Month: January 2025

The VTC4 gene has been reported to be a bifunctional enzyme, also catalysing conversion of D-myoinositol 3-phosphate to myoinositol in myoinositol biosynthesis . The myoinositol can be converted to UDP-glucuronic acid , which is a common but cell wall-specific biochemical precursor for cell wall bio-genesis . The bifunctional VTC4 enzyme facilitates formation of AsA and cell walls . Therefore, the VTC4 may be a candidate gene for enhancing nutrition and delaying softening via influencing of the AsA production and cell wall formation in tomato fruit. Tomato has three VTC4 homologue genes SlIMP1, SlIMP2, and SlIMP3 . The SlIMP3, which has the highest expression level in tomato fruit, was selected for this investigation as it also has bifunctional enzyme activity, similar to that of the VTC4 in Arabidopsis. Overexpression of SlIMP3 in tomato increases the AsA content in multiple tissues. Overexpression of SlIMP3 increased the myoinositol accumulation, cell wall thickness, and altered cell-wall composition. Overexpression of SlIMP3 markedly delayed fruit softening and enhanced fruit resistance to Botrytis cinerea. The results demonstrate a critical role for SlIMP3 in AsA biosynthesis and cell wall bio-genesis and provide new method of delaying fruit softening and extending shelf-life of tomato.Three IMP genes have been identified in tomato . Alignment and sequence analysis revealed that the IMP amino acid sequences contained signature motifs . A phylogenetic analysis of tomato IMP sequences, together with Arabidopsis and tobacco IMP-related genes, large plastic planting pots was carried out using the neighbour-joining method on mega6. The results indicated that SlIMP3 was most closely related to NtIMP3 and clustered with SlIMP1, NtIMP1, and AtVTC4 into one subfamily .

The expression pattern of three SlIMPs in vegetative and reproductive tissues was carried out using the online TomExpression platform. The SlIMPs had ubiquitous expression in all tested tissues, including roots, shoots, leaves, flowers, and fruits. It was interesting that SlIMP3 gene had highest expression levels during fruit development and ripening . A qRT-PCR test was performed to confirm the expression patterns of SlIMPs in tomato plants. The results were consistent with the TomExpression data, with the highest expression levels of SlIMP3 in fruits . The expression pattern of SlIMP3 was also explored through a transgenic tomato plant in which GUS reporter gene was driven by the SlIMP3 promoter. Consistent with the qRT-PCR results, the GUS staining revealed the ubiquitous expression pattern of SlIMP3 in leaves, stems, buds, flowers, and fruits at different developmental stages, with strong expression in immature green fruit. The expression of SlIMP3 was also decreased in the ripening stages, though weakly expression in orange fruits .It is established that the VTC4 gene encodes a bifunctional enzyme that influences myoinositol and ascorbate biosynthesis . We expressed and purified recombinant SlIMP3 to analyse the catalytic character. The open reading frame of the SlIMP3 gene was cloned into a pGEX-4T-1 vector to generate translational fusion with glutathione-S-transferases . The recombinant protein was expressed in E. coli and purified with affinity chromatography. The molecular mass of the GST-SlIMP3 fusion protein was estimated to be 55 kD , similar to the predicted molecular mass. Mg2+ is necessary for myoinositol monophosphatase activity.

Optimal SlIMP3 activity was obtained by examining the optimum MgCl2 concentration for enzyme activity from 1 to 30 mM of MgCl2. The 3.5 mM of MgCl2 concentration was the most effective at a pH of 7.0 . The Arabidopsis VTC4 enzyme has been reported to use L-Gal 1-P and D-Ins 3-P as substrates . Therefore, the catalytic properties of SlIMP3 enzyme were analysed using L-Gal 1-P and D-Ins 3-P as substrates. In the reaction mixtures of 3.5 mM of MgCl2, pH 7.0, and 2 mg of enzyme, the apparent Km for L-Gal 1-P was 0.29 mM and that for D-Ins 3-P was 0.28 mM . The apparent Vmax values of SlIMP3 for L-Gal 1-P and D-Ins 3-P calculated were 6.0 and 8.0 units, respectively. LiCl was an important inhibitor, which inhibited the catalytic effect of IMPs on the substrate. In previous studies, LiCl inhibited the catalytic activity of VTC4 for D-Ins 3-P, and the half-maximal inhibitory concentration was 0.08 to 0.1 mM . The inhibition of SlIMP3 for D-Ins 3-P by LiCl was also tested . The IC50 of SlIMP3 with LiCl was 0.03 to 0.05 mM when the reaction contained 0.5 mM of substrate. The relationship between AsA biosynthesis and SlIMP3 gene function, transgenic lines expressing either sense or anti-sense SlIMP3 constructs under the control of the cauliflower mosaic virus 35S promote were further characterized. A qRT-PCR was conducted to analyse the expression levels. The independent overexpressed or anti-sense lines, which displayed substantial altered gene expression by comparison with the wild type plants, were used for further analysis . Total AsA and reduced AsA content quantification were conducted using these transgenic plants. Interestingly that altered SlIMP3 expression led to dramatic AsA content change in different tissues of the transgenic lines .

The AsA content quantification in leaf, stem, and different fruit developmental stages indicated that SlIMP3-overexpressed fruits accumulated higher amounts of AsA than the WT plants, whereas the downregulated fruits had lower AsA content than the WT plants. In addition, qRT-PCR results indicated that SlPGI, SlGMP1, SlGMP3, SlGGP1, SlGGP2, and SlGalLDH were significantly upregulated in SlIMP3-overexpressed fruits . We also found that SlGalLDH was significantly down-regulated in downregulated fruits . These results indicated that the SlIMP3 gene regulated AsA biosynthesis in tomato plants.Postharvest fruit storage was conducted to test the influence of SlIMP3 overexpression on fruit softening. The WT and SlIMP3- downregulated fruits were shrivelled after 26 d of storage versus the well maintained pericarp quality of the SlIMP3-overexpressed fruits . The fruit water loss of the SlIMP3-overexpressed fruits was less than that of WT and SlIMP3-downregulated fruits . The SlIMP3-overexpressed fruits also were firmer throughout fruit development versus the downregulated fruits and WT fruits, which were again similar . Ethylene production in SlIMP3-overexpression and SlIMP3-downregulated fruits did not change significantly compared with the WT fruits . Overexpression of SlIMP3 had no effect on fruit yield and weight . In addition, both the up-regulation and down-regulation of SlIMP3 did not affect the fruit development and maturation . Transmission electron microscopy was used to examine the cell wall thickness to determine the effects of altered SlIMP3 expression on cell walls. The SlIMP3-overexpression fruits had thicker cell walls than those of WT fruit but not of those of SlIMP3-downregulation fruits . At the same time, we measured the expressions of cell wall metabolism-related genes as previously described . qRT-PCR results indicated that SlEXP1, SlPG2, SlPL, SlTBG4, SlXYL1, and SlXTH5 were significantly down-regulated in SlIMP3-overexpressed fruits . Moreover, like fruits, the cell wall in SlIMP3- overexpression leaves and stems were also increased . Collectively, these results suggested overexpression of SlIMP3 increased the cell wall thickness, delayed the fruit softening, and enhanced tomato shelf-life.To gain more insight into the mechanism by which cell wall thickness was impacted in SlIMP3-overexpressed plants, biochemicals related to cell wall bio-genesis was analysed. Measurement demonstrated overexpression of the SlIMP3 gene increased myoinositol in mature green and red fruits versus no effect of the downregulation of SlIMP3 gene . Uronic acid content was measured in the cell wall of mature green and red fruits by high-performance liquid chromatography . The uronic acid content increased markedly in SlIMP3-overexpressed red fruits but was unchanged in SlIMP3-downregulated fruits and WT fruits . Various neutral sugars, rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose were also measured. The rhamnose, xylose, mannose, black plastic planting pots and galactose content of SlIMP3-overexpressed red fruits were significantly higher than that of WT . Collectively, these results suggested that overexpression of SlIMP3 improved cell wall bio-genesis in tomato fruit.Because myoinositol is related to cell wall bio-genesis, the WT tomato plants were treated with 10 mg/L, 100 mg/L, and 1 g/L of myoinositol to examine the role myoinositol in cell wall bio-genesis and fruit softening. When treated with 10 mg/L of myoinositol, the fruit cell wall thickness increased slightly, but the fruit softening and water loss were unchanged versus the control.

