Development of a Rooted Cutting Propagation Method for Selected Arbutus unedo L. Types and Seasonal Variation in Rooting Capacity

In this study, the rooting abilities of semi-hardwood cuttings from 8 Arbutus unedo L. (strawberry tree) types were evaluated. For this purpose, cuttings were taken at two different vegetation periods in July and November. The collected cuttings were treated with IBA (2, 4, 6, 8 and 10 g/l) and placed into a misting system in July and bottom-heated system in November in the greenhouse. The rooting rate, survival rate and root quality were determined during the study. The cutting collection period significantly affected rooting ability, and according to the results obtained the best rooting performance was obtained with July cuttings except for types 1 and 8. There were differences among the A. unedo L. types in rooting ability; Type 4 had the highest rooting rate (87.01% in July and 70.71% in November) among the types studied. The percentage of rooted cuttings ranged from 0% to 100% for the control and IBA treatment for both periods and cuttings taken in November required higher concentrations of IBA for rooting than cuttings taken in July. The survival rate of cuttings varied according to type in the acclimatization stage, the best types being type 3 and type 4. July cuttings of all types obtained shoots in the rooting media.


Introduction
Arbutus unedo L. (strawberry tree) is an evergreen small tree or shrub largely found as a native in the Mediterranean region.The consumption of locally grown wild edible plants has been important for most human cultures, especially in the Mediterranean region, making an important contribution to the health of local communities (Heinrich, Müller, & Galli, 2006).The Arbutus berries are also used for local vine production and other food applications Seidemann (1995); Pawlowska, De Leo, and Braca (2006); medicinal uses (Bnouham, Mekhfi, Legssyer, & Ziyyat, 2002), landscaping and as a complement in the cut flower industry (Metaxas, Syros, Yupsanis, & Economou, 2004).All populations, mainly near roads and coastlines, have been severely damaged due to deforestation and over-collecting for road-building studies during recent decades.Heterogeneity in the plantations is an important reason for the low-quality fruits and the edible use of the strawberry tree is currently limited.Restricted growing area and lack of commercial plantations leads to destruction of natural populations.
There are some studies to select good quality fruits for A. unedo L. (Songlin et al., 1995;Cai-Huang, 1997;Çelikel, Demirsoy, & Demirsoy, 2008;Şeker, Akçal, Sakaldaş, & Gündoğdu, 2010).Sulusoglu, Cavusoglu and Erkal (2011) evaluated pomological and chemical characteristics of 37 types of A. unedo L. in Samanli mountain locations in Marmara region of Turkey and according to the results; five types were selected as being superior.Vegetative propagation of these selected types is necessary to provide material for commercial use and orchard design and to reduce the natural habitat degradations.Generally A. unedo is characterized as a difficult-to-root plant (Hartmann, Kester, Davies, & Geneve, 2002;Şeker, Akçal, Sakaldaş, & Gündoğdu, 2010).Rooting of cuttings depends on the genotypes, age of the source tree, degree of hardening of the cuttings, injury and heat treatments of the cuttings, the collection date and the treatment concentrations of auxin-like compounds (Metaxas, Syros, Yupsanis, & Economou, 2004;Pignatti & Crabeddu, 2005); Metaxas, Syros, & Economou, 2008;Şeker, Akçal, Sakaldaş, & Gündoğdu, 2010).Among the Mediterranean scrubs, A. unedo is known for intensive activity in spring (Gratani & Ghia, 2002) while in the Mediterranean climate a second period of growth starts with the first storm rains in August (Pignatti & Crobeddu, 2005).This species bears fruit in late autumn and blossoms at the same time for the following year's fruits.Morphological and physiological characteristics of the stock plants at time of cutting collection affected the cutting rooting, and the optimal time for rooting should be established individually for each plant (Davies, 1984;Hansen, 1990;Howard, 1996;Klein, Cohen, & Hebbe, 2000).
The primary objectives of this research were the evaluation of the rooting performance of the selected superior genotype and to clarify the importance of genotypes, concentration of auxin and cutting collection time on rooting of A. unedo L. Thus the wild growing genetic material will be protected, an alternative fruit crop will be obtained for growers, and abundant high quality fruits will be supplied to consumers.

Plant Material
The investigations were conducted with the native grown A. unedo L. types selected in previous studies (Sulusoglu, Cavusoglu, & Erkal, 2011) from the eastern part of the Marmara region in Samanli Mountain locations of Turkey.Some characteristics of types were given in Table 1.Semi hardwood cuttings were collected on the 10 th of July in 2010 and 2011 and 10 th of November in 2009 and 2010.Immediately after collection, the cuttings were cut into 15-cm pieces and were cleaned from the leaves, leaving only a half leaf at the top portion of the cuttings.Then the cuttings were treated with a commercial fungicide (benomyl) by dipping in a liquid solution for 2 minutes.After surface drying, the bases of the cuttings were dipped for 10 s in one of five concentrations of IBA (2, 4, 6, 8 and 10 g/1 IBA), which is dissolved in a solvent composed of 50% ethanol and 50% tap water.The control cuttings were treated with the 50% ethanol only.

Rooting Medium and Rooting Conditions
The cuttings were then planted in mist beds by inserting their bases about 5 cm into a rooting medium composed of perlite in an un-heated greenhouse.The cuttings were maintained in the system using an automatical fogging system (three minutes of fogging every hour during the lighted period) in July.The greenhouse had an evaporative cooling system (cooling pads and fan) to prevent the negative effects of high temperatures.In November the mist bed was covered with a polyethylene sheet for protection against the cold, and a bottom heating system was run.
Additionally hydration was performed manually in this season.

