avocado trees, a process known as top working. This publication is a brief introduction to budding and grafting for the home gardener. For more information, consult the materials listed at the end of this publication or contact your local Cooperative Extension office.To germinate citrus or avocado seed, plant them in a shallow container such as a nursery flat or a pan with drainage holes in well-drained commercial potting mix. Plant the seed two to three times deeper than their length. For example, a citrus seed about 1 ⁄4 inch long should be planted about 1 ⁄2 to 3 ⁄4 inch deep. Keep the seed in a warm place—between 70° and 80°F —and keep the soil moist. Covering the nursery flats with clear glass or plastic will help maintain the proper humidity. Avocado seed can also be germinated by suspending them in water. Place toothpicks horizontally into the seed near the top. Suspend the wide end of the seed in a small container of water with the toothpicks resting on the edge of the container. Place it in indirect light and refresh the water at least weekly. After germination , replant the seedlings into a larger container of good-quality commercial potting mix. Good choices for containers include a cardboard milk carton cut horizontally in half or a one-gallon can. Punch drain holes in the bottom of the container. The seedling will be ready for budding or grafting when it has grown to 24 to 30 inches tall.Budding and grafting are vegetative propagation techniques in which a single bud or stem of a desired plant is attached to a rootstock plant. In budding, a single bud with its accompanying bark is used as the scion. In grafting, part of a stem or branch is used as the scion. One of the most important keys to successful budding and grafting is properly positioning the scion on the rootstock. In order for the scion and rootstock to grow together, the thin greenish plant layer just under the bark of the scion and rootstock must be aligned so that they touch each other. If they do not touch each other, the bud or graft will fail. Within 10 to 15 days, a successful bud or graft forms a hard whitish tissue where the two cambium layers grow together. Always use sharp cutting or grafting instruments and make clean, even cuts. Options include a budding knife, a sharp kitchen knife, or a single-sided razor blade. Do not allow the cut surfaces of the scion or rootstock to dry out: immerse cut scions in a pail of water, wrap them in plastic, or graft them immediately after cutting. Also, remove any leaves from scions after cutting to help keep the scions from losing water. Keep the scions in a cool place during the work.Budding and grafting are best done in the spring or fall when the bark is easily separated from the wood. It should be timed to be early enough so that warm weather will help ensure a good bud union, yet late enough so that the bud will not begin to grow and callus will not grow over the bud itself. Citrus budded or grafted in the fall must be protected from frost. Avocados are best grafted in the spring when the bark is easily separated from the wood.Budding is the standard method used to propagate citrus. Aside from being the easiest method, it allows a large number of plants to be propagated from a small amount of scion wood and is suitable for trees, rootstocks, or branches from 1 ⁄4 to 1 inch in diameter. Budwood should be taken only from high-producing, disease-free trees . The best citrus budwood is located just below the most recent flush of new growth; the best avocado budwood is located near the terminal end of shoots that have fully matured, leathery leaves.Cultivated alfalfa is a major forage crop that plays a key role in global livestock production. Alfalfa hay is a relatively cheap, high-quality forage which is high in protein and thus an ideal feed for ruminant animals . Often referred to as the ‘Queen of forages’, alfalfa is one of California’s most important crops, driving production of the state’s most significant agricultural enterprise: dairy . In 2022, 200,000 hectares of alfalfa were harvested for hay and haylage with a total value of over $1B .

The opposite is true for Rr and no effect is found for Rs. Overall CAwr has the highest relative fecundity with the highest fruit production . Average number of fruit damaged for each lineage correlates: strongly with Rs and Rr fruit production, Rs and Rr potential reproduction and Rs and CAwr final plant weight, and moderately with CAwr fruit production, potential reproduction and Rr final plant weight. In all the cases the covariance values are positive. No correlation was found with days to germination . Debris due to granivory – Debris material due to granivory was only found under plants in unprotected plots. In general the debris due to granivory was a mix of viable and unviable seeds as well as fruit wall segments. Some seeds were found bared, with no fruit material around them, while others were either fully or partially covered by fruit wall segments. The largest amount of debris due to granivory was found under CAwr plants in unprotected plots as well as the largest number of potentially viable seeds. Most of the fruit segments were found broken and empty. The average values are listed in table 2.6. Although the debris due to granivory data was based on a small sample size, I calculated the average proportion of seeds removed for each plant by granivory in two different ways. One of the calculations was done as described in the methods. The other calculation included the subtraction of the potential “viable” seeds found in the debris due to granivory and averaged for each lineage . Subsequently, I compared both averages. In the case of both progenitors there are minimal differences. However, procona system for the hybrid derived-lineage there is a 10% reduction of seeds removed.