When treated with 100 mg/L, myoinositol fruit softening was delayed, cell wall thickness increased, and water loss decreased significantly . However, after treatment with 1 g/L of myoinositol, fruit cell wall thickness, storage life, and water loss were unaffected . The 10 mg/L and 100 mg/L of myoinositol treatments sharply increased fruit AsA concentration, but 1 g/L of myoinositol did not modulate fruit AsA production . These experimental results demonstrated exogenous myoinositol application 100 mg/L enhanced the cell wall bio-genesis and delayed softening in tomato fruits. A B. cinerea spore suspension surface applied to injured tomato transgenic tomato fruits produced significantly smaller lesion diameters in SlIMP3-overexpressed fruits versus those in WT and SlIMP3-downregulated fruits, which were approximately equal . Similarly, the biomass of B. cinerea detected with qRT-PCR was significantly lower in the SlIMP3-overexpressed fruits versus the WT and downregulated fruit, which were again approximately equal . Moreover, the expression levels of pathogen-related genes were significantly up-regulated in SlIMP3-overexpressed fruits but were unchanged in SlIMP3-downregulated fruits and WT fruits. This demonstrated overexpression SlIMP3 in tomato significantly improved the tolerance to B. cinerea.In Arabidopsis, the VTC4 gene encodes a bifunctional enzyme that catalyses conversion of L-Gal 1-P to L-galactose in AsA biosynthesis and catalyses conversion of the D-Ins 3-P to myoinositol. In tomato, three SlIMP isoforms are lithium-sensitive enzymes that catalyse the myoinositol biosynthesis from myoinositol monophosphate. In this study, the SlIMP3 gene that had highest expression level among the SlIMP genes was expressed and the SlIMP3 protein purified. The SlIMP3 showed high affinity with the L-Gal 1-P and D-Ins 3-P, and was sensitive to lithium, consistent with enzymatic properties of the Arabidopsis VTC4 . Overexpression of the SlIMP3 gene increased the AsA content, while downregulation of the SlIMP3 gene decreased the AsA content in tomato .Overexpression of the SlIMP3 gene also increased the myoinositol content in tomato fruit . Our data indicated the SlIMP3, which functions like the VTC4, is involved in the biosynthesis of AsA and myoinositol in tomato. But downregulation of the SlIMP3 did not decrease the myoinositol in tomato fruit , which may indicate a redundancy in myoinositol biosynthesis, and suggests the SlIMP1 and SlIMP2 may supplement the enzymatic activity of SlIMP3 in tomato. Interestingly, we found that the expression level of SlIMP2 was significantly up-regulated in SlIMP3-downregulated fruits, which may compensate myoinositol biosynthesis. However, the relationship between SlIMP2 and SlIMP3 remains unknown. Perhaps, in future studies, a tertiary mutant of SlIMPs can be generated using CRISPR/Cas9 methods and used to study their precise function in the biosynthesis of AsA and myoinositol in tomato. In Arabidopsis, overexpression of the myoinositol oxygenase increased AsA content, suggesting that myoinositol can act as a precursor for ascorbate biosynthesis. However, it has also been reported that the MIOS controls the myoinositol level, but does not contribute to AsA biosynthesis . In this study, treating fruits with 100 mg/l of myoinositol not only increased the cell wall bio-genesis, but also increased the fruit’s AsA content . The myoinositol was converted into D-Glucuronic acid by MIOS and the D-Glucuronic acid was catalysed into L-gulonate, which can be converted into the AsA by a multistep process in animals. Our results support that the hypothesis that the myoinositol oxidation pathway contributes to AsA biosynthesis in tomato fruit. Perhaps rigorous radiotracer experiments could elucidate the pathway of myoinositol conversion into AsA in tomato fruits.Myoinositol has been reported to play an important role in cell wall formation . Myoinositol is catalysed by inositol oxidase to D-glucuronic acid, a precursor of pectin and hemicellulose in cell wall biosynthesis . Radioactive myoinositol injected into ripening strawberry fruits was converted to the Dgalacturonosyl residue of pectin and D-xylosyl residues of hemicellulose . In this study, overexpression of SlIMP3 markedly increased the myoinositol, uronic acid and neutral sugar content, and fruit cell wall thickness . Simultaneously, treating fruits with 100 mg/L of myoinositol produced a similar phenotype as that of SlIMP3-overexpressed fruits . The data presented here proved that myoinositol participates in tomato fruit cell wall bio-genesis. Silencing of the PL gene reinforced the tricellular junction in the fruit cell wall and delayed the tomato fruit softening . Mutations in the GA2-oxidase gene improves cutin and wax biosynthesis and increases tomato fruit firmness and shelf-life . In this study, overexpression of SlIMP3 gene increased cell-wall thickness and fruit firmness, delayed fruit softening, and prolonged tomato fruit shelf-life . These results support that improving cell wall bio-genesis is an effective strategy for delaying fruit softening and extending fruit shelf-life.

With fruit sizes reaching almost 300 grams, it is likely that thinning may have been excessive and crop load per tree could have been increased while still reaching above minimum fresh market size requirements. The strong yield for high density plantings of August Flame during the 2017 harvest supports reports of higher density plantings reaching full cropping sooner than low density systems . By 2018 the Nema Quad systems were able to produce fruit of similar size compared to systems with size-controlling rootstocks, but fruit were still not the largest. Large fruit sizes indicate that the amount of thinning could, again, have been reduced. In 2019 there was noticeable water stress in the field due to some irrigation problems, but the magnitude of the problem was not documented. It is likely the water stress was a reason for some of the smaller fruit sizes compared to previous years. Most interesting about the 2019 season was the performance of the C-9 Quad system and how after producing significantly smaller and fewer fruit in both previous seasons, it now had the largest mean fruit size. Overall, systems with size-controlling rootstocks performed well and on par compared to the Nema Quad system giving confidence that reduced hydraulic conductance associated with size-controlling rootstocks does not necessarily reduce fruit size in either early or late bearing cultivars such as June and August Flame.In addition to fruit size, plastic pot plant containers number of fruits produced was not diminished in systems using size controlling rootstocks compared to the Nema Quad system.

The 2017 harvest for the June Flame cultivar was the only harvest that the Nema Quad system produced significantly more fruit per hectare than all other systems. These results differ with previous studies where KAC_V plantings reached full cropping at the same time as trees on vigorous rootstocks but, systems with size-controlling rootstocks pruned to an open-vase lagged behind more vigorous rootstocks . By 2018 the C-6 Quad and C-6 V systems produced more fruit per hectare than the Nema Quad system while the C-9 system had a substantially reduced yield compared to all other systems. Fruit count could have been increased had thinning been more consistently managed but since fruit sizes were also similar, results would not likely have changed in terms of differences between systems. 2019 was by far the most productive harvest for June Flame, with strong yields in the C-6 Quad, C-6 V, and Nema Quad systems while the C-9 Quad was less productive. Due to the lack of significant differences among systems there is no evidence that a reduction in either fruit size or fruit count would be expected in an orchard system using size-controlling rootstocks compared to a system with more vigorous rootstocks, when using appropriate management practices and planting densities adjusted for the reduced tree size. Results from the 2017 harvest of August Flame were much more aligned with previous studies where systems with high-density plantings reached maximum yield capacity earlier than in low-density systems . It is possible that if the amount of thinning in the C-6 Quad and C-6 V systems had not been as severe, they could have produced significantly more fruit than the Nema Quad system. The C-9 Quad system had the lowest fruit count but, with such a large fruit size, could have potentially produced a fruit count similar to the Nema Quad system if thinning had been done more precisely. In 2018 the fruit count was similar in the C-6 Quad, C-6 V, and Nema Quad systems while the C-9 Quad system had half the crop load as the other systems.

Since the most size-controlling rootstocks produced yields on par with the Nema Quad system, it is probable that the C-9 Quad system was under some stress that hindered production rather than its reduced fruit count being a result of reduced hydraulic conductance in the rootstock. It is likely in 2019 that all systems were under stress. Not only was fruit sizesignificantly smaller than previous years, fruit count per hectare was also fewer than that of even the earlier bearing cultivar for all systems except the C-9 Quad. In this study the relationship between crop load and fruit size was similar among systems with high density plantings on size-controlling rootstocks and the system with lower planting densities on a vigorous rootstock. Results were as expected, as crop load increased fruit size diminished. Although the relationship between fruit size and fruit produced per hectare was not significantly different among systems, appropriate crop load per tree and fruit size was influenced by planting density. The larger crop load that trees in the Nema Quad system could hold while maintaining similar fruit size as trees from other systems with significantly reduced crop load per tree indicate that trees with size-controlling rootstocks planted at a higher density may not be able to maintain as large of fruit size with larger crop loads compared to trees with more vigorous rootstocks at wider spaced plantings, this concurs with findings from Inglese et al., .

