Outcomes of Autologous Osteochondral Transplantation With and Without Extracellular Matrix Cartilage Allograft Augmentation for Osteochondral Lesions of the Talus

Background: Autologous osteochondral transplantation (AOT) using a cylindrical graft in the treatment of osteochondral lesions of the talus (OLTs) is typically indicated for patients with larger lesions. However, with lesions that are irregular in shape, the AOT graft may not completely replace the lesion. For these lesions, we utilize extracellular matrix cartilage allograft (EMCA) augmentation in AOT to act as a physiologic grout at the host-graft interface. Purpose: To determine if the combination of EMCA with concentrated bone marrow aspirate (CBMA) would improve integration of the host-graft interface and subsequently reduce postoperative cyst formation after AOT. It was also hypothesized that EMCA in conjunction with CBMA would demonstrate improved MOCART (magnetic resonance observation of cartilage repair tissue) scores and functional outcome scores at a minimum 2 years after surgery. Study Design: Cohort study; Level of evidence, 3. Methods: A retrospective analysis was performed comparing patients treated with AOT/CBMA alone and AOT with CBMA/EMCA. Clinical outcomes were evaluated with the Foot and Ankle Outcome Score. Magnetic resonance imaging appearance was evaluated with the use of the MOCART (magnetic resonance observation of cartilage repair tissue) score. Cyst formation was also evaluated on postoperative magnetic resonance imaging. Results: A total of 26 patients were included in the AOT + CBMA/EMCA group (10 male, 16 female), and 34 patients were included in the AOT/CBMA group (17 male, 17 female). The mean Foot and Ankle Outcome Score significantly improved in both groups ( P < .001) across all subscales (symptoms, pain, activities of daily living, sports activities, and quality of life), but there was no significant difference between groups at final follow-up. There was no significant difference in mean MOCART scores between the groups ( P = .118). In the AOT/CBMA group, 3 patients (8.8%) complained of knee pain, and 1 (2.9%) required additional surgery (hardware removal). In the AOT + CBMA/EMCA group, 2 patients (7.7%) complained of knee pain, and 6 patients (23%) required additional surgery (3 hardware removals and 3 arthroscopic debridements of scar tissue in the ankle). Conclusion: We found that while EMCA with CBMA has benefit in regeneration and repair of OLT treated with bone marrow stimulation, there appears to be little benefit of EMCA over CBMA alone as a physiologic grout at the graft-host interface in OLT treated with AOT.

Physiological and Molecular Characterization of New Apricot Cultivars Grafted on Different Prunus Rootstocks

In the last years, an important renewal of plant material from different breeding programs is taking place in apricot in order to improve resistance to biotic stresses, extension of the harvest season, fruit quality, and productivity. However, the graft compatibility of many of these cultivars with most popular Prunus rootstocks is unknown, and this is an essential agronomical trait for their better performance and longevity. Hence, the introduction of new cultivars requires knowledge of the extent and nature of incompatibility reactions before releasing these cultivars on the market. In this study, the determination of graft compatibility was carried out in 13 new apricot cultivars grafted on four Prunus rootstocks: ‘Marianna2624′ (P. cerasifera ×x P. musoniana), ‘Miragreen’ (P. cerasifera × P. davidiana), ‘Mirared’ (P. cerasifera × Nemared), and ‘Montclar’ (P. persica L. seedlings) at early stages of development. By combining cytomorphological, anatomical, and phenylalanine ammonia-lyase (PAL) gene expression analysis at the graft interface, as well as different vegetative parameters, the results highlighted ‘Miragreen’ and ‘Mirared’ as promising rootstocks for apricot, showing the highest degree of compatibility with more than 90% of the apricot cultivars. These results provide useful information for breeders and growers by selecting the most suitable scion-rootstock for efficient orchard design by planting compatible graft combinations.

A CLEM approach to access to the ultrastructure at the graft interface in Arabidopsis thaliana

Despite recent progress in our understanding of the graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy approach (CLEM) to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed the efficient targeting of the grafting interface for under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the call wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these failed PD gives us insights into the process of secondary PD biogenesis. We showed that the thinning of the cell wall and the endoplasmic reticulum-plasma membrane tethering seem to be required for the establishment of symplastic connections between the scion and the rootstock. The resolution reached in this work shows that our CLEM method offer a new scale to the study for biological processes requiring the combination of light and electron microscopy.

Cell-to-Cell Connection in Plant Grafting – Molecular Insights into Symplasmic Reconstruction

Grafting is a means to connect tissues from two individual plants and grow a single chimeric plant through establishment of both apoplasmic and symplasmic connections. Recent molecular studies using RNA-sequencing data have provided genetic information on the processes involved in tissue reunion, including wound response, cell division, cell-cell adhesion, cell differentiation, and vascular formation. Thus, studies on grafting increase our understanding of various aspects of plant biology. Grafting has also been used to study systemic signaling and transport of micro- and macromolecules in the plant body. Given that graft viability and molecular transport across graft junctions largely depend on vascular formation, a major focus in grafting biology has been the mechanism of vascular development. In addition, it has been thought that symplasmic connections via plasmodesmata are fundamentally important to share cellular information among newly proliferated cells at the graft interface and to accomplish tissue differentiation correctly. Therefore, this review focuses on plasmodesmata formation during grafting. We take advantage of interfamily grafts for unambiguous identification of the graft interface and summarize morphological aspects of de novo formation of plasmodesmata. Important molecular events are addressed by re-examining the time-course transcriptome of interfamily grafts, from which we recently identified the cell-cell adhesion mechanism. Plasmodesmata-associated genes upregulated during graft healing that may provide a link to symplasm establishment are described. We also discuss future research directions.

Sugars promote graft union development in the heterograft of cucumber onto pumpkin

The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

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.

Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of β-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the β-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.

Cell–cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for vegetative propagation in plants, whereby a piece of living tissue is attached to another tissue through establishment of cell–cell adhesion. Plant grafting has a long history in agriculture and has been applied to improve crop traits for thousands of years1. Plant grafting has mostly relied on the natural ability of a plant for wound healing. However, the compatibility of cell–cell adhesion typically limits graft combinations to closely related species2–4, and the mechanism by which cell–cell adhesion of injured tissues is established is largely unknown. Here, we show that a subclade of β-1,4-glucanases secreted into the extracellular region facilitates cell–cell adhesion near the graft interface. Nicotiana shows a propensity for cell–cell adhesion with a diverse range of angiosperms, including vegetables, fruit trees, and monocots, in which cell wall reconstruction was promoted in a similar manner to conventional intrafamily grafting5–7. Using transcriptomic approaches, we identified a specific clade of β-1,4-glucanases that is upregulated during grafting in successful graft combinations but not in incompatible grafts and precedes graft adhesion in inter- and intrafamily grafts. Grafting was facilitated with an overexpressor of the β-1,4-glucanase and, using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. Our results demonstrate that the mechanism of cell–cell adhesion is partly conserved in plants and is a potential target to enhance plant grafting techniques.

Metabolite profiling during graft union formation reveals the reprogramming of primary metabolism and the induction of stilbene synthesis at the graft interface in grapevine

Background Grafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruits and vegetables. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine 1 month after grafting. Table-top grafting was done on over-wintering stems (canes) of grapevine and the graft interface tissues (containing some woody stem tissues and callus) were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses. Results A total of 54 compounds from primary and secondary metabolism (19 amino acids, five primary and 30 secondary compounds metabolites) and the activity of two enzymes were measured. The graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Some amino acids and stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues. Conclusions This study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes.