Phenotypic heterogeneity of ARF in hardwood cuttings
The rooting of hardwood cuttings taken from node 3 to node 5 (N3, N4 and N5) of current canes of Shine Muscat (SM) and Summer Black (SB) was investigated, respectively (Fig. 2(a) and (c)). Cuttings of two cultivars were rooted after 22 DAC (F = 22 DAC). The ARF phenotype of SB was more consistent than SM, USB = 90~98% and USM = 53~75% at 38 DAC. The rooting rate of SM was different in nodes. The N5 was higher than N3 and N4. From the first root induced to the stable rooting rate, SSB and SSM were taken 16 days (S=16 days). In addition, the standard deviation (E) of rooting rate among samples was large. Some of them were more than 22%. At the same time, there were also inconsistent rooting and sprout in cuttings from the same node (Fig. 2(b) and (d)).
Differences in ARF phenotype among various types of explants
Field and tissue culture materials were used to obtain different types of explants to study the phenotypic heterogeneity of ARF. Young and mature leaves (Blade or LP) were taken from top to base of current canes of SB and cultured on medium A6 or A6 with 0.1 mg·L-1 IBA (A6+IBA0.1) under light condition (Light: 16h-photoperiod, 4000lux). After 60 days till the leaves were scorched, no ARs appearance. AR induction was also attempted on leaves (N8, N6, N4 and N2) from infantile two-years-old SB vines but was also unsuccessful (Fig. 3(a)). Field leaves cultured on medium A6 with 10g·L-1 sucrose (A6+S10) were easily contaminated with bacteria or fungi, and statistical results could not be obtained.
By distinguishing the different nodes and culturing under the light condition in WPM+S10+AC3, the results showed that rooting rate of the first and second nodes (N1~N2) was low (U=34.44~46.67%), while the rooting rate of N3~N4 and terminal bud (NT) was 80.00%~88.89% at 45 DAC (Fig. 3(b)). Heterogeneity of N1~N2 samples was larger than that of N3~N4, with a partial error (E) was more than 15% (Fig. 3(b)). These results indicated that explants from different nodes exhibit different rooting abilities.
Subsequently, six types of explants were selected from N3~N6 (the basal two nodes and NT were not taken) of plantlet with relatively consistent growth potential and these ARF phenotype were compared (Fig. 3(c)). The explants were cultured on A6 medium at 25℃ under light condition (Light: 16h-photoperiod, 4000lux) and results showed that petiole (P) and stem segment (SS) did not regenerate root even after 60 days of continuous culture. The SBS-L, LP and SBS began to root at 7-10 DAC, faster than the blade (F=11 DAC). The highest rooting rate of explants was LP (67.78%±14.99% at 20 DAC). The rooting rate of SBS-L was 44.4% (44.44%±10.30% at 20 DAC), SBS was 10.0% (10.00%±2.70% at 20 DAC), respectively (Fig. 3(d)). However, only 1-2 explants of the blade (B) rooting occasionally after 20 DAC culture (N>90). Different types of explants were further cultured on A6 medium under dark condition. After 20 DAC, only LP rooting was observed (17.80%±15.00% at 20 DAC) (Fig. 3(e)), and no rooting was observed till 30 DAC in other explants (Fig. 3(e)).
Then, different explants were cultured on medium A6+S10 under dark condition. The results showed that P, B and SS did not root after continuous observation of 30 DAC. However, after culture of 45 DAC, 37.78% (37.78%±22.19%) of SS developed roots (Fig. 3(c)). These indicating that neither leaves nor buds were necessary for ARF in this condition, while sugar and darkness were important factors promoting rooting. The rooting efficiency of SBS-L and LP on A6+S10 medium under dark was significantly greater than that of A6 medium under light, and almost all the explants of SBS-L and LP could root in 13 DAC (U=100%). The rooting rate of SBS was also increased significantly and reached 73.33%±5.40% at 20 DAC (Fig. 3(g)).
