The bacterium causes disease by transforming the host cell with sets of oncogenes leading to uncontrolled cell division. These oncogenes encode biosynthetic genes for the production of plant hormones auxin and cytokinin. The endogenous plant genes and transferred oncogenes share no sequence homology making the bacterial genes an ideal target for RNA homology-dependent silencing . Arabidopsis and tomato plants were transformed with constructs containing direct inverted repeats of the auxin and cytokinin oncogenes . In planta, these tandem inverted repeats generate dsRNA molecules that in turn induce RNA homology-dependent silencing of the transformed Agrobacterium oncogenes. In the resulting transgenic tomato and Arabidopsis, Agrobacteriummediated transformation was not prevented but the formation of galls by oncogene expression was abrogated completely. This was confirmed by the lack of detectable RNA from the bacterial oncogenes. The transgenic plants did not show any developmental phenotypic variation indicating that endogenous hormone production was not altered by the presence of the silencing construct .Fruit quality is a measure of the desired traits in a certain kind of fruit.Scientists turn fruit quality into a complex matter, attempting to quantify many separate horticultural, environmental, physiological,ebb and flow trays and biochemical components of a fruit, which all contribute on some level to fruit quality.

Although desired traits can vary between individuals, citrus fruit quality is often based upon size, shape, color, peel, uniformity, organic compounds, acidity, flavor, ease of peeling, and seed content of the fruits. Each of these factors needs to be considered during citrus breeding programs. The relative importance of each quality component depends on its intended use and this can vary among producers and consumers. To producers, high yield, good appearance, ease of harvest, and long shelf life are the most important. Consumers, on the other hand, judge quality of fresh fruit based on the appearance and firmness at the time of purchase. Subsequent purchases will be based on the flavor quality and nutritional value of the fresh fruit4 . Maturity at harvest is the most important factor that determines final fruit quality. The main indicators of citrus maturity are coloration, sugar and acid levels, and percent juice. Maturity indices are used to decide when a product should be harvested to ensure an acceptable quality for the consumer. California’s mandatory quality standards for fresh fruits include objective indices of maturity to guarantee the minimum acceptability of their flavor quality to consumers. Enforcement of these standards is the responsibility of the fruit growers and is monitored by the Agriculture Commissioner in each county, representing the California Department of Food and Agriculture. In citrus, these indices differ by variety.Citrus plants grown for fruit production are rarely grown from seed. Virtually all commercial citrus is propagated by grafting and the root stock has a significant impact on the physiological traits of the citrus scion cultivars. Grafting is the process by which a part of one plant is attached to a cut made on a plant with a healthy root system. The plant with the healthy root system is called the root stock, and the part of the plant being attached to this system is called the scion.

The scion confers the properties that are desired by the breeder or grower, while the root stock nourishes the plant. One of the many advantages of grafting is shortening of the juvenile phase, allowing for the trees to produce fruit many years earlier than producing from seed.Due to the large variation in growing conditions and climate in California, different citrus root stocks are required to improve fruit quality in numerous diverse climates, as well as resist various pests and diseases. Root stocks impart certain traits onto the scion and the effects of root stocks can be large. Castle reviewed root stock effects on fruit quality in citrus. In the review, he states that citrus root stocks affect many external and internal fruit characteristics including size, shape, peel thickness, juice content, and juice soluble solids concentration. The most significant impacts are on growth and vigor, tree nutrition, stress resistance, and fruit quality. The magnitude of root stock effects on fruit quality factors ranges from 4% to 24%, depending on the scion-root stock combination. In the present study, four root stocks were chosen from a root stock trial with Washington navel orange scion in Riverside, CA to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich trifoliate orange. These root stocks have imparted significant phenotypic differences on overall growth and fruit quality in many previous studies. Generally speaking, rough lemon root stocks produce the highest yield and fruit size, but this fruit is often of lower quality.Trifoliate orange root stock, on the other hand, produces high quality fruit, with high yield on smaller trees. Carrizo citrange root stocks produce intermediate yield with good fruit quality.

Sweet orange root stocks produce good quality fruit but are very susceptible to various citrus diseases, so sweet orange is rarely used as a root stock for commercial growth. A general explanation for the effects that varying root stocks have on quality is their ability to uptake and utilize water and nutritional elements that play crucial roles in citrus quality and productivity. These effects have been well documented in a variety of crops, including pepper, tomato, pear, grape, watermelon, apple, and citrus. However, these studies still leave many unanswered questions about the mechanisms by which root stocks impart these traits, especially at the regulatory level. In order to elucidate effects of root stocks, previous studies in apple, grape, sweet cherry, and other fruit crops have examined transcriptome changes in various root stock-scion combinations. In citrus,4×8 flood tray gene expression profiling has been used to understand root stock effects and responses to biotic and abiotic factors. These studies look at transcriptome changes in grafted citrus in response to fungal inoculation, cold stress, and boron deficiency. In these studies transcriptomes of leaves from the same scion grafted onto differing root stocks were subjected to RNA-seq in order to observe root stock responses to biotic and abiotic stressors. In another study, expression studies of leaves from mandarin grafted onto various root stocks were analyzed in order to explain root stock effects on the growth of scions. Additionally, many transcriptomic studies have also been performed in citrus to explain mechanisms of fruit ripening and development in commonly grown citrus cultivars. Many of these studies take advantage of the many citrus bud mutations with altered aspects of fruit quality in order to compare gene expression to that in the nonmutant ancestor cultivar. Several of the papers utilize late ripening mutants to assess genes that are important for ripening. Genes involved in hormone-signaling pathways are the main variations associated with ripening in the majority of these reports. Others use mutations in acid and sugar accumulation to explain the molecular mechanisms underlying those processes. Yet to date, none of these reports have linked the genetic effects of citrus root stocks to fruit quality. There have been some previous studies showing changes in the transcriptome of various root stock genotypes, especially in response to biotic and abiotic stressors. These types of changes have been shown in Arabidopsis, corn, mulberry, tomato, and poplar. Though when it comes to root transcriptome studies in citrus, knowledge is extremely limited. A small number of studies have been performed that evaluated trifoliate, trifoliate hybrid, and mandarin root transcriptomes in response to citrus diseases, but these studies each assessed only one genotype. Even in an RNA-seq based approach to establish a reference transcriptome for citrus, 28 samples were used for the study and only two were obtained from roots.

