Transport of mRNA molecules coding NAC domain protein in grafted pear and transgenic tobacco

Grafting is an important cultivation method and recent research on the mechanism of interactions between rootstock nad scion is focused on the long-distance transport of mRNA and small RNAs in the phloem. Among these transportable molecules, NACP gene coding NAM, ATAF1/2, CUC2 (NAC) domain protein might be involved in apical meristem development. Here, we report the transport of NACP mRNA between Chinese pear (Pyrus bretschneideri) cv. Yali (scion) and the wild Pyrus betulaefolia Bunge (rootstock). Our results indicated that NACP mRNA can be transported in both directions from the 3^rd to 10^th day after micro-grafting. It can also be transported to the shoot apex 30 to 70 cm away from graft-union in 2-year-old grafted trees. For further investigation, transgenic tobaccos with 35S: P. betulaefolia-NACP construct were grafted on wild-type tobaccos (Nicotiana tabacum L. cv. Samsun). The sustainable transport of Pyrus-NACP mRNA through the graft-union occurred from the 15^th day after grafting.     

The role of horticulture in plant invasions: how grafting in cultivars of Callery pear (Pyrus calleryana) can facilitate spread into natural areas

Although few plant species escape from cultivation, some horticultural practices used with woody ornamentals may enhance their invasion potential into natural areas. One such procedure is grafting, in which individuals are propagated for commercial sale by joining a clone of a desired cultivar (the scion) with rootstock obtained from a different individual. If the rootstock can sprout and flower, it can potentially cross-pollinate the scion, leading to fruit production in a self-incompatible species. The effect of grafting on invasion success was examined in the Callery pear, Pyrus calleryana, one of the most popular landscaping trees in the United States. Using nine microsatellite markers, scion and rootstock were genotyped for 57 cultivated trees to characterize rootstock genotypes and the rootstock gene pool. Invasive populations were then sampled to determine if rootstock genotypes have contributed to their formation. In no case were scion and rootstock genotypes identical for any given cultivated tree and rootstock genotypes were genetically variable, although some cloned rootstock genotypes were detected. Rootstock genotypes were also observed in invasive populations, with up to 17% of wild individuals having at least one rootstock parent. These results indicate that rootstock can contribute to the formation of invasive populations of the Callery pear through cross-pollination with other available genotypes. Future investigations of woody ornamentals propagated by grafting should consider this horticultural practice as a potential contributor to invasiveness. Furthermore, plant breeders are encouraged to assess the ability of rootstock to sprout as well as its cross-compatibility with the scion or other cultivar genotypes growing nearby.     

Apple phloem cells contain some mRNAs transported over long distances

Apple (Malus × domestica Borkh.), like many fruit trees, cannot be propagated clonally from seed and is instead propagated by the grafting onto rootstocks. Rootstocks affect the growth of scions, but it is not known why. The circulation of some mRNAs throughout the phloem has recently been shown. To clarify whether RNAs are transported long distance through the graft union of apple trees, we analyzed cDNAs derived from shoot phloem cells by laser capture microdissection. We detected several mRNAs that have already been reported as phloem-transported RNAs in other plants. One of them, MpSLR/IAA14, was probed to transport a long distance through the graft union in grafted apple plant. These results suggest that a phloem RNA transport system may be involved in the effects of rootstocks on scion growth and cropping.     

Influence of rootstocks on growth,yield, fruit quality and leaf mineral element contents of pear cv. ‘Santa Maria’ in semi-arid conditions

Background

Rootstocks play an essential role to determining orchard performance of fruit trees. Pyrus communis and Cydonia oblonga are widely used rootstocks for European pear cultivars. The lack of rootstocks adapted to different soil conditions and different grafted cultivars is widely acknowledged in pear culture. Cydonia rootstocks (clonal) and Pyrus rootstocks (seedling or clonal) have their advantages and disadvantages. In each case, site-specific environmental characteristics, specific cultivar response and production objectives must be considered before choosing the best rootstock. In this study, the influence of three Quince (BA 29, Quince A = MA, Quince C = MC) and a local European pear seedling rootstocks on the scion yield, some fruit quality characteristics and leaf macro (N, P, K, Ca and Mg) and micro element (Fe, Zn, Cu, Mn and B) content of ‘Santa Maria’ pear (Pyrus communis L.) were investigated.

Results

Trees on seedling rootstock had the highest annual yield, highest cumulative yield (kg tree⁻¹), largest trunk cross-sectional area (TCSA), lowest yield efficiency and lowest cumulative yield (ton ha⁻¹) in the 10^th year after planting. The rootstocks had no significant effect on average fruit weight and fruit volume. Significantly higher fruit firmness was obtained on BA 29 and Quince A. The effect of rootstocks on the mineral element accumulation (N, K, Ca, Mg, Fe, Zn, Cu, Mn and B) was significant. Leaf analysis showed that rootstocks used had different mineral uptake efficiencies throughout the early season.

