Growing Tomato Seedlings Suitable for Mechanical Grafting under Regulated Light Regime

Abstract

The uniformity of growth and mechanical properties of grafted seedlings affect the quality of mechanical grafting operations. The growth uniformity of grafted seedlings in a greenhouse will be poor due to the uneven and unstable light and temperature conditions. Plant factories can cultivate grafted seedlings in the most suitable environment by regulating environmental parameters such as light and temperature. The aim of this study was to investigate the impact of the light conditions on tomato seedlings in plant factory and to develop an optimal cultivation light formula. The effects of light intensity (50, 100, 150, 200, and 250 μmol m⁻² s⁻²) and photoperiod (10, 12, 14, 16, and 18 h a day(h/d)) on the morphological and mechanical properties of tomato seedlings were experimentally investigated. Orthogonal experiments were conducted involving light quality (R:B = 75:25, R:B = 50:50, and R:B = 25:75), light intensity (150 μmol m⁻² s⁻², 200 μmol m⁻² s⁻², and 250 μmol m⁻² s⁻²), and photoperiod (14, 16, and 18 h/d) as independent variables to determine the optimal combination. Finally, a comparative grafting experiment was conducted between the seedlings cultivated using the optimal light formula and commercially available seedlings. The result showed that increasing light intensity inhibited hypocotyl length and promoted seedling stem growth, and excessive light intensity decreased seedling mechanical properties. The optimal light intensity for rootstocks is 200 μmol m⁻² s⁻², and the optimal light intensity for scions is 250 μmol m⁻² s⁻². Shortening the photoperiod would promote hypocotyl growth and inhibit seedling stem elongation. Different photoperiods had a significant impact on the mechanical properties of tomato seedlings. The most suitable photoperiod for rootstocks was 18 h/d and for scions was 16 h/d. The most suitable light formula was R:B = 50:50, 250 μmol m⁻² s⁻², 18 h/d. By analyzing the experimental results, the mechanical properties of seedlings grown by the regulated light environment were better than those of commercially available seedlings, and the success rate of mechanical grafting was 7% higher. Overall, in plant factories compared to commercially available tomato seedlings, tomato seedlings cultivated by the regulated light environment were more suitable for mechanical grafting. This research result provides theoretical support for subsequent research on grafting machinery.

1. Introduction

Tomatoes are a significant food crop, cultivated extensively globally [1,2]. Grafting is a common practice in tomato cultivation, employed to enhance quality and yield. It is estimated that hundreds of millions of grafted seedlings are produced each year [3,4]. Currently, grafting operations are predominantly manual, which can result in high labor intensity and low work efficiency [5,6]. Further, seedling grafting is a labor-intensive process and accounts for more than 40% of the total cost of seedling production in China [7]; consequently, the utilization of machinery for grafting operations is imperative.

The development of automatic grafting robots has been a research topic in numerous countries since the 1990s, with significant advancements in this field [8,9]. Liang et al., in 2023 [10], designed a 2TJGQ-800 grafting machine for watermelon seedlings, which is capable of smoothly grafting rootstocks and scions. However, the machine failed to graft the scion because the hypocotyl length was insufficiently long, resulting in the scion falling off. Fu et al., in 2022 [11], designed a grafting device for whole trays of melon seedlings. This device works by positioning the whole tray of rootstocks and scions and cutting the hypocotyl. For this procedure to be successful, the hypocotyl must be of a specific height and diameter and exhibit uniformity. Chang et al., in 2012 [12], designed a grafting machine for chili seedlings and demonstrated that machine-grafted seedlings are subjected to tremendous graft shock, which can result in slower growth. The experimental analyses of various grafting machines have demonstrated that the quality and morphology of the seedling have a direct and significant influence on the success of the effectiveness of the grafting machine [9,13]; the rootstock and scion must always be of the same size and specification required by the grafting machine [14]. In greenhouses, tomato rootstocks and scions grown on a large scale have seedlings with different morphological sizes and poor uniformity, which is the main reason that hinders the large-scale diffusion of grafting robots [8]. The workflow of an existing automatic grafting machine is shown in Figure 1. The diagram is time-oriented. At the beginning of the grafting work, the scion and rootstock go through the feeding, clamping, and homing processes simultaneously and are cut simultaneously. The pith-cambium grafting process is then completed by clamping the interface with grafting clips [15,16]. In the process of machine grafting, the seedlings will be damaged by external forces [16], therefore, seedlings of cultivars with good mechanical properties are favorable for grafting quality.

To produce seedlings suitable for mechanical grafting, it is necessary to regulate the seedlings’ morphological parameters and mechanical properties. Light is the most critical factor in plant growth, and many scientists in China and abroad have studied its effect on crops. Meiramkulova et al., in 2021 [17], showed that LEDs are more favorable than high-pressure sodium lamps for the growth of tomato seedlings under greenhouse conditions. Zhang et al., in 2022 [18], found that tomato seedlings in the white + red supplemental light combination had the highest plant height, and the stem diameter of tomato seedlings in the red + blue supplemental light combination was significantly higher than that in the white + red supplemental light combination. Liu et al., in 2018 [19], showed that far-red light plays a dominant role in the effects on tomato seedling and hypocotyl length when combined with blue light or UV-A. Kim Hye Min et al., in 2019 [20], conducted a study to examine the impact of different LED light qualities on tomato plug seedlings. The results demonstrated that seedlings exhibited greater growth in monochromatic red light compared to monochromatic blue light. Additionally, the findings indicated that a combination of red and blue light could potentially restrict the elongation of seedlings. Kim Eun-Young et al., in 2015 [21], studied the effect of monochromatic LED light quality on cherry tomato seedlings and showed that there was no significant difference in above-ground fresh biomass between seedlings treated with red and blue LEDs. Kong et al., in 2023 [22], studied the effect of blue light on plant growth and showed that a combination of red and blue light was more effective in inhibiting plant elongation than pure blue light. Hwang et al., in 2020 [23], investigated the impact of far-infrared LEDs on the growth and morphology of tomato seedlings in a plant factory setting. The findings indicated that far-red light exerts a discernible influence on the development of tomato seedlings, with notable inter-varietal differences. Khoshimkhujaev et al., in 2014 [24], conducted a study investigating the impact of UV-A radiation on the growth of tomato seedlings. The results demonstrated that UV-A radiation at 376 nm was conducive to the growth of tomato seedlings. Zheng et al., in 2021 [25], reported that using a light ratio with a red-to-blue ratio of 1.2, a red/far-red ratio of 16, and a light intensity of 150 μmol m⁻² s⁻¹ during the healing process of grafted tomato seedlings favored growth healing.