Granivore behavior – We observed and filmed house finches actively manipulating and feeding on fully formed Raphanus fruits. No other avian species were observed on Raphanus plants although there are at least five other granivores regularly occurring in this location . At the different plots, the birds visited the plants daily in an almost predictable fashion. In all cases I observed males, easily recognizable for their characteristic red coloration around the face and head. The peak of house finch foraging activity corresponded to the period in which the fruits were fully formed but still green. We never observed the house finches feeding out of the debris on the ground; they appear to only choose fruits attached to the plants.The house finch Carpodacus mexicanus, an avian granivore, affected the reproductive patterns of three Raphanus progenitors and hybrid lineages in dissimilar ways. When exposed to the granivore, the hybrid-derived CAwr had proportionally more fruits damaged relative to its progenitors . Overall relative fecundity of CAwr was significantly reduced compared to protected plants of the same lineage . When exposed to the granivore, CAwr had lower fecundity compared to the wild progenitor Rr. However, due to its high overall number of fruits and seed production relative to both progenitors, CAwr was not negatively affected by granivory. Under conditions where granivory is excluded, CAwr has the highest relative fecundity compared to both progenitors , concurring with results from a prior study . The fecundity of protected Rr fruits was comparable to the high fecundity of protected CAwr fruits. A potential explanation for this result might be that Rr fruits detach easily from the mother plant. I found a large number of dropped Rr whole fruits relative to whole attached fruits . Finches perch when attempting to feed, with the result that many Rr fruits fall to the ground before they can be damaged on the branch.

Because the birds did not forage on the ground, the seeds in fallen fruits remained viable. Easily detachable fruits were also observed occasionally on CAwr plants but not as frequently as in the wild Rr progenitor . Fecundity values of the cultivated Rs lineage did not differ between unprotected and protected treatments. Rs exhibited low fecundity values overall, which is in agreement with other studies .However, the reproductive output of Rs may be even lower than reported because our measurement protocol likely led to an underestimate of the negative impact of the granivore for this lineage. Observations by us and other researchers indicated that birds open entire fruits of the cultivar Rs and consume all the seeds inside . Fully eaten fruits often appear similar to an empty pedicel and thus may not be counted as damaged at all. An additional caveat is that by planting the three lineages close together I may have influenced the granivore’s choice to feed more often on Rr and CAwr, which are bigger and more productive compared to Rs. Density-dependence has been observed in other studies of granivory and it is expected that birds make foraging choices to maximize their own fitness . Plant invasion often involves multiple introductions followed by a time lag during which invader populations overcome small population effects such as genetic drift and inbreeding causing Allee effect, Founder Effect, and bottlenecks . In addition, other mechanisms favor invasiveness like gene flow, potentially leading to interspecific hybridization, . Some invasive plants are a result of successful interspecific hybridizations, presenting defense traits that may be identical, different or additive relative to one or both progenitors, defining their susceptibility or resistance . Hybrids can be more susceptible: a comparison of the densities of herbivore and pathogen species on two progenitors species, Salix sericea and S. eriocephala and hybrid willow shrubs, revealed significantly higher herbivore densities on the hybrids . Althoughthe trend in the Fritz et al. study and others that he and collaborators reviewed do not support higher resistance in hybrids, other studies have found evidence for this. For example, the hybrid derived Helianthus annuus texanus acquired resistance to two types of herbivores from both ancestors H debilis and H annuus annuus increasing its relative fitness . In the case of invasive plants, hybridization can contribute to the evolution and success of invasive lineages . In addition, after establishment of long-standing populations in the community in a novel environment, invasive plants evolve and with them their potential enemies . And as it is the case for all ecological interactions, invasive species exist in a complex network, where new abiotic and biotic conditions imposing a new selective pressure on the plants that may cause an adaptive response from the plants . Considerable effort has been given to the idea that plant colonists have the advantage to be unknown by their new biotic enemies, i.e. herbivores, frugivores and pathogens. This idea, initially known as the “enemy release hypothesis”, was proposed by Elton in 1958 but can be traced back to Charles Darwin in 1859 and subsequently with an added evolutionary context became the Evolution of Increased Competitive Ability hypothesis by Blossey and Nötzold . The EICA predicts that invaders have the opportunity to evolve a competitive advantage compared to native taxa by re-allocating on plant growth instead of on plant defenses. Aspects of this hypothesis have been tested empirically multiple times at many levels, at both single sites and continent-wide scales,but the results have been equivocal .