Evaluations of Results
The rooting process was monitored after the beginning of the experiments for the presence of callus, root and shoot.Cuttings were removed from rooting unit after 12 weeks for July cuttings and 15 weeks for November cuttings and rooting data was taken.
The experiment was conducted in a completely randomized plot design.Each treatment was comprised of an average of 30 cuttings (three replications of ten plants).Each experiment was repeated at least once.Rooting response (as rooting percentage, root number, root length), shoot and callus formation were measured.The arcsine square root transformation (Steel & Torrie, 1960) was applied to the data to provide a normally distributed data set for the ANOVA.Data were subjected to ANOVA using Minitab Software (MINITAB Inc.) and the means were separated by Duncan's Multiple Range Test (P≤ 0.05).

Results
Types used in this experiment had large variations in rooting ability, with rooting percentages ranging from 0-100 percent according to IBA concentrations and cutting collection time.A significant interaction was observed between the cutting harvesting time and IBA concentrations for the all observed criteria.
Survival rates of type 1 cuttings were high in July but rooting rate, root length and root number were higher in November according to average values.Maximum rooting percentages were observed with 4 g/l IBA concentrations in both cutting collection times.The longest roots (3.12 cm) were obtained with 4 g/l IBA concentrations in November while the root number (5.67) was significantly increased with 8 g/l IBA, again in November (Table 2).Callus formation was significantly high in November (Figure 1).Acclimatization rate was also better in cuttings to which was applied 8 g/l IBA for rooting in November (Figure 2) and this type displayed the lowest survival rate in the acclimatization stage.
Survival rates increased in July-collected cuttings of type 2. Type 2 needed high concentrations of IBA; the maximum rooting rate, root number and length were recorded in 8 g/l IBA concentration in July and 10 g/l IBA concentration in November (Table 3).Cuttings rooted with these IBA concentrations also survived better in the acclimatization stage (Figure 2).
Survival rates of type 3 cuttings were significantly higher with 6 g/l IBA concentration in July.At the end of the experiment the percentage of rooted control cuttings of type 3 reached 69.84% in July and this effect was statistically similar to the effects of IBA concentrations except 10 g/l IBA.Increased concentrations of IBA negatively affected the rooting capacity; the lowest rooting rate was recorded with 10 g/l IBA.November cuttings showed a different response to hormone concentrations and maximum rooting was recorded with 8 g/l IBA concentrations (Table 4).In the acclimatization stage, the cutting survival rate was higher in July except for cuttings rooted with 8 g/l IBA (Figure 2).
The best quality rooted and survived cuttings were taken from type 4. Rooting was observed in control cuttings at both cutting collection times.All cuttings treated with 8 and 10 g/l IBA survived and rooted (100%) in July (Table 5).Rooting was significantly increased with higher concentrations of IBA in July, while it decreased in November.It is shown that IBA concentration effects changed seasonally the rooting performance of A. unedo L. cuttings.The average root number was higher in November than in July and all IBA concentrations increased the root number in this time, the result being statistically important as well.Callus formation was very limited in July and November (Figure 1).Survival rates were quite high among the types in the acclimatization stage, and planted cutting numbers were at a satisfactory level (Figure 2).
Survival and rooting rates decreased with increasing concentrations of IBA in both cutting collection times (July and November) for type 5. Rooting was observed in untreated control cuttings in July.The best rooting was observed with 4 g/l IBA-applied cuttings at July and November cutting collection times (Table 6).The longest root (9.88 cm) was obtained with 10 g/l IBA in July.A high amount of callus formation was observed in the July cuttings (Figure 1).The survival rate of July cuttings rooted with high concentrations of IBA was low in the acclimatization stage for this type of A. unedo L. (Figure 2).
In the case of type 6 cuttings, increasing concentrations of IBA caused the death of cuttings in July and this effect is statistically important as well.The maximum rooting was achieved with 4 and 6 g/l IBA treated cuttings in July while untreated cuttings or cuttings treated with 10 g/l IBA did not root.Rooting was achieved with high concentrations of IBA in November; rooting was observed in 8 and 10 g/l IBA applied cuttings (Table 7).The survival rate was high for cuttings taken in July; only cuttings treated with 8 g/l IBA could continue to growth after transfer to the acclimatization stage in November (Figure 2).
With all concentrations of IBA the cutting survival rate was higher in July than in November for type 7, but the highest rooting rate (61.11%) was obtained with 8 g/l IBA concentrations in November (Table 8).This concentration of IBA also increased the number of roots at significant rate (20 roots per cutting).Cuttings collected in July and rooted with 6 g/l IBA fared better after transfer to the soil mixture in the acclimatization stage (Figure 2).
The proportion of type 8 cuttings that rooted was relatively high in November, compared to July.Only 4 g/l IBA obtained rooting in July but rooting percentages (18.89%) and quality (root number is 3.25 and root length is 2.50 cm) was very low so that November is the better time for cutting collection and rooting for this type (Table 9).Cuttings collected in July did not develop any callus, while a very high level of callus was observed on cuttings collected in November (Figure 1).The survival rate was very low and all cuttings were dead after transfer for acclimatization except those rooted with 4 g/l IBA in July and 6 g/l IBA in November (Figure 2)., 3, 4, 5, 6, 7, 8, and 9, values in the same column (IBA doses) with different lower-case letters and values in the same row (cutting collection time) with different capital letters are significantly different at P≤0.05.

Table 1 .
Characteristics * of cutting collected types

Table 2 .
Survival and rooting characteristics of type 1