Plant factories can be employed to cultivate tomato seedlings to produce seedlings that meet the requirements of grafting machines. This is achieved by precisely regulating environmental parameters, which allows for the control of morphological parameters. To identify the most appropriate light formulation for cultivating seedlings suitable for mechanical grafting, a detailed study of the changes in growth and mechanical properties observed in tomato seedlings subjected to different light quality, light intensity, and photoperiodic conditions is essential. A series of comparative tests were conducted with commercially available seedlings with the objective of determining the advantages of light regulation in seedling production. A similar paper of the same main author was published recently [26], and the effect of light quality on the mechanical properties of grafted tomato seedlings was studied in that paper, and the most suitable light quality ratio was discussed. But the previously mentioned paper added more data to the experiment and is relevant for the scientific community, and the mechanical grafting process was analyzed systematically, and the optimal light formula was derived further. This study determined that red light/blue light ratio = 50:50 is the most suitable light quality ratio for cultivating mechanically grafted tomato seedlings based on the prequel experiment. This paper only selects the optimal light formulation and does not study the mechanism.

2. Materials and Methods

2.1. Plant Material and Treatment Design

Both the rootstock seeds and the scion seeds were purchased from Glseed Company (Guangzhou, China). Rootstock seeds (Glseed T17-2) and scion seeds (Glseed 2) were soaked in warm water at 50 °C for five hours and then placed in an electrically heated thermostat incubator (Sunne, Shanghai) at 26 °C for germination. The average germination time was 3 days for rootstock seeds and 4 days for scion seeds. Germinated seeds were planted into 72-hole trays (540 × 280 × 50 mm, L × W × H) with a substrate ratio of grass charcoal/perlite/vermiculite = 7:2:1. The trays were incubated in an LED dark room in the plant factory of South China Agricultural University (the temperature was 23 ± 1 °C, the relative humidity was 75 ± 5%, and the concentration of CO₂ was 580 ± 10 ppm). The light quality was set to R:B = 50:50 (Figure 2). The PPFD of all treatment plots was set with the optical spectrometer (LI-180, Li-cor, Lincoln, NE, USA) nearly 30 cm above the ground.

During the experiment, the tomato seedlings were irrigated every two or three days with the nutrient solution. The nutrient solution was based on the following Japanese horticultural formula (mg/L): KNO₃, 808; Ca (NO₃)₂·4H₂O, 944; MgSO₄·7H₂O, 492; NH₄H₂PO₄, 153; DTPA-Fe-7, 42.9; H₃BO₃, 2.82; MnSO₄·H₂O, 1.54; CuSO₄·5H₂O, 0.08; ZnSO₄·7H₂O, 0.22; (NH₄)6Mo₇O₂₄·4H₂O, 0.03, respectively [25].

In the light intensity experiment, the light intensity was set to 50, 100, 150, 200, and 250 μmol m⁻² s⁻¹ under the same photoperiod of 18/6 h/d. The photoperiod experiment was set to 10, 12, 14, and 16 h/d under the same light intensity PPFD of 150 μmol m⁻² s⁻¹. In the photoperiod test, the photoperiod was set at 10, 12, 14, 16, and 18 h/d under the same light intensity PPFD of 150 μmol m⁻² s⁻¹. Subsequently, the groups of different light intensities were referred to as 50, 100, 150, 200, and 250, and the groups of different photoperiodic treatments were referred to as 10, 12, 14, 16, and 18 h/d.

2.2. Growth Measurements

2.2.1. Determination of Morphological Traits and Fresh and Dry Biomass

The rootstocks and scions were sown on 23 February 2024. The hypocotyl length and stem diameter of seedlings are the criteria for their use in automated mechanical grafting [7]. Seedlings from different treatments were randomly selected on days 5, 10, 15, 20, 25, and 30 after emergence, and stem diameter and hypocotyl length were measured using Vernier calipers (range 0.02 mm), with 20 seedlings selected each time. Fresh and dry biomass were determined on 30th day, with a selection of 10 samples per measurement. Seedlings were removed from the substrate, washed with water and drawn surface water, and weighed fresh using an electronic balance (range 0.01 g). The washed seedlings were dried at 105 °C for 30 min in a stove (Yiheng, Shanghai, China) and then at 75 °C to a constant biomass. The moisture content and seedling index were calculated by employing the formulae described by Liu et al. [27] Measurements of hypocotyls, plant height, and stem diameter of the commercially available seedlings (Glseed) were taken using Vernier calipers (range 0.02 mm), with 20 plants selected each time.