The root samples collected for this study were sour orange and trifoliate genotypes, but samples were grouped by organ to perform differential expression and subsequent analyses. To our knowledge, there have been no comparative studies of citrus root transcriptomes between genotypes during fruit development. As highlighted above, RNA sequencing has become a very powerful and widely used technology to profile the transcriptome. This has provided valuable information for gene identification and defining their potential roles in the grafting process as well as during fruit development and maturation. However, additional regulators need to be discovered to better understand the regulatory network that controls these processes. One regulatory factor that could contribute to root stock effects on the scion are plant small RNAs. One type of small RNA, microRNAs , are 21-24 nucleotide RNAs that are products of genes in plants. Generally, miRNA genes are transcribed into primary transcripts , which are processed into the stem-loop precursor molecule by a DCL protein. These pre-miRNAs are further processed by DCL1 into a miRNA and miRNA* duplex. In plants, the mature miRNA sequence can bind to transcripts of target mRNAs based on perfect or near-perfect complementarity, leading either to cleavage-induced degradation of the mRNA or translational inhibition. Increasing evidence has shown that the miRNAs in plants and animals consist of a set of conserved, ancient miRNAs, as well as many recently evolved species-specific miRNAs. The availability of next generation sequencing technologies provides high throughput tools to discover species-specific miRNAs in a variety of plants, such as Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Triticum aestivum, Zea mays, Medicago tuncatula, Lycopersicon esculentum, Gossypium hirsutum, Taxus chinensis, Vitis vinifera and Citrus trifoliata. Plant miRNAs act as master regulators and control genes involved in various biological and metabolic processes and are thus imperative to proper plant development. Recently, there has been an emergence of studies demonstrating miRNAs have a critical role in the regulation of fruit development and maturation. For example, in strawberry, miR159 acts as a ripening regulator by targeting a MYB transcription factor, which plays a critical role during the transition from development to ripening. Additionally, miR73 was found to be involved in regulation of strawberry fruit ripening by targeting a gene that affects the abscisic acid-signaling pathway. Application of this high-throughput technology to miRNA-related research has identified numerous miRNAs involved in fruit development and maturation in many fruit producing species, including apple, grape, peach, blueberry, date palm and tomato. In citrus, many miRNAs have been identified in different tissues, such as leaf, flower, fruit, and callus. A comparative study was performed between a spontaneous late-ripening sweet orange mutant and the wild-type sweet orange cultivar to better understand the role of miRNAs in citrus fruit ripening. In this study, csi-miR156k, csi-miR159, and csi-miR166d were found to suppress specific transcription factors that are supposed to be important regulators involved in citrus fruit development and ripening. While grafting is known to induce many phenotypic differences, including those involved in fruit quality, there have been very few studies to assess the involvement of miRNAs in the regulation of graft-induced physiological events. Comparisons of miRNA expression profiles in various root stock-scion combinations have been investigated in a few crops such as grapevine, watermelon, cucumber and tomato. In general, it was found that grafted plants exhibit differential expression of miRNAs compared to non-grafted plants. Furthermore, expression profiles of miRNAs were altered when plants were grafted onto differing root stocks, suggesting they play a role in regulating biological and metabolic processes resulting from grafting. In citrus, the hypothesis that changes in activity of specific miRNAs is one of the mechanisms involved in the physiological effects of grafting was tested by determining the expression of miRNAs in different scion-root stock combinations. Changes in expression of the miRNAs tested was associated with the reduction of juvenility and micronutrient requirements of the grafted plants96. However, the precise mechanisms could not be elucidated. Taken together, it is hypothesized that miRNAs in diverse citrus root stocks are differentially expressed in response to grafting and can influence processes related to fruit development and ripening. My dissertation study was originated with the goal of identifying small RNAs that are likely causing changes in fruit quality in grafted citrus. To do this, an integrated study of miRNA and mRNA transcriptomes of sweet orange scions grafted onto varying root stocks was performed. Transcriptomes and miRNomes of fruit and root tissues from four different scion-root stock combinations at four time points throughout fruit development were obtained. These results were correlated with the changes in fruit quality observed when fruit are grown on these genetically diverse root stocks.