*3.2. Plant Growth Parameters*

SPAD and canopy size were measured at 33 and 41 DAG in the 2019 experiment (Table 1). There was a significant difference in SPAD among treatments at 33 DAG, but no difference was found at 41 DAG. At 33 DAG, non-grafted pac choi showed lower levels of leaf SPAD values than nonand self-grafted daikon radish. The lack of difference in SPAD between MQ/BK, MQ, and MQ/MQ indicated that BK as a rootstock did not impair accumulation of chlorophyll by MQ. The similar canopy size between MQ/BK, MQ, and MQ/MQ at both 33 and 41 DAG (Table 1) suggested that grafting with daikon radish did not reduce the leaf growth and expansion of pac choi.


**Table 1.** Relative chlorophyll content and canopy size of grafted, self-grafted, and non-grafted pac choi and daikon radish plants at 33 d after grafting (DAG) and 41 DAG in the 2019 experiment.

<sup>z</sup> BK = Non-grafted 'Bora King' daikon radish; BK/BK = Self-grafted 'Bora King' daikon radish; MQ = Non-grafted 'Mei Qing Choi' pac choi; MQ/MQ = Self-grafted 'Mei Qing Choi' pac choi; MQ/BK = 'Mei Qing Choi' pac choi grafted onto 'Bora King' daikon radish. <sup>y</sup> Mean ± SE (standard error); means followed by the same letter are not significantly different at *p* ≤ 0.05 according to Fisher's LSD test.

As shown in Figure 1B, above-ground pac choi and below-ground daikon radish taproot developed normally in MQ/BK plants. We observed cavities in the vascular bundle connections at the graft union area in grafted pac choi plants with the daikon radish rootstock but not in self-grafted daikon radish plants (Figure 1E,F), similar to what was reported in grafted Chinese cabbage/turnip plants [8]. In our experiments, grafting was carried out at 16 d after sowing (DAS) for daikon radish and 23 DAS for pac choi. According to Liu et al. [23], as early as 16 DAS, tuberization began in turnip (*B. rapa* subsp. *Rapa*) and the center part of the upper hypocotyl which accounted for about 50% of the cross section area was occupied by pith cells, with an actively dividing cambium circle and a thin xylem ring. When pac choi was grafted at 23 DAS, more than 50% of the center part of the upper hypocotyl of pac choi consisted of secondary xylem cells with highly lignified cell walls. This discrepancy in hypocotyl structure between scion and rootstock seedlings likely resulted in the formation of the cavity inside the graft union during healing as observed in our study (Figure 1E), especially considering that the cavity did not exist in self-grafted daikon radish (Figure 1F) or pac choi (Figure 1G).

Leaf number and taproot length and diameter were measured at harvest in both experiments (Table 2). In the 2016 pilot study, MQ/BK had more leaves than non-grafted BK, but did not differ significantly from MQ. In the 2019 experiment, MQ/BK had 35% and 26% more leaves than selfand non-grafted BK, respectively, but no difference was found between self- and non-grafted BK. Similar leaf numbers were observed for MQ/BK, MQ/MQ, and MQ. Total leaf area was also measured in the 2019 experiment and MQ/BK had smaller leaf area than all other treatments. In both 2016 and 2019, MQ/BK produced significantly shorter and smaller taproots than non-grafted BK and in 2019, MQ/BK was also smaller in taproot diameter than BK/BK (Table 2 and Figure 1D). Our results were consistent with Zheng et al. [24], who reported that the diameter of turnip was significantly smaller when grafted with rapeseed (*B. rapa* subsp. *oleifera*) than self-grafted turnip. BK/BK did not differ significantly from BK in taproot length but was 7% smaller in taproot diameter.



<sup>z</sup> BK = Non-grafted 'Bora King' daikon radish; BK/BK = Self-grafted 'Bora King' daikon radish; MQ = Non-grafted 'Mei Qing Choi' pac choi; MQ/MQ = Self-grafted 'Mei Qing Choi' pac choi; MQ/BK = 'Mei Qing Choi' pac choi grafted onto 'Bora King' daikon radish. <sup>y</sup> Mean ± SE (standard error); means followed by the same letter are not significantly different at *p* ≤ 0.05 according to Fisher's LSD test.