Moisture content (MC) = (fresh biomass − dry weigh)/fresh biomass

(1)

Seedling index (SI) = (stem diameter/seedling height + root dry biomass/shoot
dry biomass) × seedling dry biomass

(2)

2.2.2. Measurement of Mechanical Properties

A total of five samples were selected for each measurement, with the rootstocks and scions having a stem diameter of 3 ± 0.2 mm. This was carried out in accordance with the working requirements of the grafting machine [7]. The axial compression force (ACF), radial compression force (RCF), shear strength (SS), and bending strength (BS) of tomato seedlings were measured using a mass spectrometer (TMS-Pilot, FTC, Sterling, VA, USA). Shear strength and bending strength were calculated according to the formulae proposed by Zong et al. [28]. The same measurements were taken for commercially available seedlings (Glseed), and five samples were selected for each measurement.

$${\sigma }_m={\displaystyle \frac{960000F_{max}L}{\pi D^3}}$$

(3)

$${\mathsf{\epsilon }}_{\mathrm{n}}={\displaystyle \frac{2{\mathrm{f}}_{\max }}{{\mathsf{\pi }\mathrm{D}}^2}}$$

(4)

where ${\sigma }_m$ is the bending strength in MPa, and ${\epsilon }_n$ is the shearing strength in MPa. F_max is the maximum applied load in the bending testing in N, and f_max is the maximum applied load in the shearing testing in N. L is the space distance of the two fixed supports; it is 30 mm. D is the diameter of the middle stem. In calculations, the cross-section of the specimen is approximated to a circle.

2.3. Statistical Analysis

One-way analysis of variance (ANOVA) and then the Duncan’s multiple range test were carried out using SPSS 19.0 software (lBM, Inc., Chicago, IL, USA) to compare the means between treatments. The Duncan’s multiple range test was used to determine the significant differences at the 0.05 significance level (p < 0.05). The graphing was conducted by Origin 2021.

2.4. Grafting Experiment Comparing Commercially Available Seedlings and Light-Regulated Seedlings

Commercially available seedlings and light-regulated seedlings were tested on an automatic grafting machine at the Glseed Seedling Farm (Sanshui, China). The grafting machine was designed and manufactured by Xie [7]; the grafting angle between rootstock and scion was 30°. There were 10 trays of commercially available seedlings, including 5 trays of rootstocks and 5 trays of scions. There were 10 trays of light-regulated seedlings, including 5 trays of rootstocks and 5 trays of scions. The trays used had 72 holes. The rootstocks and scions were supplied into the grafting machine separately for the grafting test (Figure 3). Grafting was judged successful if the rootstock and scion did not fall off after the grafting operation. The grafted seedlings were placed in a chamber for healing. The environmental parameters of the healing chamber were 22 °C temperature and 95% humidity, with shading for the first three days, followed by gradual intermittent light supply. After 7 days, if the interface was healed without obvious cracks, and there were no adverse symptoms on the leaves, the healing was considered successful. The grafting success rate and post-grafting survival rate were calculated using Equations (5) and (6).

$${\delta }_s=\left|{\displaystyle \frac{n_s-n}{n}}\right|\times 100\%$$

(5)

$${\delta }_a=\left|{\displaystyle \frac{n_t-n_s}{n_s}}\right|\times 100\%$$

(6)

where ${\delta }_s$ is the grafting success rate. $n_s$ is the number of successfully grafted seedlings. $n$ is total number of grafted seedlings; it is 350. ${\delta }_a$ is the post-grafting survival rate. $n_t$ is number of seedlings surviving after grafting.

3. Results

3.1. Single-Factor Experiments

3.1.1. Light Intensity Experiment

Effects on Morphology and Longevity

As illustrated in the accompanying figure (Figure 4), the morphology of tomato seedlings exhibited a pronounced response to light intensities. The significant differences between hypocotyl and plant length evidenced this. Furthermore, rootstocks and scions exhibited disparate morphological characteristics under identical lighting conditions.

It can be observed that a reduction in the length of the hypocotyl of the rootstocks accompanies an increase in light intensity (Figure 5A). The diameter of the stem exhibited a progressive increase with increasing light intensity (Figure 5C). On the 30th day, the rootstock grown at 250 micro mol m⁻² s⁻¹ light intensity showed the shortest hypocotyls. (Figure 5A). However, the hypocotyls did not differ significantly from those of rootstocks with light intensity 200 μmol m⁻² s⁻¹ (Figure 5A), and the rootstocks with light intensity 250 μmol m⁻² s⁻¹ exhibited the most pronounced stem diameter (Figure 5A). The greater the light intensity, the shorter the length of the scion hypocotyls (Figure 5B). However, no significant differences were observed between the treatments, except the treatment with light intensity 50, which exhibited approximately double the height of the hypocotyls observed under the light intensity 250 μmol m⁻² s⁻¹ treatments (Figure 5B). From the 15th day onwards, the hypocotyl growth rate of the scion with light intensity 50 continued to increase. In contrast, the hypocotyl growth rate of the scion, along with other treatments, exhibited a gradual slowing (Figure 5B). At day 30, the scion with light intensity 50 μmol m⁻² s⁻¹ exhibited the smallest stem diameter, while the scion with light intensity 250, 200, and 150 μmol m⁻² s⁻¹ exhibited minimal differences in stem diameter (Figure 5D). The uniformity of hypocotyl growth did not differ much between the different light intensity treatments.

This study examined the effect of different light intensities on the growth and development of tomato seedlings. The water content of the scion was higher than that of the rootstock under different light intensity treatments, with the lowest water content of rootstock and scion at light intensity 150 μmol m⁻² s⁻¹ (Figure 6A). Rootstock fresh biomass was higher than scion at light intensity 50 μmol m⁻² s⁻¹ and light intensity 100 μmol m⁻² s⁻¹, and scion fresh biomass was higher than rootstock at the remaining treatments (Figure 6B). Rootstock in light intensity 250 μmol m⁻² s⁻¹ was the largest of all treatments, and scion in light intensity 150 μmol m⁻² s⁻¹ was the largest of all treatments (Figure 6B). Dry biomass (Figure 6C,D) and the seedling strength index of rootstocks (Figure 6E) and the seedling strength index of scions were the best for light intensity 150 μmol m⁻² s⁻¹, which was also an inflection point. The effect of light intensity on plant height was significant; rootstocks and scions grown under 50 μmol m⁻² s⁻¹ showed significantly higher plant height than the other treatments (Figure 6F). Light intensity has a considerable influence on the growth of grafted tomato seedlings.

