5BB anacı ve Cabernet Sauvignon (Vitis vinifera L.) üzüm çeşidinin adventif kök oluşumu üzerine Melatonin ve IAA’in etkileri

Both derived from tryptophan, melatonin (MEL) and indole acetic acid (IAA) were tested for their influence on rooting in the cuttings of two Vitis species, the 5BB rootstock and the cultivar Cabernet Sauvignon. The 2-bud cuttings were grown in a growth chamber after the basal ends were dipped in the following treatments; for 5 seconds in IAA solutions at 0 (the control), 5.7, 11.4 and 16.1 μM concentrations, and for 10 minutes in the melatonin solutions at 0 (the control), 0.1, 0.5 and 1.0 μM. The results indicated that percentages of rooting, shooting and healthy plants in both species were not statistically significant under the influence of the treatments. However, increasing concentrations of IAA resulted in increasing rooting percentages in 5BB rootstock. On the other hand, 0.1 and 0.5 μM MEL also induced rooting. In the cuttings of Cabernet Sauvignon, 11.4 μM IAA provided the highest percentage (100%). Melatonin caused a relative decrease in rooting compared to the IAA. The action of melatonin on rooting seems to be independent of IAA. Hormone treatments caused significant differences in the Research Article Article History Received : 09.01.2020 Accepted : 16.04.2020


INTRODUCTION
In woody perennial horticultural species, propagation with cuttings destined to be used in direct planting or in grafting purposes is almost compulsory to ensure the perpetuation of same genetic material over generations. In grapevine propagation, different rootstocks with varying degree of rooting ability make it necessary to treat the cuttings with auxins to increase root formation.
Using plant growth regulators on promoting cuttings to form adventitious roots has been a practice for a very long time. Among them, auxins are the one that has acquired the name root-promoting hormones. These are signals that not only affect root formation but also involve in variety of processes during plant growth and development (Woodward and Bartel, 2005). With the advent of new information regarding phytohormones and signal molecules, it is clear now that these compounds interact with each other during root formation, a process in which auxin is the major factor (Lavenus et al., 2013). In a coordinated manner, auxin acts with cytokinins, gibberellins, ethylene, abscisic acid, jasmonate and strigolactones, all of which have their own specific biosynthesis and signaling pathways. The evidence clearly indicate that root formation and growth depend on communication among these compounds (Pacifici et al., 2015).
Since the discovery in plants in 1995 (Dubbels et al., 1995;Hattori et al., 1995), melatonin (MEL), which was long thought a molecule found only in vertebrates, has drawn some attention regarding its role in regulating a plant's physiology. Particular attention was given since melatonin is an indoleamine compound, as auxins are, and both are derived from tryptophan in tryptophan-dependent IAA biosynthetic pathway. Similar molecular structure of these two molecules (IAA and MEL) led scientists to think that melatonin might have the comparable or similar stimulating effect as IAA under certain conditions (Arnao, 2014).
Considering that auxins (IAA, IBA, and NAA) are known for their inducing effects on adventitious root formation, a question regarding if MEL might have a matching effect has risen. Studies on lupin and barley (Hernández-Ruiz et al., 2004Hernández-Ruiz and Arnao, 2008) showed that MEL, depending on the concentration applied, had an influence on roots similar to IAA.
Melatonin in grapevine has been first identified by Iriti et al. (2006) in some Vitis vinifera L. cultivars.
Other studies on grape tissues (Stege et al., 2010;Boccalandro et al., 2011;Vitalini et al., 2011aVitalini et al., , 2011bVitalini et al., , 2013 have followed. Meng et al. (2014) showed that treating roots of 1-year old Riesling canes with melatonin helped better growth by stimulating waterstress tolerance. To this date, best to our knowledge, an experiment regarding determination of effects of melatonin applied during cutting preparation on root formation and growth was not performed. In studies by Sarropoulou et al. (2012aSarropoulou et al. ( , 2012b it was stated that MEL increased rooting of some sweet cherry rootstock. Pioneering this work, a study was designed to see if MEL promotes adventitious root formation as IAA does, in an American rootstock and a Vitis vinifera L. cultivar.

