Plant grafting: new mechanisms, evolutionary implications

1Grafting is an ancient, vegetative, asexual plant propagation technique. It is accomplished most commonly by connecting two plant segments, the shoot piece known as ‘scion’ and the root piece called ‘rootstock’(stock). A broad range of classical grafting techniques can be found in; recent seedling micrografting protocols have been summarized by. Grafting has been practiced for many centuries with perennials – mainly fruit trees but also some forest trees and ornamentals – but, as of the early 20th century also with vegetable crops, mainly Cucurbitae and Solanaceae species. Thus, the majority of recent grafting research concerns physiological and pathological aspects of vegetable grafting. Grafting also plays an important role in various types of physiological investigations, in particular in classical studies on the movement of the floral stimulus. An overview of the current state of the art can be obtained from several recent reviews, each emphasizing a slightly different aspect. The history of grafting has been described in detail by. Attempts to provide adequate explanations for the immediate and long-term effects of grafting have been made for generations but, according to a recent review, this plant propagation practice is still shrouded in mystery. Undoubtedly, the use of a large number of diverse plant species in grafting studies has slowed down progress in this research area. The recent introduction of the Arabidopsis model into grafting research has opened the way for more targeted, advanced grafting research. Thus, the purpose of the present, concise update is threefold: (a) To point out a few major lacunas in our understanding of grafting processes. (b) To introduce some novel, emerging graft physiology mechanisms. (c) To discuss recent evidence that suggests a role for natural grafting in plant evolution.

2There is no precise definition of ‘graft compatibility’; it generally means establishment of a successful graft union as well as extended survival and proper functioning of the composite, grafted plant. Taxonomic affinity is a prerequisite for graft compatibility. Homografts (= autografts) are presumably always compatible. In heterografts, broadly speaking, intraspecific grafts (= rootstock and scion belonging to the same botanical species) are nearly always compatible, interspecific grafts (= rootstock and scion belonging to different species of the same genus) are usually compatible, intrafamilial grafts are rarely compatible, and interfamilial grafts are essentially always incompatible. Examination of heterografts compatibility should include homograft controls, a requirement not always fulfilled. Even in homografts, where rootstock and scion share the same genetic background, certain grafting effects are evident. Differences in rootstock and scion age and juvenility markedly affect their microRNA (miRNA) expression patterns and reproductive development. In sweet pepper (Capsicum annuum L.), old-stage homografts (58 days old plants) had slower graft-take than young-stage (34 days) homografts. Old-stage grafts had a lower percentage of xylem connections and seemed to suffer from drought stress. The time needed for the establishment of a functional graft union is considerably shorter in herbaceous plants than in trees. In the recently introduced Arabidopsis micrograft system 7–12 days are required but achieved functional vasculature in Arabidopsis within 3 days after grafting. Several months are commonly required for completion of a functional union in tree grafts (e.g., ).

3Graft incompatibility may be defined as failure to form a successful graft union. Yet, despite innumerous follow-ups of graftage in various types of plants, the reasons for graft incompatibility are still vague. Initial healing of the graft union does not in itself ensure long-term compatibility. In cucurbits, apparently successful grafts proved incompatible 25 days after grafting. In certain tree stock/scion combinations incompatibility may appear only after several years.. Although incompatibility is not a measurable quantitative trait, various degrees of incompatibility may be discerned, from mild interference with the normal development of the composite plant to mortality of the stock, scion or both.

4As already pointed out by, as yet there is no evidence for a specific biochemical-immunological recognition/rejection mechanism between the graft components. This is in contrast to pollination incompatibility, where specific mechanisms have been identified. Yet, heterograft incompatibility clearly increases with genetic distance, indicating some kind of physiological rejection. On the other hand, incompatibility occurs even among related genera of the same family in a rather unpredictable fashion. Thus, within the Solanaceae, reciprocal grafts of tomato (Solanum lycopersicum L.) and pepper were considered severely incompatible, whereas tomato and eggplant (Solanum melongena L.) only moderately so, in comparison with compatible tomato homografts. Considerable variation in degree of compatibility was evident among grafts of melon (Cucumis melo L.) onto 22 Cucurbitae rootstocks and among other Cucurbitae species and among chestnut (Castanea) species. In citrus trees, visual inspection of trunks at the graft union was believed to reflect the degree of compatibility (Figure 1, reproduced from ), although some graft combinations proved to be successful despite the unsmooth graft union. Anatomical follow-ups of heterograft unions invariably disclose mild to severe interferences with the formation of a fully functional stock/scion continuum. Changes in vascular anatomy were evident in grafts of apple (Malus domestica Borkh. and sweet cherry (Prunus avium L.) onto dwarfing rootstocks and even in compatible graft unions among Pinus species. A commonly observed disturbance is a convolution of the vascular elements orientation. proposed that wound-induced changes in the normal flow of endogenous auxin, which plays a key role in vascular differentiation, might be responsible for this distortion. This hypothesis is supported by the recent study of who demonstrated the involvement of auxin in early stages of graft union formation. The oxidative stress symptoms reported by appear only at a considerably later stage and may represent a belated response to the auxin imbalance.