Interestingly, when different explants were cultured on A6+S10 medium under light condition, the rooting of SBS-L, SBS and LP were significantly slower than that under dark culture (Fig. 3(f) and 3(g)). Moreover, the rooting rate of these explants was significantly reduced at 20 DAC, only 43.3%±8.20% for SBS-L and 10.00%±5.40% for SBS, and only 4.40%±4.20% for LP (Fig. 3(f)), which was easy to root in dark culture (Fig. 3(g)). Subsequently, rooting rates of SBS increased gradually with the extension of culture time but were ultimately not as high as that under dark culture. Rooting rates of SBS-L and LP did not change with observation time. Meanwhile, P and SS did not generate root after continuous culture at 60 DAC, while B occasionally took root. These results suggest that light significantly inhibited ARF of explants with a leaf in the presence of additional sucrose (10 g·L-1), which was confirmed by subsequent experiments.
Combined with the above results, it is indicated that the ARF phenotypes of SBS-L and LP were fast, synchronous, and consistent between samples. Leaves accelerate rooting and it may act as a supplier or distributor of carbohydrates and hormones needed for ARF. The comparison of ARF results from field woody cuttings and leaves suggested that the tissue culture materials with continuous subculture was easier to root that might be because of its juvenile state, which is difficult for field young leaf samples to be simulated.
ARF phenotypic characteristics of grape LP
LP was an ideal research system for grape ARF, which excluded the influence of sprouting, and was the simplest and most readily rooted unit of the explants investigated. Wounding was the primary factor to induce AR formation in LP explants. When the petiole was picked with tweezers, the middle part of the petiole was occasionally clipped, and these wounds can form AR (Fig. 4(a)). With attention to the clamping force, LP-induced AR appears more frequently about 2mm above the cut site, rather than at the base of the cut. Observing under the stereomicroscope, the ARs formation did not undergo callus, but was direct de novo organogenesis from petiole (Fig. 4(b)).
High light intensity inhibited ARF of grape LP
In an accidental culture process, we noticed that when the light intensity was ≥6000lux, the leaves cultured on A6 medium were damaged to varying degrees within 12h under 16h-photoperiod condition, and part of the leaf margin was burnt, which seriously affected rooting (from 67.78% reduced to 12.22% at 20 DAC), although injured LP can take root occasionally (Fig. 4(c)). Therefore, the light intensity was determined as 4000lux in the subsequent experiments.
The selection and control of LP explants influenced the consistency of ARF phenotype
In tissue culture, the growth potential of the SBS propagated plantlets were not completely consistent at different ages, and the leaf size and growth state of the same plant at different nodes were also different. Usually, leaves at the basal nodes were small and some of them may become brown and senile, while the middle and upper nodes are large and healthy, and the apical leaves are young and tender (Fig. 5(a)). These are different from the leaves of 12-day-old seedlings that are most suitable for ARF study in Arabidopsis thaliana by precisely controlling seedling age (Chen et al. 2014).
Leaf size was reflected in length×width (L×W cm2) to analyze the correlation between leaf size and rooting (rooting rate and speed). In this study, the size (L×W) of healthy leaves of tissue culture plantlets ranged from 1 to 6. Cultured 20 days under dark (D) condition on A6 medium, 17.80% LP could form ARs. Forty LPs that rooted (D-R) and unrooted (D-NR) were randomly selected for statistics, and it is shown that when the average L×W was less than 1.8 (median), most of the leaves had no spontaneous rooting ability. While, in LP that could spontaneously root, the L×W was above 3.8 (Fig. 5(b)). Then LP was placed on A6 medium under light condition (L), most healthy leaves could generate root (67.8%) at 20 DAC. There was also a positive correlation between the rooting capacity and the size of leaves, which was mainly reflected in the small LP of unrooted (L-NR), with the size shrinking at about 1.6, however, the rooted LP (L-R) are widespread in a wide range of sizes (Fig. 5(b)). In addition, leaves with senescence at the base of the plant or withered patches at the leaf margin were found difficult to root under various conditions. These results suggest that the amount of nutrients stored in healthy leaves (under dark condition) and the ability to synthesize photosynthates and auxin-related to leaf photosynthetic area (under light condition) determines the ability of ARF. Therefore, leaf size and health status are important factors affecting the homogeneity of ARF.