Results from this study also demonstrate that an increased number of trees per unit area compensate for the reduced crop load per tree, thus allowing high-density plantings on size controlling rootstocks to be a viable option for commercial production, similar findings have been reported by Webster and DeJong et al., .It is well documented that an orchard’s ability to intercept photosynthetically active radiation influences yield and that the two are linearly related up to 50% light interception . Data from this trial had a similar trend for the relationship between light interception and yield. In the June Flame cultivar, yield measurements up to 50% light interception had little deviation from the linear model, above this point yield varied significantly. Above 50% light interception, differences in yield are likely caused by other limiting factors such as water or nutrients . This would also explain the poor fit for the model of the C-6 V system. With a canopy that intercepted >50% in all three seasons, yield variation was likely a result of environmental conditions. August Flame systems had more time in the first growing season to fill out their allotted space. This resulted in most systems having >50% light interception in all three seasons. Due to the higher light interception, all models had a poor fit and did not provide confident yield predictions.Plants and their secondary metabolites have been used as one of the important sources in the field of medicines or health related issues since ancient times. The role of medicinal plants in the health care had been already mentioned in the Indian holy books like “Vedas”. Recent report of World Health Organization has been estimated that approx 45,000 plants being practiced for the medicinal purposes across the globe. Presently, around 65% of Indian population directly are dependent upon the traditional medicine for their need of primary health. Secondary metabolites of these herbal plants is an alternative source broadly used in the treatment of chronic diseases. Currently, traditional medicine is broadly used in the treatment of ulcer worldwide, and has been proven as one of the best strategies for the disease management of ulcer . Ulcer is a discontinuity or break in a bodily membrane in the form of wound or sores that are slow healing or keep returning. It impedes the organ of which that membrane is a part from continuing its normal functions . It is of many forms which occur on both, inside and outside of the human body. Currently, large plastic gardening pots different types of ulcer forms are recognized in medicine such as peptic ulcer, corneal ulcer, stomach ulcer, foot or leg ulcer etc. Ulcer causing problems in digestive system and wounds appearing in the lining of digestive track in human beings are very common. The digestive track of human beings is very sensitive and the health of digestive track can be good or bad and depends on many factors. Pepsin exposed ulcers i.e., peptic Ulcers are the most common type in the gastrointestinal tract area that result from an imbalance between stomach acid-pepsin and mucosal defence barriers and more than 4 million people affected worldwide annually. In medicine, the ulcer which occurs as mucosal lesions which penetrate the muscularis mucosae layer and form a cavity surrounded by acute and chronic inflammation is defined as peptic ulcer.Adesanwo et al.studied the anti-ulcerogenic effect of Melaleuca bracteata stem bark extract and showed thatthe extract significantly reduced gastric acid secretion. They also reported that the bark extract contains two important constituents’ betulinic acid and oleanolic acid, play major role in anti-ulcer effect. In another attempt, Agrawal et al. studied the antiulcer activity of petroleum ether, alcohol and aqueous extracts of Smithia conferta. Phytochemical analysis of petroleum ether extract found to have steroids, alcohol extract constitute isoflavonoids, alkaloids and carbohydrates whereas in the aqueous extract significant amount of amino acids, carbohydrates and flavonoids were present. However, the aqueous and alcoholic extracts showed significant reduction in ulcer index compared to petroleum ether extract.

All through in our evolution, natural products have enormous eminence in the fields of medicine and health. Natural products along being the earth friendly, they are free from any adverse effect to the human health.Plant metabolites have been the most successful source of potential drugs since ancient period. However, due to the emergence of new human diseases with the changing environment, continuous screening and validation of secondary metabolites in the form of drug identification/designing needs to be updated. Different cheminformatics approaches like target identification, active site prediction, drug likeliness properties, biological activity and molecular docking of selected phytoligands are the key features for identifying for functional aspects of any drug. Secondary metabolites of the plants have been recognized to elicit beneficial effects in virulent factors of diseases. The raw materials and pharmaceuticals needed for the preparation of essential drugs are largely obtained from the local herbal plants. The revolution of metabolic engineering and the development molecular docking algorithms approaches lead to improved molecular simulations with crucial applications in virtual high throughput screening and drug discovery. Analysis with molecular docking of interactions between protein-ligand, become an emerging tool in drug design. In case of Helicobacter pylori infected individuals, the frequencies of virulent factor IFNg cells have been increased in the antrum, which induces development of gastric ulcers. Protodioscin a secondary metabolites of Asparagus racemosus is used as medicinal compounds against several diseases. The analysis by molecular docking between the virulent factor and plant metabolites showed the interaction between structural protodioscin and interferon-g , in which all residues of interferon-g exhibited hydrophobic interactions . Although, the obtained binding energy of protodioscin- interferon-g complex revealed disruptions of interferon-g integrity. These types of interactions between the virulent factors of ulcer and plants secondary metabolites open a new door in the field of designing and discovery of a new drug in the ulcer treatment.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. Fruit ripening has been studied for decades, yet there are still many unanswered questions about the timing and coordination of the biological processes related to this developmental program. Much of this research has been done in the model for fleshy fruit ripening, tomato , and has utilized the spontaneous single ripening mutants Cnr , nor , and rin . Each of these mutations produces pleiotropic defects to ripening and occur in or near genes encoding the transcription factors SPL-CNR, NACNOR, and MADS-RIN, belonging to the SQUAMOSA promoter binding protein-like , NAM, ATAF1/2, CUC2 and, MCM1, AG, DEF, SRF TF families, respectively. Each TF family functions in diverse developmental processes and have distinct spatiotemporal expression patterns . These mutants were used to study ripening under the assumption that the mutations cause a complete loss of function to the corresponding protein. Recently, it has been discovered that the nor and rin mutations produce proteins that are still functional and gain the ability to negatively regulate their targets . In nor, the two base pair deletion truncates the protein but still produces a functional DNA-binding and dimerizing NAC domain . In rin, a large deletion creates a chimeric protein with the neighboring gene MACROCALYX , producing a functional protein with suppression activity .

Several previous reports also demonstrated that low levels of IAA stimulated primary root growth. Similar to our observations, the Burkholderia sp. SSG that was isolated from boxwood leaves produced 2.9 to 4.5 µg mL−1 of IAA with tryptophan and had plant growth promotion ability in three boxwood varieties. Additionally, Burkholderia phytofirmans strain PsJN, which was isolated from onion roots, showed higher IAA production, around 12 µg mL−1 , with the addition of tryptophan and improved the growth of potato, tomato, maize, and grapevines. Other Burkholderia seminalis strains can also synthesize IAA and have been reported to increase rice and tomato seedling growth. These previous studies, along with our observations, suggest that B. seminalis strain 869T2 may be similar to other Burkholderia species and other plant-growth-promoting bacteria that utilize IAA to increase root growth, which may assist host plants in taking up nutrients from the surrounding environment and improve aerial tissue growth. Consistent with this hypothesis, we observed that plant size, height, fresh weight, dry weight, and total leaf areas of several tested plant species all significantly increased after inoculation with B. seminalis strain 869T2. It is known that the IAA can positively affect cell division, enlargement, tissue differentiation, root formation, and the control process of nutrition growth. The IAA can also function as a signal molecule to influence the expression of various genes involved in energy metabolism and other plant hormone synthesis, such as gibberellin and ethylene. Interestingly, we observed earlier flowering in the 869T2-inoculated hot pepper and okra plants, square plastic plant pots suggesting that acceleration of plant growth rates might occur in these plants.

In the future, transcriptome analysis of plant hormone response genes and energy-metabolic-related genes in the 869T2-inoculated plants might help us further decipher the possible mechanism of plant growth promotion ability of strain 869T2. From the results of our study, we observed that B. seminalis strain 869T2 had a better IAA yield at a temperature range of 25 ◦C to 37 ◦C and pH of 6 to 9. Similarly, Burkholderia pyrrocinia strain JK-SH007 reached the maximum production of IAA at 37 ◦C and pH 7.0. Several other plant-growth-promoting bacteria, including Bacillus siamensis, Bacillus megaterium, Bacillus subtilis, and Bacillus cereus, had relatively higher IAA yields at temperatures of 2–135 ◦C and pH 7–8. Three different bacteria isolated from therhizosphere of Stevia rebaudiana also exhibited greater production of IAA at a pH range of 6–9 and a temperature of 35 ◦C to 37 ◦C; these bacteria also increased the root and shoot bio-masses of wheat and mung bean. Various carbon sources are used as an energy source for IAA production and could enhance recycling of cofactors in bacterial cells. Our results revealed that IAA yields of B. seminalis strain 869T2 were slightly better when glucose and fructose were used in media. Several previous publications also indicated that the ability of plant-growth-promoting bacteria to produce IAA was different, depending on the carbon source used in the media. Results from these studies and our study demonstrated that IAA production by different plant-growth promoting bacteria can be influenced by various factors, such as temperature, pH, carbon sources, culture conditions, and bacterial species. In this study, we utilized the colorimetric method to estimate the IAA amounts of B. seminalis strain 869T2 when grown in various in vitro conditions and media.

Because the available tryptophan in the rhizosphere and root exudates of plants might be relatively lower than the tryptophan used in the media, the IAA production of B. seminalis strain 869T2 when grown in inoculated plants shall be determined with more sensitive and accurate methods, such as high-performance liquid chromatography or ultra-performance liquid chromatography systems. Apart from the IAA production ability of B. seminalis strain 869T2, this bacterium exhibited siderophore production and phosphate solubilization activities. Iron is an important element for many biological processes in plant growth and development. Most iron in soils is present in the highly insoluble ferric form, which is unavailable for plant absorption. Endophytic bacteria can yield iron-chelating agents such as siderophores, which bind ferric iron and help transport it into plant cells via root-mediated degradation of organic chelate, ligand exchange, or other mechanisms. Phosphorus is another essential macronutrient for numerous metabolism processes in plants, such as biosynthesis of macromolecules, signal transduction, photosynthesis, and respiration. Most of the phosphorus in soil is insoluble and not available for root uptake to support plant growth. In order to increase the bio-availability of phosphorus for plants, certain endophytic bacteria turn insoluble phosphate into soluble forms via the processes of chelation, ion exchange, acidification, or production of organic acids. Previous studies have also correlated siderophore production and phosphate solubilization abilities with the plant growth promotion traits of other Burkholderia species, such as the Burkholderia sp. SSG isolated from boxwood and the Burkholderia sp. MSSP isolated from root nodules of Mimosa pudica. Burkholderia cenocepacia strain CR318, which was isolated from maize roots, significantly enhanced maize plant growth by solubilizing inorganic tricalcium phosphate. Other studies have revealed that additional Burkholderia species also have the ability to solubilize inorganic phosphate to increase available phosphorous in agricultural soils and improve agricultural production.

