Quali-Quantitative Study on Phenol Compounds as Early Predictive Markers of Graft Incompatibility: A Case Study on Chestnut (Castanea spp.)

In recent years, research has focused on phenolic compounds and their putative role as markers of graft incompatibility. Thus far, no studies have been conducted on the role of phenolic compounds in chestnut (Castanea spp.). The present study investigated the content of phenolic compounds in different combinations of Castanea spp. cultivars and rootstocks. Analyses were performed on the inner and outer tissues of chestnut grafts at two phenological sampling stages. The separation, identification and quantification of the phenolic markers via HPLC were preceded by an ultrasonic green extraction. Two chromatographic methods were tested for a total of 15 phenol compounds. Flavonol compounds were not detected, while cinnamic acids were found in low concentrations. The amount of gallic acid turned out to be higher at the graft union of the incompatible combination (20.11 ± 1.47 mg/100 gFW vs. 8.94 ± 1.08 mg/100 gFW). The same pattern was observed for catechin (15.79 ± 1.83 mg/100 gFW vs. 9.63 ± 1.98 mg/100 gFW). Differences in tannin concentrations seemed to be species-specific, and were apparently not related to graft incompatibility. The present work underlines the potential application of certain phenol compounds for the early prediction of graft incompatibility in Castanea spp.

Compatibility Evaluation and Anatomical Observation of Melon Grafted Onto Eight Cucurbitaceae Species

Melon (Cucumis melo) is one of the top 10 fruits in the world, and its production often suffers due to soil-borne diseases. Grafting is an effective way to solve this problem. However, graft incompatibility between scion and rootstock limits the application of melon grafting. In this study, the melon was grafted onto eight Cucurbitaceae species (cucumber, pumpkin, melon, luffa, wax gourd, bottle gourd, bitter gourd, and watermelon), and graft compatibility evaluation and anatomical observation were conducted. Taking melon homo-grafted plants as control, melon grafted onto cucumber and pumpkin rootstocks was compatible, while melon grafted onto luffa, wax gourd, bottle gourd, bitter gourd, and watermelon rootstocks was incompatible based on the scion dry weight on day 42 after grafting. Meanwhile, we found that starch–iodine staining of scion stem base is an index to predict graft compatibility earlier, on day 14 after grafting. Further, microsection observations showed that there was more cell proliferation at graft junction of melon hetero-grafted combinations; vascular reconnection occurred in all graft combinations. However, excess callose deposited at graft junction resulted in the blockage of photosynthate transport, thus, leading to starch accumulation in scion stem base, and finally graft incompatibility. In addition, undegraded necrotic layer fragments were observed at graft junctions of melon grafted onto incompatible bitter gourd and watermelon rootstocks. The above results provide clues for the selection and breeding of compatible Cucurbitaceae rootstocks of melon and demonstrate that starch accumulation in scion base and callose deposition at graft junction is associated with melon graft compatibility.

Biochemical Characterization and Differential Expression of PAL Genes Associated With “Translocated” Peach/Plum Graft-Incompatibility

Grafting is an ancient plant propagation technique widely used in horticultural crops, particularly in fruit trees. However, the involvement of two different species in grafting may lead to lack of affinity and severe disorders between the graft components, known as graft-incompatibility. This complex agronomic trait is traditionally classified into two categories: “localized” (weak graft unions with breaks in cambial and vascular continuity at the graft interface and absence of visual symptoms in scion leaves and shoots) and “translocated” (degeneration of the sieve tubes and phloem companion cells at the graft interface causing translocation problems in neighboring tissues, and reddening/yellowing of scion leaves). Over the decades, more attention has been given to the different mechanisms underlying the “localized” type of graft-incompatibility; whereas the phenylpropanoid-derived compounds and the differential gene expression associated with the “translocated” graft-incompatibility remain unstudied. Therefore, the aim of this study was to shed light on the biochemical and molecular mechanisms involved in the typical “translocated” graft-incompatibility of peach/plum graft-combinations. In this study, the “Summergrand” (SG) nectarine cultivar was budded on two plum rootstocks: “Adara” and “Damas GF 1869”. “Translocated” symptoms of incompatibility were shown and biochemically characterized in the case of “SG/Damas GF 1869” graft-combination, 3 years after grafting. Non-structural carbohydrates (soluble sugars and starch), phenolic compounds and antioxidant activity, were significantly enhanced in the incompatible graft-combination scion. Similarly, the enzymatic activities of the antioxidant enzyme peroxidase, the phenylalanine ammonia-lyase (PAL) and polyphenol oxidase involved in the phenylpropanoid pathway were significantly affected by the incompatible rootstock “Damas GF 1869”, inducing higher activities in the scion than those induced by the compatible rootstock “Adara”. In addition, a positive and strong correlation was obtained between total phenol content, antioxidant capacity and the expression of the key genes involved in the phenylpropanoid pathway, PAL1 and PAL2. Regarding the “SG/Adara” graft-combination, there were neither external symptoms of “translocated” incompatibility nor significant differences in the biochemical and molecular parameters between scion and rootstock, proving it to be a compatible combination. The differential expression of PAL genes together with the biochemical factors cited above could be good markers for the “translocated” peach/plum graft-incompatibility.