Conclusion

The results showed that the rootstocks strongly affected fruit yield, fruit quality and leaf mineral element uptake of ‘Santa Maria’ pear cultivar. Pear seedling and BA 29 rootstock found to be more prominent in terms of several characteristics for ‘Santa Maria’ pear cultivar that is grown in highly calcareous soil in semi-arid climate conditions. We determined the highest N, P (although insignificant), K, Ca, Mg, Fe and Cu mineral element concentrations on the pear seedling and BA 29 rootstocks. According to the results, we recommend the seedling rootstock for normal density plantings (400 trees ha⁻¹) and BA 29 rootstock for high-density plantings (800 trees ha⁻¹) for ‘Santa Maria’ pear cultivar in semi-arid conditions.     

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.     

Changes in the anatomical structure of tree rings of the rootstock and scion in the heterografts of Siberian pine

The analysis of the anatomical characteristics of tree rings of the Pinus sibirica scions and Pinus sylvestris rootstocks is presented. The main anatomical features maintain the seasonal dynamics characteristic of the reference (ungrafted) trees of scion and rootstock, pointing to the stability of the genetic control of the xylem differentiation. However, various anatomical characteristics are reduced for rootstocks and increased for scions relative to the reference trees. A mechanism consistent with our results is that the graft union zone creates a barrier for the ascending and descending transport of substances, including phytohormones. The reaction of the scion and rootstock to climatic factors is weakened relative to the reaction of the reference trees. Thus, the presumed shift towards phytohormone disbalance reduces the influence of external factors on xylem differentiation. The interannual variation of the individual characteristics of the scions and rootstocks is increased relative to the reference trees, reflecting either the influence of the graft partners on each other or the non-uniform distribution of the growth regulators across the graft union. Thus, heterografts can be used to evaluate the interaction of the internal and external mechanisms of xylem differentiation.     

Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar

Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf‐eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt‐Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non‐transgenic poplar 741 and non‐transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt‐Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt‐Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt‐Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt‐Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt‐Cry1Ac protein). The results showed that Bt‐Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt‐Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.     

Association of Tobacco Ringspot Virus with a Union Incompatibility of Apple

A virus-like disease of apple was observed for the first time in Canada in 1974 in a three year old planting. The disease was characterised by stem pitting, necrosis and breaking or separation of scion/rootstock at the graft union. Foliage was sparse and leaves were chlorotic and diffusely mottled. Using both frozen and freshly harvested leaves of infected apple trees from rootstock suckers as inoculum, a virus was transmitted to herbaceous indicator hosts. Based on host range symptoms, serology and electron microscopy, this virus was identified as tobacco ringspot virus. Initial counts of nematodes in the rhizophere of apple trees affected with union necrosis showed high densities of Xiphinema americanum Cobb., a known vector of nepoviruses. Reports of similar symptoms have indicated that the disease, apple union necrosis and decline was associated with the recovery of tobacco ringspot virus from infected rootstocks. This is the first report associating a union incompatibility condition of apple and tobacco ringspot virus.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a Model System for Graft Union Development in Homografts and Heterografts

Homografting of Arabidopsis thaliana scions on stocks of A. thaliana and heterografting on other species were used to study the compatibility and the ontogeny of graft union formation. Highly compatible homografting with scions of young leafy inflorescence stems was obtained on stocks of inflorescence stems growing from large 3-month-old A. thaliana plants. Histologic analysis revealed four developmental stages of graft union formation in Arabidopsis homografting: (1) development of a necrotic layer, (2) callus proliferation in the grafted scion, (3) differentiation of new vascular tissues within the scion, and (4) a full vascular graft union formation between the scion and the stock. Vascular connections were formed within the callus bridge between rootstocks and scions 15 days after grafting. Heterografts of Arabidopsis on two members of Brassicaceae, cabbage (Brassica) and radish (Raphanus), showed partial incompatible interaction with a lower level of vascular differentiation. Arabidopsis grafting on tomato (Solanaceae) rootstock showed complete incompatibility and limited noncontinuous differentiation of new vascular tissues that did not cross the scion/stock boundary. Although lacking scion/stock vascular connections, Arabidopsis scions grafted onto tomato rootstock flowered and produced seeds. This may indicate some nonvascular functional connections between the two plants, probably of parenchyma cells, further emphasizing the usefulness of Arabidopsis as a model plant for studying various levels of the complicated scion/stock relationships expressed in grafting biology. Experiments with dye transport in the xylem showed that although in general there was an agreement between the histologic study and dye transport, in Arabidopsis homografts water transport frequency was lower than functional and histologic compatability. We conclude that homografting and heterografting of Arabidopsis inflorescence stems is a convenient and reproducible method for studying the fundamental cellular genetic and molecular aspects of grafting biology.     

Growth characteristics and phenylalanine ammonia-lyase activity in peach grafted on different Prunus spp.

