Graft Compatibility Classification within Aurantioideae Based on Biometric Traits and the Anatomy of Graft Union

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respectively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by ‘Candidatus Liberibacter asiaticus’. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their graft compatibility, as follows: fully compatible with high graft-take and plant growth; potentially compatible with high graft-take but lower plant growth; partially incompatible with lower graft-take and poor plant growth; and fully incompatible with a complete absence of graft-take.

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.

Cyanogenic glycosides content as graft compatibility indicator in pear/quince combinations

The objective of this work is to evaluate the concentration of cyanogenic glycosides CGs (amygdalin and prunasin) in the cultivar and rootstock, as an indicator of compatibility in pear grafts. The work consists of two experiments. Experiment 1 evaluated Cascatense/Pyrus Calleryana and Cascatense/EMC combinations. Experiment 2 evaluated the combinations Packham’s Triumph/Adams, Santa Maria/Adams, and Rocha/Adams. The experimental design of the two experiments was randomized blocks with four replications. The evaluations were carried out in the spring and summer of the harvest 2014/2015. The results indicated graft compatibility in the Cascatense/P. Calleryana, Packham’s Triumph/Adams, and Rocha/Adams combinations; slight incompatibility of Santa Maria/Adams, and severe incompatibility of Cascatense/EMC. The obtained results indicate that differences ≥ 20 mg g-1 of CGs between scion and rootstock were correlated with a drastic reduction in vigor, problems of continuity in graft union, low yield, and early defoliation.

Root-to-Shoot Long-Distance Mobile miRNAs Identified from Nicotiana Rootstocks

Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.

Anatomical and Histological Studies of Grafted Tomato with Interspecific Solanaceous Rootstocks

A study was carried out at the Department of Vegetable Science, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu,India during 2019-2020 to assess the anatomical and histological changes within the graft union of tomato and identify compatible rootstocks for improved propagation of the crop. This experiment involved rootstocks of three wild tomatoes including Solanum torvum, S. sisymbrifolium and S. capsicoides and scions of two tomato hybrids including TNAU tomato hybrid CO3 and Shivam. The grafting was done using the cleft grafting method. The anatomical and histological sections of six graft combinations and two tomato scion samples were viewed microscopically at 7, 14 and 21 days after grafting (DAG). Among the six tomato graft combinations, S. torvum rootstock showed complete development of vascular connection at 21 DAG followed by S. sisymbrifolium rootstock, whereas S. capsicoides rootstock showed only callus growth at 14 DAG. Scion growth dominated the rootstock growth in S. torvum rootstock leading to mismatch of scion-rootstock stem thickness and delayed epinasty symptom at the later stages of plant growth. Of the rootstocks of the three species studied, S. sisymbrifolium rootstock was compatible for tomato grafting though it exhibited delayed vascular connection between the scion and rootstock.

Comparative Vegetative, Nutritional and Anatomical Study of Two Almond Varieties Grafted on Bitter Almond and Nemaguard Peach Rootstocks

A comparative study was conducted during the 2017/2018 and 2018/2019 growing seasons for Nonpareil and Ne Plus Ultra almond cvs grafted on Bitter almond and Nemaguard peach rootstocks at the Experimental Research Station of National Research Centre at Nubaria, El Behera governorate, Egypt. The comparison was evaluated through the grafting success percentage, vegetative growth, determination of some leaf mineral composition, as well as, the anatomical examination of scion/rootstock union zone and cross-section of leaves. The obtained results clarified that the grafting success percentage was significantly affected by the different used rootstocks. Using Bitter almond rootstock recorded the highest percentage of grafting success, leaf chlorophyll content, root length, and a number of lateral root /plant in both cultivars. Using Nemaguard peach rootstock recorded significantly the highest values of scion length, stem girth above and below union zone, number of branches and leaves/plant, leaf area as well as seedlings fresh and dry weight. Leaves of both cultivars grafted on Nemaguard peach rootstock recorded the highest concentrations of leaf mineral contents as compared to those grafted on Bitter almond rootstock. Cross-sections of the graft union zone were taken after 28 days and 6 months from grafting for anatomical analysis. The callus cells developed 28 days after grafting, but cambial cells between the rootstock and scion tissues did not occur in all scion/rootstock combinations. After 6 months of grafting, cambial cells were established, vascular differentiation was observed, regular parenchymatic tissue properties and sclerenchyma bundles were seen in the graft union. There was no problem in terms of compatibility of Nonpareil and Ne Plus Ultra on Bitter almond and Nemaguard peach rootstocks. Comparing leaf cross-sections of almond grafted on both rootstocks demonstrated that the density of mesophyll cells was affected as a consequence of the rootstock–scion interaction. Nemaguard peach rootstock can be recommended for grafting Nonpareil and Ne Plus Ultra almond cultivars under Nubaria conditions to produce vigorous trees.

Rootstocks Overexpressing StNPR1 and StDREB1 Improve Osmotic Stress Tolerance of Wild-Type Scion in Transgrafted Tobacco Plants

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

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.