5Major grafting effects are readily comprehensible if viewed in the broader context of root–top relations. Thus, dwarfing rootstock effects are not surprising once the well-documented Bonsai culture and root restriction effects are kept in mind. By the same token, the invigorating effect of strong, expansive Cucurbitae rootstocks on their scions might be anticipated, although the precise physiological mechanisms involved need clarification. The rather complex natural root–top relationship is further complicated in the composite, grafted plant, which undergoes a drastic wounding/healing operation, followed by life-long interactions between different genomes.

6Most long-term compatibility studies have been conducted with fruit trees. Xylem graft union anatomy determines the hydraulic root–top conductivity in apple, thereby affecting the growth potential of rootstock/scion combinations. Phloem graft union irregularities appear to be a major source of long-term incompatibility. The pioneering studies of demonstrated a gradual build-up of biochemical poisoning reaction between pear (Pyrus communis L.) scions and quince (Cydonia oblonga Mill.) rootstocks. Prunasin, a cyanogenic glucoside, rises from the quince rootstock into the pear scion, where it is catabolized enzymatically by pear glycosidase to liberate cyanide at the graft interface. Cyanide ‘poisons’ the graft union tissues, causing cellular necrosis at the graft interface, leading to severe incompatibility. However, further studies with peach (Prunus persica L. Batsch) grafted on myrobalan plum (Prunus cerasifera L. Ehrh) suggested a more general poisoning mechanism, due to progressive impairment of the phloem carbohydrate transport and accumulation of starch above the graft union. Gradual starch accumulation is indeed a symptom of serious interference with nutrient metabolism and root starvation. These phenomena resemble the damage often caused by girdling (= removal of a ring of bark from the trunk or branch of a tree;; ). Rootstock-scion interactions persist throughout the life of the composite plant, even where satisfactory graft compatibility has been achieved. The upward supply of water and mineral nutrients as well as the downward flow of photosynthates are modified and so is the root–top interchange of hormonal signals. These mechanisms may account for many of the well-known grafting effects.

7Long-distance phloem transport of proteins and RNAs and their potential role in inter-organ signaling has become a major research domain in recent years. Trans-graft transmission of gene silencing in tobacco (Nicotiana tabacum L.), which presumably involves some kind of mobile RNA, was one of the pioneering findings in this area. A number of small RNAs and even some mRNA have been identified in phloem saps. Root-to-top as well as top-to root graft transmissible signals have been convincingly demonstrated. The involvement of miRNAs in grafting effects has been demonstrated recently by. Reduction of juvenility is one of the most conspicuous effects of grafting in trees. The juvenile-to-adult transition in trees as well as in annuals is mediated by a marked decline in the expression of miR156. Both heterograft and homograft citrus scions had reduced miR156 (and the related miR157) expression rates as compared with non-grafted control seedlings, suggesting a role for miRNAs in primary grafting effects. Changes in the expression of specific miRNAs have recently been implied in heritable chimeras of grafted Brassica sp.; however, only a few miRNAs have been shown to be graft-transmissible so far.

8There is no obvious reason why a viable, physiologically functional graft union should interfere with regular, long-distance vascular transport. However, the occurrence of a compound which supposedly originated in one organ (e.g., apical meristem), in a distant organ (e.g., roots), does not in itself prove its vascular transport, since this compound could also be synthesized in the presumably recipient organ. Therefore, grafting of genetically distinct mutants/transgenes is still being used as a principal means to distinguish between the site of biogenesis and the recipient organs. Roots of grafted Arabidopsis contained specific small RNAs that originated in the apical, source organs, although they were less abundant than in the source tissue by three orders of magnitude. Furthermore, the identification of a mobile macromolecule does not indicate in itself a physiological role. The complexities of this area have been discussed by.

9Notwithstanding, physiologically meaningful protein and RNA long-distance signaling has been convincingly demonstrated in several plant systems. The identification of the FT protein as a major, graft-transmissible component of the long-sought universal florigen is a highly significant achievement. The role of phloem mobile miRNAs in the systemic regulation of several key mineral nutrients appears to be a principal mechanism. The regulation of potato (Solanum tuberosum L.) tuberization by the graft transmissible miR156 is another recently studied case. The regulatory role of graft transmissible mRNA has now been clearly demonstrated with the Gibberellic Acid Insensitive (GAI) mRNA, including the anticipated changes in plant phenotype. Thus, we have by now ample evidence for the regulatory role of both protein and several types of RNA as long-distance, graft-transmissible signals, and further discoveries in this area can be expected.

10The role of grafting in plant disease is a “double-edged sword.” On one hand, grafting played a pivotal role in the spread of many important plant diseases. But, on the other hand, grafting has become the principal means in overcoming most hazardous plant epidemics and pests.

11Plant pathogens spread in tree communities (forests and plantations) through a variety of tracks, including natural, underground root grafts. This avenue of disease transmission has been underestimated, just as root grafting in general has been somewhat neglected. Its significance became evident with the burst of oak wilt and Dutch elm fungal disease epidemics during the midst of the 20th century. Also viral diseases are transmitted by root grafting. The frequency of intraspecific and interspecific root grafts in wild forest stands has been examined in just a few cases. Generally, there are considerable differences in the actual significance of disease spread by root grafts between crops and habitats.

12Parasitic plants such as mistletoes (= parasitic plants in the order Santalales) and dodder (Cuscuta sp.) transmit pathogens very efficiently, even among intergeneric and interfamilial plant species. Dodder is often used in experimental studies as a bridging vehicle between distant, graft incompatible plant species. Thus, the parasitic, invasive disease transmission mechanism goes beyond the phylogenetic graft compatibility barriers and these seemingly similar pathogen spread pathways cannot be simply equated.

13Grafting of virus infected plant material is another, dangerous way of disease transmission. Graft-transmission of pathogens does not require a compatible, physiologically functional graft union. Even mechanical contact, such as using a contaminated grafting device, can spread viral and bacterial diseases. Interfamilial grafts of citrus on avocado (Persea americana Mill.) transferred a citrus viroid to avocado; the grafted citrus budwood remained viable for almost a year without forming a true graft union. Periwinkle (Catharanthus sp.) seems to be unique in its ability to form distant interspecific grafts. The apple proliferation phytoplasma disease was transmitted from infected apple scions to periwinkle rootstocks. There is no evidence, however, that such periwinkle grafts form a true, functional graft union.

14The role of grafting in the management and control of a broad array of plant pests is nowadays a high priority topic; grafting of Cucurbitae and Solanaceae vegetables to control soil-borne diseases is widely practiced. The beginnings are, however, considerably older. Grafting of citrus onto sweet orange to combat the Phytophthora foot rot, and grafting of Vitis vinifera onto American (Vitis labrusca and other species) rootstocks to overcome the Phylloxera grapevine plague were adopted in the 19th century. The 20th century struggle of the citrus industry with the Citrus tristeza virus (Closterovirus) and other virus-like diseases exploited every possible graft combination. The selection of suitable fruit tree rootstock/scion combinations is dominated by disease resistance considerations even today; soil and climate adaptation also affect the preferable stock/scion selection.

15Nevertheless, most recent research focuses on vegetable grafting. Superimposed on the natural plant disease resistance comes once more the complexity of the composite, grafted plant. The diverse mechanisms involved in disease resistance of grafted vegetables have been reviewed by. These range from genetic non-host resistance and rootstock vigor effects to rootstock-induced systemic acquired resistance, which involves salicylate, jasmonate, and other plant hormone signals.

16Long-distance protein and RNA signals (see previous section) also seem to play a role in rootstock-induced disease resistance. Polygalacturonase-inhibiting proteins (PGIPs) are plant cell proteins that specifically inhibit the cell wall degrading endo-polygalacturonases of plant pathogens. Leaf extracts and xylem exudates from grapevine rootstocks transformed to express the pear fruit PGIP-encoding gene (pPGIP) had PGIP activity. PGIP was detected in xylem exudates of untransformed grapevine scions grafted onto transgenic rootstocks expressing pPGIP, indicating that the PGIP protein is graft–transmissible. Such scions revealed improved tolerance toward grapevine diseases caused by Xylella fastidiosa and Botrytis cinerea. Following these results, suggested that cultivars genetically engineered to express disease controlling, graft–transmissible proteins and RNAs, could be used as rootstocks for disease susceptible scion cultivars. Grafting genetically engineered rootstocks with non-transformed fruit tree scions would presumably result in disease-resistant cultivars bearing fruits which are not genetically modified, a desirable combination. This idea undoubtedly merits further research.