MATERIAL and METHODS
Cuttings prepared from the American rootstock 5BB, and the Vitis vinifera L. cv. Cabernet Sauvignon (CS), were obtained from The Viticulture Research Institute, Tekirdag, Turkey. These were chosen for their easy to root aspects. Cuttings were shortened to 2-bud sections, with the lower bud removed. They were cut straight at the bottom and 45° slanted at the top away from the bud. Plant growth regulator (PGR) treatments in which the basal ends (2 cm in depth) of the cuttings were dipped were carried out as follows: 5 second-dipping in IAA at 0 (the control), 5.7 µM (equivalent of 1000 ppm), 11.4 µM (eq. of 2000 ppm) and 16.1 µM (eq. of 4000 ppm) concentrations, and 10 minute-dipping in the melatonin (Sigma M5250) solutions at 0 (the control), 0.1, 0.  At the end of the experiment following characteristics were evaluated for both rooting and shooting aspects (Uzunoğlu and Gökbayrak, 2018) percentages (%) of rooting and shooting along with healthy plant ratio which was determined by assessing the healthy development of root and shoots on the same vine, root number per vine, a longest root length (cm), weights of fresh and dry roots and ash ratio (%). In addition, distribution pattern of the root emerged was also assessed as the place of the roots developed on the quadrant plane at the bottom of the vine by denoting 0-4 scale (0 being no roots emerged, 4 being roots emerged from all quadrants).

Statistical analysis
The study was conducted as a completely random trial design with three replicates (30 cuttings in total) per treatment. A total of 480 cuttings were evaluated. Data were statistically analyzed using Minitab statistics software (Release 16 for Windows, Minitab Inc.). Significant differences between the means were tested with Tukey test.

RESULTS
The effects of two indolamine compounds, IAA and MEL, on root and shoot development of 5BB and CS cuttings were evaluated in a growth chamber. The results indicate that percentages of rooting, shooting and healthy plants in 5BB rootstock were not statistically significant under the influence of the treatments ( Figure 1A). However, it was obvious that increasing concentrations of IAA resulted in increasing rooting percent in 5BB rootstock. Interestingly, cuttings of MEL control (in water only for 10 min) provided a complete and total rooting. However, 5 sec. dipping (i.e., IAA control) resulted in loss of 6 cuttings out of 100, indicating the inherent rooting capacity of 5BB. It was also intriguing to see that 0.1 and 0.5 µM MEL also gave plants with 100% roots. However, the highest concentration of MEL applications caused a decrease in rooting percentage (80%). Variations in the shooting and healthy plants stood out more compared to the rooting. With IAA applications, shooting percent showed undulations, changing from 86.7% (5.7 µM) to 100 (11.4 µM) and later 96.7% (16.1 µM). MEL-applied 5BB cuttings developed fewer shoots.
On the CS cuttings, root and shoot growth were evaluated in Figure 1B. The percentages of rooting, shooting and healthy plants were not affected by the IAA and MEL treatments. 11.4 µM IAA provided the highest ratio (100%) in all counts. The 1000 ppm IAA and 4000 ppm IAA applications gave the next best ratio in rooting, (96.7%). Shooting was supported by all the treatments at 100% level, except for the 0.5 µM MEL and the MEL control. Healthy plant percentage took a downfall in all the applications, especially being the worst with MEL control. Melatonin caused a dramatic decrease in rooting compared to the IAA but applying 0.5 or 1.0 µM melatonin still boosted rooting compared to 5 second dipping (IAA control) at least 3%.
Root aspects of the 5BB cuttings after 8 weeks of growth were also presented in Table 1. Statistically significant effect of IAA (16.1 µM) on root number was observed (19.93 roots per cutting). The number of roots in the rest of the treatments were between 12-14.5 roots. However, 0.1 and 0.5 µM MEL resulted in the longest root (10,51 and 9.70 cm, respectively). Longest root length, root distribution pattern and dry root weight, on the other hand, were not affected. Fresh root weight was greatly influenced by the applications, being the heaviest with 0.5 µM MEL (6.27 g) and the lightest with 1.0 µM MEL (3.13 g).

