# Behavior of Different Grafting Strategies Using Automated Technology for Splice Grafting Technique
## 2 Materials and Methods
### Definition of the Operating Conditions  
The research was performed at the Tecnova Technology Centre: Foundation for Auxiliary Technologies in Agriculture, Almeria (36 deg52'38'' N, 2 deg19'59'' W) between March and June 2017. The rootstock used in the experiment was the interspecific hybrid 'Maxifort' (De Ruiter SeedTM, Spain),  
Figure 3: Detailed view of the device that dispenses and places the graft clip. @ Pinching system with clip wings pressed, @ clip clamping system (at rest), @ and @ elements used in the manufacture and clamping of grafting clips from continuous plastic tube, @ overview of the equipment for making, dispensing, and securing the clips.  
while the scion used was 'Ventero' (De Ruiter SeedTM, Spain). Both seed types are commonly used in seedlings destined for manual grafting using the 'tomato on tomato' (T/T) splicing technique, so their pre-experiment compatibility to the automated grafting system has already been demonstrated.  
Prior to the grafting process, the plants were cultivated in the seedbed and nursed until the rootstock and scion achieved a similar growth state; for this to occur, the scion was planted 2 to 5 days before the rootstock so that both seedlings had similar stem diameters. The seedlings were grafted when they had 2 to 4 well-defined true leaves. We tried to pair rootstock and scion stems of similar diameters. The grafting operation should be carried out over a very short period of time. During this process, the environmental conditions in the work environment were regulated with temperatures between 20\({}^{\circ}\) and 25\({}^{\circ}\), and relative humidity conditions of between 75% and 95%, with stable lighting conditions of non-direct daylight. Splice grafting requires stringent environmental conditions during the healing process, with high relative humidity (HR) and an optimal temperature in order to reduce stem transpiration until the rootstock and the vascular tissue of the stem are healed and water and nutrient transport is restored [48]. After each of the grafting trays was completed, they were immediately placed in a small healing chamber; this comprised a tunnel slightly larger than the size of each tray, covered by a transparent film. This tunnel was placed in a chamber where climate conditions were controlled throughout the healing process. During the first 48 h, the plants were unlit to reduce transpiration and evaporation. Over the following days, the light intensity was increased, establishing a 14 h photoperiod with a value of 100 umol-m\({}^{-2}\) s\({}^{-1}\) of Photosynthetically Active Radiation (PAR), (x3000 Lux) non-direct and diffuse light, as recommended for the callus formation stage [6; 49]. This illumination was gradually increased over successive days while the temperature was controlled between 23 and 30 \({}^{\circ}\)C (an average of 26 \({}^{\circ}\)C), using a variable setting point in the healing chamber, slightly varying the daytime and night-time conditions. Relative humidity (HR) was initially established between 95% and 75% in the first phase. It was then gradually reduced over subsequent days for outdoor acclimatization.  
For the first three days, the grafts were not handled. After the fourth day, the grafts underwent a thorough visual inspection on a daily basis, evaluating the changes and the healing process in each of them, assessing the natural evolution of the grafts and analyzing other symptoms and external signs that might determine their success or failure. This follow-up was performed for 14 days after grafting. Non-destructive techniques were used for the evaluation of graft success, based on direct observation of the evolution of the grafting junction. Visual inspection of evidences was evaluated such as vigor or sagging on leaves and stems, consummation of the callus, appearance of necrotic areas, substantial differences between scion and rootstock part growth, or the progressive appearance of new axillary buds in the scion.