Effect of Light Intensities on the Mechanical Properties of Grafted Seedlings

This study aimed to ascertain the impact of varying light intensities on the mechanical properties of grafted seedlings. The results demonstrate that different light intensities exert a discernible influence on these properties.

The radial compression force of rootstock and scion seedlings was found to be significantly reduced in the case of light intensity 50 μmol m⁻² s⁻¹ (Figure 7A). There was no significant difference between the radial compression forces of scions in the remaining treatments (Figure 7A). The radial compression force of rootstocks was observed to be the greatest at light intensity 200 μmol m⁻² s⁻¹. The light intensity had a minimal impact on the axial compression force of the seedlings. The axial compression force of the rootstock and scion was observed to be at its maximum level at light intensity 200 μmol m⁻² s⁻¹ and its minimum at light intensity 100 μmol m⁻² s⁻¹, with a difference of approximately 26.2% for the rootstock and 37.5% for the scion (Figure 7B). The bending strength of the rootstock exhibited a positive correlation with increasing light intensity, with the scion demonstrating the greatest bending strength at light intensity of 150 μmol m⁻² s⁻¹, which was significantly higher than the bending strength of the rootstock and scion under other treatments (Figure 7C). The shear strength of the rootstock exhibited a “U”-shaped pattern with increasing light intensity, initially decreasing and subsequently growing. The shear strength of the scion exhibited a maximum at a light intensity of 150 μmol m⁻² s⁻¹, which was not significant under other treatments (Figure 7D).

Summary

Based on the growth rate and mechanical properties of seedling stems, and in combination with the magnitude of the solid seedling index, it was concluded that a light intensity of 200 μmol m⁻² s⁻¹ is the best suited for cultivating rootstocks for mechanical grafting, and a light intensity of 250 μmol m⁻² s⁻¹ is the best suited for cultivating scions for mechanical grafting.

3.1.2. Photoperiodic Experiment

Photoperiodic Effect on Morphology and Longevity

As illustrated in the accompanying figure (Figure 8), the morphology of tomato seedlings exhibited a pronounced response to varying photoperiod. The significant differences between hypocotyl and plant height evidenced this. Furthermore, rootstocks and scions exhibited disparate morphological characteristics under identical lighting conditions.

The effects of different photoperiods on the growth rate of rootstock hypocotyls were evident from the 15th day to the 20th day. The photoperiod 18 h/d showed a sharp increase in growth rate at the 20th–25th days. The heights of the hypocotyls under the various photoperiod treatments at day 30 were ranked from highest to lowest as 10 > 12 > 18 > 14 > 16 h/d (Figure 9A). Except for the photoperiod 18 h/d, the effects of the remaining photoperiods on the growth rate of stem diameter in rootstocks were not discernible, and the overall trend remained consistent. At the 30th day, the greatest rootstock stem diameter was observed in the photoperiod 18 d/h (Figure 9C). The effects of different photoperiods on the growth rate of scion hypocotyls were not apparent, and the overall trend was the same, with a fast growth rate from day 5 to day 15, and a significantly slower growth rate from the 15th day to the 30th day (Figure 9B). Hypocotyl length was considerably higher in the photoperiod 10 h/d than in the other treatments, and there was no significant difference in hypocotyl length between the 18 h/d and 16 h/d photoperiods. Between the 5th and the 30th days, the overall growth trend of stem diameter under the photoperiod treatments was consistent, except for the photoperiod 10 h/d, where the growth trend slowed down significantly on day 25 (Figure 9D).

This study examined the effects of different photoperiods on the growth and development of tomato rootstock and scion seedlings. The impact of different photoperiods on the water content of rootstocks and scions varied, with rootstocks having the highest water content at the photoperiod 10 h/d and the lowest at the photoperiod 12 h/d (Figure 10A). The moisture content of the rootstock significantly decreased in the photoperiods 12 h/d and 18 h/d, while that of the scion decreased in the photoperiod 16 h/d. (Figure 10A). The fresh biomass of scion was greater than that of rootstock in all cases, with the scion having the highest fresh biomass at the photoperiod 18 h/d and the rootstock having the highest fresh biomass at the photoperiod 14 h/d (Figure 10B). The overground dry biomass measurements of rootstocks were greater than those of scions, with the rootstocks at the photoperiod 16 h/d having the highest overground dry biomass as well as the most significant difference from scions and with the overground dry biomass of rootstocks and scions at the photoperiod 12 h/d being essentially equal (Figure 10C). The rootstock demonstrated the highest level of excellent underground dry biomass at a photoperiod of 16 h/d, the scion had the highest underground dry biomass at a photoperiod 14 h/d, and the scion and rootstock had a significant difference at the photoperiod 12 h/d, about 59.1% (Figure 10D). The seedling strength index of rootstock was highest at the photoperiod 14 h/d, and the seedling strength index of the scion exhibited the highest level of excellence at a photoperiod of 16 h/d (Figure 10E). The effect of photoperiod on plant height was insignificant; rootstock and scion plant heights were similar under the same photoperiod, and rootstock and scion seedlings heights were identical under different photoperiods (Figure 10F).

Effect on Mechanical Properties

This study aimed to ascertain the impact of varying photoperiod on the mechanical properties of rootstock and scion seedlings. The results demonstrate that different photoperiods exert a discernible influence on these properties.