With respect to pre-dispersal seed predation, procona valencia buckets previous studies have found that other factors are fundamental to maintain the demographics. For example, higher predation does not translate in lower fitness rates that will affect the population size or the following year’s adult density in invasive Centaurea solstitialis . Similarly, in spite of higher susceptibility to the effect of invertebrate pre-dispersal seed predation by the fly Hylemya sp. in natural and artificial hybrids crosses relative to progenitor species, Ipomopsis aggregata and I. tenuituba, flower traits and higher pollination rates compensate for seed losses . One of the fruit characteristics that has been found to reduce the negative effects of seed-eaters on fitness is multi-seeded fruits . In our study, estimated percentages of damage decreased when calculated based on estimated seeds removed compared to values based on fruit damage, diminishing the level of impact by the granivore. Thus, multi-seeded fruits reduce the effect of the granivore pressure on seed survival. Prior studies in non-invasive plants also support this idea; the impact of seed predation is reduced when plants produce multi-seeded fruits . To our knowledge, this study is the first to find evidence of the advantage of multi-seeded fruit in an invasive plant in the context of granivory. Aside from the density-dependent effect that may have distracted the birds from eating fruits of Rs, I propose that fruits of each lineage provide a different level of defense to the seeds inside. Currently it is known that fruit morphology does vary among the Raphanus lineages as does fruit mechanical defense . Invasive plants have been demonstrated to exhibit rapid evolutionary change suggesting that ~100 years of granivore interactions may have imposed substantial evolutionary pressure on the hybrid-derived fruits. This possibility coupled with the pre-existing hardened fruit wall in Raphanus raphanistrum leads to the hypothesis that fruit hardness is a genetically based trait that is favorably recombined as hybrids are formed and diversify in time . We never observed fully eaten fruits of California wild radish, but at most ~1/3 of the seeded portion. Previous work on CAwr mating system has found that the seeds inside an individual fruit are usually sired by various pollen sources and that the siring of the pollen occurs in a non-random manner , which has been related to a within-fruit seed size variation . The combination of these traits and the impact of the granivore provide a unique situation to study the combined effects of natural and sexual selection. To include the granivore’s perspective was beyond the scope of our study. Nevertheless, our observations are in agreement with other features of the physiological ecology of house finches. We only observed male house finches feeding on our experimental radish plants. According to Hill et al. house finches have a 6-month breeding season, which in their western distribution starts in January . The fruits in the experimental plants described here were fully formed in June, which is near the end of house finch breeding season and just prior to the onset of molting. Carotenoid based plumage pigmentation such as red, orange and yellow can only be acquired through diet and are known to have a significant function in sexual selection, immune system and senescence . To acquire the necessary pigments to color their plumage, male house finches need to include carotenoid rich food in their diet during the time the feathers are replaced and growing . Raphanus plants have carotenoid contents and are particularly rich in ?- carotenoids , which are the specific class of pigments that house finches most likely require to color their feathers . Female house finches prefer males with bright red plumage and the intensity of the red coloration in the males significantly correlations with: 1) an early beginning of the breeding season , 2) increase in number of brood for a given pair , as well as 3) the concentration and type of carotenoids in the food ingested . To our knowledge there is no study that has reported carotenoids extracted from radish seed endosperm or even fruit wall; most studies focus on cotyledons and leaves . However, carotenoid pigments in canola Brassica napus seeds, another Brassicaceae species have been reported . The pattern of fruit consumption described here represents a positive effect for house finches and,surprisingly, a positive effect for Raphanus. We propose a novel interpretation that granivore-fruit interactions in C. mexicanus- Raphanus are mutualistic. The results of our study provide information of an invasive hybrid derived lineage performance relative to both progenitor lineages in the context of a novel biotic interaction. The hybrid as well as the wild progenitor have a combination of traits that increases their ability to survive and persist in novel environments. High fecundity and multi-seeded fruits with mechanical defenses protect and compensate for the effect of the granivore. The wild progenitor has some advantages over the hybrid lineage at the predispersal seed predation phase. Although this result might be accurate I also suspect that our experimental design might have added some unwanted level of competition among the plants at the early growing stages affecting the hybrid performance .