In summary, both previous studies and our results suggest that the IAA synthesis, siderophore production, and phosphate solubilization abilities of B. seminalis strain 869T2 may collectively contribute to the growth enhancement observed in the several plant species tested here. We successfully inoculated and reisolated B. seminalis strain 869T2, which was originally isolated from the monocot plant vetiver grass, in several eudicot plant species of the Brassicaceae, Asteraceae, Amaranthaceae, Solanaceae, and Malvaceae families. Strain 869T2 can significantly improve the growth of both the roots and aerial parts of Arabidopsis and several leafy vegetables, including ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. These results suggest that the endophytic bacterium strain 869T2 may have a wide host range. A similar observation was reported for Burkholderia phytofirmans strain PsJN, first isolated from onion roots, which enhanced the growth of Arabidopsis, switch-grass, potato, tomato, maize, wheat, and grapevines. We did not observe significant growth improvement in hot pepper or okra plants after inoculation with strain 869T2; however, we did observe early flowering and better fruit development in these tested plants. These results suggest that the plant growth promotion abilities of strain 869T2might be more apparent in crops with a shorter life cycle or that the latter two tested host plant species might not be fully compatible with this bacterium. The plant colonization process and growth promotion abilities of endophytic bacteria seem to be active processes that are regulated by different characteristics of both the host plants and bacteria. In conclusion, our study revealed the potential of Burkholderia seminalis strain 869T2 for use as a bio-inoculant in agriculture to improve plant growth and production.Tropical fruit flies , such as the Mediterranean fruit fly from Africa, square pot plastic the oriental fruit fly from Asia and the Mexican fruit fly from the Americas, are recognized by entomologists as among the most destructive agricultural insect pests in the world. Because of tephritids’ economic importance, US states such as California—considered by both the US Department of Agriculture and the California Department of Food and Agriculture to be free of these pests, but with climates favourable to their establishment—invest heavily in measures to keep tephritids from becoming established. These steps include restricting importation of commodities that originate in regions with ongoing tephritid outbreaks, requiring post-harvest treatments for imported fruits and vegetables grown in areas where the pests are endemic or established, maintaining large-scale monitoring programmes for early detection, supporting preventive release programmes of sterile flies to pre-empt establishment, and launching eradication campaigns to eliminate pest populations once discovered. Indeed, 90% of the eradication projects initiated in California between 1982 and 2007 were directed against tropical fruit flies . The historical challenges posed by the fruit fly threat to California are similar to those posed by many other invasive insect species. For example, the propagule pressure of fruit flies resulting from the ever-increasing movement of people and products is an ongoing challenge posed by all invasive species. Similarly, global warming has resulted in the expansion of pest ranges worldwide. Fewer frost days, longer growing seasons, more heat waves and greater frequency of warm nights in California, combined with an abundance of suitable hosts in both urban and commercial environments, create ideal conditions for a wide range of species, particularly tropical tephritids, to successfully invade. Two aspects of California’s fruit fly invasions are unique, however. First, in most years and locations, fruit fly detections are extremely rare because of a combination of the slow population growth of newly introduced species and of population suppression from intervention programmes.

This combination of elements makes it difficult to decipher patterns in detections, because there are few ‘dots’ to connect, and small numbers of captures separated in both time and space may give the illusion that previously detected populations have been eliminated. Second, an unprecedented number of pest tephritids have been detected in California in recent decades, including a more than eight fold increase in the number of species , and thousands more flies have been captured in California than in all other US mainland states combined. We are unaware of any other single taxonomic group that consists of such a large number of economically important invasive species that are continually reappearing in the same region. Our broad goal in this paper is to bring principles of invasion biology, mapping techniques and quantitative methods to bear on detection and interception data to answer questions about the residency status of tropical fruit flies captured in California. We show that, despite the due diligence, quick responses and massive expenditures of government agencies to prevent entry and establishment of these pests, virtually all of the species against which eradication projects were directed have been reappearing; several species reappear annually, and several others every 2–5 years. The preponderance of evidence supports the hypothesis that at least five and as many as nine species are established in the state. Tephritids have been detected in nearly all regions of California where conditions are favourable for fruit fly establishment . Although the largest numbers of detections by far have been in the greater metropolitan areas of southern California, including the Los Angeles Basin and San Diego, a substantial number of flies were also detected in northern California, in the San Francisco Bay Area. Tephritids also began appearing in the state’s main agricultural growing region, the Central Valley, which includes the Sacramento and San Joaquin Valleys, and the Imperial Valley. The non-random pattern of the invasions is reflected in the fact that 100% of first records for all species were in southern California , all but one of which were found in two regions: Los Angeles and San Diego. These regions contain only around one-third of the state’s population, yet account for 100% of the tephritid first records. California was free of any tropical fruit fly species before the mid-1950s , despite the rapid growth of the fruit industry in the late nineteenth century and first half of the twentieth century, as well as relatively lax regulatory protocols at ports of entry. Two species were detected during the 1950s , followed by four more in the 1960s and 1970s. The tephritid situation in the state changed drastically in the 1980s because of: continued reappearances and spread of previously detected species in metropolitan Los Angeles and San Diego; seven new species detected, raising the total in the state from six to 13 species ; and first tephritid detections in northern California , including a massive, widespread medfly outbreak in the Bay Area. Three new tephritid species captured in the 1990s raised the total in the state to 16. The economic stakes were elevated to a new level when one of these, the olive fly , was declared established, and several previously detected species appeared in the Central Valley growing region. Even though only one new species has been captured during the past 12 years, nine previously detected species have been recaptured repeatedly over ever-expanding areas , including seven that have been captured multiple times during the past 3 years . The magnitude and geographical scope of the recurrent detections are evident in maps in figure 1b, showing the historical records of the hundreds of state-wide, regional and local detections of the four most frequently captured species.

Table 1.2 shows the pH values of dried fruits before and after using PBS or MilliQ water as the wet carrier and then homogenizing with PBS or MilliQ water. As shown in Table 1.3, no difference was observed between pH measurements taken from samples inoculated with PBS or MilliQ water and homogenizing with PBS or MilliQ water for most of the samples. The only significant difference in pH measurement was observed from dried peaches that were inoculated with PBS and dried for 48 h. When these samples were homogenized with PBS or MilliQ water for pH measurement, the pH taken from samples homogenized with MilliQ water were higher than the pH taken from samples homogenized with PBS . Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. The efficacy of recovering Salmonella from inoculated sand by stomaching or manually shaking was evaluated. Inoculated sand samples were taken right after inoculation and after 48 h of drying in the oven. The concentration of the Salmonella cocktail was 11.07 ± 0.04 log CFU/mL. Since 20 g of sand was mixed with 1 mL of liquid culture, the theoretical inoculation level in sand is 9.77 log CFU/g. As shown in Table 1.4, 9.51 ± 0.04 log CFU/g of Salmonella was recovered from freshly inoculated sand by stomaching. After 48 hours of drying, 6.93 ± 0.09 Log CFU/g of Salmonella was recovered from the inoculated sand by stomaching. Drying at 40 °C for 48 h caused an approximately 2.5 log reduction of Salmonella. The differences between counts obtained from TSAR and XLT-4R indicated the formation of injured cells during drying.

Comparing the cell numbers recovered by stomaching with the cell numbers recovered by shaking methods, blueberry box no difference in TSAR counts was observed from inoculated sand that has been dried for 48 h . When looking at the XLT-4R counts, stomaching method had higher counts on XLT-4R than shaking method . Similar trend was observed from freshly inoculated sand. Based on this result, stomaching was used in the following studies for recovering pathogens from inoculated sand or sand-inoculated dried fruits.The objective of this chapter was to determine the inoculation carriers for different dried fruits, the buffer system for pH measurement, as well as the pathogen recovery method for sand-inoculated dried fruits. Based on the results, both wet and dry inoculation changed the pH of low-moisture dates, while generating no significant impact on the aw of the samples . A similar observation of aw before and after inoculation was found in high-moisture dates. Dry inoculation reduced the pH of high-moisture dates more than the wet-inoculation. However, when looking at the physical properties of the inoculated dates, wet inoculation caused the skin to start to peel from the dates. Based on these observations, dry inoculation was chosen to inoculate dates. In addition, since dates are typically grown in sandy regions where sand storms are common and some dates are cleaned by air pressure without any contact with water before packaging . For dried peaches, the wet inoculation reduced the aw slightly compared to the noninoculated ones . This reduction in aw might be caused due to the additional 48 h drying after inoculation. 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 in the original food.

Dry inoculation did not impact the aw of either dried peaches. Neither dry nor wet inoculation altered the pH of dried peaches without sulfur treatment significantly. They both reduced the pH of dried peaches made with sulfur treatment. Based on the measurement, there is no strong preference between two inoculation methods. In this case, it was decided to use both wet and dry carriers to inoculate dried peaches.For dried pluots and sundried tomatoes, wet inoculation generated no impact on their pH or aw. In addition, since pluots and tomatoes are also often processed in large quantities and washed before drying like the peaches . Using a liquid carrier could help represent possible contamination from water during washing. The impact of different inoculation methods on the behavior of inoculated pathogens can be determined by the types of products. As discussed earlier, both Blessington et al. and Beuchat and Mann did not find any significant differences between wet- and dry inoculated products. Bowman et al. monitored the survival of Salmonella on black peppercorns and cumin seeds. The tested methods included wet-inoculation with Salmonella cells grown in TSB wet-inoculation with Salmonella grown on TSA biofilm inoculation , and dry inoculation with TSAgrown-Salmonella-inoculated sand. Their results showed that the biofilm inoculation method had the least Salmonella reduction. When comparing the dry inoculation method vs. the wet inoculation methods , the dry inoculation method had no difference with wet inoculation method on black peppercorn but had less reduction of Salmonella on cumin seed. One potential explanation for these differences might be the content and release of different amounts of antimicrobials from the food during wet inoculation . Deng et al. investigated the impact of aw, pH and temperature on the survival of E. coli O157:H7 in a commercial dry infant rice cereal. Results showed that much better survival was seen when the pH was at 6.8 than 4.0. In the current study, if PBS is chosen to carry out the wet inoculation, how it impacts the pH and pH measurement needs to be understood before the experiment. PBS is more often used as a carrier than MilliQ water because of its ability to prevent cells bursting due to osmosis .

