There were no many studies performed on the graft incompatibility mechanism and its prediction methods in sweet cherry trees. Sweet cherry is mainly grown byamateur growers, and they are very rarely planted in commercial orchards (Lanauskas et al., 2023). This study aimed to investigate the changes in some metabolites and techniques needed to understand this mechanism better.Four-five days after grafting, callus tissue is formed at the junction of the rootstock and scion to the graft, while the lack of callus tissue formation at the intersection leads to graft failure (Porika et al., 2016).
The grafts' degree of compatibility showed that when the degree of compatibility is less than 1, the grafts are incompatible. if, the degree of compatibility is more than 1, the grafts are compatible. The highest degree of compatibility was recorded in ‘Takdaneh’ and ‘Siyah Mashhad’ cultivars on ‘Gisela-6’ rootstock. The degree of compatibility in ‘Gisela-5’ rootstocks was less than ‘Gisela-6’, which indicates that the ‘Takdaneh’ and ‘Siyah Mashhad’ cultivars are more compatible with ‘Gisela-6’.
Plants rapidly increase their antioxidant capacity when stress occurs to increase resistance and tolerance to the conditions created. It can be said that the plant's ability to disperse excess energy and neutralize free radicals is impaired, thus increasing the antioxidant capacity to increase the resistance to stress, so in incompatible grafts increases phenolic compounds and peroxidase to minimize oxidative damage compared to compatible grafts (Zarrouk et al., 2010; Assuncao et al., 2016; Baron et al., 2019). Since ‘GF-305’ rootstock was an incompatible control in this experiment, higher phenol content was observed in grafting sweet cherry cultivars on ‘GF-305’ rootstock. Also, total phenol content in sweet cherry cultivars on ‘Gisela-6’ rootstock, which was a compatible control, was lower than that of the incompatible control. The amount of total phenol in the transplants performed on ‘Mahaleb’ (M-168) rootstock was higher than the compatible control and less than the incompatible control. In general, the amount of total phenol in the grafts made on ‘Gisela-6’ (compatible control) and ‘Gisela-5’ rootstocks was less than ‘GF-305’ (incompatible control).
‘GF-305’ is incompatible with sweet cherry cultivars, and it seems that phenolic compounds, can be used to pre-screen incompatible grafts. According to high storage theories, phenolic compounds such as catechins above the graft union can be used as a biochemical marker in the diagnosis of graft incompatibility (Canas et al., 2015; Baron et al., 2019). Pina et al. (2017) reported that the amount of phenolic compounds at the top and bottom of compatible and incompatible grafts was significantly different. Also, Hudina et al. (2014) have reported phenolic compounds as markers to evaluate the grafts' compatibility between the rootstocks and scion.
Peroxidase levels in incompatible grafts were higher than compatible grafts. It seems that the peroxidase enzyme can be used to predict incompatible grafts quickly. Peroxidases are enzymes that play essential biochemical and physiological roles in plants, including plant growth, differentiation and development, auxin catabolism, ethylene biosynthesis, plasma membrane regeneration, cell wall development, ligninification, and response to pathogens (Pandey et al., 2017). Preliminary analyses of peroxidase indicate that this enzyme is essential in forming cell wall constituents and ligninification. The next step, by creating cross-linking between cell wall phenolic polymers, reduces cell wall flexibility (Hatfield et al., 2017). This process causes irreversible hardening of the cell wall (Suchy et al., 2010).
The result of present study showed that the f peroxidase enzyme activity in grafting cultivars on ‘GF-305’ rootstocks that were incompatible was higher than grafting cultivars on ‘Gisela-6’ (compatible control), ‘Gisela-5’, and Mahalb. Many studies have shown the role of peroxidase in inhibiting growth and reported that peroxidase levels in incompatible grafts are higher than incompatible grafts (Zarrouk et al., 2010; Pina et al., 2017; Baron et al., 2019). The researchers reported that the lack of similarity in the isoperoxidase composition between the rootstock and scion could lead to abnormal ligninification and absence of vascular attachment at the graft union, leading to incompatible grafts (Zarrouk et al., 2010).
Some research findings on the relationships between peroxidase isoenzyme patterns and graft incompatibility in different plants suggest that the matching of isoperoxidase grafts between the rootstock and the scion can be used as an indicator of graft proliferation (Dogra et al., 2018). In the present study, starch accumulation at the union’s grafting did not have a good performance for predicting the incompatibility of graft. Different results have been reported for starch accumulation at the union’s grafting (Deng et al., 2019).
In some of them, starch accumulation occurred in incompatible grafting, Due to genetic differences and cultivar type, starch accumulation was not a good sign of graft incompatibility as in the present study. Accumulation of starch above the graft union and its absence or lack at the bottom of the graft union causes damage to the phloem vessel, resulting from the starch collection in the phloem vessel (Deng et al., 2019). Researchers have shown that the grafts union's total starch content changes dramatically (Hudina et al., 2014). The relative starch content of rootstock and scion tissues is affected by the type of graft composition. In incompatible compounds, the relative starch content of scion wood is higher than rootstock wood. Accumulation of starch and soluble sugars in scion has also been observed in transmitted incompatibility. Low accumulation of starch at the top and bottom of the graft causes better boiling of the graft and adequate transfer of nutrients to the roots and vice versa (Karimi and Hassanpour, 2017). However, in the grafting of cherries on the ‘Mahaleb’ (M-168) rootstock, although signs of incompatibility were seen, no difference was found between the starch at the graft union's top and bottom. Therefore, it is true that in some cultivars, starch accumulation does not occur at the top of the graft, but there are signs of incompatibility in them, which may indicate their incompatibility (Garner, 2013).
The result of callus fusion experiment showed that hormones (2,4-D and BAP) are necessary for callus production, and callus was not produced in the culture medium without hormones. Similar results have been recorded by researchers in several plant species (Ahmad et al., 2016).
Using the callus fusion technique is a quick way to predict grafts compatibility or incompatibility. The rootstock and scion callus are fully integrated in the compatible grafts after about 10 days, but the calluses do not merge if the graft is incompatible. The present results showed that ‘Gisela-6’ rootstock grows more than other rootstocks with scion in culture medium. It seems that in compatible sweet cherry grafts, a growth- promoting substance is secreted to which the compatible rootstock reacts. Determination of graft incompatibility using callus culture in some plants was reported by Gainza et al. (2015). Callus tissue formation at the graft union is the first positive reaction in the graft (Porika et al., 2016). Researchers used the callus fusion technique to determine early graft incompatibility in apricots and reported that callus integration in vitro can be used to initially predict graft compatibility (Pina et al., 2017). Irisarri et al. (2015) used callus fusion to predict early graft compatibility in apricot and lemon trees. Early and accurate prediction of transplant incompatibility is essential because of the avoidance of incompatible compounds and selecting compatible ones (Zarrouk et al., 2010). However, the metabolic and physiological mechanisms involved in maladaptive responses are not well understood and require further research.