The introgression region was split into 18 loci and a representative primer pair was made for each locus and these were sequenced using GT-Seq . Whether the region was from M82 or S. pennellii was determined by the presence of SNPs and can be found in Table S8. All F3 and F4 plants were grown in the greenhouse and compared to M82. The bHLH032 promoter region in S. pennellii is 200-bp larger than that of M82. This region was sequenced to determine if the lines contained the M82 or S. pennellii version of the gene and all data binned by genotypes determined by sequencing. Leaf shape for the select lines showed that those line containing the S. pennellii version of the bHLH032 had rounder leaves while those with the M82 version were closer to the M82 control although still significantly different . Likewise, the vascular density showed that those plants which had the S. pennellii bHLH032 had lower vascular density than those with the M82 version, which had a density the same as the M82 control plants . For both leaf shape and vascular density the heterozygous plants were more like the S. pennellii homozygous bHLH032 plants than the M82 containing plants . The BY for the S. pennellii and M82 bHLH032 containing plants was higher than the control M82 plants, suggesting that other parts of the introgression were still present and likely played some role in the increase in BY . The plants heterozygous for bHLH032 had a BY similar to the M82 control plants but did have a wider range of values. The trait measurements and the genotype of the bHLH032 gene present suggested that there was a likely role for bHLH032 in the regulation of BY through modulation of leaf shape and vasculature. To confirm this possible correlation, blueberry in pot we made bHLH032 CRISPR mutants in the M82 background.

Two RNA guides were made within the bHLH032 gene for CRISPR mutation, one in the first exon on the 5’ end of the bHLH domain and the second at the 5’ end of the second exon . We obtained ten CRISPR lines, however only the second guide in exon 2 was successful in creating mutations. Of these ten lines we chose lines 05, 06, and 10 to carry forward for further characterization. bHLH032-05 has a one base pair deletion near the guide site that creates a stop codon and truncates the gene early Similarly bHLH032-10 has a two base pair deletion which also creates an early stop codon, but it is located 20 amino acids further downstream than that found in bHLH032-05, and it alters the amino acid sequence between the guide site and the truncation stop codon . bHLH032– 06 was unique from the other mutations as it had a three base pair deletion which created a two amino acid deletion and one amino acid insertion . The T1 plants from these lines were grown up in the greenhouse and their leaf shape and vasculature characterized. All three lines had significantly rounder leaflets than that found in M82 and bHLH032-06 and bHLH032-10 had significantly reduced vasculature . bHLH032-05 did not have significantly lower vasculature but it was lower than M82 following the same trend as the other mutants . All three lines were carried forward after confirming a phenotype similar to BIL 260 , however bHLH032-05 also had fruiting/seed set deffect that caused it to have difficulty making fruit or seed. Because of this phenotype we decided not to continue with bHLH05 progeny and work only with bHLH032-06 and bHLH032-10, neither of which had seed set or fruiting issues. For bHLH032-06 and bHLH032-10 the T3 and T4 plants were grown in the greenhouse for characterization of BY along with the leaflet shape and vascular density. These plants are in the process of the being sequenced to determine if they have the same mutation as the T0 parents, however a few plants have been genotyped and they will be discussed here. bHLH032-10 lines that have been genotyped so far contain the two base pair deletion resulting in a stop codon /10.