Based on our results, wet inoculation with PBS did not have a significant impact on the pH of the dried fruit and using PBS for pH measurement also did not impact the result . Thus, PBS was appropriate to use as the liquid carrier for the dried fruits as changes in pH can influence pathogen survival in dried foods. In the Blessington et al. study, in which both sand and chalk were tested as dry carriers, bigger impact on the weight of the inoculated nuts was observed from the chalk inoculated nuts, especially almonds . Based on our own lab’s data, chalk and talc are lighter and tend to fly around when conducting the inoculation. When dealing with pathogens, it is not safe. Thus, sand is a better option compared with other dry carriers. Unfortunately, sand is an abrasive substance and could potential damage cells during inoculation. Sahin used sand in their study to disrupt bacterial cell walls, indicating that sand can lead to cell abrasion if used in a particular way. When comparing stomaching and shaking by hand in this study for homogenization of samples, Salmonella was recovered at significantly higher levels from stomached samples than shaken samples . When looking at the cell counts obtained from the selective agar, stomaching recovered a higher number of Salmonella from sand samples that had been dried for 48 h. The major difference observed in this study was the injured cells formed during drying as indicated by the differences between counts on TSA and XLT-4 agars. Based on these results, stomaching was chosen for recovering pathogens from sand-inoculated samples.Sulfur treatments are often used in dried fruits to preserve color . In other food applications, blueberry package sulfur is often used as an antimicrobial but it has not been evaluated in this capacity for dried fruits. Based on a recent report , dried fruits produced in the U.S. contain free SO2 ranging from 20 to 635 ppm. Among all the products tested, dried pineapple contains the least free SO2 while dried apples contain the highest levels of SO2. Dried peaches contain 355 ppm SO2 when measured in slurry . Witthuhn et al. evaluated the sulfur dioxide content in various commercial South African dried fruits and the microbial quality associated with these products. Results showed that raisins, Clingstone peaches, apricots, nectarines, and prunes contained approximately 1,302, 866, 1,318, and 806 mg/kg of SO2 respectively. However, no correlation between the SO2 content and the microbial counts was identified. On the other hand, Karabulut et al. showed that the total number of microbes present on sulphurated dried apricots were 3 logs less compared to the non-sulfured dried apricots. Türkyılmaz et al. reported a significantly lower total microbial population on dried apricots made with the sulfur dioxide treatment compared to non-sulfured dried apricots. In the Liu et al. study , the authors have shown that pathogens survived at higher levels for a longer period on dried apricots made without sulfur treatment.

Sulfur dioxide is considered by the FDA as generally recognized as safe . The FDA does not set limits on the amount of sulfur dioxide permitted in foods. However, proper labeling is required for foods containing levels of sulfur dioxide that exceeds 10 ppm. In dried fruits, less than 100 ppm is typically found . However, when inhaled or ingested by sensitive groups, it can induce asthma, even in low concentrations . The California Office of Environmental Health Hazard Assessment has developed a proposed MADL for SO2 of 220 µg/day . Taking dried peaches as an example and assuming 26 grams of dried peaches are consumed each day, the estimated exposure to SO2 is 191 µg/day .Bacterial strains and preparation. The strains of bacteria used for this survival study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains each of rifampicin-resistant Salmonella spp., Listeria, and E. coli O157:H7 were used. The strains are summarized in Table 2.1. Preparation of inocula. Individual frozen stock cultures were streaked onto TSAR and incubated at 37 °C overnight. Each isolated colony was transferred into 10 ml of TSBR, 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, 6 plates 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 2 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 and a total of 15 mL cell suspension was recovered for each strain. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail. A 5-strain cocktail was made for each of the pathogens and was then used to inoculate dried fruits. Each cocktail was diluted and plated onto TSAR for calculating the inoculum levels. Inoculation of dried peaches with wet or dry carriers. Before inoculation, 3 samples of uninoculated dried peaches were homogenized with 100 mL of PBS. One hundred microliters of each of the homogenates were plated on to TSAR to check for the presence of rifampicin-resistant bacteria. This was done to ensure that any bacteria present on plates during the study were from the rifampicin-resistant inoculum used and not from background microbes. Once the 5-strain cocktails were prepared, they were used to inoculate the dried peaches or sand. For sand inoculation, 1 mL of each 5-strain cocktail was added for every 20 g of fine white sand and mixed thoroughly. The inoculated sand was then spread as thinly as possible across a sheet of filter paper in a metal tray. The tray was placed in a gravity oven at 40 °C to dry for 48 h. Once dry, 160 g of the inoculated sand was used to inoculate 800 g of dried peaches and massaged into the fruit for 1 min by hand. The dry inoculated peaches were transferred into Ziploc bags and placed in a plastic container for storage at ambient or refrigerated temperature for 6 months. For wet inoculation, the remaining 5-strain cocktail was added to enough PBS to make a 1:10 dilution.Dry-inoculation, due the cell loss during the drying of inoculated sand, had lower initial inoculation levels. On Day 0 , there were 7.15 ± 0.11 log CFU/g of Salmonella on dried peaches. Storage temperatures directly impacted the die-off pattern of Salmonella.

All sensor signals were logged at 20min intervals and averaged every hour. The results presented are the averages of four replications. Xylem sap uptake into the fruit was determined based on the daily average xylem sap flow measurements from 15 and 30 DAP.Extractions were accomplished at the end of the irrigation cycle, before 08:00h. Soil solution was extracted by adding 450ml of the nutrient solution, without Ca2+, to each plant pot, and collecting the drained leachate. After collecting the soil leachate, plants were decapitated 15 cm above the soil level and the pots containing the roots plus stem stump were used to extract the stem xylem sap. Two fruit and two middle leaves were harvested at the end of the irrigation cycle from each replicate plant with the entire peduncle and pedicel attached and kept in a sealed plastic bag for xylem sap extraction. Xylem sap extraction was accomplished by placing the plant pot, fruit, or leaf blade inside a pressure chamber , while the cut end of the stem, peduncle, or pedicel was exposed to the outside of the chamber through a seal. After sealing, the pressure inside the chamber was increased up to 0.8MPa with N2. The initial xylem sap moving out of the stem, peduncle, or pedicel cut end was blotted dry to reduce Ca2+ contamination from the cut. The following 100 μl was collected over a period of 5min and used to determine the Ca2+ concentration in the xylem sap . The plant pots were pressurized in a custom-built chamber large enough to hold a 9.5 litre pot, with a two-part lid in order to allow assembly around the stem of an intact plant .

The custom-built chamber was also used to pressurize the roots of whole plants to induce guttation on leaf blades, growing bags which were collected for Ca2+ quantification. The guttation samples represent the xylem sap extracted from leaf blades without any contamination from a cut surface. Apoplastic water-soluble Ca2+ was extracted from the blossom-end pericarp tissue of tomato fruit as previously described by De Freitas et al. .The Ca2+ concentration in the soil solution, xylem sap, and apoplastic solution was determined with an Ultra-M micro Ca2+-selective electrode . A standard Ca2+ calibration curve was used to determine the Ca2+ concentration in the samples. The Ca2+ concentration in leaf and fruit tissues was determined in freeze-dried leaf blades, as well as pericarp tissues manually cut from the peduncle and blossom regions of the fruit. Freeze-dried samples were subjected to microwave acid digestion and analysed by inductively coupled plasma atomic emission spectrometry . Calcium accumulation was quantified by subtracting the total middle leaf and fruit Ca2+ contents observed at 15 DAP from the total middle leaf and fruit Ca2+ contents observed at 30 DAP. Calcium accumulation was also estimated by multiplying the quantified xylem sap Ca2+ concentration observed in the middle leaf pedicel and fruit peduncle by its respective daily average xylem sap flow rate observed at 15 and 30 DAP.BER was completely suppressed by spraying the whole plants weekly with ABA during fruit growth and development, compared with water-sprayed fruit that reached a 36% incidence of BER at 30 DAP . Dipping the fruit in solutions containing ABA prevented BER development at 15 DAP, but ABA-dipped fruit reached a 16% incidence of BER at 30 DAP. Control fruit dipped in water had a 39% incidence of BER at 30 DAP. The electrolyte leakage of fruit pericarp tissue was lower in response to whole-plant and fruit-specific ABA treatments at 15 DAP . At 30 DAP, only the whole-plant ABA treatment had lower electrolyte leakage in fruit pericarp tissue. SWP was less negative in response to whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments .

