**2. Materials and Methods**

#### *2.1. Definition of Operating Conditions*

The experimental study was carried out at the Tenova Technological Center: Foundation for Auxiliary Technologies for Agriculture (Centro Tecnológico Tecnova: Fundación para las Tecnologías Auxiliares de la Agricultura) in Almería between March and June 2017. Almería's surroundings correspond to a model of agricultural exploitation of high technical and economic performance of greenhouse herbaceous fruit crops, especially for the tomato crop (Figure 1).

**Figure 1.** Working procedure of the experimental study to determine the combined importance of random rootstock and scion diameters at different cutting angles on splice grafting success.

For the study, rootstocks of the interspecific hybrid KNVF (*L. esculentum x L. hirsutum*) were used since it is the most used stock for tomato grafting [42–44]. The commercial rootstock Maxifort was used because of its strong roots and vigour and good performance at lower temperatures and in high salinity conditions. It presents high resistance (HR): ToMV: 0–2/Fol: 0,1/For/PI/Va: 0/Vd: 0; intermediate resistance (IR): Ma/Mi/Mj [45]. Likewise, the Ventero variety has been used as a grafting scion as an indeterminate tomato hybrid for truss harvesting, and it presents medium vigour, with good foliar coverage, very uniform fruits, slightly flattened of good red colour and deep shine, very good cracking and micro-cracking tolerance, and compact and well-formed clusters. It presents high genetic resistance (HR): ToMV: 0–2/Ff: B, D/Fol: 0,1/Va: 0/Vd: 0; and intermediate resistance (IR): TYLCV/Ma/Mi/Mj [39]. Both varieties are commonly used for manual grafting using the "tomato on tomato" (ToT) splicing technique, which demonstrated their compatibility prior to the experiment.

The working environment during the study was maintained under stable and controlled environmental conditions throughout all grafting experiments, with temperatures oscillating between 20◦ and 25◦, relative humidity conditions occasionally forced between 75% and 90% and stable brightness conditions of natural in-direct light. Oda [39] indicated that grafting must be performed in the shade in an area protected from the wind and the sun to avoid wilting of grafted seedlings. Grafting was performed at the lowest period of plant transpiration during morning hours [46], between 8 h and 12 h to maintain transpiration similar among the experiments and at the time period when the transport of water from the roots to the leaves is slowest, which makes the graft less susceptible to water stress and therefore to water loss. Other parameters with possible influence, such as atmospheric CO2 and other air contaminants, were not been controlled.

In the nursery, the plants were cultivated and attended to from sowing until 25 to 35 days after, and the scions were sown 2 to 5 days before the rootstock seeds. This variability of days is determined by the growth rate since different plants require different germination periods [47], and such periods are directly related to climatic conditions of the month of growing.

For the experiment, the plants were considered mature and ready for grafting when they had 2 to 4 well-defined true compound leaves [32], preferably with little foliage, thus decreasing the transpiration demand and post-grafting stress. The peat root ball remained wet but not soggy at all times during the grafting process, thus ensuring proper root respiration. The substrate used was 80% black peat with 10% perlite and 10% mulch. The experiments were always conducted with seedlings whose stems were still green and tender (herbaceous and non-woody).

For the splicing method, the seedling stems diameters should be at least 1.5 mm [48], and not too thick but with some natural and random variation. In the study, the diameters in the area close to the cut have varied from 1.5 to 2.5 mm for the scion and 2 to 3 mm for the rootstock.

The matching of the rootstock and scion samples was established randomly among plants that were healthy, had an acceptable anatomy and growth and presented diameters between the established reference limits. Seedlings with anomalous growth and diameters outside the established range were discarded. Diameters were measured using a digital calliper with a resolution of 1 dmm (0.1 mm) and repeatability of 1 dmm in the areas close to where the cut was performed both for the rootstocks and for the scions. The cut in the rootstock was always performed below the cotyledons, whereas the cut in the scion was performed above the cotyledons.

The complete experiment consisted of 10 individual events of 150 grafts each distributed over 4 months. Each graft consisted of 10 series of 15 grafts per tested angle. Therefore, 10 representative angles of the possible cutting range were selected: 0◦, 10◦, 20◦, 30◦, 40◦, 50◦, 60◦, 70◦, 80◦ and 85◦. Once each of the experiments was performed, grafts were grouped for healing on a single grafting tray of 15 × 10 cells, placing each group of grafts of a given angle in each of the 10 rows of the tray. Each of the grafted plants was rotated within their row for every tested angle eliminating in this way the position factor and its possible effects (ventilation and luminosity among others).