Leaf shape analysis shows that none of these lines are significantly different from M82, however homozygous plants had significantly lower vascular density than that found in M82 or the heterozygous plants . Figure 5d shows example leaflets and vascular images all three genotypes, and while the homozyogous leaflet trends towards rounder than M82 it is still very similar. The vascular density of homozygous mutant plants was much lower, replicating the phenotype seen in BIL 260, despite not having round leaflets . The BY for the homozygous mutant plants was significantly higher than either M82 or heterozygous plants, indicating that the previously identified correlation between decreased vascular density and the total output of the plants holds in these lines . Thus, the pattern of inverse relationship between vascular density and BY seen in the original BIL 260 plants was replicated in the bHLH032-10 CRISPR plants .The primary focus of crop improvement, specifically yield, has been on improving photosynthesis as photosynthetic rates tend to correlate strongly with total biomass . However, more recent studies have shown that photo assimilates, when available in large quantities as would be found with high photosynthesis, are primarily converted to vegetative biomass in tomato as well as rice . Other characteristics such leaf shape were explored here as well as in previous studies, that revealed a potential correlation between BY and the shape of leaflets found in tomato . However, while BIL 260 and sub IL 4-3-4 showed a marked increase in BY and yield respectively, other lines such as BIL 267 and 139 also had rounder leaflets than that found in M82 but did not show an increase in BY . While models such as the PLS-Path Model seen in Rowland et al. indicate a direct relationship between leaf shape and BY, there are likely other genetic or physiological factors that confound this correlation. Photosynthetic rates in BIL 260 were slightly elevated over M82 and sub IL 4-3-4 which suggests that an increase in photosynthesis, as has been previously suggested, could result in increased BY as seen here . However, the large variability in photosynthesis rates creates some doubt as to the efficacy of this claim, as the BIL 260 photosynthetic rate is 27 µmols m-2 s -1 , compared to that for M82 at 25 µmols m-2 s -1 . The opposite, however, appears to be true with lower photosynthesis, and therefore lower photo assimilates, resulting in lower yields and BRIX values . All these data together suggests that while photosynthesis is necessary to increase BY there may well be a hard cap on how much it can improve crop output. If photosynthesis itself does not improve BY beyond a certain point, then research should investigate other areas such as sugar transport and storage. The sugar and starch concentrations in the leaves of BIL 260 and sub IL 4-3-4 show an increased mobilization of starch during night hours but no coinciding increase in sugar, unlike M82 where the same starch mobilization led to an increase in leaf sugar during the same time period . This points to mobilization and export of sugar as a likely reason for the increased BY seen in BIL 260 and sub IL 4-3-4. Leaf sugar in BIL 260 is lower than that for both other lines during the 1 to 5 am time period, plastic planters wholesale and correlates nicely to the fact that BIL 260 has a higher BY than either other line . How leaf shape relates to sugar export could be tied to the vascular density found in the leaves, as leaf shape and vasculature are closely linked during development .

The vascular density of BIL 260 is significantly lower than that of M82 or sub IL 4-3-4, indicating that a lower amount of vasculature per mm2 results in increased sugar export, a trend seen in previous studies . How decreased vascular density increases export and what is regulating these changes remain open questions. To address these questions, we performed WGS and identified three transcription factors introgressed from S. pennellii unique to BIL 260 . Of these three only bHLH032 showed up as DEG in RNA-seq data for SAM, mature leaves, and young leaves . Analysis of gene expression using dimensionality reduction methods, showed the Cluster 19 from young leaves was enriched in sugar related genes and contained the bHLH032 transcription factor. This suggests bHLH032 may be a regulator of sugar related metabolism, similar to the mobilization and export processes seen previously . bHLH032 is peripherally connected to the network in M82, which means it acts as an input or regulator to the network, working through a gene similar to the Arabidopsis genes BDX/DGR2 which regulate leaf and vascular development . bHLH032 is not present in the BIL 260 network, likely due to low expression levels precluding it from being an input into the network, and causing alterations to vascular development, leaf development through the tie to vascular development, and subsequent sugar export and processing .To identify if bHLH032 was an important part of this pathway we generated back crosses of BIL 260 with M82 to break up the introgression region, and indeed those lines in the F3 population that contained the S. pennellii version of bHLH032 had rounder leaves, lower vasculature density, and higher BY . These data strongly suggest that bHLH032 is a master regulator of these morphological characteristics which lead to increased BY. In fact, the CRISPR knockout mutants of bHLH032 in the M82 background show a similar trend, with lower vascular density and increased BY . Interestingly, the leaf shapes of these CRISPR lines were not different from M82, suggesting that the mutation in bHLH032 separated the normal link between leaf shape and vasculature, and pointing to vascular density as the