DISCUSSION
The studies involving adventitious root formation in Lupinus albus L. (Arnao and Hernández-Ruiz, 2007) and Arabidopsis (Koyama et al., 2013)  In this study where a growth chamber was utilized, 1.0 µM Melatonin better supported rooting in CS than in 5BB, indicating that the response was genotype specific. Arnao and Hernández-Ruiz (2018) reported that the inhibitory effect of Melatonin on root growth surfaces when the concentration is above 10 µM. In the current study, this negative effect appeared at considerably low concentrations, such that 1.0 µM in 5 BB and 0.1 µM in Cabernet Sauvignon cuttings. This might have been the result of the growth conditions and the state of the plant material. IAA and melatonin maintained a good shooting performance in the cuttings of both 5BB and cv. CS. However, the reaction of CS cv was contingent on the concentration. In contrast to our findings, shooting in pomegranate cuttings treated with single concentration of Melatonin (Sarrou et al., 2014a) was  Root growth aspects of the CS cuttings were under the influence of the hormones (Table 2). Root number was considerably high in the cuttings treated with IAA. However, the differences in the longest root length was not as distinct among the applications. IAA-treated cuttings tended to develop roots from all quadrants with high fresh and dry weights.
Mean number of roots in the cuttings used in the experiment were influenced by the PGR's, in agreement with the study by Sarropoulou et al. (2012b), which revealed that cherry rootstocks produced the highest number of roots at 0.5 and 1.0 µM, following the control. In the cherry rootstock, PHL-C, Sarropoulou et al. (2012a) found the decreasing effect of Melatonin on root number. In this study, 5BB cuttings significantly developed more roots with IAA than with Melatonin. However, with cv. CS, this difference was less prominent, spiking at 16.1 µM IAA and being at similar levels in the rest of the applications. Effects of MEL on the length of the longest root was evident in 0.5 and 1.0 µM MEL. Even though Sarrou et al. (2014) presented the mean length of roots, they still reported the supportive influence of 0.1 and 1.0 µM MEL treatments. Sarrou et al. (2014) also showed that Melatonin caused a prominent increase in the length of the roots formed on the pomegranate cuttings. Hernández-Ruiz et al.
on the other hand, showed that in monocot species IAA and MEL decreased root length.
Fresh weight of roots was generally low in 5BB and cv. Cabernet Sauvignon with melatonin, however 0.5 µM MEL caused a peak in 5BB. Sarropoulou et al. (2012a) stated that low MEL concentrations (0.05-1.0 µM) increased fresh root weight of PHL-C rootstock.
The same tendency was also found with Gisela 6 and MxM 60, but not with CAB-6 P. These contradictory results might be due to genotype differences and sensitivities. The scientists stipulated that the increase could be from the volume increase in old cells and/or cell extension in new cells. Since dry weight did not increase in a parallel way to the fresh weight it led us to think that the increase was due to a volume increase by accumulating more water in the cells.

CONCLUSION
Results indicated that effects of Melatonin on adventitious root formation in the 5BB and Cabernet Sauvignon cv. need more extensive studies. Its effect is not the same or as much extent of IAA and depend on its concentration. Both genotypes certainly had a better response to IAA applications. Further studies involving the underlying mechanism which Melatonin might exert on root formation would enable us to find out more about the crosstalk among signal molecules. There might be a possibility of using Melatonin in practical applications since it is inexpensive and easy to obtain in pure form.

Statement of Conflict of Interest
Authors have declared no conflict of interest.

Author's Contributions
The contribution of the authors is equal.