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.     

Almond brown line and decline: a new disease probably caused by a mycoplasma-like organism

Young almond (Prunus dulcis, cvs Carmel, Peerless and Price) orchards established on the plum rootstock Marianna 2624 (P. cerasifera×P. munsoniana) contained trees that exhibited poor terminal shoot growth and wilted, chlorotic leaves. The scion/rootstock graft union showed an external splitting of the bark and an internal line of necrotic bark tissues that extended into the woody cylinder of the union, which was deeply pitted. Affected trees declined. The disease was named almond brown line and decline (ABLD). Incidence of ABLD ranged up to 55% per cultivar in some orchards. Numerous attempts to graft-transmit orchard collections of ABLD to healthy almond/Marianna 2624 indicators failed. Also, ABLD does not appear to be soil-borne. However, ABLD was serendipitiously determined to be bud-perpetuated when infected scion buds from an apparently healthy appearing Peerless almond/peach tree located in a foundation orchard were grafted onto healthy rooted cuttings of Marianna 2624 to produce yearling trees. Also, graft-inoculations on the almond scion portion of healthy trees, but not the plum rootstock portion, with the peach yellow leafroll mycoplasma-like organism (PYLR-MLO) caused symptoms resembling ABLD. Laboratory and glasshouse assays of several symptomatic trees did not detect tomato ringspot virus and two ilarviruses. These results suggest that an MLO, possibly PYLR-MLO, may be the causal agent of ABLD and that Marianna 2624 is probably resistant to the PYLR-MLO.     

<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.     

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'.     

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.     

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.     

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.     

Application of Electrical Wave Transmission in Basic Research of Grafting

Electrical wave (EW) transmission from scion to stock across the grafting interface was related to the histological changes during the development of graft union. Variation wave (VW) could not be transmitted to stock from scion before isolation layer broken and callus interdigitation. As plasmodesmata formed secondarily at the interface between stock and scion where the isolation layer had disappeared, VW could be transmitted from scion to stock, but its velocity was not rapid until the vascular bridges form between two partners of graft union. Hence, the authors could deduce the degree of graft union formation by measuring whether VW could be transmitted from scion to stock or not and its velocity. EW trans mission method was a new tool for quickly detecting the formation of graft union.     

电波传递在嫁接基本理论研究中的应用

研究发现电波从接穗跃过嫁接面向砧木传递与嫁接的组织学变化相关。砧木和接穗愈伤组织细胞突破隔离层互相嵌合之前,接穗中产生的电波不能传到砧木。砧木和接穗细胞间产生次生胞间连丝后,电波即可沿接穗传至砧木,但此时传递速度慢。当砧木和接穗间产生维管束桥时,电波下传的速度加快。由此可见,通过检测电波跃过嫁接面与否及下传的速度,就可推断嫁接接合部组织学变化状况。电波传递法是一种快速鉴定嫁接植株发育过程的新方法。     

The Anatomy of the Developing Bud Union and its Relationship to Dwarfing in Apple

Light microscopy has been used to study the effect of dwarfingand semi-dwarfing apple rootstocks on the early developmentof bud-unions with 'Gala', and the anatomy of 2-year-old bud-unionsbetween 'Bramley' and the same rootstocks. The bridging of thecut edges of the cambia of bud and rootstock was achieved bydifferentiation of callus formed at an early stage in budding.New cambial cells were aligned at right angles to the pre-existingcambia, with their long axes horizontal. Subsequently-formedxylem adopted this arrangement, so that fibres and vessels werearranged obliquely to the axis of the stem. At the interfacebetween the bud and dwarfing rootstocks vessels with smallerthan normal diameter were formed, indicating the presence ofelevated levels of auxin in this region. In addition, littlexylem was produced in the adjacent rootstock tissue. In thecase of semi-dwarfing rootstocks, the rootstock produced normalxylem after a brief interruption. We suggest that failure ofauxin to cross the bud-union interface in the case of the dwarfingrootstocks leads to reduced rootstock xylem formation, and hencea poor supply of water and minerals to the scion, and this underliesthe dwarfing effect.Copyright 1994, 1999 Academic Press Apple, budding, dwarfing, anatomy, graft union     

Possible roles of auxin and zeatin for initiating the dwarfing effect of M9 used as apple rootstock or interstock

Endogenous indole-3-acid (IAA) and zeatin (t-Z) may play important roles in the dwarfing mechanisms of rootstocks. The concentrations of IAA and t-Z, as well as the expression of the genes PIN1 and IPT3 were measured in leaves, barks, and roots from nine treatments: M9, Malus X micromalus Makino, Red Fuji/M9/Malus X micromalus, Red Fuji/M9, Red Fuji/Malus X micromalus, M9 rootstock substitution above and below the original graft union, interstock bridging, and interstock bark substitution of M9. The results show that there were greater amounts of t-Z and expressions of IPT3 in invigorated trees (Malus X micromalus and Red Fuji/Malus X micromalus) when compared with dwarfing trees (M9, Red Fuji/M9/Malus X micromalus and Red Fuji/M9) during the period of fast shoot growth (early June to mid-August). Moreover, the variation of IAA contents and PIN1 expressions shared the same pattern with t-Z contents and IPT3 expressions in all the tested locations. IAA content was extremely high in the bark of M9 interstock when compared with that of the scion and the rootstock, yet PIN1 expression in corresponding tissues was very low. After M9 rootstock were substituted above and below the original graft union, contents of t-Z, IAA, and PIN1 expressions in leaves and branch-barks recovered gradually to standard tree levels. However, there is no significant difference between the two treatments. We conclude graft union has no obvious influence on hormone transport. After M9 interstock and its bark were substituted, the hormone measurement of every index was consistent with that of rootstock substitution.     