driving factor behind increased BY . The decrease in bHLH032 causes changes both leaf shape or vasculature which correlate with BY directly . This also breaks the long known trend of BRIX and yield being inversely correlated to allow for increase in both. Thus bHLH032 could be an important regulator of BY, opening an avenue for tomato improvement. It and other transcription factors functioning in the regulation of leaf shape and vascular density provide a target for future research to continue tomato improvement.Gas exchange measurements were made in the field on attached leaves after the plants had recovered from transplanting. For the 2015 field season measurements were made weekly from week 10 through week 13 , week 14 through 17 , and 18 – 21 , on approximately 5 plants per week. For the 2014 and 2016 field seasons terminal harvest was performed on 60, and 20 plants, respectively. For all field seasons measurements were made on leaves from the upper and lower portion of the plants to eliminate positional bias within the plant and measured for three leaves per plant. The A , gst , transpiration, and ɸPS2 of a 6 cm2 area of the leaflet was measured using the LI-6400 XT infra-red gas exchange system , and a fluorescence head . The chamber was positioned on terminal leaflets such that the mid-vein was not within the measured area. Light within the chamber was provided by the fluorescence head at 1500 µmol m-2 s -1 PAR, and the chamber air flow volume was 400 µmols s-1 with the chamber atmosphere mixed by a fan. CO2 concentration within the chamber was set at 400 µmols mol-1 . Humidity, leaf, and chamber temperature were allowed to adjust to ambient conditions, however the chamber block temperature was not allowed to exceed 36°C. Measured leaflets were allowed to equilibrate for 2 to 3 minutes before measurements were taken, allowing sufficient time for photosynthetic rates to stabilize with only marginal variation.For the 2015 field season three plants per cultivar were destructively harvested each week. The final yield and fresh vegetative weight of each plant harvested, was measured using a hanging scale in the field. Five leaves were collected at random from the bottom and top of the plant to capture all canopy levels, and approximately nine fruit were collected for BRIX measurements.

In fleshy fruits, soluble sugars, including sucrose, fructose, and glucose, are not only essential for fruit growth and development but also central to fruit quality. Fruit taste and flavor is closely related to the composition and concentration of sugars and their balance with acids . As the composition and concentration of sugars at fruit maturity is determined by metabolic and transport processes during fruit development, understanding these processes and their regulation is important for fruit quality improvement. At the center of sugar metabolism in sink cells is the Sucrose cycle, previously named the Sucrose–Sucrose cycle or the futile Sucrose recycle, which consists of the breakdown of sucrose by invertase and sucrose synthase, the phosphorylation of the resulting hexoses and the interconversion between hexose phosphates and UDP-glucose, and the re-synthesis of sucrose via sucrose-6-phosphate synthase and sucrose-6- phosphate phosphatase. This metabolic system connects sugar metabolism with many other metabolic pathways such as glycolysis and tricarboxylic acid cycle, starch synthesis, and cellulose synthesis, and its coordination with the sugar transport system on the tonoplast is expected to determine the partitioning of sugars between metabolism in the cytosol and accumulation in the vacuole . In fleshy fruits, collection pot the concentration and distribution of sugars in parenchyma cells are affected via this cycle by developmental processes and environmental factors.

However, the biochemical regulation of the cycle and the associated transport system is not fully understood.Apple is one of the most economically important deciduous tree fruits worldwide. In apple and many other tree fruit species of the Rosaceae family, sorbitol is a primary end product of photosynthesis and a major phloem-translocated carbohydrate, accounting for 60–80% of the photosynthates produced in apple leaves and transported in the phloem. In source leaves, sorbitol is synthesized from glucose-6-phosphate in a two-step process: G6P is first converted to sorbitol-6-phosphate via aldose-6-phosphate reductase , then followed by dephosphorylation of S6P to sorbitol via S6P phosphatase. The loading of both sorbitol and sucrose into the companion cell-sieve element complex in the phloem is passive and symplastic in apple, but their phloem unloading in fruit involves an apoplastic step. Once released from the SE-CC complex of the phloem in apple fruit, sorbitol is taken up into the cytosol of parenchyma cells by plasma membrane-bound sorbitol transporters and then converted to fructose by sorbitol dehydrogenase ; sucrose is either directly taken up into parenchyma cells by sucrose transporters , or first converted to glucose and fructose by cell wall invertase and then transported into the parenchyma cells via hexose transporters. Compared with plants that transport and utilize only sucrose, such as Arabidopsis, tomato , and poplar , apple is unique in that both sorbitol and sucrose are transported in the phloem and are metabolized in sink organs. It is estimated that >80% of the total carbon flux goes through fructose in apple . Once taken up into parenchyma cells of fruit, both sorbitol and sucrose feed into the Sucrose cycle to meet the carbon requirement for fruit growth and development while excess carbon is converted to starch for storage in plastids or transported into vacuole by sugar transporters for accumulation.