17Of all grafting issues, the least understood and most controversial is the ‘graft hybrid’ concept. According to this concept grafting may involve stock to scion transfer of genetic material (= graft transformation), leading to heritable changes in the scion. The scion which has acquired certain heritable traits from the rootstock is regarded as a ‘graft hybrid.’ However, graft transformations occur only under ‘Mentor grafting’ conditions, which presumably enforce the transfer of genetic material from stock to scion (Figure 2).

18In ‘Mentor grafting,’ young seedlings shoots serving as ‘scions’ are grafted onto mature, flowering plants used as ‘stocks.’ To make sure that the scions fully depend on the stock for the supply of nutrients, leaves of the scions (except for two or three leaves at the top) are removed throughout the experiment. Stock fruits are also removed in attempt to maximize flow of substances from the stock to the scion. Interestingly, the ‘Mentor grafting’ technique is the same as that used in classical flowering research, where removal of leaves from the scion was expected to promote stock to scion movement of the floral stimulus.

19The graft hybrid concept, which was developed and demonstrated by the Soviet horticulturist, appeared to be in contrast with Mendelian genetics and was rejected (and almost forgotten) by Western scientists who opposed and distrusted the Soviet biology led by Lysenko. Several published studies (and most probably many unpublished) could not confirm the appearance of graft hybrids. Yet, reports supporting the occurrence of graft transformations appeared from time to time, and increasingly so in recent years. Proponents of the graft hybrid concept reviewed the supporting evidence and called for reassessment of the graft transformation hypothesis that has been neglected for several decades.

20Within the ‘graft hybrid’ supportive evidence some specific characteristics can be defined. (a) The frequency of the appearance of variant plants is highly variable, sometimes below 1%. Thus, the argument that ‘no variants were found’ must be based on a very large number of replicate grafts. Indeed, some of the ‘negative’ reports used several thousands of grafts. (b) The graft hybrid experimental evidence rests almost exclusively on intra- and interspecific grafting of Solanaceae, in particular pepper, which is somehow more amenable than other plant species to rootstock-induced scion transformation. (c) The alleged rootstock to scion transmission of genetic material is the most mysterious part of it all. The initial belief of that genes can move between rootstock and scion has been refined by who presented histological evidence that masses of chromatin are moving via the vascular system from the older rootstock across the graft union to the apical primordia or flower buds of the younger, mentor-grafted scion. The model of amplified plasmodesmatal macromolecular transport toward apical meristems has been cited in this context but no further evidence in support of this mechanism has been presented.

21In their review, indicated that the emerging concept of graft-transmissible gene silencing signals may hold the key for a new approach to the graft transformation riddle. Recent research further extends this view in conjunction with probable involvement of epigenetic inheritance mechanisms.

22Epigenetics refers to reversible heritable changes in genome function that occur without a change in the DNA sequence and may have morphological, physiological, and ecological consequences. Changes in DNA methylation are presumably among the principal, ubiquitous epigenetic mechanisms although their heritability requires further elucidation. DNA methylation during plant gametogenesis, in particular, appears to involve epigenetic, heritable changes. Plant DNA methylation has been shown to be regulated by siRNAs; ‘siRNA-mediated epigenetic modification’ is currently an acceptable term.

23As already discussed, graft-transmissible RNA gene silencing signals have been demonstrated in both the upward and the downward direction. Changes in DNA methylation in the recipient organs have been detected; these changes are regarded as epigenetic modifications. Partially heritable, locus-specific alteration of DNA methylation patterns have recently been found in scions of interspecific grafts of Solanaceae. think that their research paves the way for resolution of the graft hybrid controversy. However, further, rigorous research is desperately needed, in order to unequivocally elucidate the graft hybrid – graft transformation issues.

