Explants taken from tissue immediately adjacent to the graft junction were able to grow on selective media for both constructs and fluorescence from nuclei and plastids was detected. This outcome was not due to cellular fusion but rather to the exchange of large sections of plastid DNA. However, the study did not exclude the possibility that entire organelles were transferred. While this effect was restricted to a few cell layers near the graft junction, it, nevertheless, challenges the idea that the root stock and scion strictly maintain their individual genetic identities. It has been suggested that exchange of genetic material might occur during graft healing as cell walls and vascular systems are being remodeled. The formation of new plasmodesmata could allow the root stock and scion cells to become symplastic and, perhaps, exchange organelles ; this would thus accomplish transfer of organellar genes. It is important to emphasize that the resulting chimera was not due to cellular fusion, because through single nucleotide polymorphism genotyping and partial sequencing, scion cells were shown to have incorporated only a large piece of the root stock plastid DNA. While it is extremely unlikely that genomic or organellar DNA would be mobile over long-distances,blueberry production as suggested by some researchers , it is possible that heritable changes induced by epigenetic modifications of genomic DNA may occur as a result of movement. Heritable changes can result from RNA mediated silencing mechanisms; siRNA can induce epigenetic effects such as sequence-specific DNA methylation .

Our more recent understanding of heritable epigenetic influences might explain earlier claims of graft hybridization that alleged phenotypic changes in grafted pepper progeny due to mobility of DNA through the graft junction and into the seeds . Although grafting applications that take advantage of epigenetic modifications have not been developed, epigenetic changes present an opportunity to endow progeny with characteristics that result from transcriptional down-regulation or gene silencing without introduction of heritable transgenic DNA. Furthermore, based on previous epigenesis experiments , subsequent generations could revert back to non-silenced phenotypes, thereby limiting the duration of the original modification to the plant of interest, while providing a potential containment against the spread of transcriptionally modified progeny.Evidence of a highly regulated and selective process involving long distance trafficking of mRNA has been demonstrated. Observations have been made of differential localization and accumulation of transcripts in sink tissues, presence of mRNA-binding proteins in phloem sap, and sequence-specific motifs of mobile mRNAs that interact with transcript-binding proteins. Messenger RNAs encoding transcriptional regulators and cell fate/cycle-related, hormone response, and metabolic genes have been identified in pumpkin and tomato sieve tube elements. For example, the transcripts of pumpkin CmNACP, a member of the family of NAC transcription factors that are involved in apical meristem development and leaf senescence, have been identified in scion tissues from pumpkin root stock–cucumber scion plants. This observation supports the idea of long-distance transport and accumulation of CmNACP RNA in vegetative, floral, and root meristematic tissues. Data for this experiment were gathered using in situ RT-PCR and confirmed by in situ hybridization studies. Further experiments with seven other phloem sap-localized transcripts gave similar results, demonstrating the existence of delivery systems of specific transcripts to shoot and root apices .

In another pumpkin root stock/cucumber scion heterograft experiment, a phloem-mobile pumpkin RNA,CmPP16,was found in stems, leaves, and flfloral tissues of the scion. It was determined that the translated protein product CmPP16 bound sense and antisense CmPP16 transcripts and, thus, mediated the transport of its own mRNA into the phloem translocation stream . Due to this self-mobility characteristic, the protein was termed a “plant paralog to viral movement protein.” In a grafted tomato example, a line carrying the dominant mutation, Mouse ears , which causes rounded and unlobed leaflets, was used as the root stock and grafted to a semi-dominant Xanthophyllic mutant scion with yellow, lobed leaves. Eleven of 13 grafted plants demonstrated the Me phenotype in the scion. Interestingly, the Me gene is a fusion of two separate genes, PFP and LeT-6, that produces two transcript splice variants, but only the longer transcript is in-frame with the Let-6 homeodomain and only this transcript was detectable in the scion. Fluorescent in situ RT-PCR confirmed accumulation of the longer Me transcript that had been detected in scion phloem sieve tubes and associated companion cells by Northern blots and confocal imaging. It was concluded that the Me phenotype of the scion was caused by movement of the Me transcript from the root stock. The authors suggested that patterns of transcript accumulation observed by in situ experiments may not be entirely due to promoters expressing locally, but also may be attributed to transport of transcripts . In a follow up experiment, the Me tomato genotype was used as a heterografted root stock with potato as the scion. Again, leaf morphological changes in the scion were observed and DNA gel blot analysis of the RT-PCR products demonstrated translocation of the Me transcript across the graft junction . Two mutant transcripts from the GRAS gene family, CmGAIP and GAI, were used to examine processes underlying mRNA mobility in pumpkin and Arabidopsis. In pumpkin these genes influence responses to gibberellin hormones. The pumpkin CmGAIP transcript, with a deleted DELLA domain, and the equivalent Arabidopsis mutant gai, with a non-functional DELLA domain, were analyzed because the DELLA domain mutations offer an easily trackable semi-dominant, dark-leafed, dwarf phenotype.