Although we have characterized the genes and proteins involved in sugar metabolism and accumulation in apple, it remains unclear how apple trees adjust the Sucrose cycle and the transport system in response to altered supply of sorbitol and sucrose from source leaves. In transgenic apple trees with anti-sense suppression of A6PR, leaf sorbitol concentration is dramatically decreased, whereas sucrose concentration is significantly elevated in the source leaves, but neither leaf CO2 assimilation nor plant vegetative growth is altered. The decreased sorbitol synthesis leads to significant changes in the expression profile of key genes in leaf starch metabolism and many stress response genes. In addition to being a key metabolite in carbohydrate metabolism, sorbitol also acts as a signal regulating stamen development and pollen tube growth and resistance to Alternaria alternata in apple. In the shoot tips of the A6PR transgenic plants, both the activity and transcript level of SDH are down regulated, whereas those of sucrose synthase are upregulated in response to a lower sorbitol but higher sucrose supply. Teo et al. reported that fruit of the transgenic apple trees accumulated a higher level of glucose and lower levels of fructose and starch at maturity, but no significant difference was detected in the activity of key enzymes in sugar metabolism, CWINV, neutral invertase , fructokinase , hexokinase , or SPS between the transgenic lines and the untransformed control . Considering that anti-sense suppression of A6PR has drastically decreased leaf sorbitol level and increased sucrose level, leading to less sorbitol but more sucrose being transported in the phloem; and both transcript levels and activities of SDH and SUSY responded to the altered sorbitol and sucrose supply in the shoot tips of the transgenic plants, we predicted that the decreased supply of sorbitol and increased supply of sucrose would lead to down regulation of sorbitol metabolism and upregulation of sucrose metabolism in the transgenic fruit as well.

The discrepancy between the data obtained by Teo at al. and our predicted responses on the activities of sucrosemetabolizing enzymes in the transgenic fruit has prompted us to re-evaluate sugar metabolism and accumulation in the fruit of these transgenic plants to better understand how the Sucrose cycle and the sugar transport system respond to an altered supply of sorbitol and sucrose.Antisense suppression of A6PR significantly decreased sorbitol concentration but increased sucrose concentration while largely maintaining fructose and glucose concentrations in source leaves throughout fruit development in the two transgenic lines relative to the untransformed CK . Sorbitol concentration in the source leaves of anti-sense line A27 was decreased to ~70% initially and 13% at harvest of that detected in CK. For anti-sense line A04, sorbitol concentration was decreased to 32% initially and 10% at harvest of the CK level. By contrast, sucrose concentration in the source leaves of A27 and A04 was much higher than in CK throughout fruit development, 10 plastic plant pots with larger differences detected at later developmental stages . Concentrations of sorbitol and sucrose were also measured for source leaves, leaf petioles, and fruit pedicels at 75 days after bloom . Compared with CK, anti-sense lines A27 and A04 had lower concentration of sorbitol, higher concentration of sucrose, and lower molar ratio of sorbitol to sucrose in the source leaves, leaf petioles, and fruit pedicels. The abundance of sorbitol followed the order of source leaves > leaf petioles > fruit pedicels .Average fruit fresh weight did not differ significantly between the two anti-sense lines and CK during fruit development except for about a 10% lower value detected for A27 and A04 at 108 DAB and at harvest . Dark respiration was ~1.5–1.9-fold higher in A27 and A04 fruits than in CK fruits between 40 and 108 DAB during fruit development, but no significant difference was detected at harvest . Fruit yield per tree was significantly lower in the two anti-sense lines than in CK, largely due to lower average fruit weight at harvest as fruit number per tree was not significantly different between the two anti-sense lines and CK .Suppression of sorbitol synthesis in source leaves led to a significant decrease in sorbitol concentration in the fruit of two anti-sense lines A27 and A04 throughout fruit development, particularly in A04 . However, sucrose concentration was similar in the fruits of the two anti-sense lines and CK during fruit development with a higher level detected in the transgenic fruit only at 74 DAB. Fructose concentration showed no difference between the transgenic fruit and CK except being slightly lower at 108 DAB in the transgenic fruit. Compared with CK, concentrations of glucose and galactose were much higher throughout fruit development, with larger differences detected at later developmental stages. Concentrations of G6P and fructose-6- phosphate decreased during fruit development and were significantly lower in A27 and A04 than in CK from40 to 108 DAB . At fruit maturity , total soluble solids concentration was significantly higher in A27 and A04 than in CK . Fruit starch concentration did not show obvious difference between the transgenic lines and CK before 74 DAB but was slightly lower in A27 and A04 than in CK after 108 DAB .Our data clearly showed that sorbitol concentration was significantly lower, whereas sucrose concentration was significantly higher in the source leaves of 5-year-old transgenic “Greensleeves” apple trees with anti-sense suppression of A6PR compared with the untransformed CK throughout fruit development.