Leaf stomatal conductance progressively increased from the base, middle, to the top regions of the plant, and was lower in the whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments . Whole-plant water spraying, as well as water and ABA dip treatments, had similar stomatal conductance for the basal, middle, and top leaves. Based on the stomatal conductance analysis, the plant ABA uptake was considered high with whole-plant ABA treatment, and no significant ABA movement from the fruit into the plant was observed based on changes in stomatal conductance in response to fruit-specific ABA dip treatment . Plant water loss was reduced by the whole-plant ABA treatment at 15 and 30 DAP , but was similar in all other treatments, including the ABA fruit dip. Whole-plantat 15 and 30 DAP . The water spray treatment had the highest sap flow during most of the daylight period at 15 and 30 DAP . The average flow of xylem sap moving into the fruit during a 24h irrigation cycle was substantially higher on plants sprayed with ABA, compared with all other treatments , with the same diurnal pattern as seen in the leaves . At 15 DAP, fruit on water-sprayed plants, as well as water- and ABA-dipped fruit had a reverse flow of xylemic sap from the fruit back to the plant, starting in the late afternoon until the next irrigation cycle in the morning . The ABA-dipped treatment had a slightly higher sap flow to the fruit than the two water treatments at 15 and at 30 DAP . Fruit on plants sprayed with ABA had no reverse xylemic sap flow throughout the irrigation cycle at 15 DAP, but had the same diurnal pattern, with the lowest flows occurring during the night period . The diurnal pattern of fruit xylem flow at 30 DAP was similar to that at 15 DAP, but the magnitude was substantially reduced, again with no discernible reverse flow . Spraying tomato plants with ABA resulted in higher total fruit water uptake used for growth and lower fruit water uptake through the phloem from 15 to 30 DAP .

The estimated phloem sap solute concentration uptake into the fruit from 15 to 30 DAP was higher in ABA-sprayed plants than in non-sprayed plants .The Ca2+ concentrations in the soil solution and in the main stem xylem sap were similar among all treatments at 15 and 30 DAP. The average Ca2+ concentration in the soil solution among treatments was 1.41±0.09 mM at 15 DAP and 1.08±0.14 mM at 30 DAP. The average Ca2+ concentration in the main stem xylem sap was 0.72±0.04 mM at 15 DAP and 0.63±0.05 mM at 30 DAP. There was no statistical difference among treatments in Ca2+ concentrations in the xylem sap of basal, middle, or top leaves at 15 or 30 DAP. The same results were obtained when Ca2+ was determined on an independent set of plants under the same treatments using the leaf guttation method . The average xylem sap Ca2+ concentrations were 0.71±0.06 mM and 0.86±0.02 mM in top leaves, 0.77±0.03 mM and 0.87±0.02 mM in middle leaves, and 0.64±0.03 mM and 0.81±0.02 mM in basal leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in the peduncle xylem sap was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, nursery grow bag compared with all other treatments , and water-soluble apoplastic Ca2+ was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, compared with all other treatments . Fruit dipped in ABA solution had slightly higher water-soluble apoplastic Ca2+ than fruit dipped in water and fruit from plants sprayed with water at 15 DAP. The Ca2+ concentration in the top and middle leaves was statistically lower in response to whole-plant ABA treatment compared with all other treatments at 15 and 30 DAP . The Ca2+ concentrations in ABA-sprayed plants were 8.7±0.21 and 8.1±0.09mg g DW–1 in top leaves and 17.5±0.52 and 16.1±0.63mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentrations in all other non-ABA-sprayed plants were 13.0±0.36 and 13.0±0.15mg g DW–1 in top leaves and 25.1±0.96 and 23.9±0.81mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in basal leaves was similar in all treatments at 15 DAP , and statistically lower in plants sprayed with ABA than all other treatments at 30 DAP. The Ca2+ concentration in fruit tissue collected at thepeduncle and blossom ends of the fruit was higher in ABAsprayed plants at 15 and 30 DAP . Fruit dipped in ABA had a higher Ca2+ concentration at the blossom-end tissue at 15 DAP, compared with water-dipped fruit and fruit of water-sprayed plants . Ca2+ accumulation was lower in the leaf and higher in the fruit of ABA-sprayed plants than in the other treated plants and fruit from 15 to 30 DAP . Ca2+ accumulation in leaf and fruit quantified by tissue analysis was similar to the estimated Ca2+ accumulation based on the Ca2+ concentration in the xylem sap and xylem sap flow rates into leaf and fruit tissues . The average relative humidity and air temperature from 15 to 30 DAP inside the greenhouse, where the tomato plants were grown, oscillated from 58.2% and 27.8 °C during the day up to 77.8% and 18.2 °C during the night, respectively . The VPD increased from 0.5 kPa at 05:30h to 1.6 kPa at 14:30h, decreasing thereafter . The number of Safranin-O-stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossomend regions of the fruit was higher in response to whole-plant and fruit-specific ABA treatments at 15 DAP . The number of stained vascular bundles decreased in all treatments from 15 to 30 DAP, and all treatments showed a similar number of stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossom-end regions of the fruit at 30 DAP . The fruit growth rate was higher in ABA-sprayed plants compared with all other treatments at 15 and 30 DAP . All treatments showed a positive fruit growth rate during a 24h period at 15 and 30 DAP . For all treatments, the fruit growth rate was higher at 15 DAP than at 30 DAP . The average fruit weight was also higher in ABA-sprayed plants at 15 and 30 DAP . Fruit Ca2+ uptake, both directly quantified and estimated based on the product of fruit xylem sap uptake and fruit peduncle xylem sap Ca2+ concentration, was 6-fold higher in ABA-sprayed plants compared with water-sprayed controls . A much smaller increase in Ca2+ uptake was found in ABA-dipped fruit, but, again, this was consistent for both directly quantified and estimated values . The sprayed and dipped ABA/water ratios for fruit growth rate were 1.41 and 1.15, respectively .Previous studies showed that weekly spraying of tomato plants with ABA prevented BER development in the fruit, while water-sprayed plants reached a 30–45% incidence of BER at 40–45 DAP . At that time, possible mechanisms through which ABA increased fruit Ca2+ concentration and reduced fruit susceptibility to BER were suggested based on estimations of fruit xylem sap uptake and Ca2+ concentration in the xylem sap .Water uptake in leaves comes exclusively from xylem vessels, while water uptake into the fruit comes from both phloem and xylem vascular tissues . Treating the whole plant with ABA reduced stomatal conductance, which resulted in lower plant water loss, lower soil water uptake and xylemic water movement into the leaves, as well as higher SWP and increased xylemic water movement into the fruit. Considering that Ca2+ concentrations in the soil solution and stem xylem sap were similar among all treatments, the observed lower Ca2+ accumulation in ABA-sprayed plants was due to lower soil solution uptake triggered by lower leaf transpiration rates . Our results also estimate a higher solute concentration in the phloem sap moving into the fruit of ABA-sprayed plants . Although ABA reduced stomatal conductance and this would be expected to decrease leaf photosynthesis , the improved plant water status associated with ABA application may have caused compensatory physiological effects in other areas, such as reduced carbon partitioning to roots and/or improved carbon transport rates, resulting in higher solute concentration in the phloem sap, compared with the other treatments. The non-ABA-sprayed plants had an average fruit phloem sap uptake of 1.04ml fruit–1 d–1 and an average phloem sap solute concentration of 144.3mg ml–1 .

Fruit ripening has been studied for decades, yet there are still many unanswered questions about the timing and coordination of the biological processes related to this developmental program. Much of this research has been done in the model for fleshy fruit ripening, tomato , and has utilized the spontaneous single ripening mutants Cnr , nor , and rin . Each of these mutations produces pleiotropic defects to ripening and occur in or near genes encoding the transcription factors SPL-CNR, NAC-NOR, and MADS-RIN, belonging to the SQUAMOSA promoter binding protein-like , NAM, ATAF1/2, CUC2 and, MCM1, AG, DEF, SRF TF families, respectively. Each TF family functions in diverse developmental processes and have distinct spatiotemporal expression patterns . These mutants were used to study ripening under the assumption that the mutations cause a complete loss of function to the corresponding protein. Recently, it has been discovered that the nor and rin mutations produce proteins that are still functional and gain the ability to negatively regulate their targets . In nor, the two base pair deletion truncates the protein but still produces a functional DNA-binding and dimerizing NAC domain . In rin, a large deletion creates a chimeric protein with the neighboring gene MACROCALYX , producing a functional protein with suppression activity . The Cnr mutation is also thought to be a gain of function mutation, although the mechanism has yet to be understood . The Cnr mutation results from hypermethylation upstream of the gene near the promoter and has been shown to inhibit the genome-wide demethylation cascade associated with normal tomato ripening .