The CmGAIP transcripts were found in stem, leaf, and floral tissues of heterografted plants with pumpkin root stocks, particularly in the stem CC and SE. Long-distance trafficking of these transcripts influenced development and leaf morphology in the scion. While the CmGAIP transcripts could be found in floral tissues, they were never detected in maturing fruit tissue; thus, it was concluded that tissue sink strength did not necessarily affect localization and delivery. To confirm specificity and selectivity and to rule out promoter effects, enhanced GFP was transformed into root stocks under the companion cell-specific SUC2 promoter. Although fluorescent signal from the eGFP protein could be detected in grafted scions, the eGFP transcript was not detected,blueberry in container suggesting that inherent properties of particular transcripts were likely responsible for their mobility or lack of mobility. That is, CC were able to retain eGFP transcript, but allowed the eGFPprotein product to enter the phloem and the CC did not retain the CmGAIP transcripts. The observations suggested a complex, regulated, and cell/tissue-specific process underlying mRNA phloem mobility . Furthermore, the 3 untranslated region of the GAI transcript was shown to be necessary and sufficient to target GFP RNA for long-distance movement . A mutated, movement-deffectiveGAI transcript could be partially rescued by restoring nucleotides involved in the formation of predicted stem-loop structures. Thus, in addition to the nucleic acid sequence, the macromolecular structure of the mRNA may also contribute to its ability to be mobilized . Aside from studies of individual transcripts, large scale experiments have identified families of mobile RNAs. Transcripts within the extracellular apoplastic compartments would be candidate mobile RNAs, particularly in the vascular fluids. Out of 1830 expressed sequence tags isolated from melon phloem exudate and sequenced, 986 were shown to be unique and many transcripts associated with biotic responses, stress and defense responses, metal-ion binding, and signal transduction were detected. Only three of the 1830 ESTs were identified as encoding Rubisco or chlorophyll-related proteins. Thus, the authors of this experiment concluded that the results were not due to contamination from surrounding cells. Heterografting with cucumber root stocks revealed that 43 of the 986 unique transcripts were mobile and translocated through the vascular system into the pumpkin scion, perhaps suggesting conservation among these RNA trafficking motifs, at least within the Cucurbits . Despite specific experimental examples, the general mechanism behind RNA trafficking motifs is not well understood. However, studies using non-protein-coding viroids offer evidence that the tertiary structure of viroid RNA is a requirement for mobility across cellular boundaries as well as through the phloem . In addition to the heterografting studies, there is evidence for cross species mRNA mobility in the parasite–host interaction between Cuscuta and tomato . RT-PCR and microarray analyses showed the presence of over 400 tomato transcripts in Cuscuta tissue. Earlier studies had shown that one of the transcripts, LeGAI, was mobile in tomato phloem . It is clear that RNA sequences specify their mobility. Both the 3 and 5 UTRs appear to contain cis-acting sequences termed “zip codes” that provide competence for mRNA vascular transport, transcript stability, and translational regulation .

It is known that mobile mRNAs can influence phenotypes and, at least in one case, this was demonstrated to be the direct result of translation of the mobile mRNA . The experimental evidence makes it clear that mRNAs are present in the vascular stream and can be transported with a high degree of specificity. There have been no studies yet that demonstrate the non-regulated mobility or diffusion of mRNA into the vasculature. Given the relative instability of nascent mRNAs and the identification of protein binding regions in mobile mRNAs , it is generally believed that mRNA transport is mediated via a ribonuclear protein complex and movement of isolated single-stranded RNA transcripts has not been reported . Aside from providing protection against endogenous ribonucleases, RNP proteins may provide additional information for targeting functions. Utilization of the mRNA transit mechanisms with specific anti-pathogen transcripts may be a viable strategy for improving pathogen resistance of scions, although no specific examples for this approach have been described at present. Future applications will likely involve the addition of “zip codes” to target root stock-generated transcripts to specific scion tissues or organs. Under the control of temporal, developmental, or inducible promoters in a root stock, the effects of the transgene in the scion would be evident while maintaining the shoot, as well as its seed and pollen, free from transgenic DNA. In Arabidopsis, the mean and median half-lives of mRNAs are 5.9 and 3.8 h, respectively, but this varies with mRNA function and sub-cellular localization . Given the relatively short halflives of RNA transcripts, it is possible that once fruit and other products are harvested by removal from the plant, any transgenic RNAs in the scion tissues would degrade because the conduits from the sites of RNA synthesis, the source root stocks, have been severed. Much remains to be discovered in the field of nucleic acid movement and associations before applications that can utilize mobile, scion-targeted mRNAs are sufficiently defined to permit their exploitation.Small double stranded RNAs [sRNAs, less than 200 nucleotides ] that participate in gene silencing can be divided into two major groups: siRNA and miRNA. siRNAs are generated from perfect double stranded RNAs produced by RNA-dependent RNA polymerase and can be induced by viruses, genetic constructs, or experimentally introduced. miRNAs are derived from noncoding, imperfect stem-loop RNAs and transcribed from their own promoters by RNA polymerase II. Both are processed by the RNA-induced silencing complex, but while siRNAs have a strictly silencing or quenching effect on gene expression, miRNAs are able to regulate gene expression in a much more tunable manner . The silencing effect can be cell-autonomous or nonautonomous, the latter indicating that silencing effects can be exerted over long-distances from the site of synthesis. With endogenous miRNAs, evidence indicates that most appear to be cell-autonomous . There are exceptions; for instance, the gradual spreading and accumulation of miRNA166 in phloem tissue has been observed during leaf development . In addition, regulation of transcription factors in roots and xylem patterning due to crosstalk between miRNA166 and miRNA165 and transcription factors has been observed . Additionally, long-distance movement of miRNA399 is essential for inorganic phosphate uptake in the roots of phosphate-stressed Arabidopsis, rapeseed, and pumpkin.Subsequent grafting experiments confirmed that, inaddition to its non-cell-autonomous nature, the effect could spread unidirectionally from the tobacco root stock to tobacco scions across a 30-cm WT-grafted “bridge” . Similar results were later reported in grafted sunflower using a GUS marker gene . In both tobacco and sun- flower, the silencing effect was unidirectional, from root stock to scion. Three-week-old embryos derived from self-fertilized graft-silenced scions in sunflower did not show the silencing effect, demonstrating that, at least in this case, the silencing signal was not transmitted to the progeny through the graft.