Carbohydrate provision is prerequisite in the formation of AR from LP
In order to further prove the importance of carbohydrate in ARF, N3-N6 nodes were selected according to the previous results, and the leaf size was controlled at (2<L×W<6) to compare the difference of ARF in A6 and A6+S10 culture under dark and light, respectively. After controlled blade size and node position (Controlled), the rooting rate of LP in A6 medium under dark condition (16.7%±7.80% at 20 DAC) was not significantly different from that without control (Uncontrolled) (Fig. 5(c)). The rooting rate of LP with strict selection was increased (82.8%±6.80% at 20 DAC) under light culture on A6 medium compared with that without control, and the errors were significantly smaller during the rooting process (Fig. 5(d)). When sucrose (10 g·L-1) was supplemented by the A6 culture medium, all healthy LP could root under dark condition, and the rooting rate finally reached 100% after 13 DAC (Fig. 5(e)). The leaves specially selected for senescence or withered spots at the leaf margin at the base of plantlets could hardly generate root on A6+S10 medium under dark. These results suggest that the amount of nutrients and hormones stored in healthy leaves (under dark condition) and the ability to synthesize photosynthates and hormones as determined by leaf photosynthetic area (under light condition) determines the ability of ARF. Therefore, leaf size and health status are the factors that need to be considered to affect the uniformity of ARF.
We used different mineral-containing medium without sucrose (B5, WPM and C2D) and cultured for 30 days under dark and found that only about 20% LPs could root (data not shown). However, it was difficult for LP to root on A6 medium under dark condition. In the absence of added sucrose, we did not find a culture regimen that increased LP under dark culture conditions.
The importance of auxin supply in ARF of grape LP
In this study, grape LP could generate root without adding exogenous auxin, suggesting that LP explants could provide endogenous auxin to meet the need of AR formation. In A6+S10 medium, different concentrations of NPA (0.1, 1 and 3 mg·L-1) were added and cultured under dark conditions. The results showed that the inhibition of NPA on ARF was related to concentration. With the increase of concentration, the inhibition effect of ARF became more obvious, and 3 mg·L-1 NPA could completely inhibit the rooting (Fig. 6(a)). In A6 medium, light culture showed that 0.1 mg·L-1 NPA had no noticeable inhibition on ARF, while 1 mg·L-1 and 3 mg·L-1 NPA could completely inhibit rooting (Fig. 6(b)). IBA was added to A6 medium with 0.1 and 1 mg·L-1, respectively, and cultured under darkness. The results showed that 0.1 mg·L-1 IBA significantly promoted ARF, and the rooting rate stabilized to 78.90%±10.30% after 11 DAC. However, 1 mg·L-1 IBA (36.70%±19.60%) had a less promoting effect on ARF than 0.1 mg·L-1 IBA (Fig. 6(c)). In addition, the adventitious roots produced after IBA treatment continuously was different from the control, showing an increase in the number of roots, rooting sites were not limited to the base of petiole, and AR elongation slowed down (Fig. 6(d) and 6(e)).
Light inhibited rooting in the presence of exogenous sucrose
Higher plants can integrate multiple signal transduction pathways such as light, auxin and reactive oxygen to fine-regulate adventitious root formation, to adapt to changing environmental conditions (Bai et al. 2020). Light exerts a strong influence on multiple aspects of the auxin system, controlling auxin level, transport and responsiveness (Halliday et al. 2009). Sugar and light signal transduction is associated with auxin biosynthesis and root distribution changes (Garcia-Gonzalez et al. 2021).