Identifying Molecular Markers of Successful Graft Union Formation and Compatibility

Grafting is a technique used for millennia for vegetative propagation, especially in perennial fruit crops. This method, used on woody and herbaceous plants, can improve several agronomic characteristics, such as yield or vigor, as well as tolerance to biotic and abiotic stresses. However, some scion/rootstock combinations suffer from poor graft compatibility, i.e., they are unable to form and/or sustain a successful graft union. Identifying symptoms of graft incompatibility is difficult because they are not always present in the first years after grafting and in most cases the causes of incompatibility are still poorly understood. Studies of changes in transcript abundance during graft union formation indicate that grafting responses are similar to responses to wounding and include the differential expression of genes related to hormone signaling, oxidative stress, formation of new vascular vessels, cell development, and secondary metabolites, in particular polyphenols. This review summarizes current knowledge of the changes in transcript abundance, redox status and metabolites accumulation during graft union formation and in cases of graft incompatibility. The goal of this review is to discuss the possibility of identifying marker transcripts, enzyme activities and/or metabolites of grafting success and graft compatibility which could be used to score grafting success for genetic research and in breeding programs. We highlight gaps in current knowledge and potential research directions in this field.

Construction of an Arabidopsis/Nicotiana inter-order graft for studies of scion-rootstock interaction

Background Scion–rootstock union formation is a critical step towards functional assemblage of heterogeneous plants. However, scion-rootstock interaction often results in graft incompatibility during the process of assemblage. So far, the lack of model heterografts involving both clear genetic backgrounds and taxonomically distant species greatly impedes insights into the mechanisms underlying scion-rootstock interaction. Results In this report, we established an Arabidopsis (At)/Nicotiana benthamiana (Nb) heterografting system in which the model plant At and the model plant Nb for plant bioreactor was used as scion and rootstock respectively, to explore the interaction between the two model plants. Regarding to the At scion phenotypes, the At-Nb connection can be characterized into three groups: the mild-stressed, the albino and the dormant grafts. Examination of symplastic and apoplastic flow indicated that a functional inter-order grafting was established in the mild-stressed group, but not in the dormant group. What’s more, the free GFP movement in both At/At homograft and the At/Nb graft implicated that macromolecules moved across the heterograft union of the mild-stressed graft, but congealed at the union of dormant graft. These results accentuated the role of vascular connection in the establishment of compatible heterografts. Conclusions The present study established an inter-order model graft involving Arabidopsis and Nicotiana. The interactions from these two species resulted in three distinct grafting groups, which offer us a novel vista to explore many important issues such as grafting compatibility and biomolecule movement.

Peroxidase activity and initial growth of ‘Barbosa’ peach on clonal rootstocks

In the peach nursery trees production, the use of rootstocks with unknown genetic identity obtained from peach seeds in the canning industry is frequent. The hypothesis tested was that there are rootstocks that express greater graft compatibility, enabling greater survival and growth, and that peroxidase activity can be used as indication of graft incompatibility. The aim of this study was to evaluate the survival and the peroxidase activity efficiency in identifying graft incompatibility and the trunk diameter growth of ‘Barbosa’ peach grafted onto 18 Prunus clonal rootstocks, propagated by herbaceous cuttings, compared to own-rooted scion trees. The experimental design was three randomized blocks, with one tree per plot. The first three vegetative cycles of trees conducted in double Y (5.0m x 2.0m) without irrigation were evaluated. The highest trunk diameter growth was provided by ‘Okinawa’, ‘Tsukuba-1’, ‘Tsukuba-2’, Mexico F1 and ‘Flordaguard’ rootstocks, without differing from own-rooted ‘Barbosa’ peach. Peroxidase activity differs by rootstock effect at the beginning of the dormancy period, and is higher than in vegetative growth, especially in interspecific graft combinations. Peroxidase activity is a biochemical indicator of stress, but should not be used alone and generalized to characterize graft incompatibility.