The radial compression force of rootstock was the best at a photoperiod 16 h/d and the minimum at a photoperiod 14 h/d. The scion had the most significant radial compressive force at the photoperiod 16 h/d and the least radial compressive force at the photoperiod 10 h/d, with a difference of about 30.3% (Figure 11A). The radial compressive force of rootstock was more significant than that of scion in all photoperiod treatments except the photoperiod 14 h/d. The axial compression force of rootstock and scion at a photoperiod 10 h/d had the most important difference of about 74.8% (Figure 11B). The axial compression force of rootstock was greater than that of the scion for all photoperiods. The treatments of the axial compression force of rootstock were ranked from largest to smallest as photoperiod 10 h/d > photoperiod 16 h/d > photoperiod 18 h/d > photoperiod 12 h/d > photoperiod 14 h/d, and the treatments of the axial compression force of scion were arranged from largest to smallest as photoperiod 18 h/d > photoperiod 16 h/d > photoperiod 12 h/d > photoperiod 14 h/d > photoperiod 10 h/d (Figure 11B). Except for the photoperiod 14 h/d, the bending strength of the rootstock was significantly higher than that of the scion under the rest of the treatments, with the rootstock having the greatest bending strength at the photoperiod 18 h/d and the scion having the greatest bending strength at the photoperiod 16 h/d (Figure 11C). The shear strength of the scion was higher than that of the scion under the rest of the treatments except the photoperiod 10 h/d; the shear strength of rootstock at photoperiods 10 h/d, 16 h/d, and 18 h/d did not differ much, and the shear strength of the scion at the photoperiod 16 h/d was the greatest (Figure 11D).

Summary

Based on the growth rate and mechanical properties of seedling stems, and in combination with the magnitude of the solid seedling index, it was concluded that a photoperiod 18 h/d is best suited for cultivating rootstocks for mechanical grafting, and a photoperiod 16 h/d is best suited for cultivating scions for mechanical grafting.

3.2. Multifactor Experiments

3.2.1. Light Formulation Experimental Design

The most suitable light intensity and photoperiod for growing seedlings for mechanical grafting were obtained from a one-way experiment, but the effects of the three light elements on seedlings were integrated, so orthogonal tests needed to be designed to find the optimum light formulation to cultivate seedlings for mechanical grafting. Light quality, light intensity, and photoperiod were selected as three factors, and based on the results of the single-factor test combined with the research base, the levels of light quality were determined to be red (R)/blue (B) = 75:25, R:B = 50:50, and R:B = 25:75; the levels of light intensity were determined to be 150, 200, and 250 μmol m⁻² s⁻¹; and the levels of photoperiod were determined to be 14, 16, 18 h. Based on the table of orthogonal tests, L9 (3⁴) was selected, and a total of nine groups of experiments were performed, as shown in

Table 1. Orthogonal experimental design.

Considerations Test No.	Light Quality (Red/Blue)	Light Intensity (μmol m−2 s−1)	Photoperiod (H)
1	50:50	150	14
2	50:50	200	16
3	50:50	250	18
4	25:75	150	16
5	25:75	200	18
6	25:75	250	14
7	75:25	150	18
8	75:25	200	14
9	75:25	250	16

. The seeds and test conditions of the multifactorial experiment were consistent with those of single-factor experiments, which were conducted at the plant factory of South China Agricultural University.

3.2.2. Effects on Morphology and Longevity

As illustrated in the accompanying figure (Figure 12), the morphology of tomato seedlings exhibited a pronounced response to varying light formulation. The significant differences between hypocotyl and plant height evidenced this.

The hypocotyls of the rootstocks in No. 8 exhibited the most rapid growth, while those in No. 6 displayed the slowest growth (Figure 13A). Additionally, at days 15–20, the hypocotyls of the rootstocks in No. 5 and No. 7 experienced a deceleration in growth, whereas the growth in the other light treatments remained rapid. The scion hypocotyl growth was most pronounced in No. 7, and it was significantly higher in No. 8 compared to the different treatments during days 15–20 (Figure 13B). The stem diameter growth of the rootstock and scion in No. 4 differed from the rest of the trials, characterized by a slow growth rate at days 5–15 and a sharp increase in the growth rate at days 15–20 (Figure 13C,D). At day 20, the rootstocks of No. 2 had the largest stem diameter, and the scions of No. 3 had the greatest stem diameter (Figure 13D).

The rootstock had maximum moisture content in No. 7 and minimum in No. 9, the scion had maximum moisture content in No. 6 and minimum in No. 3 (Figure 14A). The rootstock had maximum fresh biomass in No. 7 and minimum fresh biomass in No. 4; the scion had maximum fresh biomass in No. 5 and minimum water content in No. 9 (Figure 14B). The effect of different light formulation on dry biomass was significant, with the overground dry biomass of the rootstock being maximum in No. 9 and that of the scion in No. 2 (Figure 14C). The root dry biomass of the rootstock was maximum in No. 5 and that of the scion in No. 3 (Figure 14D). Different light conditions had a significant effect on the seedling strength index. The rootstock had the lowest seedling strength index in No. 4 and the highest in No. 6. The scion had the lowest seedling strength index in No. 6 and the highest seedling strength index in No. 8 (Figure 14E). The effect of different light formulation on plant height was also evident, with the rootstock plant height being the highest in No. 9 and the lowest in No. 6, with a difference of about 33.4%. Scion height was highest in No. 7 and lowest in No. 6 (Figure 14F).