Previously, these TFs were regarded as master regulators of ripening; however, nursery pots given the new information about the nature of the mutations in Cnr, nor, and rin, it is less clear the precise roles the TFs are playing in ripening . The nor and rin mutants have been utilized in breeding for developing tomato hybrids with extended shelf life or extended field harvest depending on their purpose for the fresh market and processing tomato industries . Hybrids between elite varieties and the ripening mutants have a delayed ripening progression, but with the tradeoff of decreased fruit quality attributes, such as color, taste, and aroma . Although there are some publications dedicated to evaluating the physiological characteristics of mutant or hybrid fruit , up to this point, much of what we know about the ripening mu-tations is based on controlled greenhouse experiments with limited fruit and few ripening stages examined. A complete dataset of phenotypic data produced from large-scale field trials evaluating fruit ripening and senescence is lacking to provide information relevant to breeding, particularly in the new context of the molecular mechanisms behind the nor and rin mutations. The Cnr mutant provides a unique opportunity to study the role of epigenetics in fruit ripening but is not used in breeding because the mutant phenotype is dominant. It has been suggested that Cnr fruit undergo normal growth and development ; however, fruit appear different from wild type even before ripening, with a smaller size, alterations in cell wall enzyme expression, and earlier chlorophyll degradation . To better utilize Cnr as a tool for studying fruit development and ripening, a broader understanding of the physiological and transcriptomic alterations in this mutant is necessary. These spontaneous single mutants need to be reevaluated as tools to understand the wide-ranging biological processes regulated by each TF.

Previous literature has generally assumed that the mutations block ripening, resulting in similar processes affected . This study demonstrates that each mutant has a unique ripening phenotype, resulting from a combination of inhibited and delayed developmental processes. We integrated phenotypic data with gene expression data and hormone measurements in the Cnr, nor, and rin mutants across ripening and senescence to characterize the extent and timing of the ripening defects. Tomatoes grown under field conditions were assessed for fruit traits over multiple seasons. We then performed a transcriptomic analysis to gain more definition of the timing in which mutant fruit deviated from WT in their development and to determine specific molecular functions altered in each mutant. Due to their pivotal role in regulating ripening, we focused on defects in hormone networks, including biosynthesis and accumulation. We analyzed the influence of each mutation on the expression of the other TF throughout ripening and senescence. Finally, to better understand the combined genetic effects of the mutants on fruit ripening, we generated homozygous double mutants of Cnr, nor, and rin and used phenotyping and transcriptional data to evaluate the relationships between the mutants.Tomato plants of c.v. ‘Alisa Craig’ and the isogenic ripening mutants Cnr, nor, and rin were grown in randomized plots under standard field conditions in Davis, CA, United States, during the 2016, 2017, 2018, and 2020 seasons. Fruit tagged at 10 days post-anthesis , which corresponds to 7 mm in fruit diameter, were harvested at stages equivalent to the WT fruit. Fruit were sampled at the mature green , turning , red ripe , and overripe stages, corresponding to 37, 45, 50, and 57 dpa, respectively. The term “RR” is used throughout the manuscript to refer to the 50 dpa stage of all genotypes, even when the mutant fruit do not turn red. Fruit stages for each of the mutants were further validated by external color analysis . Double mutant fruit were generated through reciprocal crosses: Cnr × nor, nor × Cnr, Cnr × rin, rin × Cnr, nor × rin, and rin × nor. Fruit were selfed after the initial cross to generate an F2 segregating generation. The double mutants were initially selected in the F2 generation through genotyping and phenotyping. At least two additional generations after F2 were obtained through selfing to ensure the stability of the double mutations and to perform the experiments in this study.

Three seasons of data were collected for the Cnr/nor fruit while only one season of data was collected for the rin/nor and Cnr/rin crosses.The mutant lines were genotyped for their respective mutations. For nor, the Phire Plant Direct PCR Kit was used to extract DNA and amplify the region of the gene containing the 2 bp mutation using the primers listed inSupplementary Table 1 . The PCRs were run on a SimpliAmp Thermal Cycler with the following conditions denaturation: 99°C for 5 min; 35 cycles of 98°C for 5 s, 56°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR products were purified using Wizard SV Gel and PCR Clean-Up System and then sequenced with Sanger technology to confirm the absence of the two nucleotides. For rin, the Phire Plant Direct PCR Kit was used to extract DNA and perform end-point PCRs using primers specific for the mutant and WT alleles . The following PCR conditions were used for the WT allele primers: denaturation 99°C for 5 min; 35 cycles of 98°C for 5 s, 55°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR conditions for the mutant allele primers were: denaturation 98°C for 5 min; 40 cycles of 98°C for 5 s, 58°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR products were visualized as bands using a 1 The Cnr epimutation was genotyped by bisulfite sequencing. Extracted DNA was treated with the Zymo Gold bisulfite kit . Bisulfite treated-DNA was PCR amplified for the CNR promoter region containing the methylation changes using the primers listed in Supplementary Table 1 . The following PCR conditions were used: 94°C for 2 min; 40 cycles of 94°C for 30 s, 54°C for 30 s, and 60°C for 45 s, plastic planters and a final extension of 60°C for 10 min. The PCR products were then Sanger sequenced and compared to the same region amplified in untreated controls with primers . The following conditions were used to amplify the untreated DNA: 95°C for 2 min; 35 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 1 min, and a final extension of 72°C for 10 min. To ensure mutants were homozygous for the locus, we confirmed the double mutants by allowing the plants to self for at least two additional generations and checking that the progeny were not segregating for any fruitphenotypes.We determined DEGs from the MG and RR stages to identify specific molecular functions altered in Cnr, nor, and rin fruit. First, we compared the ripening mutants to the WT at each stage and obtained a total of 16,085 mutation-related DEGs across all comparisons . Like the PCA suggested , Cnr MG fruit presented the largest amount of mutation-related DEGs , while nor and rin MG had considerably fewer DEGs when compared to the WT counterpart . At the RR stage, large differences between each mutant and WT were observed, with Cnr RR fruit displaying once again the largest differences in the amount of mutation-related DEGs . The large number of mutation related DEGs shown by Cnr fruit further supports our hypothesis that the Cnr mutation more broadly affects fruit development and that nor and rin appear to be more ripeningspecific mutations. We examined molecular functions based on KEGG annotations that were significantly enriched among the mutation-related DEGs for each Cnr, nor, and rin fruit at MG and RR . Large differences in enriched functions were detected in the Cnr MG fruit, which mainly corresponded to alterations in carbohydrate and amino acid metabolism, chlorophyll, and carotenoid biosynthesis, and interestingly many processes related to DNA replication and repair. The lack of green color in Cnr MG fruit could be explained by lower expression of photosynthesis and carbon fixation genes.

The nor MG and rin MG fruit showed few alterations compared to WT and were mainly noted in amino acid metabolism and plant hormone signal transduction. In contrast, at the RR stage, the three ripening mutants showed significant alterations across multiple molecular pathways that range from primary and secondary metabolism to transcription, translation, and signaling processes. We proceeded to mine the mutation-related DEGs for key genes known to affect the fruit traits evaluated in the ripening mutants: color, firmness, TSS, and acidity. We selected five carotenoid biosynthesis genes involved in fruit pigmentation, six genes encodingcell wall degrading enzymes that promote fruit softening, four genes related to sugar accumulation and transport that impact the fruit’s TSS, and one gene that regulates the levels of citric acid then affecting the fruit’s acidity . At the MG stage, we observed that Cnr fruit showed significantly lower expression than WT for several of these key genes, consistent with our phenotypic data , including firmness related enzymes and carotenoid biosynthesis genes. MG fruit from the three ripening mutants showed significantly lower gene expression in an important invertase in fruit , which may contribute to the lower levels of TSS observed in all the mutants. At the RR stage, most of the fruit trait-associated genes surveyed in the ripening mutants had a significantly lower expression than WT, in support of the phenotypic data and reinforced by the numerous functional enrichments among the mutation-related DEGs . The critical carotenoid biosynthesis gene that encodes PHYTOENE SYNTHASE 1 was significantly lower expressed than WT in the mutant fruit across all stages, accounting for the lack of red pigmentation at the RR stage. Also, downstream genes in the pathway encoding Lycopene β-cyclases were highly expressed in the mutants at the RR stage, suggesting that not only was less lycopene being produced but more was being metabolized. CWDEs were negatively affected across all genotypes, with Cnr having the most mutation-related DEGs in this category. We were interested in examining if the Cnr, nor, and rin mutant fruit displayed altered ripening progression or if they were completely inhibited or delayed in ripening events. We performed another set of differential expression analyses comparing RR against MG fruit for WT and each of the mutants to reveal ripening-related DEGs. As anticipated, WT had the largest number of ripening-related DEGs , while nor showed almost no change between the two ripening stages with only 89 DEGs detected . Cnr and rin had fewer ripening-related DEGs compared to WT but still exhibited significant changes during the transition between stages with 5,788 and 2,799 DEGs, respectively. Although Cnr showed the most differences from WT in mutation-related DEGs , it had the largest number of ripening-related DEGs in common with WT fruit .

When combined with the state’s $10.4B value of milk production, alfalfa accounts for a significant portion of California’s agricultural GDP. In addition to its advantages as a forage, alfalfa also provides a host of beneficial ecosystem services: as a legume, alfalfa requires no nitrogen fertiliser, instead it fixes atmospheric nitrogen through a symbiotic relationship with rhizobia, a nitrogen fixing bacterium; the perennial nature of alfalfa improves soil health allowing fields to recover from frequent tilling and prevents topsoil loss; long roots can access water and nutrients deep in the soil profile and increase soil organic matter throughout; and alfalfa is an important insectary, hosting a diversity of beneficial insects . Yield is the most important trait for profitable alfalfa production, yet somewhat inexplicably, yield improvement in alfalfa has stalled over the last ~30 years . In addition to the lack of yield improvement and despite the significant economic and environmental benefits of alfalfa, the area cultivated has been in steady decline both nationally and in California since a peak in 1960 . Already disadvantaged by the distribution of federal subsidies, of which commodity row crops receive billions each year, alfalfa faces stiff competition for inclusion in crop rotations. Increasing yield is therefore imperative to curb the rate of decline in alfalfa area, not only to continue to support California’s significant livestock industry, but also for the benefits alfalfa provides to agricultural ecosystems. Historically, breeders have relied on traditional breeding methods to increase biomass yield in alfalfa, with little success in recent years . However, black flower buckets with the advent of new breeding technologies, such as genomic selection and high throughput phenotyping, this lack of yield gain may be approached in a new light.