The primary root axis of radish consists of two anatomically distinct parts. The upper part originates from the hypocotyl whereas the lower part is true root tissue. Both lower and upper regions of the radish root thicken to form succulent tissue by increases in both cell number and cell size [25,26]. In this grafting experiment, the cut made on the daikon radish plant was in the thickening region of the hypocotyl as demonstrated by the longitudinal section of the graft union area of self-grafted daikon radish plant (Figure 1F). Very likely, grafting pac choi with radish shortens the hypocotyl part that could contribute to the formation of the taproot, leading to reduced taproot length compared with non-grafted radish, while self-grafting radish does not involve any loss of hypocotyl tissue. Furthermore, it has been found that in turnip the hypocotyl tissue is the main contributor to underground tuber development, and hypocotyl excision led to a lower expression level of genes controlling tuberization, leading to a substantial inhibition of tuber formation [24].

#### *3.3. Gas-Exchange Parameters*

Leaf transpiration rate, net CO2 assimilation rate, intercellular CO2 concentration, stomatal conductance, and instantaneous water use efficiency (iWUE) were compared for MQ/BK, MQ, MQ/MQ, BK, and BK/BK at 34 and 46 DAG in the 2019 experiment (Table 3). No difference in leaf transpiration rate was observed at 34 DAG, while at 46 DAG, MQ/BK had a similar transpiration rate as MQ and MQ/MQ, and all three treatments showed a 95% increase of transpiration rate on average than BK and BK/BK. Grafting significantly increased the net CO2 assimilation rate of MQ/BK compared with MQ and MQ/MQ by 15% and 28%, respectively, at 34 DAG, while no difference was observed between MQ/BK and BK/BK. At 46 DAG, MQ/BK showed a net CO2 assimilation rate that was 48% and 45% higher than MQ and MQ/MQ, respectively, but it did not differ significantly from BK/BK. MQ/BK also had a 21% higher net CO2 assimilation rate than BK at 46 DAG. Very likely, MQ/BK had a stronger sink strength than MQ/MQ and MQ, which contributed to the higher photosynthetic rate observed [27]. Interestingly, at 34 DAG, MQ and MQ/MQ had similar intercellular CO2 concentrations, which were significantly higher than that of other treatments. At 46 DAG, MQ/BK, MQ, and MQ/MQ had higher intercellular CO2 concentration than BK and BK/BK and the same trend was observed for stomatal conductance at 46 DAG although no difference in stomatal conductance was detected at 34 DAG. At 34 DAG, MQ/BK had 36% and 53% higher iWUE than MQ and MQ/MQ, but did not differ from BK and BK/BK. However, at 46 DAG, MQ/BK, MQ, and MQ/MQ exhibited a similar level of iWUE which was significantly lower than that of BK and BK/BK.

**Table 3.** Leaf transpiration rate (E), net CO2 assimilation rate (A), intercellular CO2 concentration (Ci), stomatal conductance (gs), and instantaneous water use efficiency (iWUE) of grafted, self-grafted, and non-grafted pac choi and daikon radish plants at 34 d after grafting (DAG) and 46 DAG in the 2019 experiment.


<sup>z</sup> Instantaneous water use efficiency (iWUE) = net CO2 assimilation rate (A)/transpiration rate (E). <sup>y</sup> BK = Non-grafted 'Bora King' daikon radish; BK/BK = Self-grafted 'Bora King' daikon radish; MQ = Non-grafted 'Mei Qing Choi' pac choi; MQ/MQ = Self-grafted 'Mei Qing Choi' pac choi; MQ/BK = 'Mei Qing Choi' pac choi grafted onto 'Bora King' daikon radish. <sup>x</sup> Mean ± SE (standard error); means followed by the same letter are not significantly different at *<sup>p</sup>* ≤ 0.05 according to Fisher's LSD test.

Lower leaf transpiration rate and intercellular CO2 concentration of radish compared with pac choi observed in the later growth stage could be owing to different leaf structures. Pac choi leaves are fleshy and glossy, while radish has trichomes on both upper and lower leaf surfaces [28]. It has been suggested that trichome density is negatively related to transpiration rate and CO2 diffusion rate as trichomes can increase boundary layer resistance [29–31]. The trichomes on daikon radish leaves might have affected the leaf transpiration rates and intercellular CO2 concentrations measured in this study. Further examination is needed to directly compare the intrinsic leaf structures of pac choi and daikon radish plants for their effects on gas exchange and gas exchange measurements.

Most water loss from leaves of intact plants is generally through open stomatal apertures [32], thus the higher stomatal conductance of MQ/BK was likely the driving factor for its lower iWUE compared with BK and BK/BK despite its higher net CO2 assimilation rate. It has been found in tobacco (*Nicotiana tabacum* L.) that stomatal conductance did not always parallel photosynthetic capacity changes [33,34], which could partially explain the relatively high stomata conductance, but low net CO2 assimilation rate observed in MQ and MQ/MQ.