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.     

不同砧穗组合葡萄植株对部分根区干旱的生理生化响应

利用自制木箱对嫁接在3309C、420A和110R砧木上的玛瓦斯亚葡萄（M）进行双侧根区交替灌溉（AI）和单侧灌溉（UI）两种水分胁迫处理,以探讨不同葡萄砧穗组合对干旱的适应能力.结果表明：水分胁迫导致葡萄叶片ABA浓度大幅度增加,AI和UI的3种组合平均分别提高了267.5％和394.7％,而气孔导度和蒸腾速率显著下降.UI处理的3种组合叶片SOD和CAT活性显著增加,脯氨酸（Pro）含量极显著增加,且均以M/110R增幅较大,M/3309C增幅较小;而AI处理的SOD和CAT活性增加较小,但Pro含量显著增加.干旱胁迫导致葡萄叶片质膜透性、MDA和H₂O₂含量极显著增加,以M/3309C增加最多,M/110R增加较少.不同砧穗组合适应干旱逆境的能力主要取决于砧木品种,110R适应干旱逆境的能力强于420A和3309C,其对应的嫁接植株的生长量亦呈同样趋势.与根区单侧灌溉相比,双侧根区交替灌溉对植株造成的伤害较小,是可推广的节水灌溉方式.     

Factors influencing stomatal conductance in response to water availability in grapevine: a meta‐analysis

The main factors regulating grapevine response to decreasing water availability were assessed under statistical support using published data related to leaf water relations in an extensive range of scion and rootstock genotypes. Matching leaf water potential (Ψ_leaf) and stomatal conductance (g_s) data were collected from peer‐reviewed literature with associated information. The resulting database contained 718 data points from 26 different Vitis vinifera varieties investigated as scions, 15 non‐V. vinifera rootstock genotypes and 11 own‐rooted V. vinifera varieties. Linearised data were analysed using the univariate general linear model (GLM) with factorial design including biological (scion and rootstock genotypes), methodological and environmental (soil) fixed factors. The first GLM performed on the whole database explained 82.4% of the variability in data distribution having the rootstock genotype the greatest contribution to variability (19.1%) followed by the scion genotype (16.2%). A classification of scions and rootstocks according to their mean predicted g_s in response to moderate water stress was generated. This model also revealed that g_s data obtained using a porometer were in average 2.1 times higher than using an infra‐red gas analyser. The effect of soil water‐holding properties was evaluated in a second analysis on a restricted database and showed a scion‐dependant effect, which was dominant over rootstock effect, in predicting g_s values. Overall the results suggest that a continuum exists in the range of stomatal sensitivities to water stress in V. vinifera, rather than an isohydric–anisohydric dichotomy, that is further enriched by the diversity of scion‐rootstock combinations and their interaction with different soils.     

The Function of Phloem Connections in Regenerating In Vitro-Grafts*

In order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant-grafts after application of ¹⁴C-sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls. A segmental analysis was used to determine the tissue distribution of ¹⁴C-sucrose in a graft. The ¹⁴C-profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound-phloem bridges (shown as files of aniline-blue-positive sieve plates) traverse the graft interface. In 7-d-old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, ¹⁴C-profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose. Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound-phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro-grafts is directly shown.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Amino acids as determinants of host preference for the xylem feeding leafhopper,Homalodisca coagulata (Homoptera: Cicadellidae)

Summary Homalodisca coagulata is a highly polyphagous xylem feeder with distinct seasonal patterns in it's selection of host plants. These patterns were examined in relation to the amino acid content of the xylem for four common host species; Lagerstroemia indica, Baccharis halimifolia, Prunus persica, and Prunus salicina. Xylem fluid was collected from each host species at times when numbers of feeding leafhoppers were both low and high. In each case, concentrations of amino acids were greatest when numbers were high. Similarly, comparisons between host species at given times showed that concentrations of amino acids were positively correlated with host selection. In a second study, amino acids of xylem were manipulated by budding scions of a non-preferred host (P. persica) on rootstocks of preferred (P. salinica) and non-preferred (P. persica) hosts. Morphology and phenology of the budded trees were similar to that of the scion species yet the xylem composition of amino acids was primarily dependent on the rootstock. Concentrations of amino acids and the preference of leafhoppers were roughly two-fold greater for scions of the preferred than the non-preferred rootstock. In both studies, amides (glutamine plus asparagine) were the amino acids most highly correlated with host selection. These compounds are the predominant amino acids in xylem fluid, have high nitrogen to carbon ratios, and account for a high percentage of the caloric value in xylem fluid. Many of the less abundant amino acids were positively correlated with host preference, but the correlations were less consistent and correlation coefficients were generally lower.Florida Agricultural Experiment Station Journal Series No. 9672     

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g^??1 dry wt (up to 11 mg g^??1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.