Phenylalanine ammonia lyase (PAL) is a key enzyme in the phenylpropanoid pathway responsible for biosynthesis of many secondary metabolites, such as anthocyanins, flavanols, and lignins. The objective of this work was to determine the effect of different rootstock/scion combinations on Prunus tree growth, PAL gene expression, and PAL activity in order to identify compatibility of different graft combinations. The study was performed with peach (P. persica cv. Chimarrita) grafted on two peach rootstocks (Capdeboscq and Tsukuba1) and one Japanese apricot (P. mume cv. Umezeiro). Two or three years after grafting, the growth of peach scion on Umezeiro was weak and finally the death of some trees occurred. The peach rootstocks induced vigorous growth without any tree loss through the three years of evaluation. However, PAL activity and expression of PAL encoding genes were higher in Umezeiro as compared to the other rootstocks. These results show that the differential gene expression together with the PAL activity is a promising strategy to predict graft incompatibility.     

A Study of the graft union in in vitro micrografted apple

Light microscopy was used to study graft union formation in in vitro micrografts of tissue cultured apple (Malus domestica. Borkh). Micrografts were constructed using horizontal incisions to form the grafting surface, and placing the cut ends of rootstock and scion into sterile silicon tubing to permit graft formation to occur.The outer morphological and histological development was similar for different stock-scion micrograft combinations but graft union formation was slower in heterografts than in autografts. Initial leaf expansion at the scion shoot apex occurred in all micrografted plantlets within 1–4 days and was not indicative of graft success. Progressive scion growth and development could be used as an indication of graft success by ten to fourteen days after grafting and probably was related to establisment of cell to cell contact at the graft interface. Microscopy showed initiation of callus proliferation in the vascular cumbium and the pith ten days after grafting. Differentiation was observed subsequently and this was reflected in scion development. Longitudinally orientated cambial cells began to differentiate between twenty and forty days after grafting, and formed a bridge between the vascular cylinders of scion and rootstock. The scions at this stage had as many as eight newly expanded leaves and micrografts were strong enough to permit silicon sleeve removal without damage. Continuity of new vascular elements in rootstock and scion was established around forty days. New vascular elements curved slightly in towards the pith to form a c shaped bridge across the graft union. Vascular development continued until it reached completion after six months.     

Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple

Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.     

Ultrastructural Aspects of Graft Incompatibility Between Pear and Quince In vitro

Callus cells of pear (Pyrus communis cv. ‘Bartlett’)underwent lethal cellular senescence in response to graftingwith callus cells of quince (Cydonia oblonga cv. ‘VanDeman’). Similar responses occurred when the cells werein direct contact and when they were separated by a porous membranefilter. These results indicate that direct cellular contactis not necessary to elicit graft incompatibility between pearand quince. Cydonia oblonga, grafting, incompatibility, pear, Pyrus communis, quince     

Extraction of arbutin and its comparative content in branches,leaves, stems,and fruits of Japanese pear Pyrus pyrifolia cv. Kousui

Arbutin is a tyrosinase inhibitor and is extensively used as a human skin-whitening agent. This study investigated the optimum conditions for extracting arbutin by ultrasonic homogenization from discarded branches pruned from Japanese pear (Pyrus pyrifolia cv. Kousui) trees. The arbutin content was measured in the branches and also in the leaves, stems, fruit peel, and fruit flesh.     

Agrobacterium tumefaciens AKE10-mediated transformation of an Asian pea pear, Pyrus betulaefolia Bunge: host specificity of bacterial strains

The Asian pea pear, Pyrus betulaefolia Bunge, is tolerant to several disorders in the fruit bodies caused by high humidity and dryness and is hence widely used as a rootstock for many pear plants suitable for food sources. We have now successfully transformed P. betulaefolia Bunge by an Agrobacterium-mediated gene transfer system. Among several wild-type A. tumefaciens strains examined, only AKE10 induced shoot-forming tumors at a high frequency on excised cotyledons of P. betulaefolia Bunge cultured on phytohormone-free medium. Both the nptII (kanamycin resistance) and GUS (#-glucuronidase) genes were introduced into the cotyledons by infection with AKE10 harboring a binary vector, and regenerated plants were obtained. Southern hybridization and polymerase chain reaction analyses and histochemical GUS assay indicated that morphologically normal transformed plants faithfully contained genes from the vector but not from wild-type oncogenic T-DNA. However, morphologically abnormal plants additionally possessed the 6b gene (AK-6b) of AKE10. These results show that non-disarmed A. tumefaciens is adequate to transfer genes to the Asian pea pear, P. betulaefolia Bunge.     

A graft that crafts nontransgenic and genome-edited plants

Grafting in plants facilitates the transmission of biomolecules across the union formation. Recently, Yang et al. demonstrated that inter- and intraspecific grafting in plants can be exploited for trafficking tRNA-tagged mobile reagents of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system from the transgenic rootstock to wild-type scion for genetic improvement in plants through targeted mutagenesis.