Effect of Light Formulation on Morphological Properties

Regardless of the light formulation, the mechanical properties of the rootstock were generally superior to those of the scion. The three largest radial compressive forces of the rootstock were No. 3 > No. 2 > No. 9, and the three largest radial compressive forces of the scion were No. 3 > No. 2 > No. 9, respectively (Figure 15A). The axial compressive force of the rootstock was the largest in No. 3, with a difference of about 71.1% over the least axial force, and the axial compressive force of the scion was the largest in No. 3 (Figure 15B). The three largest bending strengths of the rootstocks were shown in No. 2 > No. 3 > No. 6, respectively, where the difference between No. 2 and No. 3 was not significant, at the 5% significance level, and the difference in bending strength of scions in multiple light formulations was not significant (Figure 15C). The shear strength of rootstock was maximum in No. 9 and minimum in No. 5. The shear strength of scion was maximum in No. 3, much higher than that of rootstock in No. 3, and minimum was shown in No. 4, with a difference of about 69.5% (Figure 15D).

3.3. Comparative Grafting Experiment Results

3.3.1. Morphological and Mechanical Properties of Commercial Seedlings

The morphological indicators and mechanical properties of commercially available seedling are shown in

Table 2. Morphological and Mechanical Properties of Commercially Seedlings.

Parameter Type	Hypocotyl Length (mm)	Stem Diameter (mm)	Plant Height (mm)	RCF (N)	ACF (N)	BS (MPa)	SS (MPa)
Rootstock	32.265 ± 2.71	2.96 ± 0.23	89.89 ± 2.99	24.36 ± 0.44	1.54 ± 0.12	0.32 ± 0.06	0.35 ± 0.03
Scion	36.154 ± 1.82	3.05 ± 0.15	125.17 ± 1.83	21.48 ± 0.35	1.37 ± 0.17	0.24 ± 0.02	0.27 ± 0.03

. The mechanical properties of the scion and rootstock showed minimal differences; however, the plant height and hypocotyl length were significantly greater in comparison to the rootstock.

3.3.2. Experimental Result

The results showed that the grafting success rate of commercial seedlings was 91.1%, and the post-grafting survival rate was 90.2%. The grafting success rate of light-regulated seedlings was 98.3%, and the post-grafting survival rate was 91.1% (Figure 16). The grafting success rate of light-regulated seedlings was 7.2% higher than that of commercially available seedlings, and post-grafting survival rate was not much different from that of commercially available seedlings.

4. Discussion

4.1. Forces on Seedlings during Autografting

The rootstock and scion go through different stages as an automated grafting robot does its work. To illustrate, the grafting robot proposed by Xie [7] employs a simultaneous grafting method, whereby the rootstock and scion are transported to the grafting module separately. The dimensions required for mechanical grafting of seedlings are 3.0 ± 0.2 mm for seedling stems and greater than 30 mm hypocotyl height for rootstocks. Feeder rootstocks are crown-removed rootstocks, and feeder scions are the upper part of the scion that has finished cutting. The tray-conveying mechanism is used for transporting plug trays in which crown-removed rootstocks and grafted seedlings are loaded. The rootstock is gripped by the robotic hand, placed in a transfer cup, and conveyed to the position to be grafted. Once the rootstock and the scion have reached the grafting position, the seedling-gathering module brings them into parallel contact. Subsequently, the cutter is employed to create a slanting incision in the contact position, and the grafting clips are utilized to join the two components together, forming the grafted seedling. The grafted seedlings must be returned to the cavity tray through the transport module.

During the transfer process, the rootstock stem is not subjected to a direct force, while the paper bowl is subjected to the pressure F_b exerted by the robot hand (Figure 17A,b). The scion is fed manually and transported directly to the position to be grafted using a clamping mechanism; the clamping mechanism applies the clamping force F_a (Figure 17B,a) directly to the scion. During operation, the seedling gathering module applies a clamping force F_c (Figure 17c) to the upper part of the rootstock stem to enable it to be bent to a scion-parallel position, at which point the lower section of the rootstock is also subjected to a bending force F_d (Figure 17C,c). The scion is only subjected to radial compression forces throughout the gathering process (Figure 17c). As a cutter works, it applies a cutting force F_e to the rootstock and scion, allowing both to form a slant cut at the same time (Figure 17D,d). The grafted scion exerts axial pressure F_f (Figure 17E) on the rootstock in the vertical direction. In addition, the grafted seedling may dislodge the scion at the interface during transit due to excessive acceleration.

According to the force analysis, the rootstock needs to be subjected to a large bending force and radial pressure during the whole autografting process. In addition, it will be subjected to the axial compression force exerted by the scion. Because the scion seedling is very light in biomass, the axial compression force exerted on it is not large. Still, the larger the axial compression force, the more difficult it will be to dislodge the scion of the grafted seedling in the transport process. Therefore, rootstock seedlings with greater bending strength and radial compression are more suitable for automated mechanical grafting, and the greater the axial compression, the better. The scion is mainly subjected to clamping forces throughout the automatic grafting process, so scion seedlings with large radial compression forces are more suitable for automatic mechanical grafting. During the cutting process, the greater the shear strength of the seedling, the more pronounced the cutting blocking effect on the blade. According to the literature [29], when cutting seedlings with the same blade, the higher the shear strength of the seedlings, the greater the wear and tear on the blade, so the lower the shear strength of the seedlings, the better. However, since the force of the cutter at the moment of cutting is sufficiently high to cut off the seedling, even if the blades are worn out, shear strength is not used as a major factor in judging the suitability of a seedling to be grafted with automated machinery. Therefore, the importance of mechanical properties for rootstocks is, in descending order, bending strength > radial compression force > axial compression force > shear strength. For scions, the radial compression force is much more critical than other mechanical properties, and the bending strength and axial compression force do not need to be considered too much.