These results are consistent with those reported for the 1-year-old transgenic trees. The higher sucrose concentration in the source leaves is an indication that a larger proportion of the photo synthetically fixed carbon ends up in sucrose over a 24-h period because most of the starch accumulated during the day breaks down for sucrose synthesis at night in the transgenic plants although no difference in the carbon flux to sucrose during the day was detected. As both sorbitol and sucrose diffuse into SE-CC complex from mesophyll cells via plasmodesmata,transgenic plants. The lower concentration of sorbitol and higher concentration of sucrose in both leaf petiole and fruit pedicel and a smaller ratio of sorbitol to sucrose indicate that significantly less sorbitol but much more sucrose is translocated from leaves to fruit in the transgenic trees, which is consistent with a lower sorbitol but a higher sucrose concentration in the phloem exudates collected from fruit pedicels of these plants. The total amount of carbon translocated to fruit is expected to be very similar between the transgenic lines and the CK because all the trees had a very similar cropload and no significant difference was detected in average fruit dry weight between the transgenic lines and the CK at fruit maturity . These data clearly demonstrate that, when sorbitol synthesis is decreased in the source leaves, more sucrose is synthesized in the leaves and translocated to the fruit, thereby largely maintaining fruit growth and development. This is also consistent with the homeostasis of vegetative growth observed in the transgenic lines. The transgenic trees with decreased sorbitol synthesis grown under our experimental conditions were only slightly smaller after 5 years of growth than those of the untransformed CK . This is consistent with comparable photosynthetic rates measured in the transgenic lines and the untransformed CK throughout the growing season , with the lower rates detected only at fruit harvest being largely related to the leaf brown spots caused by Alternaria alternata in the transgenic lines. However, Teo et al. found that the transgenic trees were much smaller than the CK trees. This discrepancy is likely due to differences in growing conditions between the two locations. As sorbitol is implicated in drought-stress tolerance in apple, these trees might have experienced more drought stress under warm and dry conditions in California than under cool and humid conditions in upstate New York. In addition, as we strictly controlled cropload to a similar level each year by thinning flowers and young fruits, dry matter accumulation was not drastically different between the transgenic lines and the CK in our experiment.In response to a decreased sorbitol supply from source leaves, both the transcript level and the activity of SDH decreased in the transgenic fruit, which is consistent with previous findings in apple fruit cortex tissues fed with sorbitol and in shoot tips and fruit of the transgenic trees. As most of the fructose in apple fruit is converted from sorbitol by SDH, a significantly lower fructose level had been predicted in the transgenic fruit based on dramatically reduced import of sorbitol into the transgenic fruit and the associated lower SDH activity. However, the fructose level in the transgenic fruit was remarkably similar to that in the untransformed CK: no difference before 74 DAB and at harvest with only a slight difference detected at rapid fruit expansion between the transgenic fruit and the CK . This near homeostasis of fructose level in the transgenic fruit has clearly resulted from the response of the Sucrose cycle and the associated sugar transport system to increased availability of sucrose in the transgenic fruit, specifically, more sucrose is taken up into parenchyma cells in fruit after phloem unloading; more fructose is generated from sucrose breakdown by NINV and sucrose synthase and less fructose is phosphorylated by FK in the cytosol; and tonoplast sugar transporters are upregulated to take up more hexoses into the vacuole.