24Grafting is a natural phenomenon, wide spread between roots of the same tree, neighbor trees of the same species or even, although less frequently, among trees belonging to different species (= interspecific grafting). Root grafting could be induced artificially between potted Picea abies trees; a pair of compressed roots fused and formed a complete union within several months. Natural grafting is not confined to roots; intra- and interspecific grafts among tree branches have been described. Although anatomical evidence is scarce, it may be assumed that mutual pressure between two adjacent roots builds-up as they increase in diameter, since the surrounding soil precludes much lateral displacement of the two roots. With increasing pressure the bark of each in contact with the other wears away, bringing their cambia in contact, leading eventually to a functional graft union. Most aerial grafts occur when a branch from one tree is “caught” in the forked branch of another tree, which is analogous to the compression between adjacent roots (K. W. Mudge, personal communication, with permission).

25Assessment of the evolutionary significance of grafting involves discussion at two levels: its benefits for plant survival and its potential role in the formation of new species. The plausible ecological benefits of root grafting have been discussed by. The existence of underground graft contacts turns the individual tree into member of a cooperative community which may support each other in a variety of physical, nutritional and reproductive ways. observed that stumps sometimes persist and continue to grow for years or even decades, when root-grafted to adjacent trees but not when occurring singly, suggesting that nutrients arriving via root grafts from standing neighbor trees are responsible for the persistence of the stump. Trees that lost their crowns in a storm etc. may survive and resume above ground growth and reproduction owing to their root graft cooperation. On the other hand, as already mentioned in a previous section, there is a very real risk of pathogen transmission via natural root grafts, including fungal, bacterial, viral and mycoplasmal diseases. In as much as agriculture is nowadays considered a component of the evolutionary arena, grafting also plays a critical role in the survival of thousands of man-selected plant genotypes which are propagated only by grafting and have never existed in natural habitats.

26Addressing the significance of grafting as a potential evolutionary mechanism must take into account the ancient belief that grafting may give rise to new plant species. The ‘graft hybrid’ concept discussed in the preceding section is also related to this belief. Some of the novel species records may be attributed to graft chimeras, which are not true hybrids, but are inheritable. Adventitious buds may develop from the graft junction, and the lateral, developing chimeral shoots may retain a stable, heritable intermediate phenotype. Lateral shoots from graft junction wounds occur frequently, and a considerable percentage of those are stable chimeras.

27Recent research has brought us much closer to a potentially real evolutionary grafting mechanism. In their early report demonstrated exchange of plastid genome material between stock and scion cells in interspecific graft junction zones, later shown to consist of entire chloroplast genome transfer between distinct Nicotiana species. Interestingly, in their initial report state that their data.

28“… do not lend support to the tenet of Lysenkoism that ‘graft hybridization’ would be analogous to sexual hybridization. Instead, our finding that gene transfer is restricted to the contact zone between scion and stock indicates that the changes can become heritable only via lateral shoot formation from the graft site.”

29However, in a more recent report Bock and his team come up with a bolder title; “Horizontal genome transfer as an asexual path to the formation of new species.” Tissue cultured plants were grafted, and the tissue very close to the graft junction (some millimeters from the junction) was maintained and screened further in tissue cultures. The results show that entire nuclear genomes were transferred between plant cells in the graft junction zone, leading to the formation of novel, alloploid plant cells, and such events occurred at a considerable frequency. Plants recovered from these cells are real graft hybrids. A fertile, stable hybrid between herbaceous and woody Nicotiana species was thus obtained, tentatively named Nicotiana tabauca. Still, natural evolutionary formation of new species via this pathway would depend upon natural graft formation and emergence of adventitious lateral shoots, as previously discussed.

30The hypothetical possibility of cell fusion graft hybrids has already been mentioned by; Figure 12). Genome combinations have indeed been achieved in artificial protoplast fusion systems, and interspecific hybrid plants were obtained. However, the recent findings of Bock’s team raise the exciting option that natural grafting may have played an active evolutionary role in plant speciation. The results obtained with Nicotiana under tissue culture conditions require further confirmation with other plants in natural settings. It is difficult at the present time to estimate the actual contribution of this asexual path to plant evolution. Although seemingly dependent upon a rare sequence of events, this evolutionary niche could have been exploited as a plant survival bypass under extreme selective pressure conditions.

31While the technology of grafting has advanced tremendously, the long-term survival of grafted, composite plants is still somewhat unpredictable. The complexity of stock/scion interactions attains a new dimension when pathogenic agents enter the scenery. More enigmatic are the graft hybrid conundrum and the broader evolutionary significance of grafting. The extensive horticultural grafting research has not usually addressed these basic questions. The recently adopted Arabidopsis model has opened the way for meticulous, in-depth grafting research. The currently available molecular tools are expected to advance our understanding and eventually resolve the long standing grafting mysteries.

32The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.