**1. Introduction**

The objective of the experimental study was to determine the combined importance of random rootstock and scion diameters at different cutting angles on splice grafting success. The proposed working hypothesis suggests that both parameters have a statistically significant relationship with grafting success and an optimum working range can be defined to achieve successful grafts.

The experiment was developed as part of a larger study to optimize working conditions for the automation of grafting via the splicing technique. The study is autonomous and independent and presents sufficient and consistent results for the definition and specification of the splice grafting conditions that provide the most optimal results, whether performed manually or automated.

Grafting can be defined as a natural or deliberate fusion of plant parts by which vascular continuity is established [1], so that the resulting organisms function as a single plant [2].

The portion of the upper tissue or crown of a plant, which is also known as the stem or scion, adheres to another portion of the plant, specifically its root and lower part, which is commonly called rootstock, under stock or stock, and both parts come in contact and join with each other so that the resulting composite plant grows and develops as a single organism (graft). The callus corresponds to

the mass of parenchyma cells that develop from the plant tissue of the scion and the rootstock around the wound and where the development of vascular connections of the resulting graft union occurs [3].

Reducing the impact of pathogens is a challenge in all agricultural production systems [4], and monocultures are even more vulnerable than more diversified agricultural production systems [5]. Thus, grafting has become a tool of enormous potential to quickly enhance the efficiency of modern vegetable cultivars to promote wider adaptability or resistance to different stress situations [6].

The sequence of structural and biological events that occur in the development of a compatible graft between plants has been described in many studies, and the following development pattern is observed in which three fundamental phases can be distinguished: fusing of the rootstock and scion; proliferation of the callus around the union; and vascular re-differentiation through the interface establishing continuity between rootstock and scion [1,3,7,8]:


The success of the graft performed with a variety of compatible seedlings is determined by the three events previously described, assuming an important role of the plant hormones related to growth, such as auxin, in the grafting process [10]. Thus, the graft may originate from a combined mechanism of wound healing and conductive vessels formation [11]. Therefore, vascular connection is the last event in a successful healing process and represents the most important event because once such a vascular connection is established, water and solute transport begins from the stock to the scion, and the mechanical strength at the graft union increases [7,12]. One difficulty is to understand when the grafting process is completed [13], since a simple technique for continuous evaluation of graft development is not available [14]. Nonetheless, the assessment can be based on various techniques, including destructive and non-destructive techniques as follows:

(1) Visual estimation of the constituent seedlings and the appearance of graft growth [15]; (2) thermal camera imagery because the temperature of the leaves is 2 to 3 ◦C lower than that in a failed graft due to the transpiration of a successful graft [16]; (3) vertical cut performed on the graft surface and observation of the curvature of the vascular system formed at the union [17]; (4) measurement of the electrical resistance transferred from the scion to the rootstock through the surface area of its connection, which undergoes variations associated with histological changes during the union of the rootstock and the scion [14]; (5) assay performed to evaluate the tensile strength of the graft and analyse the strength of the graft union between rootstock and scion until breaking [18,19]; (6) displacement transducers used to perform a continuous and non-disruptive evaluation of the functional hydraulic connections within the plant [20]; and (7) NMR-based method (Nuclear magnetic resonance), that reveals water flux vectors inside individual vessels of intact plants [21].

The tomato is one of the world's most important herbaceous crops [22], and grafting of tomato plants is a widespread practice. Grafting methods among seedlings vary greatly and considerably depending upon the type of crops, farmer's experience and preferences, availability of facilities and machines, the number of grafts to perform and even the purpose and destination of the grafts, i.e., whether they are for the farmer's own uses or for sale and commercial distribution [23]. Grafting is

a common practice among herbaceous crops, and its use for Solanaceae is highlighted as follows: cleft or split grafting [21,24], splice or tube grafting [25,26], plug-in grafting [27], double-stem grafting [28,29], or pin grafting [15,30], among others.

Splice grafting, also known as tube grafting, top grafting or slant cut grafting, is the most popular [31] and widely used technique for tomato as well as eggplant [32]. The rootstock is cut below the cotyledons, thus eliminating the need to continually eliminate the sprouting of the stock over the plant's life [33]. The scion is also slant cut on a complementary angle above the cotyledons. Both parts are then placed in contact and secured by means of a tube or elastic tube-shaped clip with side slit. This method has the disadvantage of being highly demanding in terms of post-grafting microclimatic conditions, which require meticulous timing and delicate handling after the cut until healing and the maintenance of optimal temperature and humidity conditions to stimulate rooting [34]. As an advantage, the splicing method allows grafting with smaller plants, which reduces the pre-graft cultivation time and takes up less space in cultivation chambers and nurseries [35].

Velasco [12] and Villasana [36] affirmed that successful grafting is contingent upon similar stem diameters and the alignment of the vascular cambium area. However, such claims are dependent on the effect that the seedling diameter variable has on grafting success and do not consider the interrelated influence of other parameters and constraints of the grafting process.

For studying the success or failure of splice grafting, Yamada [37] established three factors of importance for its execution: (a) area of the cut surface; (b) gripping force of the union clip; and (c) smoothness of the cut surface and sharpness of the blade. For the first factor, allusive to the coincidence rate between seedlings, from coincidences of 50% graft successes of over 85% were achieved, and up to 95% success rates were observed for alignments over 80%; all this referred exclusively to a test angle of 30◦, so these results were obtained regardless of the effect of the cutting angle on the success of the process.

Furthermore, although the number of vascular bundles does not affect the grafting success, differences in diameters between the rootstock and the scion [38], that mark the alignment between them do have an effect. Thus, when a graft is performed, it is important to increase the chances that the vascular bundles from the rootstock and the scion come in direct contact, maximizing the area of the cut surfaces that are joined by pressing them together [39].

For manual and automated grafting, the alignment of diameter of both seedlings must be identified, classified, and visually paired. This is an expensive and arduous task that applies a series of calibrations and visual comparison criteria based on morphological characteristics, which may be subjective and susceptible to human error [40,41]. The present study analyses the importance of these pre-grafting tasks based on grafting success and whether an optimal working zone can be established that guarantees adequate grafting success without having to pre-sort the seedlings.