In A6 medium and under light culture, leaves could produce photosynthetic products through photosynthesis, partially satisfying ARF (67.78-82.22%, 20 DAC) (Fig. 5(d)). However, as mentioned above, damage to leaves caused by high light intensity will affect ARF (Fig. 4(c)). In the medium supplemented with sucrose (A6+S10), the light was controlled at 4000lux to prevent the damage of strong light. After 20 DAC, only a few LP explants (4.40-5.60%) rooted, and the initiation of rooting was significantly delayed (F=11 DAC) (Fig. 6(f)). From the appearance of the leaves, there were no obvious injury symptoms. Since the addition of sugar may promote auxin synthesis, it is not clear whether the difficulty of rooting is due to the influence of auxin homeostasis (possibly including high concentration or polar transport). Under light condition, in the mediums A6+S10 with 1 or 3 mg·L-1 NPA (A6+S10+NPA1 and A6+S10+NPA3), LP did not root. However, LP in A6+S10+NPA0.1 can root (66.67%±2.72% at 20 DAC) (Fig. 6(f)), which is significantly higher than the control A6+S10. Since NPA inhibits the polar auxin transport, it is speculated that the difficulty of rooting A6+S10 under light culture may be related to the excessive polar auxin transport. In conclusion, light-induced inhibition of ARF of LP explants in grapes after sugar addition, while low concentration of NPA partially relieved this inhibition, suggesting that light and sugar signal may interact, but molecular biological evidence is needed.
Investigation of PGRs affecting ARF phenotype of LP
The effect of exogenous hormone on rooting may be either promotive or inhibitory and may be dose or species dependent (Bannoud and Bellini 2021). Small changes may have the opposite effect on ARF. Due to the heterogeneity of woody cuttings, it is difficult to accurately show the phenotypic changes of ARF after exogenous PGRs treatment. Based on the established LP study system for rapid and stable ARF phenotype identification, this study preliminarily investigated the effect of exogenous PGRs on grape ARF.
Under dark condition, LP explants could not root when the A6+S10 medium added 1mg·L-1 6-BA and GA3, respectively. Meanwhile, 24-epibrassinolide (EBR) (1 mg·L-1) also showed significant inhibition of ARF. ABA had no significant affection for ARF (Fig. 7(a)). The base of LP petiole treated by 6-BA expanded significantly (Fig. 7(b)). Lower concentrations of GA3 (0.01 and 0.1 mg·L-1) were then examined, and it was found that they also inhibited rooting completely. SBS, SBS-L and woody cuttings were treated with GA3 (0.1 mg·L-1), and the rooting inhibition effect was also obvious, no root could be observed till 60 DAC. The results showed that GA3 had a significant inhibitory effect on grape ARF (Fig. 7(d)). Studies have shown that the inhibition of GA on ARF may be due to the inhibition of auxin polar transport by GA (Mauriat et al. 2014). Based on this, LP was cultured on A6+S10 with GA3 (0.1 mg·L-1) and IBA (1 mg·L-1) under dark conditions, and partial rooting of LP was found (Fig. 7(c)). It indicates that IBA (1 mg·L-1) could partially reduce the inhibition effect of GA3 (0.1 mg·L-1). The characteristics of rooting explants in this treatment were similar to those in IBA treatment: the number of adventitious roots increased, and the elongation slowed down. Therefore, it is speculated that GA3 may inhibit rooting by inhibiting endogenous growth factors in grapes.
Endogenous ethylene and jasmonic acid (JA) can be induced by wounding, and both ET and JA may promote rooting in the early stage and inhibit rooting·Later (da Costa et al. 2013). After culture on A6+S10 with 0.01, 0.1 or 1 mg·L-1 ethephon (ET) under dark condition, there were no significant effect on ARF of grapes (Fig. 7(e)). In this study, the rooting rate was reduced after culture with MeJA (0.1 and 1 mg·L-1) on A6+S10 medium under darkness (Fig. 7(f) and 7(g)). This suggests that MeJA has some inhibitory effect on ARF, at least at these two tested concentrations. After culture on A6 medium with MeJA (0.1 and 1 mg·L-1) medium under darkness, rooting rate increased, but there was no significant difference. In addition, leaves treated with MeJA in this study were uniformly yellow (Fig. 7(h)), which may be related to the initiation of systemic defense by MeJA.