This PhD project investigates the incorporation of modern breeding tools and methodologies into an existing breeding program to address the lack of yield improvement in alfalfa.Alfalfa is believed to have two centers of origin, Asia Minor/Caucasia and Central Asia . Following domestication, alfalfa quickly spread throughout the ancient world due to its adaptability to diverse climates and soils. The expansion of civilizations and the Silk Road trade routes played a pivotal role in the dissemination of alfalfa, enabling its introduction to regions such as Europe, North Africa, and eventually the Americas. Alfalfa exists naturally as the Medicago sativa complex that includes a range of diploid and autotetraploid subspecific taxa . Cultivated alfalfa primarily refers to the subspecies sativa and is an autotetraploid , perennial, outcrossing legume with polysomic inheritance . It has a basic chromosome number of eight and a genome size of 800-1000 Mb. Commercial alfalfa stands typically last three to eight years depending on variety, soil, climate, and cultural practices. Pure stands are sown at high density with typical sowing rates ranging from 16-22 kg ha-1 . Plant density starts high with up to 800 plants m-2 three months after planting and then steadily declines. In established alfalfa stands, plant density has limited effect on dry matter yield per hectare until plant numbers fall below 40 plants m-2 , wherein yields decrease and growers should consider retiring the stand. Alfalfa can grow to heights above one metre and has a deep root system that reaches beyond six metres in depth when grown in deep, well drained, moist soils . Alfalfa plants generally have a single deep taproot, with variation in the number and size of lateral roots. Following establishment, alfalfa forms a crown at the top of the root system. After defoliation, alfalfa regrowth occurs from buds located on the crown and from axillary buds at nodes from the remaining above ground stubble . This regrowth cycle allows for multiple cuttings during the growing season for up to eight years before forage yields decline below economic thresholds.

Fall dormancy is an important characteristic of alfalfa that defines a population’s fitness for specific agricultural environments. Fall dormancy is a plant’s response to decreasing photoperiod and temperature and is associated with a slowing and eventual cessation of growth through the dormant period . This trait is closely related to the ability for populations to avoid winter kill and is under complex quantitative genetic control . Alfalfa varieties are classified into different fall dormancy groups, typically ranging from 1 to 11, with lower numbers referring to dormant varieties and higher numbers representing reduced fall dormancy. Non-dormant varieties tend to be higher yielding, but may be lower quality and less persistent, therefore appropriate variety selection is of key concern to the grower.Californian agricultural regions have a broad range of climates and soils and can be divided into five main growing regions: the Central Valley, Intermountain, Low Desert, High Desert, and Coastal regions . Statewide average dry matter yields for alfalfa are 15-17 MG ha-1 from an average of 6-7 harvests per year although this varies with up to 12 cuts on stands grown in the Low Desert and as few as 3 on fields in the Intermountain region . The Central Valley is the most significant area accounting for 70 percent of the state’s alfalfa production . It has a Mediterranean climate characterized by hot, dry summers and cool winters . Rainfall totals range from 20-46 cm annually, falling predominantly in the cooler months from November through to March. The Central Valley has fertile, deep, alluvial soils, although some areas suffer from high salinity. Varieties grown in the Central Valley are predominantly semi-dormant to nondormant however, in the northern tip of the valley dormant varieties are grown for better persistence in heavy soils and greater forage quality. The Low Desert region in Southern California contains 17 percent of alfalfa production.

An area with extremely low rainfall and high temperatures . Soils are generally heavy and like in the Central Valley, high salinity is a significant issue. The Low Desert is also an important alfalfa seed production area. The final area of significance for alfalfa production is the Intermountain region in Northern California accounting for approximately 10 percent of production . This area is more temperate, located at high elevation with warm summers and cool winters . Rainfall averages about 51 cm per year falling in the cooler months. Freezing winters necessitate the use of dormant cultivars to prevent winter kill. Almost all the alfalfa grown in California is irrigated. Check-flood surface irrigation is the most common in the Central Valley and Low Desert regions, while sprinklers are the preferred method in the Intermountain area. Although a significant crop in California, the area of land cultivated in alfalfa has been in steady decline since a peak in 1960. Over the last 12 years hectarage has decreased by more than 40 percent from 390,000 ha in 2011 to 235,000 ha in 2022 . The predominant end-use of alfalfa grown in California is hay for dairy cattle. California surpassed Wisconsin as the number-one dairy state in 1993 and now produces more than 21 percent of milk in the United States . At least 75 percent of alfalfa grown in California is used to supply the dairy industry.The breeding goals for alfalfa are characteristic of most plant improvement programs. They include increasing yield, enhancing nutritive value, and improving biotic and abiotic stress tolerance . The majority of desired traits are complex and quantitatively inherited; however, some pest and disease resistance mechanisms are likely under simple genetic control.Alfalfa breeding programs are based on recurrent phenotypic selection, with or without progeny testing. They are designed to increase the frequency of desirable alleles for quantitatively inherited traits, while maintaining genetic variability for continued genetic improvement . Although self-fertilization is common, alfalfa suffers from severe inbreeding depression which is prohibitive to the production of hybrids, french flower buckets thus commercial cultivars are marketed as synthetic populations generated by crossing different numbers of selected genotypes . As a consequence of the structure of the alfalfa genome, cross pollination and severe inbreeding depression, cultivars exhibit high levels of genetic variation . Significant gains have been made in most traits of interest in alfalfa. Forage quality has improved, demonstrated by the release of new high-quality cultivars . Current cultivars exhibit resistance to a suite of pests and diseases including bacterial wilt , Verticillium wilt , Fusarium wilt , anthracnose , Phytophthora root rot , Aphanomyces root rot , root-knot nematodes , stem nematode , spotted alfalfa aphid , pea aphid , and others . However, there has been little to no improvement in yield, for which a variety of explanations have been proposed. Perennial forage breeders are at a disadvantage compared to those working with annual grain crops when it comes to yield improvement due in part to its perennial nature requiring multiple years of evaluation before selection can be made, the negative genetic correlation between forage quality and forage yield, and the inability to make gains in the harvest index that is possible in grain crops, as all above ground biomass is harvested . A lack of forage breeders and limited resources constrain the size and scope of breeding trials, reducing their efficacy in improving traits with low heritability, such as yield.

The emphasis by alfalfa breeders on pest and disease resistance and greater persistence may lead to a realization of yield potential but it is not increasing yield per se. Perhaps the most important reason for the lack of yield improvement in alfalfa is that breeders have not been explicitly selecting for increased yield potential under commercial production conditions. Yield is often selected indirectly based on evaluation of vigor on spaced plants or on short family rows rather than on measurements of yield on plots grown as a densely sown sward . Although little can be done to address issues such as the inability to increase the harvest index of alfalfa, we do have the ability to modify our trial methodology and to utilize modern technology such as genomic selection and high throughput phenotyping to improve yield potential in alfalfa. Measuring yield on plots instead of individual plants provides a better proxy of commercial production systems, and the incorporation of remote sensing, high throughput phenotyping and genomic selection allow for better allocation of resources within a breeding program and can help to speed up the rate of genetic gain through shorter selection cycles.Genomic selection is a form of marker-assisted selection where the breeding value for a given trait of a genotype can be estimated from many markers distributed across the entire genome . MAS is based upon the establishment of a tight linkage between a molecular marker and the chromosomal location of the gene governing the trait to be selected in a particular environment . This is useful when working with traits controlled by a few large-effect loci, however this is not the case for many important traits in plant breeding, including biomass yield. GS expands on this idea and guides selection based on the cumulative impact of many small-effect loci that are in linkage disequilibrium with genetic markers . To estimate breeding values for a genotype, first a model must be developed from a training population that exhibits variation for the trait of interest. This training population is both genotyped and phenotyped to develop a model which optimizes the chromosomal pattern of alleles. This model then allows breeders to estimate the genetic potential of unobserved genotypes based solely on marker information. Because it relies only on a plants genotype, which can be determined when the plant is still very young, genomic selection allows significantly shorter selection cycles than are required under recurrent phenotypic selection, thus has the potential to significantly increase the rate of genetic gain in an alfalfa breeding program. Early methods of GS in plant breeding used gene chip arrays and were mostly adopted by well-resourced breeding programs in major crop species . As high-throughput sequencing costs have decreased, sequencing based approaches have become more viable and GS is now being explored in a wide range of crops. Genotyping-by-sequencing is currently the most popular reduced-representation approach for genomic selection . It is a reduced-representation approach that uses restriction enzymes to cut the genome at fixed points to show good overall coverage. Samples are sequenced using high throughput next-generation sequencing platforms. A variety of software programs can then be used for genotype calling and identification of polymorphisms for downstream analysis . Because genome coverage is incomplete, causal loci are unlikely to be identified, but the polymorphisms are likely to be in linkage disequilibrium with loci that are causal.