4.2. Effect of Light Intensity on Seedlings

Grafted tomato seedlings showed different morphological characteristics under various light intensities. It was found that increasing light intensity effectively suppressed hypocotyl length and increased stem diameter. The higher the light intensity, the faster the seedlings grow [30,31]. Scions and rootstocks showed the same trend when subjected to light intensity. Chen et al., in 2022 [32], demonstrated that when the seedlings were exposed to various light intensities closest to their own optimal light intensity, they underwent more rapid photo acclimatization, and it took a shorter time for photo inhibition to be relieved, which directly influences the growth rate of the seedlings. This is consistent with the pattern of the scion’s stem growth rate, where the stem of a scion with a light intensity of 250 μmol m⁻² s⁻¹ grows slower than that of a scion with a light intensity of 200 μmol m⁻² s⁻¹. This explains why the growth rate decreases with higher light intensities. The research of Kim et al. in 2008 [33] showed that light intensity affects plant height and that the higher the light intensity, the lower the plant height phenomenon. However, the results of the present study suggest that this phenomenon is only found in a certain range of light intensities. There was a significant difference in plant height between rootstock and scion under the light intensity 50 μmol m⁻² s⁻¹ and light intensity 100 μmol m⁻² s⁻¹ treatments, but there was no significant change in plant height with a continued increase in light intensity. Brandon et al., in 2021 [34], showed that lower light intensity caused hypocotyl elongation in tomato seedlings, which is the same as the results obtained in this study. Light intensity can significantly affect dry matter accumulation in tomatoes, which is consistent with Zheng et al. [35], probably because too much light affects photosynthesis in plants, and if an implantation absorbs too much light energy and does not dissipate it quickly, it can lead to photoinhibition.

The most suitable light intensity for the cultivation of automated mechanically grafted seedlings is selected by taking into account the seedlings’ morphological parameters and mechanical characteristics. The maximum bending strength of rootstocks under a light intensity of 250 μmol m⁻² s⁻¹ differed by approximately 9.2% from the bending strength of rootstocks under a light intensity of 200 μmol m⁻² s⁻¹. Additionally, the maximum axial compressive force and the maximum radial compressive force were observed for rootstocks under a light intensity of 200 μmol m⁻² s⁻¹, while the minimum shear strength was found for rootstocks under a light intensity of 150 μmol m⁻² s⁻¹. As can be seen in the error bars, the uniformity of hypocotyl length of rootstocks under light intensity 200 μmol m⁻² s⁻¹ and light intensity 250 μmol m⁻² s⁻¹ did not differ much, and the uniformity of seedling stems of rootstocks under light intensity 200 was significantly better than that of those under light intensity 250 μmol m⁻² s⁻¹. Because the axial compression force and radial compression force of rootstocks under light intensity 200 μmol m⁻² s⁻¹ were greater than that of rootstocks under light intensity 250, the shear strength was less than that of rootstocks under light intensity 250 μmol m⁻² s⁻¹, and the bending strength was not much different from that of rootstocks under light intensity 250 μmol m⁻² s⁻¹. Overall, 200 μmol m⁻² s⁻¹ is the most suitable light intensity for cultivation of rootstocks for automatic and mechanical grafting. The radial compression force was highest for scions grown at 250 μmol m⁻² s⁻¹ light intensity, with a difference of approximately 10.3% from the scions grown at 200 μmol m⁻² s⁻¹ light intensity. The growth rate of scions in light intensity 250 μmol m⁻² s⁻¹ was slightly lower than that of scions grown in light intensity 200 μmol m⁻² s⁻¹. Combined with the seedling strength index, 250 μmol m⁻² s⁻¹ is the most suitable light intensity for the cultivation of scions for automatic mechanical grafting.

4.3. Effect of Photoperiod on Quality

Shorter light durations promote hypocotyl elongation and inhibit seedling stem growth [36]. The effect of different photoperiods on the growth rate of seedling stem diameter was not significant, except for rootstocks with a photoperiod 18 h/d. It could be due to the fact that the extended photoperiod had a great impact on the accumulation of organic matter inside the rootstock [37]. The dry biomass of the rootstock did not differ much in photoperiods 14 h/d, 16 h/d, and 18 h/d, and the dry biomass of the scion was greatest in the photoperiod 16 h/d and then slightly decreased in the photoperiod 18 h/d, which indicated that there exists a threshold value for the effect of light duration on the dry biomass of the seedling and that a light duration exceeding the threshold value does not promote the growth of the seedling and even reduces the development of the seedling. It could be because increasing the light duration increased the DLI (day light integral) of the seedlings, and excessive DLI led to photoinhibition, which is consistent with Hwang et al. [38]. The research results of Song et al., in 2022 [39], also showed that suitable DLI has a promoting effect on the growth and healing of grafted tomato seedlings. The photoperiod affected plant height to a certain extent, and prolonging the photoperiod reduced plant height, which was consistent with the research results of Zhang et al. [40]. In addition, the effect of the photoperiod on the internal substances of seedlings has been studied. Cao et al., in 2016 [41], found that red light break treatment of tomato seedlings decreased gibberellins in leaves and stems. And it also inhibited hypocotyl elongation. Lanoue et al., in 2021 [42], found that an alternating red and blue continuous light regime allowed for injury-free tomato production, and the parameters related to the photosynthetic performance (i.e., Pn_max, quantum yield, and F_v/F_m) of leaves grown under continuous light were similar to leaves grown under 12 h lighting.

The most suitable photoperiod for the cultivation of automated mechanically grafted seedlings is selected by taking into account the seedlings’ morphological parameters and mechanical characteristics. Rootstocks had maximum bending strength at a photoperiod 18 h/d and maximum axial compressive force at a photoperiod 10 h/d. The rootstocks at a photoperiod 16 h/d had the maximum radial compressive force, which differed from the rootstocks under a photoperiod 18 h/d by about 18.2%, and the rootstocks under a photoperiod 14 h/d had the minimum shear strength. Combined with the growth and seedling strength index, there was no difference in growth uniformity between rootstocks with a photoperiod 16 h/d and rootstocks with a photoperiod 18 h/d, and the seedling strength index of rootstocks under a photoperiod 18 h/d was greater than that of those under a photoperiod 16 h/d. Therefore, the photoperiod 18 h/d was selected as the most suitable photoperiod for cultivating rootstocks for automated mechanical grafting. Scions under a photoperiod 16 h/d had the greatest radial compression force, and the seedling strength index was greater than under other photoperiod treatments. Discussed in terms of growth, the uniformity of growth of hypocotyls and seedling stems for scions under a photoperiod 16 h/d did not differ much from other photoperiod treatments. Therefore, the photoperiod 16 h/d was selected as the most suitable photoperiod for cultivating scions for automated mechanical grafting.

4.4. Effect of Light Formulation on Quality

A three-factor, three-level orthogonal experiment was conducted to assess the impact of light quality, light intensity, and photoperiod on the outcome variable. The results of the experiment were subjected to a range analysis in order to derive the magnitude of the effect of each factor on each indicator of the seedlings (Figure 18).

The analyses showed that seedling fresh biomass was most affected by photoperiod, and seedling plant height and shear strength were both most affected by light quality. Light intensity is the most important factor affecting radial compression, axial compression, and bending strength of rootstocks. We can suppose that light is needed to regulate the mechanical properties of tomato rootstocks of different varieties so that they can meet the standards for mechanical grafting. In that case, the seedlings can be regulated mainly by adjusting the light intensity. Both light quality and light intensity are major factors influencing the radial compression force of scions. We can also suppose that light is needed to regulate the mechanical properties of tomato scions of different varieties so that they can meet the standards for mechanical grafting. For these requirements, the seedlings can be regulated mainly by adjusting the quality and intensity of light.

In terms of mechanical properties, rootstocks grown in No. 2 had the greatest bending strength, and rootstocks grown in No. 3 did not differ much from rootstocks grown in No. 2 in bending strength; rootstocks grown in No. 3 did not vary much from rootstocks grown in No. 2 rootstock in radial compression force, but rootstocks grown in No. 3 had a greater axial compression force than rootstocks grown in No. 2, which was about 29.3%. Rootstocks grown in No. 3 were lower than rootstocks grown in No. 2 in shear strength, so rootstocks grown in No. 3 were optimal in terms of mechanical properties. Rootstocks grown in No. 3 were the best in terms of mechanical properties. Combined with the growth analysis, the hypocotyl length and stem diameter were less uniform in rootstocks grown in No. 3 than in rootstocks grown in No. 2, the growth uniformity of rootstocks grown in No. 3 was better than rootstocks grown in No. 2. Thus, No. 3 was the most suitable light for cultivating automated mechanically grafted rootstocks with a combination of light quality of R:B = 50:50, light intensity of 250 μmol m⁻² s⁻¹, and a photoperiod of 18/6 h.

In terms of mechanical properties, scions grown in No. 3 had the greatest radial compression force and bending strength but did not differ much from scions grown in No. 2. The shear strength of scions grown in No. 3 was the greatest, with a difference of about 35.5% as compared to scions grown in No. 2; thus the mechanical properties of scions grown in No. 3 are optimum. Combined with the growth score analysis, scions grown in No. 3 had a greater growth rate than scions grown in No. 2 in reaching a stem diameter of 3 ± 0.2 mm. Thus, No. 3 had the most suitable light for cultivating automated mechanically grafted scions, with a combination of light quality of R:B = 50:50, light intensity of 250 μmol m⁻² s⁻¹, and a photoperiod of 18/6 h. The present findings can inform research on the purposeful regulation of mechanical properties of tomato seedlings.

4.5. Comparison of Commercial Seedling and Light-Regulated Seedlings

Morphological comparisons showed that the uniformity of the light-regulated seedlings was better than commercial seedlings. Comparison of the mechanical properties showed that the mechanical properties of the light-regulated seedlings were better than those of commercial seedlings. The results of the grafting experiment showed that the grafting success rate of light-regulated seedlings was higher than that of commercial seedlings. The growth period of light-regulated seedlings to grafting was about 20 days, and compared to commercial seedlings, the growth cycle was shortened. This indicates that seedlings cultivated under controlled lighting conditions in plant factories are more suitable for mechanical grafting than commercial seedlings.

The efficiency of artificial grafting of commercial seedlings is approximately 400 plants per hour. However, through machine grafting, the efficiency can be increased by more than two times. In the future, tomato seedlings can be controlled by light to adapt to different grafting machines, thereby promoting the process of mechanized grafting. But this is also subject to the development of new light sources.

5. Conclusions

The results of this study indicate that modulation of the light environment can change the morphological and mechanical properties of tomato seedlings. Increasing light intensity can inhibit the length of hypocotyls and promote the growth of seedling stems. The change in light intensity had no significant effect on the mechanical properties of seedlings. The shortening of the photoperiod led to an increase in hypocotyl length and decrease in seedling stem growth. The photoperiod variation had a significant effect on the mechanical properties of seedlings. By purposefully regulating the light environment, the mechanical properties of tomato grafted seedlings can be effectively improved. There was no difference in post-grafting survival rate between the seedlings cultivated by a regulated light environment and the commercial tomato seedlings, and the grafting rate was improved. Combined with the seedling growth results of the orthogonal experiment, the most suitable light for the cultivation of automated mechanically grafted tomato seedlings was R:B = 50:50, with a light intensity of 250 μmol m⁻² s⁻¹ and a photoperiod of 18/6 h.